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CORONARY STENT RESTENOSIS

CORONARY STENT RESTENOSIS

EDITORS: ION C. INTOIU JEFFREY J. POPMA JANG-HO BAE ALAIN RIVARD ALFREDO R. GALASSI GABRIEL CRISTIAN

THE PUBLISHING HOUSE OF THE ROMANIAN ACADEMY Bucharest, 2011

Copyright 2011, Editura Academiei Romne. All the rights reserved.

THE PUBLISHING HOUSE OF THE ROMANIAN ACADEMY


Calea 13 Septembrie nr. 13, sector 5, 050711, Bucureti, Romnia Tel.: 4021-318 81 06, 4021-318 81 46 Fax: 4021-318 24 44 E-mail: edacad@ear.ro Internet: http//www.ear.ro

Peer reviewers: Acad. VICTOR VOICU Prof. univ. dr. IOAN BRUCKNER

Descrierea CIP a Bibliotecii Naionale a Romniei Coronary stent restenosis / ed.: Ion C. intoiu, Jeffrey J. Popma, Jang-Ho Bae, ... Bucureti: Editura Academiei Romne, 2011 ISBN 978-973-27-2034-9 I. intoiu, Ion (ed.) II. Popma, Jeffrey J. (ed.) III. Bae, Jang-Ho (ed.) 616.12

Editorial assistants: MONICA STANCIU DOINA ARGEANU Tehnical operator: SOFIA MORAR Cover: MARIANA ERBNESCU Final proof: 10.05.2011. Format: 8/61 86. Proof in sheets: 101. D.C.L. for large libraries: 616.12(082) D.L.C. for small libraries: 616

ACKNOWLEDGEMENTS
The Editors, facing many constraints, made the difficult choice to limit the depth of topics covered in favor of widening the scope of the subject matter addressed. We hope that this approach may best serve the goal of providing a reference source for students, residents and scientists working in the field. Unfortunately there are many important scientific topics and scientists that have been omitted from this book. To this end, Editors regret not being able to accommodate many deserving and creative scientists whose contributions would have greatly enriched the book. We thank the contributing authors for their gift, and the sponsors for both the moral and financial support that helped launch this project. The Editors collectively pledge that the book will be donated to the schools, hospitals and libraries everywhere. We hope that the book will serve as a source for fertilizing and evoking new scientific thoughts and experiments something our friends in and outside Europe would liked very much. Finally, I want to thank my students, residents, fellows and colleagues for their input and particularly for their challenging questions over the years. I am grateful to my colleagues Francisca B. Clinescu, Adela Crstea, and Daniel Ni, for their work and commitment. I am most greatful to my friend and colleague, Ambrose Kibos, for his many helpful works, suggestions and comments in the preparation of this new Edition. And finally I wish to thank my family for patience, spiritual and moral support. Prof. Ion C. intoiu

FOREWORD
In-stent restenosis is a new pathology, occurred as a result of introduction in interventional revascularization of different stent types, approaching of complex lesions and different coronary vessel wall structures which have a variable response to the injury produced by stent implantation. This monograph is written by distinguished personalities with vast experience in the stent implantation and participants in different trials completed or ongoing. The structure of this book is developed in sequential stages (genesis, diagnosis, treatment) where differentiated analysis of coronary stent restenosis was made, depending of stent design and lesion particularities. Technological advances in the stents field are determined by the unresolved issue of restenosis of which initially percentage in the BMS era, reached 2040% and today is found in varying percentages (0.59%). Because BMS were the most used in the beginnings, the authors analyze their current stage in comparison with stents of new generations. The second part of this monograph presents main in-stent restenosis diagnosis methods in terms of their value, in revascularization procedures and also in determining therapeutic solutions. The treatment of in stent restenosis is difficult and dependent of many parameters: initial lesions complexity, sequential lesion, total occlusion, antiplatelet treatment and is based on multiple procedures: brachytherapy, rotational atherectomy, re-stenting strategies, balloon dilatation. New generations of stents (bioresorbable, EPC, gene therapy) are used with good results in revascularization for in-stent restenosis. Perspectives in preventing restenosis involve use of gene therapy and angiogenesis. Antiplatelet treatment is a fundamental element of restenosis and resistance to antiplatelet treatment is a clinical reality which necessitates determination measures. The existent clinical guides establish criteria for individualization of revascularizatInterventional revascularization represents an important advancement of the last two decades in the coronary lesion treatment. In the development of this therapeutic procedure complications have occurred, among which restenosis and in stent thrombosis are the most common, along with coronary lesion progression. In this monograph, important personalities of interventional cardiology analyze in detail the phenomenon of restenosis, comprising the steps of molecular and cellular genesis, diagnostics and treatment. Trials and meta-analysis were reviewed by world-renowned specialists, with panoramic view on the current state of different procedures, through the phenomenon of restenosis The book contains chapters regarding clinical reality, stent structure and design, restenosis prevention, risk factors identification, individualized treatment, the relation between stent type and the lesion suitable for dilatation, etc. These arguments make the book valuable in preparing students, physicians with interventional profile and also for device manufacturing companies. The complexity of restenosis phenomenon is well analyzed in this monograph and includes current and future stages in technology and interventional procedure, focusing on the types of stent currently used and on those tested in trials (bioresorbable stent, endothelial progenitor cells captures, etc.). Utilization of different stent types that are today on the market is viewed retroactively and through trials and metaanalysis which have proved that in-stent restenosis still exist, requiring a difficult and particular therapy (stent in stent,

ion procedure, choosing stent type and particularization of stent-lesion relationship, but often do not find their explanation in clinical reality. Preventing restenosis by correction of risk factors will probably decrease need of interventional procedures in the future. We consider that the present book represents a significant synthesis in a field of high scientific interest for both research and clinical practice, written by authors with an ample experience, thus the work will be very useful tool for those acting in this area. Victor Voicu MD PhD
Fellow of Romanian Academy Professor, Head of the Dept. Pharmacology and Clinical Toxicology University Medicine and Pharmacy, Carol Davila, Bucharest President of Romanian Society of Clinical Pharmacology, Therapeutics and Toxicology Director of the Romanian Army Center for Medical Research

rotablation, cutting balloon, drug eluting balloon, aortocoronary bypass, etc.). All these procedures have been analyzed one by one and viewed from the point of view of efficacy and restenosis. The correlation between the stent type and restenosis is confirmed by a decreased restenosis rate in DES compared with BMS, and also in bioabsorbable stents and EPCc compared with DES. The third generation of DES which differs by structure, elution mechanism and binding polymer is presented in the book as being superior to the first and the second generation. Restenosis does exist in all types of stents in various percentages and this is why research in this area will continue. Army General Vasile Cndea MD, PhD
Professor of Cardiovascular Surgery Carol Davila University of Medicine, Bucharest President of the Academy of Romanian Scientists

PREFACE
Interventional treatment of coronary lesions represents a revolutionary solution in cardiology field of the last two decades. After the pioneering stage with the first coronary dilatation performed by Grntzig in 1977 and after the initial enthusiasm, complications of this procedure appeared: restenosis and in stent thrombosis. Important research followed on many levels from the lesions suitable for revascularization to the structure and design of the new types of stents. Multiple trials and meta-analysis present restenosis as an unresolved issue, that causes further research in this area in numerous directions: risk factors analysis, better structure and design of stents based on the new concepts (bioresorbable stents, endothelial progenitor cells, etc.), all being currently in the phase of clinical evaluation. In-stent restenosis cause difficulties in clinical practice, involving the right choice of stent type according to patients characteristics and lesion type and also being dependent by antiplatelet treatment response. Considering restenosis as a healing response of the coronary vessel wall injury produced by stent implantation, researches continues sequential on each stage of this process, but unsuccessfully until now, in obtaining a neoendothelium similar to that of a coronary vessel unaffected by atherosclerosis. In this book we approached the restenosis phenomenon in its entire complexity from interpreting it as a new disease, going through current stage of stent structuring, diagnostic methods, trials and meta-analysis which studies comparisons of the process in different stents and lesions types, up to the existing therapeutic solutions. Writing this monograph we enjoyed participation of prestigious personalities of interventional cardiology from European countries, Asia, U.S.A and Latin America, whom we would warmly want to thank. We tried to approach in-stent restenosis having in view its complexity and to answer a few difficult questions: why does coexist permeable stent with in-stent restenosis, how do we choose the right stent type to prevent restenosis, which is the most efficient antiplatelet prevention treatment, how to select most adequate therapy for each restenosis pattern, what is the role of surgical revascularization for restenosis treatment, which is the most precise diagnostic method for restenosis quantification, which are the structuring principles for the ideal stent, what histology lesion elements pre and post revascularization lead to restenosis, which is the best stent type for a certain lesion type. Remains many unsolved problems in restenosis chapter which does maintain the stent war in the pipeline. In-stent restenosis represents a complicated pathology because is generated by multiple processes, some yet unclear, and because atherosclerosis efficient prevention is still an unsolved problem. The future of interventional revascularization therapy and restenosis prevention lies most probably as P.W. Serruys predicts, in the field of molecular biology. Ion C. intoiu Jeffrey J. Popma Jang-Ho Bae Alain Rivard Alfredo R.Galassi Gabriel Cristian

EDITED BY
ION C. INTOIU MD, PhD, FESC Professor of Cardiology, Carol Davila University of Medicine Professor of Medicine, Titu Maiorescu University Invasive Cardiology Department Armys Clinic Center for Cardiovascular Diseases, Bucharest, Romania JEFFREY J. POPMA, MD, PhD Professor of Medicine, Harvard Medical School, Director, Interventional Cardiology Clinical Services- Beth Israel Deaconess Medical Center, Boston, USA JANG-HO BAE MD, PhD, FACC, FSCAI Professor of Internal Medicine, Invasive Cardiology, Konyang University Hospital Heart Centre, Daejeon, South Korea ALAIN RIVARD, MD, PhD Associate Professor of Medicine, Invasive Cardiology, Central Hospital of Montral, Qubec, Canada ALFREDO R. GALASSI, MD, FACC, FESC, FSCAI Professor of Medicine Head of Catheterization Laboratory and Cardiovascular Interventional Unit Invasive Cardiology, Department of Cardiology, Ferrarotto Hospital, University of Catania, Italy GABRIEL CRISTIAN, MD, PhD, FESC Associate Professor of Cardiology, Titu Maiorescu University Director of Armys Clinic Center for Cardiovascular Diseases, Bucharest, Romania

ASSOCIATE EDITORS
AMBROSE SAMWEL KIBOS, MD, PhD Department of Cardiology, Invasive Cardiology, Armys Clinic Center for Cardiovascular Diseases, Bucharest, Romania Department of Cardiology, Bathurst Chaleur Regional Hospital,Canada MALCOLM JOHN UNDERWOOD, MB ChB, FRCS (Ed), FRCS (CTh), MD, FCSHK Professor of Medicine, Chief, Division of Cardiothoracic Surgery Department of Surgery, The Chinese University of Hong Kong Prince of Wales Hospital, Shatin, NT, Hong Kong HANS BONNIER MD, PhD, FESC, FSCAI Professor of Medicine, Invasive Cardiology, University Ziekenhuis, Brussel Belgium YOSHINOBU MURASATO, MD, Ph.D Director of Heart Center, Department of Cardiovascular Medicine Invasive Cardiology, New Yukuhashi Hospital, Japan J. WOUTER JUKEMA, MD, PhD, FESC, FACC Professor of Cardiology, Chairman Leiden Vascular Medicine Invasive Cardiology, Deparment of Cardiology C5-P, Leiden University Medical Center, Leiden, Netherlands EVELYN REGAR, M.D, Ph.D Associate Professor of Medicine, Invasive Cardiology Department of Interventional Cardiology, Clinical Head & Medical Coordinator Thoraxcenter, Erasmus MC, Rotterdam, Netherlands

CONTRIBUTORS
TAREK A.H.N. AHMED MD Departament of Cardiology Leiden University Medical Center, Leiden Netherlands AAMER ABBAS MD Texas Tech University Health Sciences Center, El Paso, Texas USA ARMAN A. ABDULLAH MD, PhD Department of Radiology Harapan Kita National Cardiovascular Center Jakarta, Indonesia TAKASHI AKASAKA MD, PhD Professor of Medicine Department of Cardiovascular Medicine Wakayama Medical University, Wakayama Japan ALBERT E. ALAHMAR MD Invasive Cardiology University Hospital of Leicester United Kingdom MARIANO ALBERTAL MD, PhD Invasive Cardiology, Department of Interventional Cardiology and Endovascular Therapeutics Instituto Cardiovascular de Buenos Aires Ciudad Autonoma de Buenos Aires Argentina ZAKARIA A. ALMSHERQI MBBch, PhD Research Scientist and Lector Department of Physiology National University of Singapore National University Hospital, Cardiovascular and Thoracic Surgery Dept. Singapore DOMINICK J. ANGIOLILLO MD, PhD University of Florida College of Medicine-Jacksonville, Florida USA YUJIRO ASADA MD Professor of Medicine Department of Pathology, Faculty of Medicine University of Miyazaki, Kihara, Kiyotake Miyazaki, Japan TAKASHI ASHIKAGA MD, PhD Clinical Professor of Medicine, Invasive Cardiology, Department of Cardiology, National Hospital Organization, Disaster Medical Center Tokyo Japan RETNO DWI ASTUTI MD Department of Radiology Harapan Kita National Cardiovascular Center Jakarta, Indonesia JANG-HO BAE MD PhD, FACC, FSCAI Professor of Internal Medicine Invasive Cardiology, Konyang University Hospital Heart Centre, Daejeon South Korea ERBAN BLNESCU MD, PhD, FESC Associate Professor of Medicine Invasive Cardiology, Cardiology Department Bucharest Emergency Hospital, Bucharest Romania JORGE A. BELARDI MD, FACC, FSCAI Professor of Medicine Invasive Cardiology, Department of Interventional Cardiology and Endovascular Therapeutics Instituto Cardiovascular de Buenos Aires Ciudad Autonoma de Buenos Aires Argentina BALZS BERTA MD Heart Center, Semmelweis University, Budapest Hungary GERARD CLAUDE BLOCH MD, PhD Professor of Cardiac Surgery Hospital Piti-Salptrire American Hospital of Paris, Paris France HANS BONNIER MD PhD, FESC, FSCAI Professor of Medicine Invasive Cardiology, University Ziekenhuis Brussel Belgium NICO BRUINING MD Department of Interventional Cardiology Thoraxcenter, Erasmus, MC, Rotterdam Netherland

14 ROSAMARIA BRUNI MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine University Magna Grcia of Catanzaro, Catanzaro Italy FRANCISCA B. CLINESCU MD Vascular Surgery Department Armys Clinic Center for Cardiovascular Diseases Bucharest Romania LIVIU CHIRIAC MD, PhD Associate Professor of Phisiology Titu Maiorescu University Invasive Cardiology Department Armys Clinic Center of Cardiovascular Diseases Bucharest Romania ADELA CRSTEA MD Invasive Cardiology Departmet Armys Clinic Center for Cardiovascular Diseases Bucharest Romania GIM-HOOI CHOO MBBS (Malaysia): MRCP (UK), FNHAM; FSCAI Invasive Cardiology Sime Darby Medical Centre Malaysia CYRIL COHEN MD Strasbourg University Hospital Louis Pasteur University of Medicine France ANDR CONSTANTINESCO MD, PhD Professor of Medicine Strasbourg University Hospital Louis Pasteur University of Medicine France STPHANE PIERRE COOK MD Professor of Medicine Head of Cardiology University Fribourg, Fribourg Switzerland GABRIEL CRISTIAN MD, PhD, FESC Associate Professor of Cardiology Titu Maiorescu University Head of Armys Clinic Center of Cardiovascular Diseases, Bucharest Romania HIROYUKI DAIDA MD, PhD Professor of Medicine Division of Cardiology, Department of Internal Medicine Juntendo University School of Medicine, Tokyo Japan DEBABRATA DASH MD, DM, FICC Senior Interventional Cardiologist Invasive Cardiology, Asian Heart Institute Bandra Kurla Complex, Mumbai India JORGE DE FRANCISCO MD Department of Neurology Hospital Universitari Mutua Terrassa, Barcelona Spain MARGREET R. DE VRIES BSc Einthoven Laboratory for Experimental Vascular Medicine, Department of Surgery Leiden University Medical Center Netherlands SERENA DI CELLO MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine,University Magna Grcia of Catanzaro, Catanzaro Italy IONEL DROC MD, PhD Head of Vascular Surgery Department Armys Clinic Center for Cardiovascular Diseases Bucharest, Romania SILVIU DUMITRESCU MD Cardiology Department Armys Clinic Center for Cardiovascular Diseases Bucharest, Romania MARK M. EWING MSc Department of Cardiology Einthoven Laboratory for Experimental Vascular Medicine, Dept of Surgery Leiden University Medical Center, Leiden Netherlands GERGELY FEHR MD, PhD Assistant Professor Department of Neurology University of Pcs, School of Medicine, Pcs Hungary ROBERTO KALIL-FILHO MD, PhD Professor of Medicine Non-Invasive Cardiology Sirio-Libanes Hospital (Chairman) Heart Institute (InCor) University of Sao Paulo Medical School Brazil

15 ALFREDO R. GALASSI MD, FACC, FESC, FSCAI Professor of Medicine Head of Catheterization Laboratory and Cardiovascular Interventional Unit Invasive Cardiology, Department of Cardiology Ferrarotto Hospital, University of Catania, Italy ALAN YEAN-YIP FONG MD Sarawak General Hospital Jalan Hospital Malaysia GIULIA GALIANO LEONE MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine University Magna Grcia of Catanzaro, Catanzaro Italy ANTHONY H. GERSHLICK MD Professor of Medicine Invasive Cardiology University Hospital of Leicester United Kingdom LUCIAN GHEORGHE MD Cardiac Surgery Department Armys Clinic Center for Cardiovascular Diseases Bucharest, Romania FRANOIS GOBEIL MD, PhD Associate Professor of Medicine Invasive Cardiology, Centre Hospitalier de lUniversit de Montral, Qubec, Canada CHRISTIAN GOETZ MD, PhD Strasbourg University Hospital Louis Pasteur University of Medicine France NIEVES GONZALO MD Department of Interventional Cardiology Thoraxcenter, Erasmus, MC, Rotterdam Netherland LAURA GRECO MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine, University Magna Grcia of Catanzaro Catanzaro Italy JAKOB HEINZ MD, PhD West German Heart Center Department of Thoracic and Cardiovascular Surgery Essen Germany SONIA HUERTAS MD Department of Neurology Hospital Universitari Mutua Terrassa Barcelona Spain JUEY-JEN HWANG MD, PhD Professor of Medicine Cardiovascular Division, National Taiwan University Hospital and College of Medicine Tatian Taiwan Clinic Taiwan SAHOKO ICHIHARA MD, PhD Department of Human Functional Genomics, Life Science Research Center Mie University, Tsu Japan HIDEKI ISHII MD, PhD Associate Professor of Medicine Department of Cardiology Nagoya University Graduate School of Medicine Japan ZOLTN JAMBRIK MD Heart Center, Semmelweis University, Budapest Hungary ZAIN JAMIL MD Department of Interventional Cardiology Thoraxcenter, Erasmus, MC, Rotterdam Netherland MYUNG-HO JEONG MD, PhD, FACC, FAHA, FESC, FSCAI Professor of Medicine Invasive Cardiology, Director of Cardiovascular Research Institute, Chonnam National University Hospital South Korea J. WOUTER JUKEMA MD, PhD, FESC, FACC Professor of Cardiology Chairman Leiden Vascular Medicine Invasive Cardiology, Department of Cardiology C5-P, Leiden University Medical Center, Leiden Netherlands WOONG-CHOL KANG MD, PhD Associate Professor of Medicine Invasive Cardiology,Gil Medical Center Gachon University, Guwol-Dong, Incheon South Korea

16 IOANNIS KARALIS MD Department of Cardiology Leiden University Medical Center, Leiden Netherlands JACCO C. KARPER MSc Einthoven Laboratory for Experimental Vascular Medicine,Department of Surgery Leiden University Medical Center Netherlands AMBROSE SAMWEL KIBOS MD, PhD Department of Cardiology Invasive Cardiology, Armys Clinic Center for Cardiovascular Diseases, Bucharest, Romania DONG-BIN KIM MD Invasive Cardiology Bucheon St. Mary Hospital The Catholic University of Korea Sosa-dong, Wonmi-gu, Bucheon City South Korea HYUN-KUK KIM MD Invasive Cardiology Chonnam National University Hospital South Korea HEE-YEOL KIM MD, PhD Associate Professor of Medicine Invasive Cardiology, Bucheon St. Mary Hospital The Catholic University of Korea Sosa-dong, Wonmi-gu, Bucheon City South Korea HYO-SOO KIM MD, PhD Professor of Medicine Department of Internal Medicine Director of Cardiac Catheterization Laboratory & Coronary Intervention, Director of National Research Laboratory for Cardiovascular Stem Cell Seoul National University Hospital South Korea YOUNG-JO KIM MD Division of Cardiology Department of Internal Medicine Yeungnam University Hospital South Korea MASASHI KIMURA MD Assistant Director of the Imaging Core Laboratory Toyohashi Heart Center Japan KAZUHISA KODAMA MD, PhD, FACC, FAHA, FJCC Cardiovascular Division Osaka Police Hospital Japan BON-KWON KOO MD, PhD, FACC Associate Professor of Medicine, Invasive Cardiology, Cardiovascular Center, Seoul National University College of Medicine South Korea JERZY KRUPINSKI MD, PhD, DSc Professor of Clinical Neurology Head Cerebrovascular Diseases Department of Neurology Hospital Universitari Mutua Terrassa, Barcelona Spain ONANONG KULAPUTANA MD Associate Professor of Medicine Invasive Cardiology, Cardiac Center King Chulalongkon Memorial, Bangkok Thailand TAEK-GEUN KWON MD Assistant Professor Konyang University Hospital, Daejeon South Korea PEDRO A. LEMOS MD, PhD Professor of Medicine Invasive Cardiology, Sirio-Libanes Hospital Heart Institute (InCor) University of Sao Paulo Medical School, So Paulo Brazil LIAN-YU LIN MD, PhD Assistant Professor Cardiovascular Division Department of Internal Medicine National Taiwan University Hospital Taiwan JOSEF LUDWIG MD, PhD Professor of Medicine Medizinische Klinik II Universitt Erlangen-Nrnberg Germany JOSE MARIANI Jr. MD Senior Interventional Cardiologist Sirio-Libanes Hospital Heart Institute (InCor) University of Sao Paulo Medical School, So Paulo Brazil

17 ALEXIS MATTEAU, MD Invasive Cardiology Centre Hospitalier de lUniversit de Montral Quebec, Canada DEBORAH MAZZAFERRO MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine, University Magna Grcia of Catanzaro Catanzaro Italy GARRY McDOWELL PhD, FRCPath Senior Lecturer in Health Science Faculty of Health Edge Hill University, Lancashire United Kingdom BLA MERKELY MD, PhD, DSc, FESC Professor of Medicine Director of Heart Center, Semmelweis University, Budapest Hungary LAMPROS K. MICHALIS MD, MRCP, FESC Professor of Medicine Invasive Cardiology, University of Ioannina Greece TAKASHI MIYAKE MD Department of Clinical Gene Therapy, Graduate School of Medicine Osaka University, Osaka Japan MASATO MIZUKOSHI MD Invasive Cardiology Department of Cardiovascular Medicine Wakayama Medical University, Wakayama Japan KYOICHI MIZUNO MD, PhD, FAHA, FACC, FSCAI Professor and Chairman Division of Cardiology Nippon Medical School, Tokyo Japan SHINYA MIZUNO PhD, DVM Division of Virology, Department of Microbilogy and Immunology, Osaka University Graduate School of Medicine, Suita Japan IANCU MOCANU MD, PhD Head of Cardiac Surgery Department Armys Clinic Center for Cardiovascular Diseases Bucharest, Romania RYUICHI MORISHITA MD, PhD Professor of Medicine Department of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, Osaka Japan DEBABRATA MUKHERJEE MD Chief, Cardiovascular Medicine Texas Tech University Health Sciences Center, El Paso, Texas USA MUHAMMAD MUNAWAR MD, PhD, FACC, FESC, FSCAI Binawaluya Cardiac Center Department of Cardiology and Vascular Medicine, Faculty of Medicine University of Indonesia, Jakarta Indonesia TOYOAKI MUROHARA MD, PhD Professor of Medicine Department of Cardiology Nagoya University Graduate School of Medicine Nagoya, Japan YOSHINOBU MURASATO MD, Ph.D Director of Heart Center Department of Cardiovascular Medicine Invasive Cardiology, New Yukuhashi Hospital Japan PAOLA NACCARATO MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine, University Magna Grcia of Catanzaro, Catanzaro Italy KATERINA K. NAKA MD, PhD Invasive Cardiology, University of Ioannina Greece TOSHIKAZU NAKAMURA PhD Professor Emeritus Kringle Pharma Joint Research Division for Regenerative Drug Discovery Center for Advanced Science and Innovation Osaka University Japan GAKU NAKAZAWA MD Associate Professor Tokai University, School of Medicine, Ischara, Kanagawa Japan

18 TAMOTSU NISHIDA PhD Department of Human Functional Genomics,Life Science Research Center Mie University, Tsu Japan DANIEL NI MD Invasive Cardiology Departmet Armys Clinic Center for Cardiovascular Diseases, Bucharest Romania MARKUS OBERHNSLI MD Department of Cardiology University Fribourg, Fribourg Switzerland DONG JOO OH MD Cardiovascular Center, Korea University Guro Hospital Seoul South Korea MICHAIL J. PAPAFAKLIS MD, PhD Invasive Cardiology University of Ioannina Greece ELD PAPP MD, PhD, FESC Head Physician Division of Cardiology Zala County Hospital, Zalaegerszeg Hungary JONG-SEON PARK MD, PhD Division of Cardiology Department of Internal Medicine Yeungnam University Hospital, Daegu South Korea ALESSANDRA PASCALE MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine, University Magna Grcia of Catanzaro Catanzaro Italy ANTONIO PATA MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine, University Magna Grcia of Catanzaro Catanzaro, Italy FRANCESCO PERTICONE MD, PhD Professor of Internal medicine Cardiovascular Disease Unit Department of Clinical and Experimental Medicine, University Magna Grcia of Catanzaro, Catanzaro Italy FLORINA PINTE MD, PhD, FESC Head of Catheterization Laboratory Armys Clinic Center for Cardiovascular Diseases Bucharest, Romania KANHAIYA LAL PODDAR MBBS, MD Cardiovascular Center Korea University Guro Hospital, Seoul Korea JEFFREY J. POPMA MD, PhD Professor of Medicine Harvard Medical School Director, Interventional Cardiology Clinical Services Beth Israel Deaconess Medical Center Boston, USA BRIAN J. POTTER MD Invasive Cardiology Centre Hospitalier de lUniversit de Montral Canada KUSHAL PUJARA MD Invasive Cardiology University Hospitals of Leicester United Kingdom PAUL H.A. QUAX PhD Professor in Experimental Vascular Medicine, Einthoven Laboratory for Experimental Vascular Medicine Department of Vascular Surgery, Leiden University Medical Center, Leiden Netherlands EVELYN REGAR MD, PhD Associate Professor of Medicine Invasive Cardiology Department of Interventional Cardiology Clinical Head & Medical Coordinator Thoraxcenter, Erasmus MC, Rotterdam Netherlands SEUNG-WOON RHA MD, PhD, FACC, FAHA, FESC, FSCAI, FAPSI Associate Professor Internal Medicine, College of Medicine, Cardiovascular Center, Department of Cardiology, Korea University Guro Hospital, Seoul South Korea ALAIN RIVARD MD, PhD Associate Professor of Medicine Invasive Cardiology, Central Hospital of Montral Qubec Canada

19 LUIS RODRIGUEZ-MENOCAL MD, PhD Scientist University of Miami Miller School of Medicine Interdisciplinary Stem Cell Institute, Miami USA PABLO SALINAS M.D Department of Interventional Cardiology La Paz University Hospital, Madrid Spain ANGEL SANCHEZ-RECALDE MD Tutor Chief in Cardiology, Assistant Professor of Medicine, Invasive Cardiology, La Paz University Hospital, Madrid Spain IASUHIRO SATOH MD, PhD Clinical Professor of Medicine Invasive Cardiology Department of Cardiology National Hospital Organization, Disaster Medical Center, Tokyo Japan DANNY SCHOORS MD, PhD Invasive Cardiology University Ziekenhuis Brussel Belgium BENJAMIN SCOTT MD Invasive Cardiology University Ziekenhuis Brussel Belgium OSCAR SEMERARO MD Invasive Cardiology University Ziekenhuis Brussel Belgium KWEN-CHUL SHIN MD, PhD Associate Professor of Medicine Invasive Cardiology, Gil medical Center Gachon University, Guwol-Dong Namapng-gu, Incheon South Korea DOO-SUN SIM MD Invasive Cardiology Chonnam National University Hospital South Korea OANA MARIA SIMION MD Invasive Cardiology Centre Hospitalier de lUniversit de Montral Quebec, Canada SANDEEP SINGLA MD Fellow in Cardiology University of Arkansas Medical Center Little Rock, Arkansas USA MARK SLEVIN PhD, FRCPath Professor of Medicine Dean of Medical Sciences British Institute of Technology and E-Commerce, ICCC Chair in Clinical Biomedicine St. Paul Hospital, Barcelona United Kingdom/Spain GIJS VAN SOEST MD Department of Biomedical Engineering Thoraxcenter, Erasmus, MC, Rotterdam Netherland SILVIU STANCIU MD, PhD Department of Internal Medicine Carol Davila Central Emergency Military University Hospital,Bucharest, Romania HIROMASA SUZUKI MD Division of Cardiology, Department of Internal Medicine Juntendo University School of Medicine Tokyo, Japan MASAMICHI TAKANO MD, PhD Assistant Professor of Medicine Cardiovascular Center, Chiba-Hokusoh Hospital Nippon Medical School, Chiba Japan CORRADO TAMBURINO MD, FESC, FSCAI Cardiovascular Interventional Unit Department of Cardiology Ferrarotto Hospital, University of Catania Italy ELIEZER J. TASSONE MD Cardiovascular Disease Unit Department of Clinical and Experimental Medicine ,University Magna Grcia of Catanzaro Italy ION C. INTOIU MD, PhD, FESC Professor of Cardiology Carol Davila University of Medicine Invasive Cardiology Department Armys Clinic Center for Cardiovascular Diseases Bucharest, Romania

20 SALVATORE D. TOMASELLO MD Cardiovascular Interventional Unit Department of Cardiology Ferrarotto Hospital, University of Catania University of Florida, College of Medicine-Jacksonville, Florida Italy/USA HUNG-FAT TSE MD, PhD Professor of Cardiology Academic Chief, Cardiology Division Department of Medicine,Queen Mary Hospital University of Hong Kong Hong Kong WASAN UDAYACHALERM MD, FAPSIC Associate Professor of Medicine Invasive Cardiology, Cardiac Center, King Chulalongkon Memorial, Bangkok Thailand YASUNORI UEDA MD, PhD, FACC, FESC, FJCC Cardiovascular Division Osaka Police Hospital Japan MALCOLM JOHN UNDERWOOD MB ChB, FRCS (Ed), FRCS (CTh), MD, FCSHK Professor of Medicine Chief, Division of Cardiothoracic Surgery Department of Surgery The Chinese University of Hong Kong Prince of Wales Hospital, Shatin, NT Hong Kong ROBERTO VAZQUEZ-PADRN MD University of Miami Miller School of Medicine Interdisciplinary Stem Cell Institute, Miami USA GUMPANART VEERAKUL MD, FSCAI Invasive Cardiology Cardiovascular Research and Prevention Center, Bhumibol Adulyadej Hospital Thailand JEFFREY J.W. VERSCHUREN MD Department of Cardiology, Leiden University Medical Center, Leiden Netherlands SONG WAN MD Professor of Medicine Chinese University of Hong Kong Prince of Wales Hospital, Shatin Hong Kong MAGGIE MEI WANG MD, PhD Cardiology Division, Department of Medicine, Queen Mary Hospital, Research Center of Heart, Brain, Hormone and Healthy Ageing, Li Ka Shing Faculty of Medicine. The University of Hong Kong, Hong Kong DANIEL WENDT MD, PhD West German Heart Center Department of Thoracic and Cardiovascular Surgery Essen Germany SHINICHIRO YAMADA MD, Ph.D Division of Cardiology Himeji Cardiovascular Center Japan YOSHIJI YAMADA MD, PhD, FAHA Professor and Director Department of Human Functional Genomics, Life Science Research Center, Mie University, Tsu Japan ATSUSHI YAMASHITA MD Department of Pathology Faculty of Medicine University of Miyazaki Kihara, Kiyotake, Miyazaki Japan HAN-MO YANG, MD Invasive Cardiology Cardiovascular Center, Seoul National University College of Medicine South Korea KAI-HANG YIU MBBS Assistant Professor Cardiology Division Department of Medicine, Queen Mary Hospital, Research Center of Heart, Brain, Hormone and Healthy Ageing, Li Ka Shing Faculty of Medicine. The University of Hong Kong, Hong Kong

CONTENTS
PART I RESTENOSIS NEW DISEASE

Chapter 1. In-Stent Restenosis and Unresolved Issues Ambrose S. Kibos and Ion C. intoiu (Canada/Romania) ..................................................... Chapter 2. Pathogenesis of Restenosis Hiromasa Suzuki and Hiroyuki Daida (Japan)........................................................................ Chapter 3. Pathophysiology of Restenosis after Percutaneous Coronary Intervention erban Blnescu (Romania) .................................................................................................. Chapter 4. Biological Role of Extracellular Matrix in Restenosis Wasan Udayachalerm and Onanong Kulaputana (Thailand) ................................................. Chapter 5. Predictive Genetic Factors of Restenosis after Coronary Stenting and Plain Old Baloon Angioplasty Takashi Ashikaga and Yasuhiro Satoh (Japan) ...................................................................... Chapter 6. Endothelial Dysfunction and Inflamation Mark Slevin and Garry McDowell (United Kingdom) ............................................................ Chapter 7. Shear Stress and Stent Restenosis Michail J. Papafaklis, Katerina K. Naka and Lampros K. Michalis (Greece) ....................... Chapter 8. Hystological Predictors and Myocardial Regeneration Jang-Ho Bae and Taek-Geun Kwon (Korea) ......................................................................... Chapter 9. The Anatomical Origin of Cells in Arterial Restenosis Luis Rodriguez-Menocal and Roberto I. Vazquez-Padrn (USA) ........................................... Chapter 10. Lesion Complexity, Quantification, Predictive Factors Kwen Chul Shin and Woong Chol Kang (Korea) .................................................................... Chapter 11. Thrombus Formation on Disrupted Plaques Atsushi Yamashita and Yujiro Asada (Japan)......................................................................... Chapter 12. Stent Thrombosis Alfredo R. Galassi, Salvatore D. Tomasello,Corrado Tamburino and Dominick J. Angiolillo (Italy Florida USA) ......................................................................... Chapter 13. Restenosis in Chronic Total Occlusion Percutaneous Coronary Intervention Albert E. Alahmar, Kushal Pujara and Anthony H. Gershlick (United Kingdom) .................. Chapter 14. Relation Between Coronary Bifurcation and Restenosis Shinichiro Yamada, Yoshinobu Murasato and Gaku Nakazawa (Japan) ................................ A. Stent Restenosis in Bifurcation Lesions from Clinical Trials ........................................ B. Insight from Bench Study ................................................................................................. C. Pathology of Bifurcation Stenting .................................................................................... Chapter 15. Small Vessel Stenting. The Best Approach Seung-Woon Rha and Dong-Joo Oh (Korea) .......................................................................... Chapter 16. (Late) Stent Malapposition in the Bare-Metal Stents and Drug Eluting Stents Era Jeffrey J.W. Verschuren, Tarek A.H.N. Ahmed, Ioannis Karalis, Paul H.A. Quax and J. Wouter Jukema (Netherlands) ............................................................................................. Chapter 17. Stent Fracture and Restenosis Jong-Seon Park and Young-Jo Kim (Korea) ........................................................................... Chapter 18. Atherosclerosis, Vascular Damage and Renal Disease Deborah Mazzaferro, Serena Di Cello, Paola Naccarato, Antonio Pata, Eliezer J. Tassone, Alessandra Pascale, Laura Greco, Giulia Galiano Leone , Rosamaria Bruni and Francesco Perticone (Italy) ..................................................................................................... Chapter 19. Drug-Eluting Stent Implantation for High Risk Patients (Diabetes Mellitus) Masashi Kimura (Japan) ......................................................................................................... Chapter 20. Impact of Chronic Kidney Disease on Restenosis and Cardiovascular Events after Percutaneous Coronary Intervention Hideki Ishii and Toyoaki Murohara (Japan) ...........................................................................

27 47 61 75 91 99 109 121 129 141 151 167 191 201 201 209 219 235 245 253

259 275 285

22 Chapter 21. Stem Cells and Intravascular Restenosis Zakaria A. Almsherqi (Singapore) ........................................................................................... Chapter 22. Small Animal Models to Study Restenosis and Effects of (Local) Drug Therapy Mark M. Ewing, Jacco C. Karper, Margreet R. de Vries, J.Wouter Jukema and Paul H.A. Quax (Netherlands) ............................................................................................... Chapter 23. Genome-Wide Association Studies of Coronary Heart Disease Yoshiji Yamada, Tamotsu Nishida and Sahoko Ichihara (Japan)............................................ Chapter 24. Hepatocyte Growth Factor: Cardiotrophic Roles and Potential Therapeutics for Cardiovascular Diseases Shinya Mizuno and Toshikazu Nakamura (Japan) .................................................................

295 313 329 341

PART II

DIAGNOSTIC METHODS

Chapter 25. Clinical Evaluation, Electrocardiography, Holter Monitoring Lian-Yu Lin and Juey-Jen Hwang (Taiwan) ............................................................................ Chapter 26. Stress Echocardiography Evaluation after Percutaneous Coronary Intervention Kai-Hang Yiu, Maggie Mei Wang and Hung-Fat Tse (Hong Kong) ....................................... Chapter 27. Magnetic Resonance Imaging for Coronary In-Stent Restenosis Muhammad Munawar, Retno Dwi Astuti and Arman A. Abdullah (Indonesia) ...................... Chapter 28. Coronary Computed Tomography Angiography Masato Mizukoshi and Takashi Akasaka (Japan).................................................................... Chapter 29. Optical Coherence Tomography Evelyn Regar, Zain Jamil, Nico Bruining, Nieves Gonzalo and Gijs van Soest (Netherland) ... Chapter 30. Gated-SPECT and Coronary Intra-Stent Restenosis Christian Goetz, Silviu Stanciu, Cyril Cohen, Andr Constantinesco and Silviu Dumitrescu (France/Romania).................................................................................................................... Chapter 31. Intravascular Ultrasound Mariano Albertal (Argentina)................................................................................................. Chapter 32. Coronary Endoscopy Masamichi Takano and Kyoichi Mizuno (Japan) .................................................................... Chapter 33. Invasive Assessment of Coronary Stenosis With a Special Scope on Fractional Flow Reserve Benjamin Scott, Oscar Semeraro, Danny Schoors and Hans Bonnier (Belgium) ................... Chapter 34. A. Quantitative Coronarography Kanhaiya Lal Poddar and Seung Woon Rha (Korea) ............................................................. B. Quantitative Coronary Angiography for In-Stent Restenosis Daniel Ni, Florina Pinte, Gabriel Cristian, Liviu Chiriac, Adela Crstea, Iancu Mocanu, Lucian Gheorghe and Francisca B. Clinescu (Romania) ......................................................

363 369 379 387 397 417 429 437 447 457 473

PART III

STENT TYPES, COMPARATIVE STUDIES AND NEW DIRECTIONS


487 501 523 535 549 567

Chapter 35. Bare Metal Stent Gumpanart Veerakul (Thailand) and Josef Ludwig (Germany) .............................................. Chapter 36. Drug-Eluting Stent Markus Oberhnsli and Stphane Pierre Cook (Switzerland)................................................. A. In-Stent Restenosis in Drug Eluting Stents Bla Merkely, Zoltan Jambrik and Balzs Berta (Hungary).................................................... B. Drug-Eluting Stent in Patient and Lesion Subset Aamer Abbas and Debabrata Mukherjee (USA) ...................................................................... Chapter 37. Comparative Studies A. Bare-Metal Stents versus Drug-Eluting Stents on Restenosis and Thrombosis Hyun-Kuk Kim, Doo-Sun Sim and Myung-Ho Jeong (Korea) ................................................. B. Drug-Eluting Stents versus Drug-Eluting Stents Pablo Salinas and Angel Sanchez-Recalde (Spain) .................................................................

23 C. Drug-Eluting Balloons versus Drug-Eluting Stents for the Treatment of Coronary In-Stent Restenosis Debabrata Dash (India)........................................................................................................... D. Optimal Treatment for Myocardial Revascularization: Surgery or Stenting? Song Wan and Malcolm John Underwood (Hong Kong)......................................................... Chapter 38. Conceptual Perspectives Jorge A. Belardi and Mariano Albertal (Argentina)................................................................ Chapter 39. What Next? Yasunori Ueda and Kazuhisa Kodama (Japan)......................................................................

579 589 599 609

PART IV PART

IV

TREATMENT

Chapter 40. Adjunctive Phamacotherapy Han-Mo Yang, Bon-Kwon Koo and Hyo-Soo Kim (Korea) ..................................................... Chapter 41. A. Antiplatelet Treatment Resistance Jorge de Francisco, Sonia Huertas and Jerzy Krupinski (Spain) ............................................ B. Clinical Studies Based on Optical Aggregometry Gergely Fehr and Eld Papp (Hungary) ............................................................................... Chapter 42. Clinical Approaches to Attenuate the Inflammation Gim-Hooi Choo and Alan Yean-Yip Fong (Malaysia)............................................................. Chapter 43. Vascular Brachytherapy and Rotational Atherectomy in the Treatment of Coronary In-Stent Restenosis Brian J. Potter, Franois Gobeil and Alain Rivard (Canada) ................................................. Chapter 44. Balloon-Based Strategies Oana Maria Simion, Franois Gobeil and Alain Rivard (Canada)......................................... Chapter 45. Management of In-Stent Restenosis: Stent-Based Strategies Alexis Matteau, Alain Rivard and Franois Gobeil (Canada)................................................ Chapter 46. Catheter-Based Treatment of Drug-Eluting Stent Restenosis Jose Mariani Jr., Roberto Kalil-Filho and Pedro A. Lemos (Brazil)...................................... Chapter 47. Bioresorbable Stent Evelyn Regar (Netherland) ...................................................................................................... Chapter 48. Endothelial Progenitor Cell Dong-Bin Kim and Hee-Yeol Kim (Korea) ............................................................................. Chapter 49. Gene Therapy for Angiogenesis and Restenosis Takashi Miyake and Ryuichi Morishita (Japan) ..................................................................... Chapter 50. Coronary Artery Bypass Grafting for In-Stent Restenosis Ionel Droc, Gerard Bloch, Daniel Wendt, Jakob Heinz, Florina Pinte and Francisca B. Clinescu (Romania/ France/ Germany)............................................................

619 627 645 667 683 697 711 723 729 749 755

769

PART V

FROM GUIDELINES TO CLINICAL REALITY

Chapter 51. Percutaneous Coronary Intervention: From Guidelines to Clinical Reality Sandeep Singla and Jeffrey J. Popma (Harvard, USA) ...........................................................

781

24

PART IV

PART I

RESTENOSIS NEW DISEASE

CHAPTER 1

IN-STENT RESTENOSIS AND UNRESOLVED ISSUES


AMBROSE S. KIBOS and ION C. INTOIU

Scale of the Problem................................................................................................................. Need of Stent Evolution............................................................................................................ Stent Changes Better Stents ................................................................................................... DES in Special Conditions ....................................................................................................... New Devices Should Treat an old Problem .............................................................................. Too Many Stents from Structure to Cover ............................................................................. The Ideal Stent ...................................................................................................................... Conclusions .............................................................................................................................. References ................................................................................................................................

27 29 31 33 33 35 38 40 41

SCALE OF THE PROBLEM


Coronary atheromatosis is an important element in the genesis of myocardial impairment considering the involved stenotic, obstructive and thrombotic complications, that triggers myocardial injury causing different degrees of ischemia and frequently tissue necrosis. Multiple studies on the prevention and regression of coronary stenosis caused by atherosclerosis are trying in several ways to solve the etiological problem of the coronary determination, among which the most important is the control of the risk factors. There are multiple revascularization possibilities for significant coronary artery lesions, with a considerable increase in the recent decade, of which interventional revascularization is the most common method of treatment for coronary stenosis, but we still need to face some unresolved issues: in-stent restenosis and thrombosis. The first interventional procedure was the percutaneous coronary dilatation (PTCA), which due to complications caused by coronary trauma, vessel response to injury, negative remodeling and finally restenosis was abandoned, making way for another procedure that prevents remodeling, coronary stent implantation with bare metal stent (BMS). The initial bare metal stents gained significant value in the early 1990s but lost ground due to the occurrence of restenosis in 2030% of cases1.

28

PART I. RESTENOSIS NEW DISEASE

As the unique phenomenon responsible for neointimal hyperplasia and the generator of restenosis, stent structure research continued through the introduction of substances that hinder this process. New stent types were developed: drug eluting stent (DES). Even if neointimal hyperplasia inhibition was achieved, a new problem raised: persistent thrombosis and restenosis. Multiple variants of the DES structure (stent, polymer, active substance) are now used, resulting lower restenosis rates (<10%)2 but not its disappearance. The most frequent causes of restenosis in DES are under-expansion, stent fracture, asymmetric stent distribution, drug or resistance failure, persistent vascular trauma, polymer distribution or drug hypersensitivity3. These factors led to research aimed at reducing or eliminating them. The need for change is imposed by another DES implantation complication: thrombosis. The factors associated with an increased risk for stent thrombosis include the procedure itself (stent malapposition, number of implanted stents, stents length and dissections), patient and lesion characteristics, stent design, premature cessation of antiplatelet drugs, polymer or drug hypersensitivity, clopidogrel (aspirin) resistance3,4. The concept of bioabsorbable stents was initially introduced as a new option to prevent in-stent restenosis and thrombosis. Additionally, improvements were made regarding the stent surface, drug carrier, stent platform for complex lesions, dual-drug strategy, etc. Currently, there are only a few studies in progress on stenotic or thrombotic complications of bioabsorbable stents. The new rationale and goal of bioabsorbable stents is to revascularize the vessel like a metallic drug-eluting stent prior to its resorbtion, leaving no permanent implant and no permanent scaffold. These new stents can restore natural vascular response to physiological stimuli and potentially permit late lumen expansion. Biodegradable stents that are currently in development fall broadly into one of two categories: those manufactured entirely from polymer and devices that are made from bioabsorbable metals. Among the polymeric stents, poly L-lactic acid (PLA) is attracting the greatest interest4. The absorbable metal stents (AMS) are manufactured from alloys consisting of 93% magnesium and 7% rare elements that degrades much quicker than polymer technologies4. Endothelial Progenitor Cell (EPC) capturing stents represent a new concept that relies on the prevention of restenosis by supporting the natural endothelization process. This is achieved by presenting specific antibodies on the stent wall that bind the EPC, cells that make endothelization possible.5 Progress was made regarding the polymer matrices used in manufacturing DES allowing a sustained drug delivery. Slow/sustained drug release is essential because drug distribution from DES to the arterial wall is non homogenous6 and about 85% of the stented vessel wall is not covered by the stent struts resulting in low tissue concentration of the antiproliferative agent in the vessel wall7, incomplete endothelization of the stent struts8 and incomplete suppression of neointimal hyperplasia at the stent margins or between the struts9. For these considerations drug-eluting balloon (DEB) represents a new option for the treatment of coronary stenosis. The key feature of DEB is to facilitate the transfer of the antiproliferative agent from the balloon surface to the vessel wall7. This might lead to significant improvements in the treatment of patients with stent restenosis, small vessel, lesions of bifurcations etc7. In conclusion the issues of restenosis and thrombosis are still unresolved and the interventional scientific community opened a stent war between the revascularization procedures: PTCA vs. BMS vs. DES vs. DEB. When trying to assess these rapid technological developments in the field of PCI we can see that Hippocrates maxim to help or at least, to do no harm is more pertinent than ever.10

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

29

NEED OF STENT EVOLUTION


The first study about the possibility to revascularize the atherosclerotic lesion was published by Charles Theodor Dotte and Melvin P Judkins in 1964.11 Starting from the idea that Coronary bypass surgery is an operation too traumatic for treatment of localized obstruction Andreas Grntzig12 developed in 1977 the technique of percutaneous coronary angioplasty (PTCA) that became the most used procedure. The drawback is that PTCA is also a traumatic procedure for the coronary wall and this injury causes modifications of the subendothelium cellularity by means of structure dilatation and protrusion of atherosclerotic plaque in the lesional and perilesional space. Restenosis represents a lumen reduction post-revascularization which leads to significant obstruction and recurrence of coronary symptoms, thus remaining the Achilles heel of intracoronary therapy. The mechanisms generating restenosis are complex and sequential: atherosclerotic plaque invasion due to coronary wall injury (de-endothelization), platelet aggregation and activation which leads to thrombus formation, vascular smooth cell activation, migration and proliferation, neointima formation and neointimal hyperplasia (Figure 1.1).
PCI Vascular injury De-endotelization Thrombus formation VSMC activation VSMC migration and proliferation Neointima formation

Neointimal hyperplasia

Re-endotelization

Restenosis
Figure 1.1. Pathogenic sequentiality of re-stenosis comprises: vascular injury (de-endothelization), thrombus formation, VSMC activation, proliferation and migration, extracellular matrix synthesis and neointima formation. These processes are achieved in the initial context of positive remodeling, then the reconstruction of the damaged vessel for the patients with re-stenosis is performed through early and negative remodeling (VSMC-vascular smooth muscle cells).

30

PART I. RESTENOSIS NEW DISEASE

A new device was introduced in 1990, which replaced plain old balloon angioplasty (POBA) based on angiographic and clinical outcomes. This device was the bare metal stent (BMS) that led to a reduced rate of restenosis by preventing negative coronarian remodeling. The autoexpandable BMS stents were introduced for the first time in clinical practice by Sigwart and co.13 providing in the short term, restenosis prevention and negative remodeling. After few years restenosis and thrombosis was observed in 18% of BMS cases14. Encouraged by the successes obtained using BMS in revascularization procedures, and disappointed by the results of retrospective studies showing high rates of acute occlusion and restenosis post POBA, interventional cardiologists abandoned this technique in favor of BMS. In stent restenosis (ISR) angiographic definition is a recurrent percent diameter stenosis (>50%) at the stent site and its edges (proximal and distal segments) 5 mm. Angiography is able to describe different morphological patterns: focal (pattern I) diffuse (pattern II,) proliferative (pattern III), total occlusion (pattern IV)15. The unexpected complications of BMS determined practitioners to reintroduce POBA, recognizing that the new stents were not competitive. After publication of the results of the first trials which used BMS, BENESTENT (Belgian Netherland Stent)16 and STRESS (Stent Restenosis Study) trial17 conclusion was that using antiplatelet treatment in absence of anticoagulants decreases restenosis and late thrombosis rate, fact that re-launched the use of stents in clinical practice especially for coronary artery stenosis18. Thus, after 1999, stent use became the dominant procedure for the second time (84.29%)19. Several studies proved a decreased risk of subacute thrombosis in BMS revascularization, but also highlighted a new problem: neointimal hyperplasia20,21. Intra-stent restenosis is the result of an exaggerated healing process that occurs in 20% to 30% of cases22. Inhibition of neointimal hyperplasia was addressed in several different studies by adding special additives to the stents structure, in order to reduce this process. This research led to the development of a new device known as Drug-Eluting Stent (DES). Comparative studies proved DES to be superior to BMS for the treatment of native coronary artery narrowings, resulting lower rates of restenosis and this lead to an exponential increase in the use of DES. In 2007, after Barcelona we were witnessing a decrease in the use of DES (52%) due to the identified complications of the procedure (thrombosis, fatal myocardial infarction and major adverse cardiac events MACE) (Figure 1.2).
3500 3000 2500 2000 1500 1000 500 0 2003 2004 2005 2006 -52% 2007 2008 2009 2010 8% -7% 6% -3% -6% 64% 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6

Market Size

Growth Rate

Figure 1.2. DES use between 20032010. A significant decrease is evident after Barcelona 2006 without reaching again the maximum values of 2004. Source: Global Coronary Stent Market Report 2010 Edition-Market Publishers2010 (modified).

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

31

In light of the new complications of DES, especially in-stent thrombosis, comparative studies of different revascularization procedures have been repeated and added to the existent trials and meta-analyses23. These studies continued, focusing especially on complications encountered in interventional revascularization. Analyzing the results of different stent types in the restenosis triangle might solve the problem of restenosis and in-stent thrombosis (Figure 1.3).

POBA vs. BMS vs DEB vs. vs. NON-DES vs. DES


RESTENOSIS THROMBOSIS

DES vs. DES

REINTERVENTION (Re-PCI, CABG)

Figure 1.3. Restenosis triangle shows comparative studies regarding restenosis and in-stent thrombosis between different revascularization techniques: POBA, BMS, DEB, DES, non- DES (POBA-plain old balloon angioplasty, BMS-bare metal stent, DES- drug eluting stent, DEB-drug eluting balloon, Non-DES (bioresorbable stent, endothelial progenitor cells, capture stent, etc.), CABG-coronary artery by-pass graft, re-PCI-re-percutaneous coronary intervention.

STENT CHANGES BETTER STENTS Sirolimus Eluting Stent (SES)


Sirolimus-eluting stent using rapamycin (macrolid antibiotics) as a pharmacological agent which inhibits cytokine and growth-factor-mediated proliferation of lymphocytes and SMC resulted in decreased neointimal proliferation.23 The SIRIUS (Sirolimus-Eluting Stent in de Novo Native Coronary Lesions) trial which enrolled 1058 patients showed a decrease in TVR, MACE when compared with BMS after 5 years of study.24 The differences between SES (Sirolimus-eluting stents) and the old BMS were studied in the following trials: C-SIRIUS25, E-SIRIUS26, DESERT27, SCORPUS28, SESAMI29, PYPHOON30, PRISON II31, GISSOC II-GISE32, SES-SMART33 and RRISC34. The purpose of those trials was to analyze differences between SES and BMS in complex situations such as: bifurcation lesions, chronic total occlusion (CTO), small coronary vessels, saphenous grafts (SVG), diabetic patients, primary PCI for ST-segment elevation myocardial infarction, and the results demonstrated significant reduction ISR in SES compared to BMS23.

32

PART I. RESTENOSIS NEW DISEASE

Paclitaxel-Eluting Stent (PES)


There are two types of stents using paclitaxel: TAXUS EXPRES and TAXUS LIBERTE, the difference between them consisting in homogenous drug distribution35. The trials TAXUS I36, TAXUS II SLOW37, TAXUS IV38, TAXUS V39, TAXUS VI40, HORIZONS-AMI41, PASED42, PASSION43 made an analysis of the differences between PES and BMS in various situations, using for comparation restenosis, late loss, MACE, death, myocardial infarction, TLR and stent thrombosis. These trials have demonstrated a significantly lower rate of ISR, MACE, target lesion revascularization (TLR), target vessel revascularization (TVR) with PES compared with BMS. This difference was consistent among different patient group including single lesions, complex lesion, unprotected left main (UPLM), STEMI23. The trials SIRTAX44,45, SORT-OUT II46, TAXI47, PROSIT48, ISAR- LEFT MAIN49, ISAR-DESIRE 250 demonstrated a greater delayed late loss (LL) with SES, but there was not longer significant difference in LL between SES and PES.

Zotarolimus Eluting Stent (ZES)


Zotarolimus is the polymer for the Endeavor stent ZES that caused less inflammation compared with the polymers on the SES stent23. In the E Five-Registry clinical trial (7.822 patients) follow -up at 24 months, the results confirm the advantage of using ZES (death 2,9%, myocardial infarction 1,5%) target lesion revascularizations (TLR) 5,1 %, target vessel failure (TVF) 7,9% and definite or probable stent thrombosis in 0,7%.51 Randomized ENDEAVOR II study in 1.197 patients reported significantly lower angiographic restenosis at 5 years follow-up in ZES compared with BMS, but MI and ST remained comparable52,53. The PROTECT (Patient Related Outcomes With Endeavor versus Cypher Stenting Trial) study which has randomized 8,800 patients will report primary and point of stent thrombosis at 3 years follow-up54 and compare ZES with PES. In the ENDEAVOR III study after 5 years follow- up, the 2.8% absolute difference in TLR between ZES and SES at 1 year was reduced to 1.6% at 5 years55,56.

The Xience V everolimus eluting stent (EES)


EES has a platform with nonerodable biocompatible polymer everolimus (a synthetic derivative of sirolimus) and its mechanism is inhibition of mTOR. Real world registries and randomized trials compared EES to BMS (SPIRIT FIRST)57,58 and EES to PES (COMPARE59, SPIRIT II60, SPIRIT III61, SPIRIT IV62, SPIRIT V63, EXCELLENT64, X-SEARCH65) and demonstrated the superiority of the EES over BMS (3.8 vs. 5.3-p<0.0552) in TLR and EES vs. PES (2.0 vs. 5.3-p<0.001) in TVR and definite/probable ST (0.7 vs. 2.6; p<0.05)59.

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

33

DES IN SPECIAL CONDITIONS


Reports indicates that DES are used in 75% of PCI procedures in USA66 and confirm that BMS are still used in contemporary PCI23. Recent data indicate that only 49% of PCI used DES in Arkansas and 35% for rural hospitals23,67. The decreased use of DES is due to their risk of early and very late thrombosis68,69. Meta-analyses and real world studies confirm that DES are not significantly reducing restenosis compared to BMS but instead increase the risk of stent thrombosis23. Stent selection is a complex problem: for lesions in vessels < 3 and > 3 mm in diameter in the BASKET trial there was an absolute difference in rate of repeat revascularization between DES and BMS of 9,1% and 2.0% respectively70. In diabetic patients there is a significant lower rate of TVR in DES compared with BMS for the lesions > 20 mm long and vessels < 3 mm in diameter (DES vs. BMS7.2% vs. 17.6%, HR:0.38, 95%CI :0.24 to 0.60, p< 0.001)71. In nondiabetic patients there is a significant difference in TVR between DES and BMS when lesions are <20 mm long and vessels < 3 mm in diameter (DES vs. BMS5.3 vs. 5.9, HR:0.87, 95CI: 0.52 to 1.47, p=0.61)70,71. There are concerns that DES might lead to higher rate of stent thrombosis, particularly beyond the first year after implantation72. Large meta-analysis data suggest that DES might be associated with a 2-to4-fold increased risk of very late thrombosis73. An incremental risk (<1%) of thrombosis in DES might be sufficient to outweigh the benefit of restenosis prevention and favor BMS for overall PCI23.

NEW DEVICES SHOULD TREAT AN OLD PROBLEM Endothelial Progenitor Cell Capture (EPCc)
The mechanism of stent endothelization is unclear while the phenomenon of vascular healing after bare-metal stent implantation is linear and predictable74. In DES the pattern of healing is delayed as a consequence of interaction between different factors. The paradox in development of DES technology is that beneficial effect of drug elution is the inhibition of stent endothelization75. Previous experience with stents with immobilized antihuman CD34 antibody on the device surface has shown feasibility of enhancing stent endothelization in the clinical studies. Effectiveness of CD34 covered stent was tested in a series of studies performed under the HEALING programme. The HEALING-FIM demonstrated feasibility and safety of EPC capture by CD34 covered stent76. The HEALING II and the HEALING IIB trials and ultimately the real-world e-HEALING multicenter registry of GenousTM prohealing EPC capturing stent have been performed. The e-HEALING registry established that the rates of CD, MI and TLR with CD34 covered stents were low and comparable to the early TAXUS studies77,78. Primary PCI with AMI implantation of these stents was associated with a relatively low rate of MACE: 1.6% in hospital, 4.2% at 30 days, 5.8% at 6 months and 9.2% at 1 year79. In the TRIAS study CD34-covered stents were as effective as PES in preventing restenosis during 12 month follow-up80.

34

PART I. RESTENOSIS NEW DISEASE

When used in high risk patients (33% with diabetes, 73% with ACS, 9% with multivessel intervention, 56% type B2/C lesions) 14 months follow-up revealed an event rate of 13% for repeated PCI and 4% for de novo lesions81.

Bioabsorbable stent
The reason and goal of bioabsorbable stents is to revascularize the coronary stenosis like a metallic drug-eluting stent, then to resorb naturally leaving no permanent metallic implant and no permanent scaffold. Researchers are seeking to understand if these new stents can restore natural vascular response to physiological stimuli. Currently projects on fully biodegradable stents have concentrated on two main categories: polymer based stents and absorbable metallic stents. Poly-L lactic acid (PLA), the focal point of interest among polymers, is made from repeating lactide units. Hydrolysis of bonds between lactide molecules produces lactic acid that enters the Krebs Cycle to be metabolized to carbon dioxide and water in a period of 12 years. The Igaki-TamaiR stent made of PLLA filament with a zigzag design and deployed with a heated balloon had good results in the pilot study showing late loss comparable with BMS and also in a larger study of 50 patients with 63 lesions82 where restenosis rates were 21% at 6 months and 19% at 12 months. Further in the development of these stents, everolimus was added to a PLA platform by Abbott Vascular creating the bioabsorbable everolimus-eluting coronary stent (BVS) which has the capacity to elute 80% of the drug in the first 30 days. The PLA is mixed with everolimus to form a matrix and the drug is eluted from a bioabsorbable coating. The BVS design has a uniform strut distribution, which should lend itself to a more even support of the vessel wall. The stent is metabolized in 18 months, leaving no drug behind once the degradation was completed. The results from multiple imaging methods of the ABSORB trial (a bioabsorbable everolimus-eluting coronary stent system)83,84 which enrolled 30 patients with simple lesions were excellent in simple lesions, showing no thrombosis and late loss of 0,40 mm at 6 months and one ischemia-driven major adverse cardiac event, no stent thrombosis, no cardiac deaths, smooth endolumen struts absorbed at 2 years. Other PLA-base devices in development are: GenousTM Bio-engineered cobalt-chromium R-stent which has the surface coated with immobilized EPC-capturing antibodies; the REVA stent (Boston Scientific) made from the modified amino acid L-tyrosine, showing complete endothelization at 30 days30, upgraded further in the evolution of its design with a paclitaxel-eluting surface. The IDEALTM stent (Bioabsorbable Therapeutics Inc) coated with everolimus and using salicylic acid integrated in a polyanhidric ester (PAE) was shown to reduce inflammatory effects when compared to BMS and reduced angiographic restenosis at 30 days follow-up85. Biodegradable metallic stents technology is based on magnesium which is metabolized to inorganic salts within 3 months. The attributes that make magnesium a good choice are its biocompatibility, low biocorrosion and lack of artifacts in non-invasive imaging due its non-visibility on X-ray examinations. Biotroniks absorbable metal stent (AMS) features a metal alloy (93% magnesium and 7% rare elements) that degrades much quicker than the polymer technologies (completely degrades in three months) and was first tested in the PROGRESS-AMS86 trial which enrolled 63 patients with single de novo lesions. The trial

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

35

succeeded in demonstrating both safety and efficacy but there were concerns about high rates of restenosis. Angiography at 4 months showed an increased diameter stenosis (17%) and the target lesion revascularization rate was 45% at 1 year follow-up. Intravascular ultrasound examinations showed only remnants of the original strut. Researchers have tried to use iron as a basis for bioabsorbable metallic stents but because of its high toxicity and slow metabolization further projects stopped. It is very important to emphasize that biodegradable polymer is not fully biocompatible and there are studies indicating that PLA could generate an inflammatory respose87. Deposition of calcium in the vessel wall during magnesium degradation appears to be another problem. More work is needed in improving bioabsorbable stents in terms of their natural absorption, full metabolization without complications or emboli production and lack of inflammatory or nonthrombogenic effects.

Drug-Eluting Balloon (DEB)


DES implantation is inherently a stressful event on the blood vessel resulting incomplete endothelization and risk of stent thrombosis. In DES a sustained drug release is essential because drug distribution to the anterior wall is inhomogenous7. About 85% of the stented vessel wall area is not covered by the stent struts resulting in low tissue concentrations of the antiproliferative drugs in this area7. A key feature of the DEB is to facilitate the homogenous transfer of the drug from the balloon surface to the vessel wall7. The return to the balloon solution originates from the negative consequences (thrombosis) of DES and the high local drug concentrations (higher than bolus administration) reached with this procedure. Clinical experience with DEB began in Germany with treatment of in-stent restenosis by Paclitaxel-coated Balloon Catheters (PACCOCATH) ISR 188 trial. This first in-human study compared paclitaxel-coated balloon (PCB) and standard POBA in the treatment of in stent restenosis. The patients treated with DEB had significantly better results: late lumen loss was 0.003 mm vs. 0.74 mm for uncoated POBA (p=0.002). PACCOCATH ISR II trial confirmed these results for longer follow-up. Treatment of Coronary In Stent Restenosis (PEPCAD II study) compared paclitaxel eluting stent and paclitaxel coating balloon. Follow-up data demonstrated that clinical outcomes after treatment with DEB alone were at least as good as DES (angiographic late loss was 0.17 mm vs. 0.38 mm, p=0.03)89. The preferred sites for treatment with DEB are small vessels and bifurcation lesions. PICCOLETO trial conclusions were that DEB failed to show equivalence to DES regarding angiographic end points90. The use of drug balloon for the treatment of bifurcation lesions was analyzed in DEBUIT study. Differences between DEB and DES plus conventional balloon strategy were less obvious91.

TOO MANY STENTS FROM STRUCTURE TO COVER


The general principles for stent classifications are determined by the main features of the stents. These features are92:

36

PART I. RESTENOSIS NEW DISEASE

Mechanism of expansion (balloon-expanding or self-expanding) Materials (cobalt-based alloy, stainless steel, tantalum, nitinol) Coating system (inert coated, active coating or bioabsorbable) Manufacturing methods (laser cut, photo-etching, water-jet cutting, kniting vapor) Geometrical configuration (coil, slotted tube, mesh structure, ring, custom design or multi-design) Addition to stent (radio-opaque markers, coating etc.) The design and structure of stent development can be represented (Figure 1.4) as concentric circles comprising the main components of the individualization of each type of stent.

future additions
geometry fabrication form materials

Figure 1.4. Stent design steps.

Materials from the balloon expandable stent are: stainless steel, tantalum, platinum iridium, polymers niobium and cobalt alloys, and those for self-expanding are: nickel-titanium (nitinol), cobalt alloy and stainless steel. The starting point is the material used and distinguishes between balloon-expandable and self expanding stents. The stents must exhibit excellent corrosion resistance and biocompatibility. Balloon expandable stents are made from materials that can be plastically deformed through the inflation of the balloon. The ideal material for these stents therefore has a low yield stress, high elastic modules and is work hardened through expansion for high strength. Self-expanding stents are manufactured in the expanded shape then compressed and constrained in a delivery system93. New materials: Biotronik has developed a stent that is not drug-eluting and combines cobalt struts with a silicone carbide layer. The BENE study comprised 202 patients and angiographic follow-up (6 month) showed no cardiac death, IM or stenosis and thrombosis94. Form. The second step of the stent steps is the form which may be: tube (the majority), wire, sheet and ribbon. A large majority of the balloon expandable and self-expanding are made from wire or tubing. Fabrication method depends on the raw material used and can be: laser cutting, photo chemical etching, water jet, braiding and knitting vapor deposition.

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

37

The majority of coronary stents are produced by laser cutting from tubing. The fabrication method depends mainly on the raw form used. A large majority of balloon-expandable and self-expanding stents are made from wire or tubing while very few are made from sheet metal. The simplest shape for wire stents is a coil made from nitinol and is self-expanding.93 Geometry. The evolution of stent design led to the development of a large variety of stent geometry which can be classified into five main categories: coil, helical spiral, woven, individual rings and sequential rings93. This sequential classification accounts for the majority of commercially available stents. These geometries can be further refined according to the manner in which the structural elements are connected and the nature of resulting cells: regular connection, periodic connection and peak-peak connection or valley connection. The connection between the structural elements can be made in two arrangements: closed cell and open cell. The advantages of closed cell design reside in an optimal scaffolding and the uniform surface. The U,V,S, or N shaped elements can accommodate much easier the changes in shape. Open cell category describes construction where some or all of the internal inflection points of the structural members are not connected by bridging elements. This design allows periodic peak-to-peak connections, peak-to-valley connections and mid-strut connections, as well as innumerable hybrid combinations93. The open cell design and the unconnected structural elements contribute to longitudinal flexibility and periodically connected peak-to-peak designs are common among self-expanding stents and balloon-expandable stents93. Additions. Stents made from stainless or nitinol are sometimes hard to see on fluoroscopy and narrow struts. To improve x-ray visibility, markers made from platinum, tantalum or gold are often attached to the stents93. Coatings. Several active compounds have been used to cover stents in order to increase their biocompatibility and to interact with different cells and molecules present in the blood and vessel wall. Among the different compounds tested, heparin was the first tested because of its mode of action is to reduce the coagulation cascade. Other coatings such as phosphorylcholine and silicon-carbide have been used in order to reduce platelet activation and adhesion to the stent struts during the initial phase of stent re-endothelization93. A PTFE layer was added between two stents or one stent was covered by an inner and outer layer of PTFE in order to have full coverage of the lesion (stent/artery ratio is 100%). Another benefit is that the PTFE layer might entrap all the material possibly protruding from the stent struts93. Drugs. Drug delivery stents require a combination of advanced metal stent design and drug delivery technology. Stent-based drug delivery can be accomplished through various mechanisms:93 metal stent can have a drug bound on their surface or embedded within macroscopic fenestrations or microscopic nano-pores thus providing more rapid drug delivery. metal stents coated with an outer layer of polymer (bio-absorbable or non-bio-absorbable) can be drug loaded thus providing more controlled and sustained drug delivery, which might allow more effective drug-tissue interaction. stent utilizes a drug-release system with reservoirs on the stents outer surface. bio-absorbable polymer stents can be loaded with a drug that is eluted slowly over time. endothelial progenitor cells (EPCc) recruitment stent is achieved through surface immobilized antibodies direct toward EPCc in surface antigens of the stent95. antisense oligonucleotide-eluting stents are designed to interfere with the information transfer from the gene to the protein96.

38

PART I. RESTENOSIS NEW DISEASE

THE IDEAL STENT


Following the first generation of sirolimus and paclitaxel-eluting stents, a variety of new stents designs, coatings and locally delivered agents are emerging and most likely will become available in the future. Development of more biocompatible, bioabsorbable and endothelial progenitor cell capturing stents is expected in the near future and may offer an alternative therapeutic approach to optimally abolish restenosis. Despite the initial enthusiasm, we now realize that all types of stents are effective but they are not a panacea for restenosis. Some issues still remain open with DES: non complementary satisfactory results in some sub-groups such as bifurcation lesions and diabetics, long term safety (mainly in terms of the occurrence of late stent thrombosis), importance of basic design and structure of the underlying stent platform. The ideal properties of an antirestenosis drug are complex (Table 1.1) and the characteristics of the ideal DES depend on stent structure, delivers-olimus drug, thrombus-resistant luminal surface and features allowing antiplatelet treatments (Table 1.2).
Table 1.1. The ideal properties of an antirestenosis drug97 (modified) Lipophilic with good tissue retention, Strong binding/slow elution from polymer, Antiproliferative without inducing cell death, Potent inhibitor of vascular smooth muscle cells (VSMCs) but not endothelial cells, specific to intimal VSMCs, Antimigration action on VSMCs, Anti-inflammatory action, Antiplatelet action, Pro-healing (optimal healing response and accelerated functional endothelium)

Table 1.2. Characteristics of the perfect DES98 (modified) Very flexible, conformable (hybrid open-cell) Very low-profile stent delivery system (<0.030 inch) High-pressure balloon, suited for direct stenting with minimal balloon lengthening at a high pressure Adequate radiopacity and radial hoop strength (metallic +/- better than bioabsorbable) Drug delivery for approximately 60 to 90 days, then complete absence of the drug Very thin (homogenous) surface of bioabsorbable (noninflammatory) polymer Elution from a microporous, nonpolymeric surface with Cypher-like elution kinetics Thrombus-resistant luminal surface Promotion of early re-endothelization Very low late loss ( 0.2 mm) Features allow antiplatelet treatment for 3 months

Interventional revascularization represented a revolutionary strategy of coronary stenosis treatment and had a fast evolution in development of different techniques. Each procedure stage had a growth component, stagnation and decrease due to arising complications, especially restenosis and thrombosis. This curve was determined by the appearance of new revascularization procedures which gradually replaced the old techniques.

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

39

Over years PTCA was replaced with BMS which in turn gave way to DES and these will probably be replaced by new types of bioresorbable stents and EPCc (Figure 1.5).
Ideal

Decreasing Incidence of Restenosis

Future
Bioresorbable EPCc Drug Eluting Stents Bare Metal Stents

stent

PTCA

1997

1986

1999

2006

2008

Time

Figure 1.5. The S curve in stent evolution. The initial increase of use of each revascularization procedure is followed by a stage of stagnation and decline generated by a new type of device, depending on restenosis rate99 (modified).

In the future we can assist important changes, because restenosis and thrombosis have not been resolved yet, although there have been significant decreases of these complications. Anticipative studies of using various types of stent shows a stagnation of DES use compared with bioresorbable stents and a mild increase of BMS (Figure 1.6). BMS return can be explained by the new concepts for the structuring of these stents. For prevention of in-stent restenosis the 3P dilemma must be solved which involves the study of individual patient parameters and also of coronary lesions suitable for revascularization, using of new implantation techniques (IVUS) and post-procedural research long time development (Figure 1.7). In clinical practice the interventionalist must decide which stent is better for the patient according to lesions characteristics. The general characteristics of the ideal stent are the following: flexible, low unconstrained profile, trackable, radio-opaque, thrombo-resistant, biocompatible, reliable, expandable, with high radial strength, circumferential coverage, low surface area, hydrodynamic compatible93. New stent platforms (biodegradable stents or endothelial progenitor cell capturing stents may provide in the future more physiological response to stent implantation in reducing vascular injury and vessel healing93. The ideal design of antirestenotic stents needs to have a large surface area contact with vascular wall, minimal inter-filament gaps, robust radial support and symmetrical expansion to ensure uniform drug eluting. In conclusion a stent without restenosis and thrombosis is still a dream.

40

PART I. RESTENOSIS NEW DISEASE

100 90 80 70 60 50 40 30 20 10 0

Future
? Molecular Biology

2008 2010 2012 2014 2015 2016 2017


Drug-Eluting Bare Metal Bioabsorbable

Figure 1.6. Anticipative evolution of stent market: 20082017. Source: AarkStore Enterprise oct. 2010, Global Stent Market (Report 2010-Edition) (modified).

Patient

Graft restenosis ASA intolerance or resistance Clopidogrel variability and resistance Hypercoagulability Malignancy, hemodialysis, diabetes STEMI, NSTEMI

Provider
Increasing post-market surveillance New formulations New drugs and combinations New concept New material

Procedure
Optimized stent deployment Avoid certain lesion subsets Avoid large vessels/short lesions, bifurcation, CTO, complex lesions Utilize IVUS

Figure 1.7. The 3P dilemma for stent evolution.

CONCLUSIONS
Restenosis and thrombosis remain the most frequent complications after interventional revascularization and DES used before 2006 in most cases. After 2006 there was a decrease in

CHAPTER 1. IN-STENT RESTENOSIS AND UNRESOLVED ISSUES

41

the use of DES because of the observed fatal complications and the publication of studies stressing the need for DES design improvement. The causes of stent failure are dependent on stent design and its effects on the healing processes, vascular injury, drug-related issues, dual drug strategy etc. (Table A). The evolution in stent design is depending on a better understanding of the wall response to the stent, refinement of DES components, lesions complexity and prevention medication. The most important modifications for acquiring better DES are: stent changes, stent platform for complex lesions, new stents (bioabsorbable and endothelial progenitor cells capture), drug-related issues, surface changes and drug currier system, dual drug strategy. Present trends in stent evolution: 1) the strut stents with radioopacity are becoming the standard due to improved profile flexibility, deliverability and reduced vessel injury; 2) stent geometry must favor optimized homogenous drug distribution and may require geometry modifications because of stent fracture complications; 3) dedicated stents for complex lesion (bifurcations, small vessels, long lesions); 4) new materials for increased stent recoil; 5) self-expanding stents Two open questions still remain: will fully bioabsorbable stent dominate the future and there is no necessary stent changes? Drug-related issues are a new problem fueling the need for evolution in stent structures and this depends on optimized pharmacokinetics, hydrophobic drugs, reduced drug doses and pro-healing pharmacology. Drug carrier systems and surface changes are another components of a new direction in stent structure evolution. This is dependent on a better understanding of inflammation and hypersensitivity reactions after vessel injury during the healing process. Specialized durable polymers with optimized properties are under investigations and show much promise. Restenosis and in stent thrombosis remain an unresolved issue demanding future studies because the ideal stent to resolve these complications was not yet created. Fast advances in stent structures technology, improvements in implantation techniques and prevention medication entitles us to believe that this problem will be solved in the future, possibly based on cellular and molecular biology principles and thus interventional revascularization will be performed without complications. There are many unresolved issues including long term effects, efficacy in complex lesions, small vessels, diabetics and the ideal antiplatelet therapy. A number of clinical trials are being designed or already in progress to answer many of these questions. Despite difficulties, we have strong reasons for optimism in the progress of PCI management of patients with coronary artery disease.

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53. van Leeuwen F. Randomised trial comparing the ENDEAVOR zotarolimus-eluting stent and the Driver bare metal stent in single de novo native coronary artery lesions: five-year clinical follow-up of ENDEAVOR II. Paper presented at: EuroPCR; May 1922, 2009; Barcelona, Spain. 54. Camenzind E, Wijns W, Mauri L, et al. Rationale and design of the Patient Related OuTcomes with Endeavor versus Cypher stenting Trail (PROTECT): randomized controlled trial comparing the incidence of stent and clinical events after sirolimus or zotarolimus drug-eluting stent implantation. Am Heart J 2009; 158:902905. 55. Kandzari D, Mauri L, Popma JJ et al. ENDEAVOR III: 5 years final outcome. Paper presented at: The American College of Cardiology Scientific Sessions: March 1416, 2010 Atlanta. 56. Eisenstein EL, Leon MB, Kandzari DE, et al. Long-term clinical and economic analysis of the Endeavor zotarolimus-eluting stent versus the Cypher sirolimus-eluting stent: 3-year results from the ENDEAVOR III trial (randomized controlled trial of the Medtronic Endeavor drug [ABT578] eluting coronary stent versus the Cypher sirolimus- eluting coronary stent in de novo native coronaryartery lesions). J Am Coll Cardiol Intv 2009; 2:11991207. 57. Serruys PW, Ong ATL, Piek JJ, et al. A randomised comparison of a durable polymer everolimus-eluting stent with a bare metal coronary stent: the SPIRIT FIRST trial. EuroIntervention 2005; 1:5865. 58. Wiemer M, Serruys PW, Miquel Hebert K, et al. Five-year long-term Rclinical follow-up of the XIENCE V everolimus eluting coronary stent system in the treatment of patients with novo coronary artery lesions: the SPIRIT FIRST trial. Catheter Cardiovasc Interv 2010; 75:9971003. 59. Kedhi E, Joesoef KS, McFadden E, et al. Second-generation everolimus-eluting and paclitaxel-eluting stents in real-life practice (COMPARE): a randomised trial. Lancet2010; 375: 201209. 60. Serruys PW, Ruygrok P, Neuzner J, et al. A randomised comparison of an everolimus-eluting coronary stent with a paclitaxel-eluting coronary stent: the SPIRIT II trial. EuroIntervention 2006; 2:286294. 61. Stone GW, Midei M, Newman W, et al. Comparison of an everolimus-eluting stent and a paclitaxel-eluting in aptients with coronary artery disease: a randomized trial. JAMA 2008; 299:19031913. 62. Stone GW, Rizvi A, Newman W, et al. Everolimus-eluting versus paclitaxel-eluting stents in coronary artery disease. N Engl J Med 2010; 362: 16631674. 63. Cheavalier B. SPIRIT V single arm study: 2 year follow-up. Paper presented at: EuroPCR; May 2528, 2010; Paris, France. 64. Park KW, Yoon JH, Kim JS et al. Efficacy of Xience/Promus versus Cypher in rEducing Late Loss after stENTing (EXCELENT) trial study design and rationale of a Korean multicenter perspective randomized trial. Am Heart J. 2009; 157:811-817. 65. Onuma Y, Kukreja N, Piazza N et al. The everolimus-eluting stent in real-world patients: 6 month follow-up of the X-SEARCH (Xience V Stent Evaluated at Rotterdam Cardiac Hospital) registry. J Am Coll CARDIOL 2009; 54: 269276. 66. Malelunda G, Lemesle G, Waksman R. A critical appraisal of the safety and efficacy of drug-eluting stents. Clin Pharmacol Ther 2009; 85: 474480. 67. Reuters. Drug-Eluting Stent Penetration Lowest in Arkansas, Outnumbered by Bare- Metal Stents. September 21, 2009. Available at: http://www.reuters.com/article/pressRelease/idUS75669+21-Sep2009+prn20090921?sp=true. Accessed October 24.2009. 68. Barragan P, Sainsous J, Silvetri M, et al. Ticlopidine and subcutaneous heparin as an alternative regimen following coronary stenting .Catheter Cardiovasc Diagn 1994; 32:133138. 69. Schoming A, Neumann FJ,Kastroti A, et al. A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary-artery stents. N. Engl. J. Med 1996; 334: 10841089. 70. Pfisterer m, Brunner-La Rocca HP, Rickenbacher P, et al. Long-term benefit-risk balance of drug eluting vs. bare-metal stents in daily practice: does stent diameter matter? Three-year follow-up of BASKET. Eur Heart J 2009; 30:1624. 71. Tu JV, Bowen J, Chiu M, et al. Effectiveness and safety of drug-eluting stents in Ontario. N Engl J Med 2007; 357:13931402. 72. PfistererM, Brunner-La Rocca HP, Buser PT, et al. Late clinical events after clopidogrel discontinuation may limit the benefit of drug- eluting stent:an observational study of drug- eluting stent versus bare-metal stent. J. Am. Coll. Cardiol 2006; 48:25842591. 73. Stettler C, Wandel S, Allemann S, et al. Outcomes associated with drug-eluting and bare metal stent: a collaborative network meta-analysis. Lancet 2007; 370:937948.

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74. Baneryee S, Brilakis E, Zhung S, et al. Endothelial progenitor alls mobilizationafter percutaneous coronary interventions. Atherosclerosis. 2006; 189:7075. 75. Grewe PH, Deneke T, Machraoui A, et al. Acute and chronic tissue response to coronary stent implantation: pathologic findings in human specimen. J. Am. Coll. Cardiol. 2000; 35:156163. 76. Aoki J. Serruys PW, van Beusekom H. Endothelial progenitor cell capture by stents coated with antibody against CO34: the HEALING-FIM(Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First in Man) J.Am.Coll Cardiol 2005; 45:15741579. 77. Duckers HJ, Soulie T, Heijer P et al. Accelerated vascular repair following percutaneous coronary intervention by capture of endothelial progenitor cells promotes regression of neointimal growth at long term follow-up: final results of the HEALING II trial using an endothelial progenitor capture stent (Genous R stent). Eurointervention 2007; 112:35. 78. Duckers H, Onuma Y, Benit E et al. Final results of the HEALING 2B to evaluate a bioengineered CD34 antibody Coated stent (GenousTM Stent) designed to promote vascular healing by capture of circulating endothelial progenitor cell in CAD patients. Circulation 2008; 1185:1042510435. 79. Co M, Tay E, Lee CH, et al. Use of endothelial progenitor cell capture(Genous Bio-Engeneered R Stent) during primary percutaneous coronary intervention in acute myocardial infarction: intermediate to long term clinical follow-up. Am. Heart J.2008; 155:128132. 80. Klamp M, Beijk MA,Verouden N.J, et al. Investigators; design and rationale of the TRI-stent adjudicatio study (TRIAS) program. Am. Heart J 2009; 158:527532. 81. Miglionico M, Patti G, DAmbrosio A, et al. Percutaneous coronary intervention utilizing a new endothelial progenitor cells antibody-coated stent: a prospective single- center registry in high- risc patients. Catether Cardiovasc Interv. 2008; 71:600604. 82. Tamai H, Igaki K, Kyo E, et al. Initial and 6-months results of biodegradable poly-L lactic acid coronary stents in humans. Circulation. 2000; 102(4):399404. 83. Ormiston JA, Serruys PW, Regar E, et al. A bioabsorbable everolimus-eluting coronary stent system for patients with single de novo coronary lesions (ABSORB): a prospective open-label trial, Lancet, 2008; 371: 899907. 84. Serruys PW, Ormiston JA, Onuma Y, et al. A bioabsorbable everolimus-eluting coronary stent system (ABSORB): 2-years outcomes and results from multiple imaging methods. Lancet. 2009; 373(9667): 897910. 85. Ramcharitar S, Serruys PW. Fully biodegradable coronary stents: progress to date. Am J Cardiovasc Drugs. 2008; 8(5): 305314. 86. Erbel R, Di Mario C, Bartunel J, et al. Temporary scaffolding of coronary arteries with bioabsorbable magnesium stents: a prospective, non-randomised multicentre trial. Lancet. 2007; 369(9576):18691875. 87. van der Giessen WJ, Lincoff AM, Schwartz RS, et al. Mrkerd inflammatory sequelae to implantation of biodegradable and non-biodegradable polymers in porcine coronary arteries. Circulation. 1996; 94(7): 16901697. 88. Scheller B. PACCOCATH ISR 1 and 2 A prospective randomized trial of a paclitaxel-eluting balloon in stent restenosis: 2 years results. TCT, 2007, 24 oct. Washington-USA. 89. Unverdorben M. The paclitaxel eluting-balloon catheter in coronary artery disease: PEPCAD I, PEPCAD II-ISR. TCT. 2007 october 24 Washington DC, USA. 90. Cortese B, Micheli A, Picchi A, et al. Paclitaxel-coated balloon versus drug-eluting stent during PCI of small coronary vessels, a prospective randomized clinical trial. The PICCOLETO Study. Heart 2010; 96: 12911296. 91. Results of the DEBUIT Drug Eluting Balloon in Bifurcation Trial. Late-breaking trial presentation. EuroPCR, Paris, France 26 May 2010. 92. Stoeckel D, Bonsignore C, Duda S et al. A survey of stent designs. Min Invas Ther & Allied Technol 2002; 11:137147. 93. Sanjorge G, Melzi G, Agostani P et al. Engineering aspects of stent design and their translation into clinical practice. Ann Ist Super Sanita 2007; 43(1):80100. 94. DiMario C, Griffiths H, Goktekin O et al. Drug-eluting bioabsorbable magnesium stent. Journ. of Interv. Cardiol., 2004; 17(6):391395.

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95. Duckers E. Endothelial progenitor cell capture (EPC): update and perspectives. EuroPCR. Paris France, 2005 96. Kipshidze N, Overlie P, Dunlap T et al. First human experience with local delivery of novel antisense AVI 4126 with infiltrator catheter in de novo native and restenotic coronary arteries: 6-month clinical and angiographic follow-up from AVAIL study. Am Heart Association Scientific Sessions, New Orleans USA, 2004. 97. Bennett M R. In stent restenosis: pathology and implication for the development of drug eluting stents. Heart 2003; 89(2):218224. 98. Fischell TA, Dishman D, Elhaddi et al. The perfect drug-eluting stent. Goals for stent, polymer and drug development. Cardiac Interv today 2009; 2936. 99. Kim J, Parikh N and White R. Future of the Coronary Stent Market: Who Will Win and Why? Spring 2005 Project. Massachusetts Institute of Technology: MIT OpenCourseWare http://ocw.mit.edu.

CHAPTER 2

PATHOGENESIS OF RESTENOSIS PATHOLOGICAL ASPECTS OF CORONARY HIROMASA SUZUKI and HIROYUKI DAIDA INSTENT RESTENOSIS
Introduction ............................................................................................................................... Platelet Aggregation and Thrombus Formation ........................................................................ Platelets-Leukocytes Interaction ............................................................................................... Endothelial Progenitor Cells for Endothelial Regeneration ...................................................... Growth Factors and Neointimal Hyperplasia ............................................................................ Proteoglycans ............................................................................................................................ In-Stent Restenosis by Bare Metal Stent (BMS) ....................................................................... Late Thrombosis and Delayed Vascular Healing in DES .......................................................... Conclusions ............................................................................................................................... References ................................................................................................................................. 47 48 50 51 53 55 56 56 57 57

INTRODUCTION
Restenosis occurs in response to vascular injury after coronary intervention and causes re-narrowing of the blood vessel. Restenosis reflects a complex cascade of molecular and cellular interaction within the injured wall. Vascular injury leads to the activation of various biological cells and the release of numerous vasoactive, thrombogenic, and mitogenic factors in the restenotic process1-4. Restenosis is histologically characterized by a combination of neointimal hyperplasia and vascular negative remodeling as the lumen loss due to vessel constriction by both elastic recoil and adventitial fibrous thickening. Neointimal hyperplasia is found not only in restenotic lesions of patients with coronary intervention but also in de novo lesions of some patients with vasospastic angina, unstable angina or acute myocardial infarction5. The mechanism in that vasospasm or plaque rupture may cause vascular injury in primary coronary lesion. Neointimal hyperplasia results from smooth muscle cell (SMC) proliferation and migration from the media into the intima of blood vessel with the accumulation of extracellular matrix (Figure 2.1). The restenotic process involves several different steps of development which involves SMC activation, proliferation, migration and phenotypic switching to synthetic phenotype and the productive accumulation of proteoglycan-rich extracellular matrix. Migration and proliferation of activated SMCs results from a complex interaction of numerous growth factors, cytokines, inflammatory adhesion molecules, hormonal factors and mechanical factors.

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PART I. RESTENOSIS NEW DISEASE

Intimal hyperplasia

EvG

Azan

EvG

Intimal hyperplasia

HE

Figure 2.1. Autopsy of coronary restenosis 3 months after balloon angioplasty. Coronary arterial restenosis site shows histologically intimal hyperplasia formation inside the old fibrous intimal thickening. Arrow signal shows the rupture site of old fibrous plaque due to the mechanical vascular injury by balloon angioplasty.

PLATELET AGGREGATION AND THROMBUS FORMATION


Mechanical Vascular injury by balloon angioplasty causes initially endothelial denudation with intima-medial fissuring which results in luminal exposure of thrombogenic subendothelial components such as tissue factor, von Willebrand factor, prostaglandins and leads to extensive platelet activation, adhesion, aggregation and secretion. Activated platelets bind to cell adhesion molecules, de-granulate and release of several platelet activators6,7. Activated platelets can recognize adhesive substrates and rapidly attach to the damaged vascular surface. The platelets release numerous cytokines and other bioactive substances. The platelet secretion results in the local release of intracellular granule constituents, including diphosphate, serotonin, thromboxane A2, fibrinogen, fibronectin and von Willebrand factor. The platelets finally induce the binding of the glycoprotein IIb/IIIa integrin to fibrinogen, von Willebrand factor, and fibronectin8. These substances contribute to the activation of neighboring platelets and induce aggregation and final thrombus formation. Other released substances also include chemokines and mitogens such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-) and basic fibroblast growth factor (b-FGF). These growth factors stimulate SMCs migration and proliferation (Figure 2.2).

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49

Adhesion molecules

Endothelial progenitor cells patrol for injured endothelium in blood vessel.

Leukocytes

P-selectin

Platelets
aggregation activation

Mural thrombus

Attach Rolling Migration ThromboxaneA2 PDGF, TGF,IL-1,6,8 Thrombin, P-selectin

Injured endothelium

Vascular repair

Endothelium

Intima

Inflammation
Growth factors Cytokine Chemokine

Mitogenic SMCs

proliferation migration

Media

Adventitia
Figure 2.2. Vascular injury with intima-media fissuring leads to platelet aggregation, inflammation, and SMC proliferation.

Platelets express two 3 receptors, not only IIb3 (GP IIb-IIIa), but also v3 (fibronectin receptor). GP IIb-IIIa complexes are confined to platelets and cells of the megakaryocyte lineage, whereas v3 is heavily expressed by numerous cells, including endothelial cells, monocytes, SMCs9-11. One of the most prevalent integrins, V3 integrin is widely expressed on vascular cells and is present on luminal and surfaces of endothelial cells, as well as SMCs, fibroblasts, macrophage, and platelets. The v3 integrin is thought to play a major role in the adhesion and migration of SMCs and endothelial cells on extracellular matrix, and consequently in the restenotic process. This integrin appears to bind several extracellular matrix proteins (vitronectin, fibronectin, thrombospondin, laminin, collagen I and IV), and antiserum against this receptor inhibits SMC migration on all substrates12. The process of restenosis formation is also attributed to the inflammatory response induced by vascular injury1-4. The local inflammatory response is critically dependent on several cell adhesion molecules such as selectins, integrins, cadherin and immunoglobulin. In particular, P-selectin has an important role in the inflammatory response and growth of neointimal formation after vascular injury13-16. P-selectin is located in both the -granule of platelets and Weibel-Palade body of endothelial cells. P-selectin upregulates tissue factor in monocytes and leads to leukocyte accumulation in the site of vascular injury associated with thrombosis and inflammation. P-selectin mediates the rolling and migration of leukocytes on the surface of the endothelium and initiates the attachment of leukocytes circulating in blood to

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PART I. RESTENOSIS NEW DISEASE

the aggregated platelets, other circulating leukocytes and endothelial cells at sites of vascular injury and inflammation (Figure 2.2). Furthermore, P-selectin could be involved in vascular inflammatory processes on the adventitial side as well as on the injured endothelial side. A recent experimental study has demonstrated that the inhibition of P-selectinmediated leukocyte recruitment prevents negative remodeling with adventitial fibrosis as well as neointimal formation in an arterial balloon-injury model17. It is of interest that P-selectin plays a key role in the restenotic processes after balloon injury and restenosis after angioplasty might be modified by the attenuation of inflammatory factors through antiP-selectin treatments.

PLATELETS-LEUKOCYTES INTERACTION
The platelet activation and thrombin secretion induce leukocyte recruitment and migration at the site of vascular injury. Leukocytes attach and roll onto the endothelial surface while undergoing activation via the interaction of leukocytes surface P-selectin glycoprotein ligand (PSGL-1) and P-selectin on the bound adherent platelet18-20. Activated leukocytes then migrate into the site of vascular injury and this process is closely mediated by adherence molecules such as monocyte chemotactic protein (MCP-1), intercellular adhesion molecule 1(ICAM-1), and Mac-121-23. Several recent studies have documented that after arterial injury, not only endothelial cells but also vascular SMCs express ICAM-1and vascular cell adhesion molecule 1 (VCAM-1). VCAM-1 interacts with the integrin 41 [very lateacting antigen 4 (VLA-4)] that is constitutively expressed on the surface of leukocytes, and the blockade of the VCAM-1/VLA-4 pathway reduces monocyte infiltration. Experimental studies revealed that the blockade of MCP-1, integrins VLA-4 and molecules responsible for infiltration and activation of monocytes/macrophages, attenuated neointimal hyperplasia and negative remodeling in different animal models2427. Moreover, a carotid artery injury model study also demonstrated that apolipoprotein Edeficient mice with targeted disruption of the P-selectin gene exhibited dramatic decreases in monocytes infiltration into the arterial wall, which result in significant decrease of neointimal hyperplasia28. In addition, VCAM-1, which is thought to act primarily or only on leukocyte chemotaxis, may also play an important role in the migration of SMCs, very likely via VLA-4 identified on SMCs27. Otherwise, coagulation proteins such as thrombin, plasminogen activator inhibitor and tissue factor, may also be implicated in the response of SMCs. These findings suggest that expression of adhesion molecules on SMCs influences their proliferation, differentiation, and migration and therefore, may actively contribute to the inflammatory neointimal formation process in the vascular wall after balloon injury. The degree of leukocyte migration could be an important factor to regulate the level of neointimal thickening at the vascular healing site29. It is no doubt that stent induces to activate platelets and promotes leukocyte recruitment to the injured vessel wall and leukocyte-platelet aggregates in blood, as well as elevated plasmatic levels of monocytes related cytokine interleukin-6. The activated monocytes may contribute to neointimal thickening30 by generating reactive oxygen species through the production of growth and chemotactic factors31 binding to a broad repertoire of ligands32 or by matrix metalloprotease production capable of degrading matrix constituents and consequently facilitating cell migration33. Clinical study has demonstrated that circulating monocytes increase after stent implantation and the peak monocyte count correlates to the in-stent neointimal volume. Circulating monocytes could play a significant role in the process of in-stent neointimal hyperplasia34 (Figures 2.3 and 2.4).

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Many monocytes attached nearby the stent-struts implanted 3 days in coronary artery of a porcine model (2000).

Intimal hyperplasia at 4 weeks in a porcine model of coronary in-stent restenosis.

Figure 2.3. Electron -micrograph and photo-micrograph of the stent-implanted coronary artery in a porcine model.

Foreign body type giant cells emerge.


Figure 2.4. In-stent restenosis in coronary bypass grafting of saphenous vein: Neointimal hyperplasia (arrow) with foreign body type giant cells (circle) emerged nearby the stent-struts in human. Intimal hyperplasia with adventitial fibrous thickening is found in the stent-site.

ENDOTHELIAL PROGENITOR CELLS FOR ENDOTHELIAL REGENERATION


In order to inhibit inflammation and SMCs proliferation at the injured wall, prompt re-endothelialization after vascular damage is a crucial mechanism limiting neointimal formation35. It was long believed that endothelial regeneration is a local process achieved by proliferation and immigration of endothelial cells from adjacent, intact parts of the intima. However, it has become evident that bone marrowderived endothelial progenitor cells exist in the blood36,37. Current main attention has focused on a novel mechanism of vascular repair involving a bone marrow-derived precursor or stem cell: the endothelial progenitor cell (EPC)

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PART I. RESTENOSIS NEW DISEASE

(Figure 2.1). This evolving concept has been confirmed primarily through studies of bone marrow transplant recipients in whom appropriately differentiated cells of donor origin integrate into host structure, such heart and lung38,39. Contrary to the traditional paradigm of re-endothelialization, it is clear that this principle applies to accelerate re-endothelialization of injured vascular endothelial denudation40. It has been proposed that EPCs are mobilized in response to vascular injury and promptly emerged to the vascular site of endothelial denudation36 and facilitate promptly re-endothelialization37. Notably, infusion of EPCs attenuates neointimal hyperplasia after vascular injury, resulting in reduction of restenosis41. Experimental study have demonstrated that mobilization of EPCs to the circulation by statins therapy is closely associated with an enhanced re-endothelialization and significantly decreased neointimal formation, resulting in reduction of restenosis42. Moreover, Estrogens also increase the numbers of EPCs by their anti-apoptotic effects, leading to accelerated vascular repair and decreased neointimal formation after vascular injury43. Recent study has demonstrated evidence of interest that the platelets activated by endothelial denudation, especially when induced by thrombin/collagen, seed numerous specific membrane vesicles of platelet microparticles (PMP) which amplify the vasoregenerative capacity of EPCs to support the maintenance of vascular integrity after arterial injury44. It is of great interest that PMPs modulate functional feature of endothelial progenitor cells crucial for their regenerative potentials, including recruitment and migration to sites of vascular injury, differentiation, and release of angiogenic factors stimulating resident endothelial cells (Figure 2.5). However, the pathophysiological relevance or therapeutic potential of EPCs as well as PMPs remains unclear in detail and further more studies are needed to determine their biological values and therapeutic benefits (Figures 2.5 and 2.6).
Endothelial progenitor cells . Synthetic SMC Re-endothelial cell

Dead endothelial cells are replaced by endothelial progenitor cells.

Endothelium
Spindle-shape, synthetic state SMCs produce proteoglycan-rich ECM in the neointima.

Intima

ECM (proteoglycan)

Media

Adventitia
Figure 2.5. Neointimal formation and endothelial regeneration: Interaction among SMCs, ECM, Endothelial progenitor cell and Platelet microparticles.

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53

Leukocytes
Rolling & migration Growth factors Cytokine Chemokine

Promotor Inhibitor
( on the restenosis process)

Endothelial denuation

Platelets
Aggregation & secretion Platelet microparticles

Neointima formation

SMCs
Proliferation & migration

ECM synthesis

Endothelial progenitor calls

Re-endothelium

Figure 2.6. Restenosis process of neointima formation: From endothelial denudation to re-endothelium.

GROWTH FACTORS AND NEOINTIMAL HYPERPLASIA


Neointimal hyperplasia is absolutely essential in restenosis. After injury, the activated platelets release numerous mitogens, various growth factors, including thromboxane A2, serotonin, PDGF, b-FGF, and insulin-like growth factor, stimulate cell proliferation via different signaling mechanisms after vascular injury (Figure 2.2). In particular, PDGF and VEGF promote a switch of the medial SMCs phenotype from the quiescent to mitogenic and the proliferation of SMCs45-49. Angiogenesis is a complex process. Vascular endothelial growth factor (VEGF) is unique among angiogenic growth factors. VEGF plays an important role in angiogenesis and is also an endothelial cell chemoattractant. In addition, certain VEGF isoforms implicate in the normal formation of vascular smooth muscle cell. VEGF, as a strong endothelial cell mitogen, can promotes re-endothelization45, reduces intimal thickening and thrombus formation after balloon endothelial denudation and stent implantation, and consequently may plays a protective role against restenosis. Multiple growth factors and cytokines lead to the activation of SMCs which is associated with a shift from a contractile to synthetic phenotype and result in the restenotic process of SMCs49. TGF- stimulates both myofibroblast and SMCs differentiations of mesenchymal progenitors, and is also likely to be important for these phenotypic changes in neointimal hyperplasia. Proliferated SMCs shift mitogenic phenotype to synthetic-type and begin to produce extracellular matrix (ECM) molecules in the neointimal lesion soon. Co-regulation system between SMCs and endothelial cells exists in vascular wall. Intact endothelium inhibits proliferation of vascular SMC. Therefore, the end of SMCs proliferation and ECM production in restenosis process is signaled directly by their contact with the excess of ECM formed or by the re-growth of normal endothelium. To promote endothelial repair is crucial to limit neointimal formation after vascular injury.

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PART I. RESTENOSIS NEW DISEASE

In particular, as shown in Figures 2.2, 2.7 and 2.8, proliferation of medial SMCs starts within 24 hours immediately after mechanical vascular injury and continues for at least 2 weeks. The activated medial SMCs migrate to the intima through the ruptured internal elastic membrane. In this early phase (Figure 2.1 and 2.2), Proliferative cell nuclear antigen (PCNA)-positive SMCs are abundantly present in the coronary restenosis lesion within 1 month after angioplasty. PCNA-positive cell is consistent with a cofactor for DNA polymerase delta50. Therefore, shown in Figure 2.6, SMCs with large-round nuclei express principally during S phase of the cell cycle and could be mitogenic in the neointimal lesion. However, in the early phase, the ECM produced by proliferated SMCs is negligible. After 3 to 4 months after angioplasty, the degree of neointimal thickening peaks to maximal and the ECM accumulation is dramatically increased. The lesion volume increases abruptly due to the adjunctive synthesis and the accumulation of proteoglycan-rich ECM, produced by synthetic SMCs. Finally after 6 months or over, synthetic SMCs return to a contractible phenotype and no further significant increase in neointimal hyperplasia occurs, otherwise the ECM is produced much and collage tissue fibers in the neointima start to emerge. Restenosis lesion is characteristically myxedematous because the property of ECM is hydrophilic. Therefore, restenosis lesion is quietly different from primary atherosclerotic lesions: comparatively, significantly greater SMCs hyperplasia and less densely layered collagen with much proteoglycan have been observed in restenotic lesions (Figures 2.2 and 2.8).

Immature-type, mitogenic-type SMCs with large-round nuclei proliferate (in early phase).

Spindle-shape, syntetic-type SMCs produce proteglycan-rich ECM (in late phase).

Figure 2.7. Atherectomy-derived coronary specimens of restenosis show 2 types of SMCs in neointima hyperplasia.

Vascular SMC growth is redox sensitive. Oxygen-derived free radical (ROS) also plays an important role in restenosis. ROS increases after stimulation with a various agonists such as thrombin, angiotensin II, PDGF, and other growth factor and ligands in injured vascular wall. These growth factors and ligands bind to trans-membrane receptors on the surface of SMCs that initiate intracellular signaling cascades. There is a link between angiotensin IIinduced NADH/NADPH oxidase activity, increases in intracellular H2O251, p38 mitogen activated protein kinase activity52, and vascular SMC hypertrophy. It is suggested that the opposing relationship between NO and angiotensin II creates a balance between SMC proliferation and apoptosis53.

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55

1 month after PCI

PCNA immuno-stain

6 month after PCI

Old fibrous plaque

-actin & alsian-blue stains

Azan stain

Figure 2.8. Comparison of histological findings of intimal hyperplasia in early and late phase of restenosis with old fibrous plaque in de novo lesion.

PROTEOGLYCANS
The extracellular matrix provides a structural framework essential for the functional properties of vessel walls. The extracellular matrix molecules are composed mainly of elastin, collagens, proteoglycans and structural glycoproteins. In restenosis lesion, the proliferative-synthetic phenotype SMCs produce abundantly extracellular matrix which consists of proteoglycan (vesicant, billycan, and decorin), hyaluronan, and collagen (type I and III). It is certain that although most attention has been focused on SMC proliferation, the ECM volume is main factor of restenosis and accounts for >50% of volume of neointimal hyperplasia54. The ECM plays various important roles in the developing neointimal formation, including the growth process of SMCs. Proteoglycan and hyaluronan are synthesized by SMCs and participate in regulation of vascular permeability, lipid metabolism, and thrombosis. On the contrary, the extracellular matrix molecules are hydrolyzed by proteasesmatrix metalloproteinases, leukocyte elastase. The synthesis of hyaluronan and versican is highly regulated and influenced by pro-inflammatory growth factors such as PDGF and transforming growth factor-beta (TGF-beta)55. Versican has a hydrophilic property and can expand the volume neointimal hyperplasia to be myxedematous and watery-state by binding hydrogens56. The characteristic morphology of neointimal hyperplasia is SMCs proliferation with water-entrapping matrix macromolecules composed of hyaluronan and versican (Figures 2.2 and 2.8).

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PART I. RESTENOSIS NEW DISEASE

IN-STENT RESTENOSIS BY BMS


Compared with balloon angioplasty, the treatment of coronary artery disease with percutaneous placement of bare metal stent is now well-established as clinical useful therapeutic strategy to be used as permanent vascular internal framework to maintain or increase the lumen of a blood vessel to prevent arterial dissection and to eliminate vessel recoil after balloon angioplasty. Although it is certain that stent reduces restenosis as well as the procedural complication rates compared with balloon angioplasty, stent placement has failed to achieve acceptable restenosis rate because of in-stent restenosis especially in several subgroups of patients, including subgroups with diabetes mellitus, small coronary vessel, saphenous vein graft and long lesions57-59 (Figure 2.6). Coronary stenting is occasionally accompanied by medial damage or penetration of the stent into a lipid core and then induces increased arterial inflammation, which is closely associated with increased neointimal growth. Therefore, although stent reduces early elastic recoil and lumen loss due to negative remodeling by adventitial fibrous thickening, they do not decrease neointimal hyperplasia60. The neointimal hyperplasia of in-stent restenosis is extremely different from that of restenosis due to balloon angioplasty. Several pathological studies revealed that chronic inflammatory cells such as macrophages and lymphocytes are seen in not only early phase but late phase (>6 months) after stenting60-62. Arterial medial disruption and lipid core penetration by stent struts are both associated with chronic inflammation. It is possible that the constant motion of the semi-rigid stent-struts damages directly the vascular wall, and then results in local hemorrhage and fibrin deposition61. Moreover, thrombotic fibrins, neovascularization, and macrophages as well as foreign-body multinucleated giant cells are found around the stent-struts in in-stent restenosis lesions62. These pathological findings are exactly consisting with foreign body reaction and chronic inflammation against implanted stent-struts.

LATE THROMBOSIS AND DELAYED VASCULAR HEALING IN DES


Drug-eluting stent (DES) with both anti-inflammatory and anti-proliferative drug attached via polymers on the stent surface to minimize SMC proliferation, has reduced restenosis rate and been suitable for nearly all lesions and patient subsets compared with BMS. Therefore, DES is highly effective in preventing in-stent restenosis after coronary intervention and the need for repeat revascularization are dramatically reduced. However, recent major clinical concern has arisen over increased risk of late stent thrombosis with this device64. Moreover, the local toxicity and allergic hypersensitivity of the drug on the vessel wall result in long-term endothelial dysfunction65,66. Recent pathological study of DES in human demonstrated that DES has significantly less in-stent neointimal growth, more frequent eosinophils, lymphocytes inflammation and higher fibrin deposition at the stent-struts compared with BMS64. Inflammatory reaction including eosinophils and foreign-body giant cells with focal fibrins deposition surrounding stent-struts are more frequent as hallmarks of delayed vascular healing in DES. Therefore, DES causes substantial impairment of endothelial biological property at the stent-implanted site for a long period after its stent implantation because of the late healing of vascular injury and local allergic hypersensitivity resulting in lack of complete re-endothelialization and persistent of fibrin deposits. Poor endothelialization is the most important predictor late stent thrombosis

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57

along with ratio of uncovered total stent-struts. The additional risk factors for late stent thrombosis placement includes stent-penetration of necrotic core, malapposition, overlapping stent placement, excessive stent length, and bifurcation lesions64,65.

CONCLUSIONS
Drug-eluting stents (DES) are currently widely used because in-stent restenosis and the need for repeat revascularization has been dramatically reduced. DES is widely established as useful therapeutic tool in preventing the rate of restenosis. However, the infrequent but worrying problem of late thrombosis and delayed healing of DES has changed the clinical focus from 6 months after coronary intervention to nearly life-long follow-up. Because the time course of vascular healing process after DES placement may vary from patient to patient, all patient at high risk for late thrombosis should receive dual anti-platelet therapy for prolonged periods of time. DES can inhibit neointimal hyperplasia but lack complete re-endothelium covering the stent-struts. Therefore, accelerating re-endothelium promptly at the stent-site is an important crucial issue and the therapeutic potential of endothelial progenitor cells is of great interest. The use of endothelial progenitor cells further enlarges the range of therapeutic options because they can be genetically engineered to express proangiogenic cytokines or other therapeutically useful molecules. To use endothelial progenitor cells effectively as new strategy combined with DES therapy for prevention of stent-related complications is expected.

REFERENCES
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38. Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, Kajstura J, Leri A, Anversa P. Chimerism of the transplanted heart. N Engl J Med. 2002; 346(1):515. 39. Suratt BT, Cool CD, Serls AE, Chen L, Varella-Garcia M, Shpall EJ, Brown KK, Worthen GS. Human pulmonary chimerism after hematopoietic stem cell transplantation. Am J Respir Crit Care Med. 2003; 168(3):31822. 40. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275:964967. 41. Minami E, Laflamme MA, Saffitz JE, Murry CE. Extracardiac progenitor cells repopulate most major cell types in the transplanted human heart. Circulation. 2005; 112(19):29518. 42. Werner N, Priller J, Laufs U, Endres M, Bohm M, Dirnagl U, Nickenig G. Bone marrow-derived progenitor cells modulate vascular reendothelialization and neointimal formation: effect of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition. Arterioscler Thromb Vasc Biol. 2002; 22:15671572. 43. Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, Nishimura H, Losordo DW, Asahara T, Isner JM. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation.2002; 105:30173024. 44. Strehlow K, Werner N, Berweiler J, Link A, Dirnagl U; Priller J; Laufs K; Ghaeni L, Milosevic M, Bhm M, Nickenig G. Estrogen Increases Bone MarrowDerived Endothelial Progenitor Cell Production and Diminishes Neointima Formation. Circulation. 2003; 107:r119r125. 45. Mause SF, Ritzel E, Liehn EA, Hristov M, Bidzhekov K, Mller-Newen G, Soehnlein O, Weber C. Platelet microparticles enhance the vasoregenerative potential of angiogenic early outgrowth cells after vascular injury. Circulation. 2010; 122(5):495506. 46. Asahara T, Bauters C, Pastore C, Kearney M, Rossow S, Bunting S, Ferrara N, Symes JF, Isner JM. Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation. 1995; 91: 27932801. 47. Lazarous DF, Shou M, Stiber JA, Hodge E, Thirumurti V, Goncalves L,Unger EF. Adenoviral-mediated gene transfer induces sustained pericardial VEGF expression in dogs: effect on myocardial angiogenesis. Cardiovasc Res. 1999; 44:294302. 48. Pakala R, Willerson J, Benedict CR. Effect of serotonin, thromboxane A2, and specific receptor antagonists on vascular smooth muscle proliferlation. Circulation. 1997; 96:228086. 49. Jackson CL, Raines EW, Ross R, Reidy MA. Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury. Arterioscler Thromb. 1993; 13:121826. 50. Bravo R, Frank R, Blundell PA, Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987; 326(6112):51517. 51. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy.Hypertension. 1998; 32(3):48895. 52. Chen S, Qiong Y, Gardner DG. A role for p38 mitogen-activated protein kinase and c-myc in endothelin-dependent rat aortic smooth muscle cell proliferation. Hypertension. 2006; 47:2528. 53. Tan NY, Li JM, Stocker R, Khachigian LM. Angiotensin II-inducible smooth muscle cell apoptosis involves the angiotensin II type 2 receptor, GATA-6 activation, and FasL-Fas engagement. Circ Res. 2009; 105(5):42230. 54. Wight TN. Cell biology of arterial proteoglycans. Arteriosclerosis. 1989; 9:120. 55. Wight TN. Arterial remodeling in vascular disease: a key role for hyaluronan and versican. Front Biosci. 2008; 13:49337. 56. Farb A, Kolodgie FD, Hwang JY, Burke AP, Tefera K, Weber DK, Wight TN, Virmani R. Extracellular matrix changes in stented human coronary arteries. Circulation. 2004; 110(8):9407. 57. Elezi S, Kastrati A, Pache J, Wehinger A, Hadamitzky M, Dirschinger J, Neumann FJ, Schmig A. Diabetes mellitus and the clinical and angiographic outcome after coronary stent placement. J Am Coll Cardiol. 1998; 32(7):186673. 58. Lau KW, Ding ZP, Sim LL, Sigwart U. Clinical and angiographic outcome after angiography-guided stent placement in small coronary vessels. Am Heart J. 2000; 139(5):8309. 59. Heidland UE, Heintzen MP, Michel CJ, Strauer BE. Risk factors for the development of restenosis following stent implantation of venous bypass grafts.Heart. 2001; 85(3):3127. 60. Farb A, Sangiorgi G, Carter AJ, Walley VM, Edwards WD, Schwartz RS, Virmani R. Pathology of acute and chronic coronary stenting in humans.Circulation.1999; 99:4452.

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61. Farb A, Weber DK, Kolodgie FD, Burke AP, Virmani R. Morphological predictors of restenosis after coronary stenting in humans. Circulation. 2002; 105(25):297480. 62. Inoue K, Abe K, Ando K, Shirai S, Nishiyama K, Nakanishi M, Yamada T, Sakai K, Nakagawa Y, Hamasaki N, Kimura T, Nobuyoshi M, Miyamoto TA. Pathological analyses of long-term intracoronary Palmaz-Schatz stenting; Is its efficacy permanent? Cardiovasc Pathol. 2004; 13(2):10915. 63. Finn AV, Nakazawa G, Joner M, Kolodgie FD, Mont EK, Gold HK, Virmani R. Vascular responses to drug eluting stents. Importance of delayed healing. Arterioscler Thromb Vasc Biol. 2007; 27:150010. 64. Joner M, Finn AV, Farb A, Mont EK, Kolodgie FD, Ladich E, Kutys R, Skorija K, Gold HK, Virmani R. Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk. J Am Coll Cardiol. 2006; 48(1):193202. 65. Finn AV, Joner M, Nakazawa G, Kolodgie F, Newell J, John MC, Gold HK, Virmani R. Pathological correlates of late drug-eluting stent thrombosis: strut coverage as a marker of endothelialization. Circulation. 2007; 115(18):243541.

CHAPTER 3

PATHOPHYSIOLOGY OF RESTENOSIS AFTER PERCUTANEOUS CORONARY INTERVENTION


ERBAN BLNESCU

Introduction .............................................................................................................................. Definition of Restenosis ........................................................................................................... Can We Predict the Occurrence of Restenosis? ........................................................................ Pathophysiology ....................................................................................................................... Conclusion................................................................................................................................ References ................................................................................................................................

61 62 63 64 71 71

INTRODUCTION
Percutaneous coronary intervention (PCI) is the most important progress in the treatment of coronary artery disease (CAD) in the last three decades and it is a good alternative of myocardial revascularization compared to by-pass surgery (CABG). The basic concept is to circumferentially and longitudinally fracture and sometimes ablate the atherosclerotic plaque responsible of an arterial stenosis by different devices introduced to a distant procedural site by endovascular approach. Successive technological improvements were associated with significant progress in the design of coronary stents, balloons and guide-wires and allowed to diversify the indications and the complexity of coronary lesions that can thus be treated. Soon after the validity and clinical usefulness of the procedure was recognized it became obvious that in some cases the abnormal healing process of the balloon or stent-induced vascular lesion is responsible for the recurrence of coronary stenosis at the treated site. This process was called restenosis and was considered as the most significant limitation of long term results after PCI that occurred in as high as 40% of patients, depending on the characteristics of the treated lesion or the implanted stent. If the initial lesion responsible for arterial narrowing is purely atherosclerotic, restenosis post-PCI consists mainly of intraluminal neointimal proliferation and is an exacerbation of the normal healing process. This can occur in the first 6 to 9 months after the initial procedure. Restenosis was the fundamental issue for Interventional Cardiology since its birth in 1977, after acute thrombosis could be effectively prevented by stent implantation and administration of dual anti-platelet therapy. Restenosis is responsible for the increased morbidity by recurrent ischemic events after the first coronary intervention and markedly increases medical costs in CAD by the need for target vessel revascularization.

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Due to initial unacceptably high incidence of restenosis and its costs in the last two decades an extensive worldwide medical effort was made to clarify pathophysiological mechanisms responsible for its occurrence. Concomitant complex randomized clinical trials were designed and realized in order to test the most appropriate therapy to limit the effect of this process. Cellular mechanisms of restenosis have been mostly elucidated and spectacular results were obtained by widespread use of drug eluting stents. Today the prevalence of coronary restenosis with modern drug eluting stents is in the single-digit range, but research is still going on to identify the best balance between plaque passivation and normal healing of the vessel wall.

DEFINITION OF RESTENOSIS
Restenosis can be defined by clinical and angiographic criteria. As far as recurrence of ischemic symptoms is the main criteria to suspect clinical restenosis, while the histological stenosis can progress in the absence of symptoms, angiographic restenosis is more frequent than it can be clinically recognized. Because of this all studies that investigated different methods or devices to prevent or treat restenosis needed angiographic control at pre-specified time intervals. With research purposes, to precisely quantify the restenotic process anatomically and functionally, angiography is completed with complex intracoronary investigation (i.e. intravascular ultrasound, flow measurement by intracoronary Doppler or Pressure-Wire to assess coronary flow reserve1. Clinical restenosis is defined by recurrence of myocardial ischemia after a PCI procedure, that can be recognized by typical angina and proven by functional tests (ECG exercise test, dobutamine stress echocardiography or radio-isotopic myocardial perfusion studies). This can be extended to target lesion revascularization driven by ischemia or in extreme cases an acute major adverse cardiac event (death, acute myocardial infarction or urgent target vessel revascularization). Angiographic criteria to define restenosis surprisingly suffered from imprecision and criteria polymorphism in different studies. Keeping in mind that the main method to assess an arterial stenosis is angiography, the lack of uniform criteria to define restenosis may look awkward. Different definitions relying on absolute changes of minimal luminal diameter at follow-up have been used in published trials (Table 3.1). Defining restenosis as more 50% recurrent stenosis of the in-segment treated vessel at follow-up gained wide practical recognition. As already stated above angiographic restenosis is not mandatory associated with symptoms. Discovery of angiographic restenosis in the absence of angina should not be followed by a new revascularization procedure, if functional tests cannot prove that restenosis could be held responsible for inducible myocardial ischemia.
Table 3.1. Angiographic definitions of restenosis in different studies with 6-month follow-up after PCI 1. Luminal stenosis of 30% compared with the initial result of PCI 2. Residual stenosis post-PCI < 50% which increases to 70% at follow-up angiography 3. Increase of stenosis with more than 10% of that initially obtained by PCI 4. Late luminal loss of > 50% at follow-up compared to the initial net luminal increase by PCI 5. Residual post PCI stenosis < 50% that progresses to > 50% at follow-up 6. Decrease of minimal luminal diameter of the treated segment with > 0.72 mm compared to the initial post PCI result

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The prevalence of angiographic restenosis is different with respect of the type of the interventional procedure performed initially: 1. Plain balloon angioplasty is associated with a restenosis rate of 40 to 50% 2; 2. Bare-metal stent (BMS) implantation reduces restenosis rate to 1530%. The efficacy of stent implantation to obtain immediate superior results with respect to balloon angioplasty was proven in BENESTENT I3 and STRESS (4) trials. The results of STRESS study showed a restenosis rate in native stented coronary arteries of 32%4 versus 14% in an international registry5. These results suggest a significant source of bias in some international registries that provide observational data, against those obtained from randomized clinical trials. 3. Up to widespread availability of DES, balloon angioplasty on chronic total occlusions was associated with a very high incidence of restenosis (up to 77%), and coronary reocclusion was apparent in 40% of patients6. BMS implantation lowers restenosis rate to 3040%7,8. Sirolimus-coated DES reduce the incidence of restenosis to 711% and the reocclusion rate to 4% with respect to BMS when used to treat recanalized chronic occlusions9. 4. After any PCI procedure associated with debulking of the atherosclerotic plaque (rotational or directional atherectomy) restenosis peaks a 40-60% rate10-12. The incidence of restenosis increases in parallel with the severity of vessel wall injury towards the media and adventitial layers2. 5. PCI on degenerated by-pass grafts is associated with a restenosis rate of 37% in randomized trials that used a BMS13; registries provide restenosis rates of 22% in this clinical context 14. Plain balloon angioplasty is associated with restenosis in more than 50% of cases15. Half of patients that develop angiographic restenosis (between 45 and 55%) irrespective of diagnostic criteria are asymptomatic, as it resulted from a retrospective analysis of 10 multicenter trials that enrolled 2690 patients16.

CAN WE PREDICT THE OCCURRENCE OF RESTENOSIS?


The most important predictors of restenosis are diabetes, lesion length and post-procedural minimal luminal diameter. Three main variable categories are related to an increased risk of restenosis after PCI17,18. 1. Patient related variables include diabetes, end-stage renal disease, age, male sex, systemic hypertension, hypercholesterolemia, unstable angina at the time of PCI, vasospastic angina and continued smoking after PCI1. 2. Anatomical factors of the treated lesion that are associated with an increased incidence of restenosis are: ostial lesions19, proximal LAD involvement20, long lesions > 20 mm21, recanalized chronic total occlusions22, calcified lesions and degenerated venous vein grafts13. Restenosis in vessels larger than 3 mm is lower than in vessels with a diameter lower than 3 mm20. 3. Procedural factors associated with restenosis risk are: low balloon artery ratio (i.e. 1/1), a final suboptimal result of PCI with a residual significant stenosis > 1530%21,23, the residual coronary dissection not covered with the stent and multi-vessel or multi-lesion PCI during a single procedure. An optimal final result of plain balloon angioplasty (stent-like result), defined by a residual stenosis of less than 15%, with no significant dissection and TIMI 3 flow is seldom encountered in clinical practice, in about 11% of patients23. The restenosis rate in this case is low, of only about

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14%. The suboptimal result, with residual stenosis of more than 15% needs stenting, that is generally associated with a mean restenosis rate of less than 20% with bare metal stents23.One of the main determinants of restenosis with the use of bare metal stents is the total length of the stents implanted; this is generally correlated with the total length of the lesion 24,25. This is mainly happening in small vessels with a diameter of less than 3 mm, while restenosis is lower in large vessels, over 3.54 mm. Thus implantation of very long multiple bare metal stents (full metal jacket) should be generally avoided26. In the MAGIC 5L trial the correlation between the length of the a bare metal stent and angiographic restenosis rate was investigated; this varied between 25.9% for short (9 mm) stents and 67.5% for long stents (> 43 mm)27. The BMS were implanted in vessels larger than 2.5 mm. Multivariable analysis showed that stent length is an independent determinant of angiographic restenosis and of major cardiac adverse events at 6 and 12 months after PCI. If this stands true for bare metal stent implantation, the widespread use of long drug eluting stents has dramatically reduced restenosis even in the case of very long lesions. The decrease of restenosis rate with the use of very long DES pays the price of an increased risk of late stent thrombosis28. Coronary angioscopy performed after stent implantation suggests the role of residual thrombus at the site of the treated lesion in restenosis occurrence29. Among good long term prognostic factors the most important is post-procedural minimal lumen diameter; this was postulated in the aphorism bigger is better, valid in interventional cardiology also30.

PATHOPHISIOLOGY
Pathophysiological mechanisms responsible for restenosis are particularly complex and involve close interaction between cytokines and growth factors with the cells of the vascular wall, platelets and monocytes, directly related to the extension of endovascular traumatic vessel wall injury (Figure 3.1)31. These mechanisms have been extensively investigated in experimental studies on restenosis models, as far as human restenotic tissue is impossible to be obtained in vivo. Some anatomical data was obtained in living humans by intravascular ultrasound and more recently by optical coherence tomography. Despite the fact that inflammation plays a crucial role in restenosis as it does in atherogenesis, the former is a completely distinct phenomenon. As far as restenosis is closely related to the mechanisms responsible for increasing vessel lumen diameter at angioplasty, we should shortly review these. 1. Angioplasty pioneers Gruentzig, Dotter and Judkins, explained the increase of lumen diameter obtained by balloon expansion through atherosclerotic plaque compression against the vessel wall32. This initial concept of plaque compression is hardly an explanation for the observed increase of vessel diameter by angioplasty, as far as most atherosclerotic plaques are made of a variable amount of fibrous tissue and calcium deposits that are very difficult to compress. The mechanism can be conceptually possible in the case of newly formed soft plaques, with a rich lipid core, or with thrombus containing lesions. 2. The main mechanism of increasing lumen diameter by angioplasty is longitudinal rupture or fracture of atherosclerotic plaque. This is associated with a variably deep rupture of the media under the plaque, intimal dissection and the stretch of healthy vessel wall opposite to the atherosclerotic plaque; this occurs when the plaque is eccentrically developed and does not involve the vessel wall circumferentially33.

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Dissection is frequently observed both on soft plaques that rupture leaving a prominent intimal flap into the lumen and on calcified plaques. Intracoronary ultrasound showed that the mechanisms of luminal increase are different, depending on the composition of the atherosclerotic plaque. 3. In some cases angioplasty can not be performed without the use of ablative procedures that allow direct excision of atheroma (directional or rotational atherectomy, laser photo-ablation), by scratching, polishing or photo-vaporization with or without extraction of pathologic tissue33. These ablative techniques are sometimes necessary in the case of heavily calcified lesions, eccentric bifurcation plaques or chronic total occlusions that cannot be successfully predilated. The procedure is consequently completed by balloon dilatation and stent implantation.

Figure 3.1. Schematic presentation of the sequence of main events leading to restenosis. (SMC smooth muscle cells; MMPs metalloproteinase).

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Based on the above mechanisms we can easily understand how vessel healing determined by the initial wall injury can sometimes lead to restenosis 34: 1. Early thrombus formation determined by vessel wall injury and on the surface of implanted stents as foreign bodies; platelet adhesion and aggregation, as coagulation is always present in various amounts at the angioplasty site, can initiate restenosis. Thrombus can serve as a matrix for fibroblast migration from the media and neointima formation may thus become excessive leading to restenosis. Dual antiplatelet therapy and appropriate anticoagulation during the procedure may limit significantly local thrombus formation. 2. Excessive proliferation of cellular components of the atherosclerotic plaque and/or of the vessel wall at the site of balloon/stent trauma; this is constantly associated with vessel wall remodeling. Barotrauma can activate smooth muscle cells from the media to migrate to the intima and transform into fibroblasts; these are the main cellular components of the restenotic process. There is a close relationship between the extent of media damage, activation of inflammation and restenosis35. 3. Incomplete removal of atheroma generally associated with simple balloon angioplasty is responsible for progressive elastic recoil. One of the characteristics of lesions prone to restenosis is related to the concentric or circumferential distribution of plaque. The sequence of events occurring at the site of PCI can be grouped in three distinct phases that are interlaced and that finally concur to produce restenosis (Figure 3.2)36:
Elastic Thrombus recoil formation Inflammation; proliferation Extracellular matrix synthesis

100

Maximal effect

(%)

TxA2 PDGF

TGF-1 FGF

PDGF-A PDGF-B

TGF- PDGF

1 100

14

30

80

300

thickness (%)
1

Neointima

14

30

80

300

Figure 3.2. The phases of restenosis. Upper panel presents the sequence of activation of growth factors implicated in restenosis; lower panel presents neointima thickness in the first year after PCI.

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1. The first phase, the elastic recoil, occurs in the first 24 h post PCI; this is mostly occurring in lesions treated by simple balloon angioplasty. The majority of lesions treated by modern PCI techniques involve stent placement; elastic recoil in stented lesions is minimal, so this mechanism is no more an issue in restenosis; 2. A second phase involves the formation of mural thrombus and its transformation that takes place in the first two weeks post-PCI; activated platelets and mechanic stimulation of the media can initiate an inflammatory process leading to: 3. The third phase is the most important, when cellular activation and proliferation occur and extracellular matrix is actively formed and accumulated at the site of previous vessel injury. This may last for 2 to 3 months post-PCI, irrespective of stent implantation or plain balloon angioplasty. In the case of DES implantation this phase may be shifted very late towards 12 months post-PCI or more. This is due to sustained endothelial and SMC inhibition in the first year after PCI by the active drug released from the stent surface; subsequent vessel wall activation by the late inflammatory response induced by the permanent presence of the polymer may be responsible for this so-called late catch-up phenomenon37. This late inflammatory reaction to the persistence of polymer in the structure of the vessel wall has been involved also in the phenomenon of late stent thrombosis after DES implantation38. By these mechanisms several factors can determine excessive neointimal synthesis in response to barotrauma at the level of the vessel wall. These include the residual amount of fibrin-platelet clot, the total number of activated smooth muscle cells migrated in the sub-intimal space from the injured media, and the amount of extracellular matrix produced by the neointimal cells. Therapeutic measures that can inhibit one or more of these factors could favorably influence the restenotic response. Intravascular ultrasound studies showed that restenosis after simple balloon angioplasty is mainly produced by a combination of elastic recoil and late vessel wall remodeling, while restenosis after stent implantation is mainly due to neointimal hyperplasia39.

PHASE I: Elastic Recoil


Elastic recoil occurs in minutes or hours after angioplasty and is the consequence of the dynamic properties of the vessel wall that can thus react to concentric or eccentric stretching. Other mechanisms involved are vasoconstriction due to extensive endothelial lesion and platelet adhesion and aggregation with thrombus formation that release vasoconstrictive agents. Some experimental studies demonstrated that the amplitude of elastic recoil may be an important determining factor for subsequent restenosis generation: the loss of more than 10% of the early lumen gain by elastic recoil is associated with a probability rate of restenosis of 73%. An early lumen loss of less than 10% leads to restenosis in only 9.8% of treated lesions. Elastic recoil may be the main mechanism for restenosis when the treated lesion is soft, rich in smooth muscle cells, even with minor balloon barotrauma. When the vascular lesion is extensive, local micro-thrombosis with subsequent activation of growth factors and local release of cytokines may be at the origin of neointimal proliferation. This mechanism of restenosis can be easily influenced by stent implantation, associated with larger minimal lumen diameter (larger early gain), prevents elastic recoil and provides laminar blood flow at the site of the treated lesion as opposed to simple balloon angioplasty. Plain balloon angioplasty determines smaller early gain because of elastic recoil and smaller late loss after 6 months. Bare metal stent implantation is associated with much larger early gain, avoiding elastic recoil and also with larger late loss because of the presence of

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metal in the vessel wall. However the net gain at 6 months after stenting is much larger than after balloon angioplasty; this observation was done first in the STRESS4 and BENESTENT3 trials, was subsequently confirmed in IVUS studies40 and demonstrated the utility of routine coronary stenting (Figure 3.3).
Early gain Late loss Net gain

Figure 3.3. Early gain after stenting is much higher because of avoidance of elastic recoil. The late loss is also higher after stenting because of higher neointima formation due to the presence of metal in the vessel wall. However the late net gain after stenting is much higher exactly because of superior early gain versus balloon angioplasty.

PHASE II: Platelet Aggregation, Thrombus Generation and Inflammation


Local activation of thrombosis induced by vascular injury and foreign body placement (stenting) is associated with initial recruitment of inflammatory cells. Cell-mediated inflammation plays a central role in restenosis, as in all phases of atherogenesis. Inflammation initiated in phase II will lead to the proliferative phase III. If we do not consider elastic recoil that can be avoided by stenting, restenosis can be subsequently divided in three successive episodes41 that cover phase II and phase III of our present classification: 1. The inflammatory reaction 2. The cellular proliferation phase and 3. Synthesis of extracellular matrix components. Endothelial trauma induced by balloon inflation leads to media denudation, a marked reduction of natural antithrombotic factors at the level of the treated plaque (i.e. lower NO, PGI2 and tPA synthesis) and increased synthesis of procoagulant factors (thrombin, tissue factor). Rupture of internal elastic lamina and of the media exposes subendothelial collagen, von Willebrand factor and extracellular matrix components to the action of platelets and leads

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in a few minutes to platelet adhesion and aggregation (through integrin receptors GP Ib and IIb/IIIa) with thrombus generation. The amplitude of thrombus formation depends on the severity and the surface of vascular lesion, plaque content and local rheological conditions (i.e. laminar or turbulent flow with post-procedural residual stenosis). Activated platelets synthesize vasoconstrictive, chemotactic and pro-aggregant mediators, from their granules. By this mechanism thrombin, thromboxane A2, serotonin, PAI-1, PDGF, TGF-, b-FGF, IGF-1 and interleukin-1 are locally generated at the site of the vascular lesion (Table 3.2). These factors are responsible for neointimal proliferation by stimulating migration and multiplication of smooth muscle cells from the media in the subintimal space31.
Table 3.2. Extracellular factors implicated in the pathogenesis of restenosis Angiotensin II Collagen fibers and Collagenase Colony stimulating factors (CSFs) Elastin Endothelins Epidermal growth factor / transforming growth factor (EGF/TGF-) Acid and basic fibroblast growth factor (a-FGF, b-FGF) Heparinoids Heparin-bound epidermal growth factor (HB-EGF) Insulin-like growth factor (IGF-1) Gamma interferon (IFN-) Interleukin 1 (IL-1) Oxidized LDL (ox-LDL) Monocyte-macrophage colony stimulating factor (M-CSF) Monocyte chemotactic protein (MCP-1) NO / EDRF Plasmin tissue plasminogen activator and its inhibitor (tPA and PAI-1) platelet derived growth factors (PDGF-AA, PDGF-BB) Prostacyclin (PGI2) Prostaglandin E Proteoglycans Thrombin Thromboxane A2 (TXA2) Transforming growth factor (TGF-) Tumor necrosis factor (TNF-)

Platelet activation leads to an increased phenotypic expression of GP IIb/IIIa receptors on the surface membrane that mediates adjacent platelet aggregation by fibrin strands. Aggregated platelets then accelerate local thrombin generation that increases expression of adhesion molecules (like E and P selectin) on endothelial cells. This determines lymphocyte, monocyte and neutrophil adhesion to the injured endothelium and this generates inflammation. CD11b/CD18 integrin that can be found both on neutrophils and monocytes is particularly important for local leukocyte recruitment. Systemic concentration of CD11b measured in venous blood after PCI is correlated with major adverse clinical events42 and with restenosis both after balloon angioplasty43 or stenting44. This molecule was targeted with specific antibodies to prevent leukocyte adhesion to the endothelium in order to avoid the ischemia-reperfusion lesion after primary PCI45. An important mediator of integrin activation is the soluble form of CD40 ligand (sCD40L) a proinflammatory factor produced and stored mainly by platelets. Many other proinflammatory markers have been correlated with an increased restenosis rate in various clinical trials: interleukin-146, MCP-147, and most notably a non-specific inflammatory marker, CRP48. In experimental models balloon-induced endothelial trauma was associated with prolonged synthesis of adhesion molecules (VCAM-1 and ICAM-1)49. Neutrophils, monocytes and lymphocytes subsequently accumulate under the endothelial cells and stimulate endothelin and tissue factor synthesis, both having mitogenic effects on smooth muscle cells.

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Inflammatory cells were identified in post-PCI mural thrombus in many animal experimental models50. In tissue samples obtained by atherectomy prior to stent implantation a significant correlation between macrophage load of atherosclerotic plaque and restenosis risk was found51. The stent may be responsible to greatly amplify intra-parietal inflammatory reaction depending on the metal structure, as a foreign-body reaction52. An early leukocyte marginalization process with circulating neutrophils and monocytes can be observed after stent implantation53. In the first consecutive weeks macrophages abundantly line-up around the metallic stent struts; inhibitors of monocyte activation may decrease subsequent neointimal proliferation54. Similarly administration of anti-inflammatory agents that reduce the number of neutrophils at the site of balloon injury may reduce SMC proliferation and neointimal growth53. Cellular inflammatory reaction that continues for the first 60 days post-PCI, is involved in the normal vascular repair process after PCI and becomes abnormal only when it is excessive and leads to neointimal thickening. Fibroblast growth factors and their receptors (a-FGF and b-FGF, Table 3.2) are synthesized by the injured endothelium and SMCs after mechanical balloon trauma, stimulating instead their own proliferation. TGF- induces SMCs migration and proliferation, is the main factor of proteoglycan synthesis modulator (the main constituents of extracellular matrix), and it is an important determinant of vascular remodeling in the restenosis process55. In conclusion, the extent of the mechanical vessel injury, the amplitude of thrombotic and inflammatory response and subsequent neointimal proliferation are deeply interlaced phenomena. The constituents of the fibrin-platelet thrombus and inflammatory cells, mainly macrophages and neutrophils, release many growth factors acting as paracrine mediators of the third stage of the restenotic process.

PHASE III: Activation of The Smooth Muscle Cells and Synthesis of Extracellular Matrix
The last episode in the cure of vascular injury determined by balloon inflation is the formation of neointima by the proliferation of SMCs and accumulation of extracellular matrix. This process can be regarded as normal as far as cellular reactions are not excessive and do not lead to an extra amount of neointima that can narrow the vessel lumen. SMCs may start to proliferate starting from the first 24h up-to 2 to 3 months after vessel injury. In the first 4 days after PCI the SMCs from the media become activated through the stimulation effect of all cytokines released in the second phase. At this process participates the stretch of vessel wall determined by balloon inflation, which is a strong stimulus of activation and proliferation of the SMCs. After activation these cells lose their contractile properties and transform into secretory cells that synthesize extracellular matrix rich in chondroitin-sulphate and dermatan-sulphate. Consequently up-to 14 days after PCI, the activated SMCs migrate through the breaches in the internal elastic lamina into the subintimal space and can invade residual thrombus from the luminal surface of the vessel. The peak of SMCs proliferation occurs 5 days after PCI. As far as they become embedded at this level, they start to synthesize extracellular matrix excessively. Besides SMCs, adventitial myofibroblasts were identified to migrate in the neointima and sustain extracellular matrix synthesis. All these events are determined by the complex interaction between the growth factors, second messengers and regulatory proteins responsible for the transformation of SMCs in secretory and proliferative cells. Osteopontin, proteoglycans and hyaluronic acid, plasmin and

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its tissue activator, some metalloproteinases (MMP-2 and MMP-9) are only a few of the main protagonists of these changes. In the process of attracting SMCs from the media in the subintimal space some glycoprotein receptors from the endothelial surface, that are part of the larger integrin family, have an essential role. The endothelial receptors 3, selectively blocked by the GP IIb/IIIa inhibitor abciximab, are implicated in the migration of SMCs. Despite the theoretical advantage of blocking endothelial integrins beside potent inhibition of platelet aggregation through the use of abciximab during PCI, a definite reduction of restenosis could not be obtained in randomized clinical trials. Many growth factors, as PDGF-AA, PDGF-BB, b-FGF, IGF, EGF, FGF, TGF-, angiotensin II and endothelin-1 have stimulating effects on proliferation of the SMCs by the activation of some proto-oncogenes (c-myc, c-myb, c-fos) with a modulating effect over the cell nucleus31. Proliferation is markedly increased by lack of phenotypic expression of some inhibitory proteins, as p21, that activates apoptosis; stimulation of these proteins reduces SMCs proliferation and neointimal formation after PCI. The endothelium that normally proliferates and cover the vascular lesion synthesize NO and heparan-sulphate and both are responsible for inhibitory effects on SMCs. Later on, while activated smooth muscle cells reduce their multiplication rate they start to produce large amounts of matrix proteoglycans. The synthesis of extracellular matrix continues until 20 to 25 weeks after the initial vessel trauma. This is progressively replaced by collagen and elastin while SMCs evolve to inactive mesenchymal cells. The resulting abnormal neointima consists of a fibrous extracellular matrix with scarce cellular component responsible for progressive, variable reduction of vascular lumen.

CONCLUSION
Synthesis on the mechanisms of restenosis The vascular lesion determined by balloon angioplasty and stenting is associated with extensive endothelial denudation and atherosclerotic plaque dissection that occasionally may extend deep in the vessel wall up to the adventitia. Vascular trauma and early thrombus (consisting of platelets and fibrin strands) activates inflammation: locally generated adhesion molecules (selectins and GP Ib platelet receptor) attract smooth muscle cells and circulating leukocytes at the site. Subendothelial migration of SMCs and leukocytes leads to a proliferative phase, in which they release growth factors added to those provided by platelets. The proliferative phase leads to progressive accumulation extracellular matrix that reduces vessel lumen. In the late phase cellular components of the restenotic lesion markedly diminish and the vessel suffers a negative remodeling process (more important after plain balloon angioplasty). Drug eluting stents markedly reduced the incidence of restenosis, by reducing the amount of inflammatory cells and SMCs proliferation after PCI.

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28. Lemos P, Hoye A, Goedhart D, et al. Clinical, angiographic, and procedural predictors of angiographic restenosis after sirolimus-eluting stent implantation in complex patients: an evaluation from the Rapamycin-Eluting Stent Evaluated At Rotterdam Cardiology Hospital (RESEARCH) study. Circulation 2004;109:1366. 29. White C, Ramee S, Collins T, et al. Coronary thrombi increase PTCA risk: angioscopy as a clinical tool. Circulation 1994;93:253-8. 30. Hirshfeld J, Schwartz J, Jugo R, et al. Restenosis after coronary angioplasty: a multivariate statistical model to relate lesion and procedure variables to restenosis. J Am Coll Cardiol 1991;18:647-56. 31. Libby P, Edelman E. Restenosis: involvement of growth factors and cytokines. In: Topol E, ed. Textbook of Interventional Cardiology. Philadelphia: Saunders, WB., 1999:356-57. 32. Gruentzig A. Transluminal dilatation of coronary artery stenosis. Lancet 1978;1:263-266. 33. Waller B. "Crackers, breakers, stretchers, drillers, scrapers, shavers, burners, welders, melters" - the future treatment of atherosclerotic coronary artery disease? A clinical morphologic assessment. J Am Coll Cardiol 1989;13:969-987. 34. Schwartz R. Animal models of human coronary restenosis. In: Topol E, ed. Textbook of Interventional Cardiology. Philadelphia: Saunders, WB, 1999:358-78. 35. Farb A, Weber D, Kolodgie F, et al. Morphological predictors of restenosis after coronary stenting in humans. Circulation 2002;105:2974. 36. Narins C, Topol E. Approach to restenotic lesions. In: Topol E, ed. Textbook of Interventional Cardiology. Philadelphia: Saunders, WB, 1999:417-32. 37. Shiode N, Shirota K, Tsunoda F, et al. Late progression after sirolimus-eluting stent implantation for de novo lesions. Comparison with bare metal stent implantation. Circ J 2010;74:1104-10. 38. Cook S, Ladich E, Nakazawa G, et al. Correlation of intravascular ultrasound findings with histopathological analysis of thrombus aspirates in patients with very late drug-eluting stent thrombosis. Circulation 2009;120:391-9. 39. Mintz G, Popma J, Hong M, et al. Intravascular ultrasound to discern device-specific effects and mechanisms of restenosis. Am J Cardiol 1996;78:18-22. 40. Hoffmann R, Mintz G, Dussaillant G, et al. Patterns and mechanisms of in-stent restenosis. A serial intravascular ultrasound study. Circulation 1996;94:1247. 41. Forrester J, Fishbein M, Helfant R, et al. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol 1991;17:758. 42. Mickelson J, Lakkis N, Villareal-Levy G, et al. Leukocyte activation with platelet adhesion after coronary angioplasty: a mechanism for recurrent disease. J Am Coll Cardiol 1996;28:545. 43. Inoue T, Sakai Y, Morooka S, et al. Expression of polymorphonuclear leukocyte adhesion molecules and its clinical significance in patients treated with percutaneous transluminal coronary angioplasty. J Am Coll Cardiol 1996;8:1127-33. 44. Inoue T, Uchida T, Yaguchi I, et al. Stent-induced expression and activation of the leukocyte integrin Mac-1 is associated with neointimal thickening and restenosis. 2003;107:1757-63. 45. Faxon D, Gibbons R, Chronos N, et al. The effect of blockade of the CD11/CD18 integrin receptor on infarct size in patients with acute myocardial infarction treated with direct angioplasty: the results of the HALT-MI study. J Am Coll Cardiol 2002;40:1199-204i. 46. Pietersma A, Kofflard M, de Wit L, et al. Late lumen loss after coronary angioplasty is associated with the activation status of circulating phagocytes before treatment. Circulation 1995;91:1320-5. 47. Cipollone F, Marini M, Fazia M, et al. Elevated circulating levels of monocyte chemoattractant protein-1 in patients with restenosis after coronary angioplasty. Arterioscler Thromb Vasc Biol 2001;21:327-34. 48. Gaspardone A, Crea F, Versaci F, et al. Predictive value of C-reactive protein after successful coronary-artery stenting in patients with stable angina. Am J Cardiol 1998;82:515-8. 49. Tanaka H, Sukhova G, Swanson S, et al. Sustained activation of vascular cells and leukocytes in the rabbit aorta after balloon injury. Circulation 1993;88:1788-803. 50. Wilensky R, March K, Gradus-Pizlo I, et al. Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation 1995;92:2995-3005. 51. Moreno P, Bernardi V, Lopez-Cuellar J, et al. Macrophage infiltration predicts restenosis after coronary intervention in patients with unstable angina. Circulation 1996;94:3098.

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52. Kornowski R, Hong M, Tio F, et al. In-stent restenosis: contributions of inflammatory responses and arterial injury to neointimal hyperplasia. J Am Coll Cardiol 1998;31:224-30. 53. Welt F, Edelman E, Simon D, Rogers C. Neutrophil, not macrophage, infiltration precedes neointimal thickening in balloon-injured arteries. Arterioscler Thromb Vasc Biol 2000;20:2553-8. 54. Mori E, Komori K, Yamaoka T, et al. Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits. Circulation 2002;105:2905-10. 55. Komatsu R, Ueda M, Naruko T, et al. Neointimal tissue responses at sites of coronary stenting in humans: macroscopic, histological and immunohistochemical analyses. Circulation 1998;98:224-33.

CHAPTER 4

BIOLOGICAL ROLE OF EXTRACELLULAR MATRIX IN RESTENOSIS


WASAN UDAYACHALERM and ONANONG KULAPUTANA
Introduction .............................................................................................................................. Role of ECM in Normal Vessels............................................................................................... Roles of ECM in Vessel Restenosis.......................................................................................... Degradation of ECM and Restenosis. Roles of ECM Proteinases............................................ Roles of ECM Macromolecules in Restenosis ......................................................................... Conclusions ............................................................................................................................. References ................................................................................................................................ 75 76 77 78 81 84 85

INTRODUCTION
Since the introduction of stent in 1986, the incidence of restenosis after PCI was markedly reduced but its changed the classical 3 major mechanisms of restenosis after balloon angioplasty; elastic recoil, vascular remodeling and intimal hyperplasia; to a more difficult to manage In Stent Restenosis (ISR). This ISR which predominantly cause by intimal hyperplasia has multiple contributory factors and clear understanding of all underlying mechanisms is mandatory to prevent this occurrence of restenosis. Unfortunately, the actual cellular mechanisms of this ISR are still unclear and numbers of cytokines, and matrix substances are under investigation Overall ISR were reported around 1535% and the process start immediately after stent implantation and continue for months. In 1991, Forrester et al. proposed a paradigm for restenosis based on the vascular biology of wound healing and suggested 3 phases in the process: an inflammatory phase, a granulation or cellular proliferation phase, and a phase of remodeling involving extracellular matrix (ECM) protein synthesis. Platelet aggregation, medial smooth muscle cell (SMC) migration and proliferation, proteoglycan deposition, extracellular matrix remodeling, and also thrombosis were identified as the major milestones in the sequences of this process. Later on, the mechanisms leading to ISR may divided into and early (days to weeks) and a late (weeks to months) phase. Each phase is uniquely regulated by molecular and cellular events, the overall control of which is the result of interaction among growth factors and their receptors, cytokines, secondary messengers, and proto-oncogenes involved in transcription, translation, and post-translation cascades. Vascular remodeling or healing process after injury of endothelial cells are initiated after stent implantation and resulted in migration and proliferation of media SMCs and also some

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element of thrombus formation. These cells are the predominant secretors of the ECM which involved in several known and unidentified cell signaling pathways coupled to the cell cycle. Fibrin and platelet deposition at the site of injury provides the foundation for the inflammatory aggregate. There is increased leukocyte trafficking to the injury site and subsequent migration into the vessel wall. The predominant cells being monocyte derived macrophages. After balloon injury to vascular wall, the healing response results in the formation of a neointima. The neointima after balloon in jury is composed of SMC that migrate from the media into the intima. With the formation of intimal lesions, the phenotype of SMC changes from a contractile state, in which the SMC are filled with myofilaments and contain a relatively poorly developed Golgi apparatus and rough endoplasmic reticulum, to a synthetic phenotype characterized by an abundance of rough endoplasmic reticulum and Golgi apparatus with few and sometimes no evident myofilaments112. In addition to various structural proteins including collagens, elastin, and proteoglycans constitute ECM, several proteinases are located in the matrix. Degradation of the ECM components and the activation or release of growth factors are required during neointima development. The function of proteinases on ECM proteolysis is crucial in vascular cell proliferation, migration, remodeling, and the processing of ECM proteins and adhesion molecules65,32,54,115. ECM degradation facilitates migration and proliferation of VSMCs and allows inflammatory cells to infiltrate the arterial wall during the process of vascular remodeling. During SMC migratory process, the ECM proteins are degraded and breached in order for the SMCs to escape the surrounding ECM-embedded environment of tunica media54. In addition to breaking down ECM, new matrix is being synthesized in order to maintain vessels integrity.

ROLE OF ECM IN NORMAL VESSELS


In normal blood vessel walls, the major compositions include endothelium, smooth muscle cells and extracellular matrix, varying within the different layers of the wall. ECM within the intima enriches in proteoglycans and hyaluronan83. In the tunica media, smooth muscle cells are embedded in an ECM constitutes elastic elements, collagen and proteoglycans, while the ECM in the adventitial layer comprises mainly of fibrillar collagen83. Collagens type I and III are predominately present in blood vessel and formed into cross-banded fibrils, providing tensile strength for the vessel, while type IV, VI and VIII are nonfibrillar collagens83. In the tunica media, an assembly of type IV and VIII collagens into 3 dimensional networks125,99 provides an anchoring substrate and assists a permeability barrier formation. In addition, being expressed in normal media, collagen XVIII serves as the precursor of the endothelial cell inhibitor endostatin72. Collagen XVIII is also associated with elastic fibers in multiple elastic membrane of large arteries72. While collagens are responsible for tensile strength, elastic recoil of the vessel wall is provided by elastic fibers which are assembled by elastin94. Deposition of elastin and assembly of elastic fibers are supported by scaffolding of a microfibrillar network of fibrillin and other ECM components86. In addition, elastogenesis is believed to be related to expression of another extracellular matrix glycoprotein, called emilin (elastin microfibril interface located protein), which can be detected prior to elastin deposition8. Emilin is associated with elastic fibers at the interface between amorphous elastin and microfibrils8. Proteoglycans and hyaluronan, present as one of the general compositions of the ECM, are synthesized by SMCs and participate in regulation of vascular permeability, lipid metabolism, and thrombosis. Proteoglycans have a core protein that is linked to polysaccharide(s), with

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diversified functions in regulating connective tissue structure and permeability46,93. Hyaluronan, a large ECM polymer belonging to the family of glycosaminoglycans (GAGs), comprises the major fraction of carbohydrates in ECM52. It contains several repeats of a simple disaccharide stretched end-to-end, providing a backbone for huge proteoglycan complexes28. Hyaluronan has gained much attention within the scientific community due to its multitude of functions. It can normally bind a substantial volume of water to form a viscous hydrate gel that enables the resistance to compression force of the matrix. Besides matrix-matrix interactions, proteoglycan and hyaluronan can also interact with vascular cells. Extracellular matrix of normal blood vessel contains adhesive glycoproteins fibronectin and laminin. The molecular sizes of these glycoproteins are very large95,113. These adhesive glycoproteins connect between other ECM and cells through integrin receptors. Fibronectin, with a size of approximately 450 kDa, can bind collagen, fibrin, and proteoglycans via specific domains as well as vascular cells via specific integrins95. Laminin, a very large size (820 kDa) trimeric glycoprotein, presents in endothelial and SMC basement membrane113. Similar to fibronectin, laminin interacts not only with other ECM constituents, for examples, collagen type IV and heparin sulphate, but also with vascular cells via specific integrin receptors113. Matricellular proteins are another class of secreted glycoproteins6, consisting of osteopontin, osteonectin (also called SPARC), tenascin and thrombospondin97. They interact with other ECM components, several specific cell surface receptors, and growth factors, to modulate cell-matrix interactions97. In normal blood vessels, the expression of these glycoproteins is very limited.

ROLES OF ECM IN VESSEL RESTENOSIS Role of ECM Accumulation in Lumen Narrowing


Restenosis after balloon angioplasty or stent placement is characterized by local, accumulation of mainly vascular smooth muscle cells and the synthesis of extracellular matrix molecules. Coordinated ECM synthesis by phenotypically modified SMCs is responsible for the increasing volume of intimal tissue. Over the period of several months following stent procedure, there is a tendency towards greater ECM synthesis as opposed to SMC proliferation76,30. The volume of the neointimal hyperplasia is largely composed of ECM proteoglycans and collagens, with a relatively small contribution of cellular elements92. ECM synthesis can be importantly regulated by TGF-56. In VSMC, endothelial cells, and fibroblasts, TGF- increases the synthesis of ECM proteins, such as fibronectin, collagens, and PAI-1114,56. Via Smad activation,TGF- upregulates the transcription of several genes important for ECM formation, such as procollagens, fibronectin, connective tissue growth factor (CTGF), and PAI-119,13,48,91. In VSMCs, over expression of Smad7 inhibits TGF--induced fibronectin, collagen and CTGF expression91, and over expression of Smad7 in adventitial cells attenuates collagen deposition, remodeling contribution of adventitial fibroblasts to neointima formation after balloon angioplasty66. However, ectopic expression of Smad3 stimulates TGF--induced fibronectin synthesis by SMC96. By using an inhibitor of growth factor-induced collagen synthesis, pirfenidone, neointimal formation is inhibited as a result of substantial reduction of local collagen deposition in ECM2. It should be noted that the drug was no effect on proliferative activity within the neointima2. By inhibiting the production and deposition of ECM components, loss of luminal diameter following vascular injury can be reduced, therefore, enhancing the important role of ECM accumulation in restenosis.

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DEGRADATION OF ECM AND RESTENOSIS. ROLES OF ECM PROTEINASES Matrix Metalloproteinases and Their Inhibitors
Matrix metalloproteinases (MMPs), so called matrixins, are a family of structurally related proteolytic enzymes that selectively digest individual components of the ECM31. MMPs constitute a multi gene family of zinc-and calcium-dependent endopeptidases with extensive sequence homology115. Base on their structure and substrate specificities, members of the MMP family are classified as collagenases, gelatinases, stromelysins, membrane-type MMPs, and others61. Various tissues and cell types, such as endothelial cells, smooth muscle cells, and fibroblasts are capable to produce MMPs33,82. After synthesis, MMPs may be secreted from the cells or anchored to the cell membrane, where they are bound with heparin sulfate glycosaminoglycans82. Under physiological conditions, MMPs are regulated at the level of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPs). The interplay between the regulating processes prevents excessive degradation of ECM. However, in pathological conditions, an imbalance between MMPs and TIMPs may lead to excessive increase in MMP activity and subsequently causes an abnormal change of vessel wall structure. Although substrates for the specific MMPs are not entirely discovered, substrate specificity to the MMPs is mostly determined by hemopexin domain77. MMPs are able to digest a large number of substrates that constitute ECM components, for examples, fibronectin, vitronectin, laminin, entactin, tenascin, aggrecan, myelin basic protein82. MMPs are known to degrade collagen type I, II, III, IV, V, VI, VII, VIII, IX, X, and XIV with varying efficacies. Hence, complete degradation of ECM proteins may required a cooperative action of MMPs82. MMPs contribute to the development of post angioplasty restenotic lesions. MMPs levels are elevated in intimal regions following a balloon injury to rat carotid arteries118,49. In carotid pig arteries, balloon injury has been shown to increase MMP-2 and MMP-9102. Similar to findings in humans, increased MMP-2 expression and activity have been found in the coronary circulation following angioplasty42. Moreover, a significant positive correlation between post-angioplasty MMP-2 levels and the degree of angiographic restenosis42. Evidence of importance of MMPs and SMC accumulation in neointima regions has been demonstrated extensively via in vitro and in vivo experiments. Previous studies reported that using an inhibitor of MMPs resulted in a decrease of the intimal hyperplasia and collagen accumulation15,58. In vitro models for SMC migration from the tunica media to intima showed that the synthetic nonselective MMP inhibitors as well as TIMPs significantly inhibited SMCs migration through reconstituted basement membrane or type I collagen lattice78,51. Another study showed that administration of Batimastat, a nonselective MMPs inhibitors, reduced neointimal hyperplasia up to 75 days after balloon injury in rats67. Administration of doxycycline, a potent MMPs inhibitor, showed a reduction in the activity of MMP-2 and MMP-9 in arterial wall and an inhibition of intimal hyperplasia after balloon injury in rat carotid arteries4. Inhibition of SMC migration and neointimal area with TIMP-1 or TIMP-2 transfection has been reported in injured rat carotid artery model14,20. In addition, mice with TIMP-1 deficiency showed enhanced neointimal area in response to vascular injury in the femoral artery64. However, the use of both synthetic MMP inhibitors and genetically modified natural inhibitors in aforementioned studies are not selective, thus affecting the activity of many

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MMPs. Hence, identifying particular enzymes that play critical roles in neointima formation requires another experimental model. The availability of genetically modified mice with MMP deficiency has enhanced the body of knowledge regarding the contribution of particular MMPs in various processes of restenosis after vascular injury. It has been shown that stromelysin (MMP-3) expression was enhanced in vascular wall after injury62. However, mice with MMP-3 deficiency had no effect on neointima development of carotid or femoral artery at one week after perivascular electric injury compared to wild-type mice63. On the contrary, using mice with MMP-9 deficiency, a crucial role of MMP-9 in response to vascular injury was demonstrated by a significant decrease in SMC migration and neointima formation16,31,55,50. Collectively, these studies gelatinases, MMP-2 and MMP-9, are involved in the SMC migration and intimal hyperplasia in mice. Constrictive remodeling worsens the effect of intimal hyperplasia on luminal narrowing. As a matter of fact, this is an inappropriate remodeling and is considered an important part of restenosis after atherectomy and angioplasty interventions81,71. There is evidence that MMPs also contribute to constrictive remodeling. An upregulated expression of collagenase IV (MMP-2) in response to low flow stage was demonstrated in rabbit injured carotid artery3. Non selective MMP inhibitors, Batimastat18 or Marimastat100, inhibited constrictive arterial remodeling and reduced luminal loss in porcine model of balloon angioplasty. Furthermore, constrictive arterial remodeling was decreased in response to balloon injury in mice with MMP-9 deficiency31.

Roles of Serine Proteases and Inhibitors


In addition to the MMP system, another important proteolytic enzyme system is the fibrinolytic (plasminogen/plasmin) system. These two enzyme systems, in concert, are capable in degrading most ECM components. While MMPs can degrade a large portion of ECM components, plasmin can only degrade laminin and fibronectin61. Importantly, plasminogen/ plasmin system is capable also in the activation of several proMMPs61,32. The consequences in plasmin inactivation in genetically deficient mice have been related to a decline in expression and activation of several MMPs10. Processes of arterial wound healing, including proliferation and migration of SMCs, and matrix remodeling, required the participation of both MMP and plasminogen/plasmin systems21,12,10. Activation of coagulation system by arterial injury induces thrombin formation and fibrinogen to fibrin conversion. Fibrin forms the cohesive network of hemostatic plugs and thrombi. Being a polymer, insoluble fibrin forms the framework of blood clots that help maintain vascular wall integrity. It also provides the temporary matrix for initial support of healing and revascularization. Intravascular fibrin deposition plays an important role in the development of intimal hyperplasia. Deposition of fibrin at the injured vascular wall provides initial and temporary matrix for VSMCs invasion allowing cell migration toward vascular lumen. Fibrin formation and degradation seem to be important in determination of structural alteration occurring with vascular wound repair. The conversion of plasminogen to plasmin activated by plasminogen activators (tissue-type and urinary-type) may be considered as the central reaction of the plasminogen activation system. Fibrin polymer is degraded by enzyme plasmin. Roles of fibrin in intimal formation have been shown via several mechanisms. Firstly, it is likely that fibrin plays an important role in supporting VSMC adhesion. VSMCs as well as other cell types bind directly to fibrin via cell-surface integrin receptors, 51 being of great importance in mediating VSMC-fibrin interactions124. Extracellular matrix fibronectin124 and vitronectin80 appear to act

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as bridging molecules between fibrin and VSMCs. In addition, in vitro experiments have shown that fibrin gel facilitates VSMC migration75. This appears to be mediated by V3 integrin receptor and independent of growth factors or cytokines75. In addition to the role on cellular migration, fibrin may also enhance proliferation of vascular cells, for example endothelial cells and fibroblasts104. The mechanism by which fibrin stimulates proliferative activity requires cleavage of fibrinopeptide B from fibrinogen by thrombin and exposure of the amino terminus of the fibrin chain104. Lastly, fibrin at the site of injury may stimulate neointima development through the effects of VEGF (endothelial cell growth factor)47. The activity of VEGF on VSMC migration appears be enhanced by fibrin. Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily, the principle inhibitor of tissue- and urinary-type plasminogen activators (t-PA and uPA, respectively). PAI-1 plays a key role in fibrin homeostasis by controlling plasmin formation by regulating the plasminogen activators. However, PAI-1 appears to play a major role in determining the proliferative response to vascular injury. It has been demonstrated that neointima formation after vascular injury was increased in Pai -/- mice, and that adenovirus-mediated over expression of PAI-1 inhibited neointima formation11. Over expression of PAI-1 in VSMCs seeded on denuded rat carotid arteries inhibited neointima formation while enhancing thrombus formation37. There are potential mechanisms explaining PAI-1 regulated vascular response to injury. One obvious mechanism is by inhibiting the degradation of fibrin and several ECM proteins by plasmin. PAI-1 also modulates vascular disease progression by plasmin independent mechanisms. PAI-1 may also involve in pathologic changes after vascular damage by alternative mechanisms, such as inhibiting activated protein C and altering interactions between VSMCs and the ECM. PAI-1 possibly modulates VSMC proliferation, by inhibiting activation of plasminogen, and consequently plasmin-dependent activation of transforming growth factor (TGF-), an inhibitor of VSMC proliferation36. It should be noted that inhibiting a conversion of plasminogen to plasmin by another mechanism has also been shown to inhibit activation of TGF-, resulting in VSMC activation in vivo35. Although PAI-1 has the effect on stimulating VSMC proliferation, it has been shown to inhibit VSMC migration. The inhibitory effect of PAI-1 on VSMC migration may be mediating by an inhibition of uPA-dependent plasmin formation and extracellular matrix degradation. Alternatively, it may exert the effect by blocking the interactions between extracellular matrix vitronectin and its receptors on VSMCs. The results from genetically modified animal model showed that adhesion of VSMCs to fibrin matrices prepared from wild-type mouse plasma was significantly greater than that observed with fibrin matrices prepared from vitronectin deficient plasma126. Vitronectin within the extracellular matrix binds to u-PA receptor (uPAR) and to integrin v3, which is expressed by VSMCs. In vitro experiments demonstrated that binding of vitronectin to these receptors promotes VSMC migration9. By binding to PAI-1, extracellular matrix vitronectin is blocked from binding to its integrin receptors in VSMCs. Subsequently, adhesion as well as migration of VSMCs and other cell types were inhibited in vitro105. This is was independent of the effect of PAI-1 on inhibiting plasminogen activator105. As vitronectin is a PAI-1 cofactor and vitronectin also interacts with VSMCs, significant inhibition of neointima formation after vascular injuries was demonstrated in vitronectin deficient (Vn-/-) mice126. Despite a great body of research on role of interactions between extracellular matrix and PAI-1 on vascular function, little is known regarding the role of PAI-1 in specific vascular remodeling after mechanical arterial injury, such as balloon angioplasty, in humans.

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ROLES OF ECM MACROMOLECULES IN RESTENOSIS


Following an injury response, ECM remodeling takes place throughout the different phases of restenosis, occurring secondary to the changes of smooth muscle cells (SMCs) and extracellular matrix (ECM). While SMCs undergo a phenotypic switch characterized by transition from a quiescent to an active/synthetic phenotype, they begin to synthesize an abundant extracellular matrix112. These phases sequentially occur and include degrade and disassembly of various ECM components and reassembly of particular components similar to what is observed during wound repair. Analysis of experimental models of balloon injury has revealed alterations in the ECM within the neointima. Different ECM environments of the SMC can modulate SMC phenotype and responsiveness. Subsequently, interactions between VSMCs with adjacent cells (cell-cell adhesion), the interaction between VSMCs and ECM (cell-matrix adhesion) influence the process of neointimal thickening73. The ECM modulates important events within the developing neointima, including cell proliferation, migration, growth factor expression, and remodeling7,34.

Collagen
Collagen is the most abundant ECM protein in the vessel wall, with the most prevalent being collagen type I and III89. The synthesis of is tightly regulated at several levels, including synthesis of procollagens, appropriate folding of polypeptides, secretion and cross-linking of mature fibers. In contrast, degradation of newly synthesized procollagen and mature collagen fibers depends on the action of MMPs. In injured arteries, collagen synthesis and degradation are associated with the intimal hyperplasia90. Collagens, particularly polymerized type I and IV surrounding SMC, promote a more quiescent and contractile SMC phenotype similar to normal SMC in tunica media in vivo53,41. There is evidence that polymerized collagen type I inhibit SMC proliferation in culture and the cells mimic various properties of medial SMC53,84. The effect on SMC phenotype shift to be more closely to that of medial SMC has been shown with culture on rigid gels of type IV collagen41. Mature polymerized type I collagen has been shown to increase the levels of p27Kip153. This suggests that SMC proliferation may be inhibited by the mature collagen type I via its regulatory effect on p27Kip1. In addition, to the effect on cell proliferation, polymerized type I collagen has been shown to suppress V3 mediated migratory activity of VSMCs45. Furthermore, fibrillar collagen type I dynamically regulates PAI-1, which may be responsible for altered V3 integrin-dependent SMC migration108. In vivo balloon catheter-mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen45. Several properties of type VIII collagen are potentially important as mechanisms used in cell migration and invasion43.Type VIII collagen is an extracellular matrix protein accumulates in variety of tissues undergoing active remodeling, including injured arteries during neointimal formation43. In vitro experiment has shown that adding of soluble type VIII collagen stimulates SMC migration and invasion43. The effects of type VIII collagen on SMC functions were mediated via integrin receptor43.It can be concluded that deposition of type VIII collagen in vascular lesions promotes SMC attachment and chemotaxis. By signaling through integrin receptors, collagen type VIII also stimulates MMP synthesis. In VSMCs isolated from rat carotid neointima after balloon injury, attachment of the cells to type VIII collagen increases MMP-2 and MMP-9 production but fails to alter MMP production in rat SMC isolated from

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normal media43. These suggested that type VIII collagen contributes to SMCs growth within neointima, but not in the normal intimal layer.

Glycosaminoglycans and Proteoglycans: Hyaluronan, Versican, Biglycan, Perlecan, Decorin etc.


Proteoglycans comprise a significant constituent of the extracellular matrix that participates in the molecular events that regulate cell adhesion, migration and proliferation. In normal blood vessels, hyaluronan and versican, extracellular matrix macromolecules, are expressed in small amounts, but substantially present in vascular pathologic conditions, including intimal hyperplasia119. In early development of neointimal lesion accumulation of versican, hyaluronan, and collagen type III has been found in response to vascular injury24. In addition, selective deposits of versican121 and biglycan and decorin88 are observed in the restenotic vascular wall of humans. Proteoglycans biglycan and versican and hyaluronan (HA) have been involved in regulation of migration and proliferation of vascular cells120. Among several ECM macromolecules, versican has been one of the most widely studied and found to play a key role in the regulation or modulation of essential events in atherogenesis and restenosis. Versican is a principal chondroitin sulphate proteoglycan in blood vessels110 that accumulates in lesions of atherosclerosis and restenosis. The principal source of versican in the vascular wall is the smooth muscle cell where its synthesis is highly controlled by specific cytokines and growth factors122. It is considered a highly interactive molecule which is able to bind growth factors, enzymes, lipoproteins, and a variety of other ECM components. Several reports have documented a significant amount of versican in stented and non-stented restenotic intimal lesions in human68,121,17. During migration and proliferation, human SMCs concomitantly express abundant HA and versican-rich pericellular matrices which appear to coordinate with cell detachment and mitotic cell rounding22. It has been suggested that abundant HA- and versican-rich pericellular matrices promote migration and mitosis by attenuating cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels22. Diminishing of HA- and versican-enrich matrix effectively enhances cellular adhesion and attenuates SMC migration and proliferation. Evidence has shown that versican is involved in restenotic lesions. The synthesis of hyaluronan and versican is highly regulated and influenced by pro-inflammatory growth factors such as PDGF and transforming growth factor-beta (TGF-beta)119. When versican expression was interrupted by using either antibodies to TGF-1123 or TGF-beta1 antisense S-oligonucleotide70, growth of neointima after injury has been suppressed. In balloon injury model, adding glycosaminoglycan HA inhibits neointimal formation by inhibiting macrophage migration25 and SMC accumulation98 in neointimal area. Versican and hyaluronan can form high molecular weight hydrophilic complexes, causing tissue swelling due to their capability in water trapping122. Therefore rapidly increased size of restenotic lesions is thought to be partly due to the swelling pressure development followed by accumulation of versican-hyaluronan complexes122. On the contrary, breakdown of the hyaluronan-versican complex is likely to result in water expulsion and tissue shrinkage with reduction in arterial diameter122. Although the exact mechanisms are unclear, interactions between versican and hyaluronan, form expanded viscoelastic pericellular matrices that are needed for proliferation and migration of arterial smooth muscle cell22,23.

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Adhesive Glycoproteins: Laminin and Fibronectin


VSMCs are normally surrounded by a simple basement membrane, comprising of collagen IV and laminin, with a small amount of fibronectin. Using electron microscopic analysis, newly deposited fibronectin, assembled into a fibrillar network associated with the surface of synthetic SMC, has been found in early developed restenotic lesion111. At 12 days after balloon injury, fibronectin assembly by SMCs in neointima has been detected in vascular tissue from rat carotid artery79. It has been found that the assembly fibronectin fibril is necessary for SMC growth. Evidence in rat aortic smooth muscle cells showed that inhibition of VSMC proliferation was not only due to an inhibition of fibronectin synthesis, but also be due to the inhibition of fibronectin matrix assembly69. Most receptors for fibronectin binding are integrins and such matrix-receptor binding provides the necessary signals for adhesion dependent growth. Laminin and fibronectin have diverse effects on smooth muscle cell phenotypic characteristics39. Laminin has been shown to inhibit the shift of cultured SMCs from the contractile phenotype, while fibronectin promotes the shift to the synthetic phenotype39. Aortic VSMCs rapidly undergo a phenotypic change to become more synthetic when cultured on a fibronectin substratum, and they demonstrated the proliferative characteristics after being exposed to appropriate growth factors39. On the contrary, when cells are cultured on laminin, they remain in a contractile state for some period of time39. The accumulative body of evidence suggests that specific ECM components have different signaling responses within the cell.

Heparin and Heparan Sulphate


Heparan sulphate species produced by SMCs have long been recognized as potent inhibitors of SMC proliferation29. Heparan sulphate-rich ECM, when bound to several growth factors107, serves as a local site of storage. When released from their heparan sulphate or matrix storage, growth factors, including IGFs, FGFs, TGF-betas, and HGF, subsequently regulate cell proliferation, differentiation, and production and remodeling of the extracellular matrix107. Heparin has been shown to be important in regulating fibroblast growth factor (FGF) function103. Heparin is essential for oligomerization of FGF and hence stimulation of cellular responses of endothelial and smooth muscle cells. PI-88, a antiheparanase compound, is capable in inhibiting interaction between heparan sulphate and FGF-2, resulting in attenuating VSMC proliferation and intimal thickening after balloon angioplasty in both rat and rabbit arteries27. This can emphasize the important role of heparan sulphate in regulating FGF-2 functions on SMC proliferation and intimal hyperplasia in vascular restenosis. Altered heparan sulfate fine structure has been shown to reduce VSMC proliferation and vascular remodeling in response to angioplasty injury1.

Osteopontin and Vitronectin


Cell surface receptor-extracellular matrix interactions are essential for cell migration. Osteopontin promotes adhesion and migration of SMC60. This has been shown to be the interaction via cell surface receptor integrins60,101. Using antibodies directed against osteopontin has been shown to inhibit neointimal thickening in response to endothelial denudation59.

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Cartilage Oligomeric Matrix Protein (COMP)


COMP expression has been found in normal ECM in human vessels38,87. Another important ECM protein that is needed to be degraded during tissue remodeling is cartilage oligomeric matrix protein (COMP). It has been shown that COMP degradation by ADAMTS-7 in response to vascular injury promoted VSMC migration and intimal hyperplasia116. A more recent study demonstrated that COMP is required for maintaining contractile phenotype of VSMC. Thus, COMP regulates VSMC as it has a protective effect on conserving the VSMC in a more quiescent stage. This protective regulation of COMP on VSMC is centrally mediated through interaction with 71 integrin117.

Elastin
The initial ECM changes involve ECM deposition of a provisional ECM, characterized by a loose, open, and watery matrix. Such properties allow for cellular invasion and repair. Subsequently, this provisional matrix is substituted by an extra fibrous ECM enriched in collagens and assorted glycoproteins. There is evidence that newly remodeled ECM lacks of elastic fibers. Elastin has been thought to solely play a structural role. However, mutation of one allele of elastin is sufficient to induced SMC proliferation of subendothelial layer. There is evidence suggesting that elastic fiber assembly is inhibited by versican40.

Other Matrix Proteins: SPARC, Tenascin and Thrombospondin


SPARC, tenascin and thrombospondin are matrix proteins which have been reported to promote antiadhesive and antiproliferative responses in endothelial cells and SMCs97. The modular domain structure of these matricellular proteins allows them to independently bind cells and matrix83. Matricellular proteins are capable in binding to several growth factors. Their ability to bind a variety of integrins and other receptors has made it difficult to determine the specific role of certain interactive bindings83. The expression of extracellular glycoprotein SPARC was minimal in most normal adult tissues but was increased after injury. The SPARC has a role in the association of PDGF-AB and -BB, but not PDGF-AA. The interaction of SPARC with specific dimeric forms of PDGF influences the activity of this growth factor. It has been suggested that concomitant expression of SPARC and PDGF-B-containing dimers following vascular injury may have a role in the regulation of the activity of specific dimeric PDGF in vivo85.

CONCLUSIONS
ECM is crucial for maintenance of vascular integrity and imparts tensile strength, viscoelasticity, elastic recoil and compressibility through the distinct properties of the different constituents. The ECM composition is altered during the formation of intimal lesions, which changes the physical properties of the ECM. Altered ECM composition changes its physical properties such as the generation of fragments with distinct activities. Alteration of the physical nature of the ECM regulates vascular cell responses. Moreover, each of the types of ECM within the local environment surrounding vascular cells creates distinct environments for the cells. Therefore, the changes of the components of ECM can also influence cellular responses. These responses, for example, cell migration and proliferation, play a critical role for vascular remodeling. Interactions among extracellular matrix, cells, and growth factors are critical to the

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regulation of directed cell migration and proliferation associated with development of wound healing, and pathological restenotic processes. Degree of vessel injury, inflammatory response to stent, vessel stretching, etc. also alter the cytokines and growth factors release from monocytes, SMCs, endothelium and various cells. Each of cytokine or growth factor effect in the whole process of neointimal formation are less well understood and to complete the jigsaws of these restenosis may require understanding of all interactions ensures to provide novel therapies for the prevention of restenosis.

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21. Dollery, C. M., J. R. McEwan, et al. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995, 77(5): 863-8. 22. Evanko, S. P., J. C. Angello, et al. Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 1999, 19(4): 1004-13. 23. Evanko, S. P., P. Y. Johnson, et al. Platelet-derived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells. Arch Biochem Biophys 2001, 394(1): 29-38. 24. Farb, A., F. D. Kolodgie, et al. Extracellular matrix changes in stented human coronary arteries. Circulation 2004, 110(8): 940-7. 25. Ferns, G. A., M. Konneh, et al. Hyaluronan (HYAL-BV 5200) inhibits neo-intimal macrophage influx after balloon-catheter induced injury in the cholesterol-fed rabbit. Atherosclerosis 1995, 114(2): 157-64. 26. Forrester JS, Fishbein M, Helfant R, et al. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol 1991, 17: 758-69. 27. Francis, D. J., C. R. Parish, et al. Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation. Circ Res 2003, 92(8): e70-7. 28. Fraser, J. R., T. C. Laurent, et al. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997, 242(1): 27-33. 29. Fritze, L. M., C. F. Reilly, et al. An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol 1985, 100(4): 1041-9. 30. Furman, M. I., S. E. Benoit, et al. Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease. J Am Coll Cardiol 1998, 31(2): 352-8. 31. Galis, Z. S., C. Johnson, et al. Targeted disruption of the matrix metalloproteinase-9 gene impairs smooth muscle cell migration and geometrical arterial remodeling. Circ Res 2002, 91(9): 852-9. 32. Galis, Z. S. and J. J. Khatri Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res 2002, 90(3): 251-62. 33. Galis, Z. S., G. K. Sukhova, et al. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994, 94: 2493-2503. 34. George, S. J. and A. Dwivedi MMPs, cadherins, and cell proliferation. Trends Cardiovasc Med 2004, 14(3): 100-5. 35. Grainger, D. J., P. R. Kemp, et al. Activation of transforming growth factor-beta is inhibited in transgenic apolipoprotein(a) mice. Nature 1994, 370(6489): 460-2. 36. Grainger, D. J., H. L. Kirschenlohr, et al. Proliferation of human smooth muscle cells promoted by lipoprotein(a)." Science 1993, 260(5114): 1655-8. 37. Hasenstab, D., R. Forough, et al. Plasminogen activator inhibitor type 1 and tissue inhibitor of metalloproteinases-2 increase after arterial injury in rats. Circ Res 1997, 80(4): 490-6. 38. Hecht, J. T., L. D. Nelson, et al. Mutations in exon 17B of cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia. Nat Genet 1995, 10(3): 325-9. 39. Hedin, U., B. A. Bottger, et al. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol 1988, 107(1): 307-19. 40. Hinek, A., R. P. Mecham, et al. Impaired elastin fiber assembly related to reduced 67-kD elastin-binding protein in fetal lamb ductus arteriosus and in cultured aortic smooth muscle cells treated with chondroitin sulfate. J Clin Invest 1991, 88(6): 2083-94. 41. Hirose, M., H. Kosugi, et al. Restoration to a quiescent and contractile phenotype from a proliferative phenotype of myofibroblast-like human aortic smooth muscle cells by culture on type IV collagen gels. J Biochem 1999, 125(6): 991-1000. 42. Hojo, Y., U. Ikeda, et al. Matrix metalloproteinase expression in the coronary circulation induced by coronary angioplasty. Atherosclerosis 2002, 161: 185-192. 43. Hou, G., D. Mulholland, et al. Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury. Am J Pathol 2000, 156(2): 467-76. 44. Hultgardh-Nilsson, A., C. Lovdahl, et al. Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture. Cardiovasc Res 1997, 34(2): 418-30. 45. Ichii, T., H. Koyama, et al. Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment. Circ Res 2001, 88(5): 460-7. 46. Iozzo, R. V. and A. D. Murdoch Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. Faseb J 1996, 10(5): 598-614.

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47. Ishida, A., J. Murray, et al. Expression of vascular endothelial growth factor receptors in smooth muscle cells. J Cell Physiol 2001, 188(3): 359-68. 48. Isono, M., S. Chen, et al. Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells. Biochem Biophys Res Commun 2002, 296(5): 1356-65. 49. Jenkins, G. M., M. T. Crow, et al. Increased expression of membrane-type matrix metalloprotein and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries. Circulation 1998, 97: 82-90. 50. Johnson, C. and Z. S. Galis Matrix metalloproteinase-2 and -9 differentially regulate smooth muscle cell migration and cell-mediated collagen organization. Arterioscler Thromb Vasc Biol 2004, 24(1): 54-60. 51. Kanda, S., M. Kuzuya, et al. Matrix metalloproteinase and avb3 integrin-dependent vascular smooth muscle cell invasion through a type I collagen lattice. Arterioscler Thromb Vasc Biol 2000, 20: 998-1005. 52. Karamanos, N. K., S. Axelsson, et al. Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans. J Chromatogr A 1995, 696(2): 295-305. 53. Koyama, H., E. W. Raines, et al. Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of Cdk2 inhibitors. Cell 1996, 87(6): 1069-78. 54. Kuzuya, M. and A. Iguchi Role of matrix metalloproteinases in vascular remodeling. J Atheroscler Thromb. 2003, 10(5): 275-282. 55. Kuzuya, M., S. Kanda, et al. Deficiency of gelatinase a suppresses smooth muscle cell invasion and development of experimental intimal hyperplasia. Circulation 2003, 108(11): 1375-81. 56. Leask, A. and D. J. Abraham TGF-beta signaling and the fibrotic response. Faseb J 2004, 18(7): 816-27. 57. Lee, R. T., C. Yamamoto, et al. Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells. J Biol Chem 2001, 276(17): 13847-51. 58. Li, C., W. J. Cantor, et al. Arterial repair after stenting and the effects of GM6001, a matrix metalloproteinase inhibitor. J Am Coll Cardiol 2002, 39: 1852-1858. 59. Liaw, L., D. M. Lombardi, et al. Neutralizing antibodies directed against osteopontin inhibit rat carotid neointimal thickening after endothelial denudation. Arterioscler Thromb Vasc Biol 1997, 17(1): 188-93. 60. Liaw, L., M. P. Skinner, et al. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest 1995, 95(2): 713-24. 61. Lijnen, H. R. Plasmin and matrix metalloproteinases in vascular remodeling. Thromb Haemost 2001, 86(1): 324-33. 62. Lijnen, H. R., F. Lupu, et al. Temporal and topographic matrix metalloproteinase expression after vascular injury in mice. Thromb Haemost 1999, 81(5): 799-807. 63. Lijnen, H. R., J. Silence, et al. Stromelysin-1 (MMP-3)-independent gelatinase expression and activation in mice. Blood 1998, 91(6): 2045-53. 64. Lijnen, H. R., P. Soloway, et al. Tissue inhibitor of matrix metalloproteinases-1 impairs arterial neointima formation after vascular injury in mice. Circ Res 1999, 85(12): 1186-91. 65. Liu, Y. E., M. Wang, et al. Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4). J Biol Chem 1997, 272(33): 20479-20483. 66. Mallawaarachchi, C. M., P. L. Weissberg, et al. Smad7 gene transfer attenuates adventitial cell migration and vascular remodeling after balloon injury. Arterioscler Thromb Vasc Biol 2005, 25(7): 1383-7. 67. Margolin, L., I. Fishbein, et al. Metalloproteinase inhibitor attenuates neointima formation and constrictive remodeling after angioplasty in rats: augmentative effect of alpha(v)beta(3) receptor blockade. Atherosclerosis 2002, 163: 269-277. 68. Matsuura, R., N. Isaka, et al. Deposition of PG-M/versican is a major cause of human coronary restenosis after percutaneous transluminal coronary angioplasty. J Pathol 1996, 180(3): 311-6. 69. Mercurius, K. O. and A. O. Morla Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly. Circ Res 1998, 82(5): 548-56. 70. Merrilees, M., B. Beaumont, et al. Effect of TGF-beta(1) antisense S-oligonucleotide on synthesis and accumulation of matrix proteoglycans in balloon catheter-injured neointima of rabbit carotid arteries. J Vasc Res 2000, 37(1): 50-60. 71. Mintz, G. S., J. J. Popma, et al. Arterial remodeling after coronary angioplasty: a serial intravascular ultrasound study. Circulation 1996, 94(1): 35-43. 72. Miosge, N., T. Sasaki, et al. Angiogenesis inhibitor endostatin is a distinct component of elastic fibers in vessel walls. Faseb J 1999, 13(13): 1743-50.

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73. Morla, A. O. and J. E. Mogford Control of smooth muscle cell proliferation and phenotype by integrin signaling through focal adhesion kinase. Biochem Biophys Res Commun 2000, 272(1): 298-302. 74. Nabel, E. G. CDKs and CKIs: molecular targets for tissue remodelling. Nat Rev Drug Discov 2002, 1(8): 587-98. 75. Nomura, H., M. Naito, et al. Fibrin gel induces the migration of smooth muscle cells from rabbit aortic explants. Thromb Haemost 1999, 82(4): 1347-52. 76. Ott, I., F. J. Neumann, et al. Increased neutrophil-platelet adhesion in patients with unstable angina. Circulation 1996, 94(6): 1239-46. 77. Patterson, M. L., S. J. Atkinson, et al. Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain. FEBS Lett 2001, 503: 158-162. 78. Pauly, R. R., A. Passaniti, et al. Migration of cultured vascular smooth muscle cells through a basement membrane barrier required type IV collagenase activity and is inhibited by cellular differentiation. Circ Res. 1994, 75: 41-54. 79. Pickering, J. G., L. H. Chow, et al. alpha5beta1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury. Am J Pathol 2000, 156(2): 453-65. 80. Podor, T. J., C. B. Peterson, et al. Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin. J Biol Chem 2000, 275(26): 19788-94. 81. Post, M. J., C. Borst, et al. The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty. A study in the normal rabbit and the hypercholesterolemic Yucatan micropig. Circulation 1994, 89(6): 2816-21. 82. Raffetto, J. D. and R. A. Khalil Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease. Biochem Pharmacol. 2008, 75(2): 346-359. 83. Raines, E. W. The extracellular matrix can regulate vascular cell migration, proliferation, and survival: relationships to vascular disease. Int J Exp Pathol 2000, 81(3): 173-82. 84. Raines, E. W., H. Koyama, et al. The extracellular matrix dynamically regulates smooth muscle cell responsiveness to PDGF. Ann N Y Acad Sci 2000, 902: 39-51; discussion 51-2. 85. Raines, E. W., T. F. Lane, et al. The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors. Proc Natl Acad Sci U S A 1992, 89(4): 1281-5. 86. Reinhardt, D. P., S. C. Chalberg, et al. The structure and function of fibrillin. Ciba Found Symp 1995, 192: 128-43; discussion 143-7. 87. Riessen, R., M. Fenchel, et al. Cartilage oligomeric matrix protein (thrombospondin-5) is expressed by human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2001, 21(1): 47-54. 88. Riessen, R., J. M. Isner, et al. Regional differences in the distribution of the proteoglycans biglycan and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries. Am J Pathol 1994, 144(5): 962-74. 89. Rocnik, E. F., B. M. Chan, et al. Evidence for a role of collagen synthesis in arterial smooth muscle cell migration. J Clin Invest 1998, 101(9): 1889-98. 90. Rodriguez-Feo, J. A., J. P. Sluijter, et al. Modulation of collagen turnover in cardiovascular disease. Curr Pharm Des 2005, 11(19): 2501-14. 91. Rodriguez-Vita, J., E. Sanchez-Lopez, et al. Angiotensin II activates the Smad pathway in vascular smooth muscle cells by a transforming growth factor-beta-independent mechanism. Circulation 2005, 111(19): 2509-17. 92. Rollins, B. J. Chemokines. Blood 1997, 90(3): 909-28. 93. Rosenberg, R. D., N. W. Shworak, et al. Heparan sulfate proteoglycans of the cardiovascular system. Specific structures emerge but how is synthesis regulated? J Clin Invest 1997, 100(11 Suppl): S67-75. 94. Rosenbloom, J., W. R. Abrams, et al. Extracellular matrix 4: the elastic fiber. Faseb 1993, J 7(13): 1208-18. 95. Ruoslahti, E. Fibronectin and its receptors. Annu Rev Biochem 1988, 57: 375-413. 96. Ryer, E. J., R. P. Hom, et al. PKCdelta is necessary for Smad3 expression and transforming growth factor beta-induced fibronectin synthesis in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2006, 26(4): 780-6. 97. Sage, E. H. and P. Bornstein Extracellular proteins that modulate cell-matrix interactions. SPARC, tenascin, and thrombospondin. J Biol Chem 1991, 266(23): 14831-4. 98. Savani, R. C. and E. A. Turley The role of hyaluronan and its receptors in restenosis after balloon angioplasty: development of a potential therapy. Int J Tissue React 1995,17(4): 141-51. 99. Shuttleworth, C. A. Type VIII collagen. Int J Biochem Cell Biol 1997, 29(10): 1145-8.

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100. Sierevogel, M. J., G. Pasterkamp, et al. Oral matrix metalloproteinase inhibition and arterial remodeling after balloon dilation: an intravascular ultrasound study in the pig. Circulation 2001, 103(2): 302-7. 101. Slepian, M. J., S. P. Massia, et al. Beta3-integrins rather than beta1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration. Circulation 1998, 97(18): 1818-27. 102. Southgate, K. M., M. Fisher, et al. Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries. Circ Res. 1996, 79: 1177-1187. 103. Spivak-Kroizman, T., M. A. Lemmon, et al. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell 1994, 79(6): 1015-24. 104. Sporn, L. A., L. A. Bunce, et al. Cell proliferation on fibrin: modulation by fibrinopeptide cleavage. Blood 1995, 86(5): 1802-10. 105. Stefansson, S. and D. A. Lawrence The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin. Nature 1996, 383(6599): 441-3. 106. Sun, J., S. O. Marx, et al. Role for p27(Kip1) in Vascular Smooth Muscle Cell Migration. Circulation 2001, 103(24): 2967-72. 107. Taipale, J. and J. Keski-Oja Growth factors in the extracellular matrix. Faseb 1997, J 11(1): 51-9. 108. Tanaka, S., H. Koyama, et al. Fibrillar collagen regulation of plasminogen activator inhibitor-1 is involved in altered smooth muscle cell migration. Arterioscler Thromb Vasc Biol 2002, 22(10): 1573-8. 109. Tanner, F. C., Z. Y. Yang, et al. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res 1998, 82(3): 396-403. 110. Theocharis, A. D., I. Tsolakis, et al. Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V(0). Atherosclerosis 2001, 154(2): 367-76. 111. Thyberg, J., K. Blomgren, et al. Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin. J Histochem Cytochem 1997, 45(6): 837-46. 112. Thyberg, J., U. Hedin, et al. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 1990, 10(6): 966-90. 113. Timpl, R. and J. C. Brown The laminins. Matrix Biol 1994, 14(4): 275-81. 114. Verrecchia, F. and A. Mauviel Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Invest Dermatol 2002, 118(2): 211-5. 115. Visse, R. and H. Nagase Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ. Res. 2003, 92: 827-839. 116. Wang, L., J. Zheng, et al. ADAMTS-7 mediates vascular smooth muscle cell migration and neointima formation in balloon-injured rat arteries. Circ Res 2009, 104(5): 688-98. 117. Wang, L., J. Zheng, et al. Cartilage oligomeric matrix protein maintains the contractile phenotype of vascular smooth muscle cells by interacting with alpha(7)beta(1) integrin. Circ Res 2001, 106(3): 514-25. 118. Webb, K. E., A. M. Henny, et al. Expression of matrix metalloproteinases and their inhibitor TIMP-1 in the rat carotid artery after balloon injury. Arterioscler Thromb Vasc Biol 1997, 17: 1837-1844. 119. Wight, T. N. Arterial remodeling in vascular disease: a key role for hyaluronan and versican. Front Biosci 2008, 13: 4933-7. 120. Wight, T. N., M. G. Kinsella, et al. The role of proteoglycans in cell adhesion, migration and proliferation. Curr Opin Cell Biol 1992, 4(5): 793-801. 121. Wight, T. N., S. Lara, et al. Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am J Pathol 1997, 151(4): 963-73. 122. Wight, T. N. and M. J. Merrilees Proteoglycans in atherosclerosis and restenosis: key roles for versican. Circ Res 2004, 94(9): 1158-67. 123. Wolf, Y. G., L. M. Rasmussen, et al. Antibodies against transforming growth factor-beta 1 suppress intimal hyperplasia in a rat model. J Clin Invest 1994, 93(3): 1172-8. 124. Yee, K. O. and S. M. Schwartz Why atherosclerotic vessels narrow: the fibrin hypothesis. Thromb Haemost 1999, 82(2): 762-71. 125. Yurchenco, P. D. and J. C. Schittny Molecular architecture of basement membranes. Faseb J 1990, 4(6): 1577-90. 126. Zheng, X., T. L. Saunders, et al. Vitronectin is not essential for normal mammalian development and fertility. Proc Natl Acad Sci U S A 1995, 92(26): 12426-30.

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CHAPTER 5

PREDICTIVE GENETIC FACTORS OF RESTENOSIS AFTER CORONARY STENTING AND PLAIN OLD BALLOON ANGIOPLASTY
TAKASHI ASHIKAGA and YASUHIRO SATOH
Background ............................................................................................................................... Remodeling ............................................................................................................................... Arterial Inflammation................................................................................................................ Endothelial Denudation............................................................................................................. Altered Lipoprotein Handling ................................................................................................... Thrombosis................................................................................................................................ Others ........................................................................................................................................ Conclusions ............................................................................................................................... References ................................................................................................................................. 91 92 92 94 95 95 96 96 97

BACKGROUND
Coronary artery disease is known as a leading cause of death and most cases have a complex, multifactorial etiology that includes a substantial heritable component. Coronary artery restenosis is a major complication that occurs after plain old balloon angioplasty (POBA) and intracoronary stent placement. Coronary artery restenosis and repeat revascularization still occurred in many patients despite technological and pharmacological advances1,2. Coronary artery restenosis is the arterial walls healing response to mechanical injury with POBA and stent. However, some differences exist in the mechanism involved in the phenomenon with POBA and stent. Arterial remodeling is the main contributor to lumen narrowing after POBA. Adventitial myofibroblasts, which are capable of collagen synthesis and tissue contraction as seen in wound healing, may play an important role in negative vessel remodeling observed in restenosis after POBA3. Whereas early thrombus formation and acute inflammation followed by neointimal hyperplasia has been thought as the mechanism of restenosis after stent placement. The migration and proliferation of smooth muscle cells (SMC) and extracellular matrix production may contribute coronary restenosis after stent placement. Clinical trials have consistently demonstrated a family history of coronary artery disease to be predictive for future cardiovascular events beyond that which would be explained by traditional risk factors. Recent advances in genotyping technology have allowed for easier identification and confirmation of susceptibility genes for complex traits across different

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cohorts via increased power of studies stemming from faster accrual of cases and control subjects and more precise genetic mapping. Gene polymorphism may lead to quantitative or functional alterations of the respective gene products4. Several lines of evidence indicate that genetic factors may explain the increased risk of restenosis independently of conventional clinical risk factors5. Genetic variations identifying patients at increased risk for restenosis may lead to patient-tailored therapy. Genetic polymorphisms may contribute to the development of restenosis by mediating exaggerated inflammatory responses of the endothelium to angioplasty-induced injury in some part. The underlying mechanism of this pathology involves endothelial denudation and mechanical injury of the vessel wall, which enhances inflammatory cell recruitment, ultimately driving excessive SMC activation and proliferation6. The application of drug-eluting stents (DES) has reduced the incidence of in-stent restenosis considerably. These drugs effectively inhibit SMC proliferation but also hinder effective reendothelialization and may delay arterial healing, pathophysiological processes implicated in late in-stent thrombosis, a potentially fatal complication7. Genetic factors related to remodeling, arterial inflammation, thrombosis and other factor are reviewed.

REMODELING Streptomelysin-1
Connective tissue remodeling and wound healing are essential process after POBA. This process regulates by major metalloproteinases (MMP), especially stromelysin-1. The transcription of human stromelysin-1 gene seems to be affected directly by a variant in the promoter region of the stromelysin-1 gene [-162(5A/6A)]8. In a subset of REGRESS study, this genetic factor was examined9. A repeat revascularization procedure was performed more often in the 6A homozygotes and heterozygotes than in the 5A homozygotes. The 6A homozygotes appeared to have a lower revascularization rate with pravastatin (15%) compared to placebo (40%). They postulated that pravastatin influences the remodeling process. Amouyel showed that 5A/6A stromelysin genotypes are related only to the restenosis with POBA10.

ARTERIAL INFLAMMATION C-reactive Protein (CRP) and IL-6


Inflammatory markers such as C-reactive protein and interleukin-6 (IL-6), increased in plasma as a response to the stimulus of coronary angioplasty. The magnitude of this inflammatory response is associated with the future risk of restenosis. Salef et al examined the relation between the single nucleotide polymorphism (SNP) of CRP and IL-6 genes, because the SNPs of CRP -286 has been associated with plasma CRP levels and IL-6 -174 were associated with plasma IL-6 concentration in healthy individuals challenged with an inflammatory stimulus in the form of vaccination. However, they could not show any association between CRP or IL-6 genotype and plasma CRP and IL-6 response to coronary angioplasty11.

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Tumor necrosis factor-alpha and Interleukin-10


Tumor-necrosis factor alpha (TNF-alpha) acts locally at sites of tissue injury induced by vessel wall damage and have many biological functions. On the other hand, interleukin-10 (IL-10) is an anti-inflammatory cytokines produced primarily by monocytes, T cell and B cells. Martinez-Rios MA examined TNF-alpha and IL-10 polymorphisms in 162 Mexican patients12. Their data showed that TNF-alpha genotype was similar with and without restenosis. However, IL-10 -819TT genotype has a protective role against in-stent restenosis after coronary stenting12. Monraats also showed that four IL-10 polymorphisms (-2849G/A, -1082G/A, and +4259A/G) were associated with restenosis13. Previous studies suggest that the different response of TNF-alpha and IL-10 in the inflammatory process after stent placement may explain pathogenesis of restenosis.

E-selectine SER128ARG Gene Polymorphism


E-selectin 128Arg allele has been found to be associated with premature atherosclerosis15. The serine (ser)-128 arginine (Arg) gene polymorphism of E-selectin has been implicated in the pathogenesis of coronary artery disease. The 128arg allele of E-selectin may be related to increased endothelial responses to injury, thereby potentially serving as a risk factor for post-angioplasty restenosis in patients with coronary artery disease. E-selectin plays a prominent role in the interactions between leukocyte, endothelial cells and platelets. Rauchhaus M demonstrated that restenosis with POBA in derivation and validation study has a higher frequency of 128Arg alle (14.81, 17.44%) than those without (5.32%, 7.14%). They concluded that E-selectin 128Arg may serve as a risk factor for the development of restenosis with POBA16.

The Nuclear Receptor Nurr 1


The nuclear receptor Nurr1 is an early response gene known mainly for its critical role in the development of dopamine neurons. Nurr1 haplotypes are shown to be associated with human restenosis risk, and Nurr1 is expressed in a human in-stent restenosis16. In SMCs, Nurr1 inhibits proliferation and inflammatory responses, which explains the inhibition of SMC-rich lesion formation in mice. They revealed the importance of the secretion of SMCs of proinflammatory cytokines and chemokines such as MCP-1 that accelerate recruitment of inflammatory cells into the vessel wall. They suggested that Nurr-1 receptor provokes a negative feedback regulation on the inflammatory response to SMCs.

Caspase-1 5352AA Genotype


Caspase-1 (IL-1 beta converting enzyme), an important factor in the inflammatory response and apoptosis, is a risk factor for the development of restenosis17. Monraats PS examined three different polymorphisms in the gene encoding caspase-1, IL-1r and PTPN22 after stent placement. They showed that 5352AA genotype of Caspase-1 gene increases the risk of restenosis with POBA and stent. However, the other two genes were not significantly related to the restenosis. Since the predominant role of catspase-1 in monocytic/ macrophagic cells is to process pro-IL-1beta to yield active IL-1beta, caspatase-1 may have divergent effect on stent restenosis via the expression of neutrophil apoptosis18.

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Toll-like Receptor (TLR-2)


Toll-like receptors are involved in the development and progression of atherosclerosis by interfering with lipid metabolism and by mediating inflammation. The development of general atherosclerosis as compared to restenosis is a slower process potentially involving endogenous mediators, such as heat shock proteins that lately have been shown to be ligands of TLR-2 and TLR-4. Screening for the TLR-2 Arg753Glu SNP may be of importance for stratifying a patients risk and for preventive and therapeutic measures. Oxidized lipids have been shown to induce innate immune responses in the host via Toll-like receptors, a family of cellular proteins19,20. Both TLR-2 and TLR-4 are up-regulated within atherosclerotic lesions and are thought to be involved in inflammatory plaque activation and arterial remodeling. Patients with an impaired ability to sense TLR-2 ligands due to a functionally relevant TLR-2 polymorphism are shown at increased risk for restenosis. Hammon et al. demonstrated the enhancement of the TLR-2 Arg753Glu SNP with restenosis after stent placement21. In addition, female carrying the TLR-2 SNP displayed an even greater risk for restenosis than male carriers. The inflammation brought about by triggering of TLR-2 apparently prevents restenosis and does not promote it. Previous report showed that TLR-2 suppresses immunity against a pathogen via IL-10 induction22.

Angiotensin Converting Enzyme (ACE) Gene


Angiotensin converting enzyme (ACE) is a key component in the production of angiotensin II and in the degradation of bradykinin, important peptides involved in cardiovascular physiology. ACE is involved in coronary thrombosis, vasoconstriction and SMC proliferation. Some investigators demonstrated the relation between ACE gene polymorphism and restenosis in patients with coronary artery disease23,24. They showed the correlation of D allele in restenosis only after stent placement. So the mechanism of ACE gene polymorphism may be thought to be not arterial remodeling but neointimal hyperplasia25.

ENDOTHELIAL DENUDATION Thrombospondin Family


The thrombospondin (TSP) family of matrix proteins are associated with premature myocardial infarction26. Connexin37, MEF2A have been shown to be related to endothelial integrity27,28. Specific delivery of agents from DES inhibits tissue growth after coronary stent implantation compared with bare metal stent29,30. The delayed and incomplete endothelialization of DES may cause the incidence of stent thrombosis compared with their bare metal stent. Although DES hinders reendothelialization in vitro and vivo, any genomics relating endothelial integrity have not still demonstrated with the association of restenosis after POBA and stent.

Von Willebrand Factor (vWF) and sVCAM-1


Impaired re-endothelialization is powerful histological predictor of late thrombosis31. Increased levels of vWF and sVCAM-1 were independently associated with late or vary late stent thrombosis, highlighting that impaired endothelial function is also linked with stent

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thrombosis. vWF is a multimeric glycoprotein exclusively expressed in endothelial cells and platelets, and its releasing is increased when endothelial cells were damaged. The triple polymorphisms (-1234C/T, -1185A/G, and -1051G/A) in the promoter of vWF gene have been reported to influence serum protein levels indicating that individuals with CC/AA/GG genotype had the highest vWF: Ag levels32. They showed that increased serum vWF and sVCAM-1 levels are associated with late stent thrombosis with sirolimus-eluting stent (SES) implantation. These data suggested that endothelial dysfunction contributes to the development of late and very late stent thrombosis. However, the genotype and allele distribution of vWF, t-PA and PAI-1 gene polymorphism were not related to coronary restenosis33.

Chromosome 9p21.3 Variation


Associations of SNPs on chromosome 9p21.3 with cardiovascular disease, as reported from genom-wide studies and subsequent replications using identified SNPs or their neighbor SNPs, may reflect an influence on the development and progression of coronary atherosclerosis in native arteries. Specific polymorphisms in the chromosome 9p21.3 region were not related to the clinical and angiographic outcomes after the placement of DES in coronary arteries. 9p21.3 is located upstream of cyclin-dependent kinase N2A, a determinant of endothelial progenitor cell proliferation and differentiation. EPCs have been implicated in vascular repair and protection from cardiovascular disease. The reason of the negative study may be suggested that sequence variation at 9p21 does not influence EPC number.

ALTERED LIPOPROTEIN HANDLING Lipoprotein


The efficacy of statins in both primary and secondary prevention of coronary artery disease has been established. Two mutations have recently been described in the cholesterol-regulated gene encoding, proprotein-convertase subtilisin/kexin type 9 (PCSK9), which is a serine protease that modulates LDL receptor levels34. Previous study showed that nonsense mutation in PCSK9 were associated with a striking reduction in low density lipoprotein (LDL) levels, myocardial infarction (MI), CAD-related death and repeat revascularization in patients with African descent. The efficacy of statins has been established in many studies. The polymorphisms that define the epsilon 2, epsilon 3 and epsilon 4 isoforms of apolipoprotein E influence low-density lipoproteins in the ability of pravastatin and atrovastatin therapy35. So recent identification of mutations in genes encoding proteins involved in the metabolism of drugs may enable the risk of drug resistance.

THROMBOSIS
Drugs released from DES exerts distinct biological effects, such as activation of signal transduction pathways and inhibition of cell proliferation. As a result, although primarily aimed at preventing vascular smooth muscle cell proliferation and migration, they also impair re-endothelialization, which leads to delayed arterial healing, and induce tissue factor expression, which result in a prothrombogenic environment. With the introduction of stents, coronary remodeling and restenosis were reduced compared to POBA. Response variability and nonresponsiveness to clopidogrel have been demonstrated in

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patients with coronary stenting36. Differences in intestinal absorption, hepatic conversion to the active metabolite through cytochrome P450 (CYP) activity and platelet polymorphisms have been suggested as mechanisms responsible for clopidogrel nonresponsiveness.

Cytochrome P450 2C19


The use of dual antiplatelet drugs is important to reduce acute and/or late stent thrombosis after stent placement. Addition of a thienopyridine to acetylsalicylic acid has been shown to reduce acute in-stent thrombosis by up to 80%37. The most commonly prescribed thienopyridine is clopidogrel. Individual variability of platelet inhibition by clopidogrel suggests a possibility for genetic factors having a significant influence on clopidogrel responsiveness. Differences in intestinal absorption, hepatic conversion to the active metabolite through cytochrome P450 (CYP) activity, and platelet receptor polymorphisms have been suggested as mechanisms responsible for clopidogrel nonresponsiveness. Recently it was demonstrated that carriers of reduced function cytochrome P450 2C19 allele had significantly lower levels of clopidogrel active metabolite, and a greater number of coronary events including stent thrombosis38,39.

Platelet Glycoprotein IIb/IIIa Polymorphism


Platelets are thought to play an important role in restenosis40. GP IIIa, the constitution of both fibrinogen and vitronectin receptors is a polymorphic protein with platelet antigen 1 (PlA1) and 2 (PlA2) as the most common allelic isoforms. Several reports showed that there is a trend toward a higher restenosis rate in PIA2 carriers41,42. On the contrary, GP IIb polymorphism is not related to in-stent restenosis43.

OTHERS Uncoupling Protein 3 (UCP 3)


UCP3 is one of a family of molecules present in the inner mitochondrial membrane and is most abundant in skeletal muscle. The T allele of -55C-T polymorphism of UCP3 has been associated with an increased BMI, an increased level of adiposity, or a greater waist-to-hip ratio. Oguri et al showed that -55C-T polymorphism of UCP3 was associated with an increased risk of in-stent restenosis and this association was not attributable to an effect of the polymorphism on lipid profile44. However, the true mechanism of this phenomenon is still unclear.

CONCLUSIONS
Restenosis is a multifactorial process in coronary angioplasty. Genetics in coronary angioplasty is a new emerging relevant research field. However, the therapeutic progress and latest technology in coronary angioplasty will increase the necessity of genomics. Hopefully, in the future, patients will undergo rapid genotyping to search a key panel of genes that will specifically quantify the risk of restenosis with POBA and stent. On the other hand, we must know that many of the studies are relatively small sample size and exhibit wide confidence intervals and larger studies showed disappointed results.

CHAPTER 5. PREDICTIVE GENETIC FACTORS OF RESTENOSIS

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1. 2. Serrys PW, Kutryk MJ, Ong AT. Coronary artery stents. N Engl J Med 2006;354:483-495. Bonta PI, Pols TWH, van Tiel CM, Vos M, Arkenbout EK, Rohlena J, Koch KT, de Maat MPM, Tanck MWT, de Winter RJ, Pannekoek HP, Biessen EAL, Bot I, de Vries CJM. Nuclear receptor Nurr is expressed and is associated with human restenosis and inhibits vascular lesion formation in mice involving inhibition of smooth muscle cell proliferation and inflammation. Circulation 2010;121:2023-2032. Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LF, Hong MK, Leon MB. Intravascular Ultrasound Assessment of the Mechanisms and Predictors of Restenosis Following Coronary Angioplasty. J Invasive Cardiol 1997;9:303-314. Damani SB, Topol EJ. Future use of genomics in coronary artery disease. J Am Coll Cardiol 2007; 50:1933-1940. Kastrati A, Dirschinger J, Schomig A. Genetic risk factors and restenosis after percutaneous coronary interventions. Herz 2000;25:34-46. Costa MA, Simon DI. Molecular basis of restenosis and drug-eluting stents. Circulation 2005;111:2257-2273. Luscher TF, Steffel J, Eberli FR, Joner M, Nakazawa G, Tanner FC, Virmani R. Drug-eluting stent and coronary thrombosis: biological mechanisms and clinical implications. Circulation 2007;115:1051-1058. Agema WRP, Jukema JW, Pimstone SN, Kastelein JJP. Genetic aspects of restenosis after percutaneous coronary interventions. Euro Heart J 2001;22:2058-2074. Jukema JW, Bruschke AV, van Boven AJ, Reiber JH, Bal ET, Zwinderman AH, Jansen H, Boerma GJ, van Rappard FM, Lie KI, et al. Effects of lipid lowering by pravastatin on progression and regression of coronary artery disease in symptomatic men with normal to moderately elevated serum cholesterol levels. The Regression Growth Evaluation Statin Study (REGRESS) Circulation 1995;91:2528-2540. Amouyel P, Bauters C, Meihaeghe A, et al. The 5A6A polymorphism in the promoter of the stromelysin-1 gene as a risk factor for restenosis. Eur Heart J 1999;20:Suppl:601. Saleh N, Kovacs A, Tornvall P. Relevance of genetic polymorphisms in inflammatory response to percutaneous coronary intervention. Scandinavian j of Clinical & Laboratory Investigation 2009;69:736-740. Matinez-Rios MA, Pena-Duque MA, Fragoso JM, Rodringuez HD, Rodriguez-Perez JM, Miranda-Malpica E, Cruz-Robles D, Cavazos-quero MM, Rodriguez-Lobato LG, Vargas-Alarcon G. Tumor necrosis factor alpha and interleukin 10 promotor polymorphisms in Mexican patients with restenosis after coronary stenting. Biochem Genet 2009;47:707-716. Monraats PS, Kurreeman FA, Pons D, Sewgobind VD, de Vries FR, Zwinderman AH, de Maat MP, Doevendans PA, de Winter RJ, Tio RA, Waltenberger J, Huizinga TW, Eefting D, Quax PH, Frants RR, van der Laarse A, van der Wall EE, Jukema JW. Interleukin10: a new risk marker for the development of restenosis after percutaneous coronary intervention. Genes Immun 2007;8:44-50. Wenzel K, Felix S, Kleber FX, Brachold R, Menke T, Schattke S, Schulte KL, Glser C, Rohde K, Baumann G, et al. E-selectin polymorphism and atherosclerosis: an association study. Hum Mol Genet 1994;3:1935-1937. Rauchhaus M, Gross M, Sculz S, Francis DP, Greiser P, Norwig A, Weidhase L, Coats AJS, Dietz R, Anker SD, Glaser C. The E-selectin SER128ARG gene polymorphism and restenosis after successful coronary angioplasty. Int J Cardiol 2002;83:249-257. Bonta PI, Pols TWH, van Tiel CM, Vos M, Arkenbout EK, Rohlena J, Koch KT, de Maat MPM, Tanck MWT, de Winter RJ, Pannekocek HP, Biessen EAL, Bot I, de Vries CJM. Nuclear receptor Nurr-1 expressed in and is associated with human restenosis and inhibits vascular lesion formation in mice involving inhibition of smooth muscle cell proliferation and inflammation. Circulation 2010;121:2023-2032. Watson RW, Rotstein OD, Parodo J, Bitar R, Marshall JC. The IL-1 beta-converting enzyme (Caspase-1) inhibits apotosis of inflammatory neutrophils through activation of IL-1 beta. J Immunol 1998;161:957-962. Monraats PS, de Vries F, de Jong LW, Pons D, Sewgobind VDKD, Zwinderman AH, Maat MPM, Hart LM, Doevendans PA, de Winter RJ, Tio RA, van der Wall EE, Jukema JW. Inflammation and apoptosis genes and risk of restenosis after percutaneous coronary intervention. Pharmacogenetics and Genomics 2006;16:747-754. Miller YI, Chang MK, Binder CJ, Shaw PX, Witzum JL. Oxidized low density lipoprotein and innate immune receptors. Curr Opin Lipidol 2003;14:437-445. Pasterkamp G, van Keulen JK, de Klejin DP. Role of Toll-like receptor4 in the initiation and progression of atherosclerotic disease. Eur J Cli Investis 2004;34:328-334. Edfeldt K, Swedendorg J, Hansson GK, Yan ZQ. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation 2002;270:121-144. Netea MG, Sutmuller R, Hermann C, van der Graaf CA, van der Meer JW, Krieken JH, van Hartung T, Adema G, Kullberg BJ. Toll-like receptor 2 suppresses immunity against candida albicans through induction of IL-10 and regulatory T cells. J Immunol 2004;172:3712-3718. Hamon M, Bauters C, Amant C, McFadden EP, Helbecque N, Lablanche JM, Bertrand ME, Amouyel P. Relation between the deletion polymorphism of angiotensin-converting enzyme gene and late narrowing after coronary angioplasty. Circulation 1995;92:296-299.

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24. Ohishi M, Fujii K, Minamino T, Higaki J, Kamitani A, Rakugi H, Zhao Y, Mikami H, Miki T, Ogihara T. A potent genetic factor for restenosis Nat Genet 1993;5:324-325. 25. Ribichini F, Steffenino G, Dellavalle A, Matullo G, Colajanni E, Camilla T, Vado A, Benetton G, Uslenghi E, Piazza A. Plasma activity and insertion/deletion polymorphism of angiotensin I converting enzyme: a major risk factor and market of risk for coronary stent restenosis. Circulation 1998;97:147-154. 26. Topol EJ, McCarthy J, Gabriel S, Moliterno DJ, Rogers WJ, Newby LK, Freedman M, Metivier J, Cannata R, O'Donnell CJ, Kottke-Marchant K, Murugesan G, Plow EF, Stenina O, Daley GQ. Single nucleotide polymorphisms in multiple novel thrombospondin gene may be associated with familial premature myocardial infarction Circulation 2001;104:2641-2644. 27. Yamada Y, Izawa H, Ichihara S, Takatsu F, Ishihara H, Hirayama H, Sone T, Tanaka M, Yokota M. Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 2002;347: 1916-1923. 28. Wang L, Fan C, Topol SE, Topol EJ, Wang Q. Mutation of MEF2A in a inherited disorder with features of coronary artery disease. Science 2003;302:1578-1581. 29. Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O'Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE; SIRIUS Investigators. Sirolimus-eluting stents versus standard stents in patients with stenosisin a native coronary artery. N Engl J Med 2003;349:1315-1323. 30. Stone GW, Ellis SG, Cox DA, Hermiller J, O'Shaughnessy C, Mann JT, Turco M, Caputo R, Bergin P, Greenberg J, Popma JJ, Russell ME; TAXUS-IV Investigators. A polymer-based, paclitaxel-elutimg stent in patients with coronary artery disease. N Engl J Med 2004;350:221-231. 31. Finn AV, Joner M, Nakazawa G, Kolodgie F, Newell J, John MC, Gold HK, Virmani R. Pathological correlations of late drug-eluting stent thrombosis, strut coverage as a marker of endothelialization. Circulation 2007;115:2435-2441. 32. Keightley AM, Lam YM, Brady JN, Cameron CL, Lippicrap D. variation at the von Willebrand Factor (vWF) gene locus is associated with plasma vWF:Ag levels: identification of these novel single nucleotide polymorphisms in the vWF gene promoter. Blood 1999;93:4277-4283. 33. Jin C, Zhu ZB, Zhang RY, Zhang Q, Du R, Ding FH, Chen QJ, Shen WF. Increased serum vWF and sVCAM-1 levels are associated with late or very late angiographic stent thrombosis after sirolimus-eluting step. 34. Cohen JC, Boerwinkle E, Mosley TH Jr, Hobbs HH. Sequence variations in PCSK9 , low LDL, and protection against coronary heart disease. N Engl J Med 2006;354:1264-1272. 35. Mega JL, Morrow DA, Brown A, Cannon CP, Saatine MS. Identification and genetic variants associated with response to statin therapy. Arterioscler Thromb Vasc Biol 2009;29:1310-1315. 36. Kastrati A, Schmig A, Seyfarth M, Koch W, Elezi S, Bttiger C, Mehilli J, Schmig K, von Beckerath N. PIA polymorphism of platelet glycoprotein IIa and risk of restenosis after coronary stent placement. Circulation 1999;99:1005-1010. 37. Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, Walker JR, Antman EM, Macias W, Braunwald E, Sabatine MS. Cytochrome p-450 polymorphisms and response to clopidogrel. N Engl J Med 2009 :360;354-360. 38. Dandona S, Stewart AFR, Roberts R. Genomics in coronary artery disease: Past, present and future. Can J Cardiol 2010;26:56A-59A. 39. Simon T, Pharm CV, Mary-Krause M, Quteineh L, Drouet E, Meneveau N, Steg PG, Ferrieres J, Danchin N, Becquemont L, for the Trench Registry of Acute ST-elevation and Non-ST-Elevation Myocardial Infarction (FAST=MI) Investigators. Genetic determinants of response to clopidogrel and cardiovascular events. New Engl J Med 2009;360:363-375. 40. Leon MB, Baim DS, Popma JJ, Gordon PC, Cutlip DE, Ho KK, Giambartolomei A, Diver DJ, Lasorda DM, Williams DO, Pocock SJ, Kuntz RE. A clinical trial comparing three antithrombotic-drug regimens after coronary-artery stenting. Stent Anticoagulation Restenosis Study Investigators. N Engl J Med 1998;339:1665-1671. 41. Abbate R, Marcucci R, Camacho-Vanegas O, Pepe G, Gori AM, Capanni M, Simonetti I, Prisco D, Gensini GF. Role of platelet glycoprotein PL (A1/A2) polymorphism in restenosis after percutaneous transluminal coronary angioplasty. Am J Cardiol 1998;82:524-525. 42. Mamotte CDS, van Bockxmeer FM, Taylor RR. Pla1/a2 polymorphism of glycoprotein IIIa and risk of coronary artery disease and restenosis following coronary angioplasty. Am J Cardiol 1998;82:13-16. 43. Bttiger C, Kastrati A, Koch W, Mehilli J, von Beckerath N, Dirschinger J, Gawaz M, Schmig A. Polymorphism of platelet glycoprotein IIb and risk of thrombosis and restenosis after coronary stent placement. Am J Cardiol 1999;99:987-991. 44. Oguri M, Kato K, Yokoi K, Segawa T, Matsuo H, Watanabe S, Nozawa Y, Murohara T, Yamada Y. Identification of a polymorphism of UCP3 associated with recurrent in-stent restenosis of coronary arteries. Int J Mol Med 2007;20:533-538.

CHAPTER 6

ENDOTHELIAL DYSFUNCTION AND INFLAMMATION


MARK SLEVIN and GARRY McDOWELL
Background ................................................................................................................................. Inflammation and Angiogenesis in Typical Atherosclerotic Plaque Formation .......................... The Inflammatory Response and Vascular Remodelling During Restenosis............................... Inflammation, Endothelial Cell Activation and Proliferation. The Key to Prevention of Restenosis? .. Novel and Emerging Strategies/Conclusions .............................................................................. References ................................................................................................................................... 99 101 103 104 106 107

BACKGROUND
Conventional based methods of catheter removal of arterial blockages formed during the process of atherosclerosis often result in production of a series of rapidly occurring events which follow the balloon catheter-induced tearing of the existing atherosclerotic plaques and concomitant arterial damage and luminal destruction and ending with significant lumen narrowing within a period of around 6 months (between 410% of cases following endarterectomy and approximately 33% of cases in coronary arteries for example; Figure 6.1).

Figure 6.1. Proliferation of CD105 positive microvessels in the adventitia (A) are associated with intimal expansion and a high concentration of active neovessels in this region Shown: A grade 6 unstable carotid plaque obtained at transplant and stained with antibodies to CD105. Arrows show adventitial active vessels (A) and intimal neovessels (I). M is media.

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The more recent introduction of stents has helped to resolve/reduce some of the problems associated with balloon angioplasty, in that it provides a scaffold which can prevent constriction from the intima, and when coated with anti-proliferative or anti-inflammatory drugs, can significantly slow down the process of in-stent restenosis. However, angiographic restenosis (>50%) and clinical symptomatic restenosis still occurs in 2030% and 1015% of patients respectively in the first year after treatment1, and evidence has shown that there is no significant difference in long-term (35 years) follow up regarding subsequent myocardial infarction and death2 . Within this pathophysiological sequence of events, thrombosis and inflammation can occur at the injury site within 1 week of treatment. These events trigger a cellular response with increased cell proliferation and migration, extracellular matrix remodeling and finally, remodeling of the arterial intima and neointima involving neovascularization or angiogenesis within 6 months (Figure 6.2)3.

Figure 6.2. Schematic representation of the major processes which occur following stent implantation beginning with endothelial damage and dysfunction in the early stages and ending with restenosis and thrombosis often within 6 months of application. EPC: Endothelial Progenitor Cells.

Extensive neovascularization is often seen in recurrent endarterectomy/coronary artery specimens, together with fibrin-rich surface thrombi in association with intraplaque thrombi, and of particular interest, it has been shown that plaques with an abundance of smooth muscle cells (SMC) are more likely to develop greater neointimal growth after surgery, compared with macrophage and lymphocyte-rich lesions4. Understanding the processes responsible for mediating endothelial dysfunction and activation and stimulation of remodeling through neovascularization may help us to design specific inhibitors to slow down the process of neointimal formation. Firstly, it is important to understand the relationship between inflammation and endothelial cell activation since these processes in combination are largely responsible for development of unstable atherosclerotic lesions which may translate to the process of restenosis.

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INFLAMMATION AND ANGIOGENESIS IN TYPICAL ATHEROSCLEROTIC PLAQUE FORMATION


The atherosclerotic process is initiated early in life, when there is already evidence of cholesterol-containing low-density lipoproteins accumulating in the intima and activation of the endothelium. Leukocyte adhesion molecules and chemokines promote recruitment of monocytes and T cells. Monocytes can differentiate into macrophages and up-regulate pattern recognition receptors, including scavenger receptors and toll-like receptors. Scavenger receptors mediate lipoprotein internalization producing foam-cells. Toll-like receptors are important transducers of activating signals that lead to the release of cytokines, proteases, and vasoactive molecules, and are considered to be an important link between inflammation and cardiovascular disease5. For example, deficiency of the toll-like receptor 4 (TLR-4) protein reduces macrophage recruitment in association with reduced cytokine and chemokine levels6. T cells in lesions recognize local antigens and mount T helper-1 responses with secretion of pro-inflammatory cytokines that contribute to local inflammation and growth of the plaque7. Vasa vasorum (VV) density, their proliferation, medial-intimal infiltration, and concurrent adventitial inflammation are strongly associated with advanced lesions suggesting a strong link between the two processes8. This is backed up by various studies conducted using animal models of atherosclerosis which, due to their rapid development times, are more akin to the processes associated with restenosis following direct arterial damage in man. Plaque neovascularization correlated with the extent of inflammation in hypercholesterolemic apoE mice and inhibition of vessel formation reduced macrophage accumulation and plaque progression9. Similarly, transfection with murine soluble VEGF-R1 inhibited early inflammation and late neointimal formation, suggesting that generation of neovessels and the inflammatory response are inter-linked and perpetuate in a continuous cycle10 (Figure 6.3). It is known that inflammatory infiltrates enhance recruitment of monocytes, secrete matrix metalloproteinases and increase the expression of -interferon (from t-lymphocytes) which may weaken the fibrous cap; similarly, they can induce synthesis of angiogenic tissue angiotensin-converting enzyme, growth factors, interleukin-8 and tissue factor11. The importance of the inflammatory response was demonstrated in vivo where oral treatment of apoE-/LDL-double knockout mice with the anti-inflammatory compound 3-deazaadenosine prevented lesion formation12. A strong correlation has been shown between macrophage infiltration, intraplaque haemorrhage and rupture-prone thin-cap lesions with high microvessel density, whilst these features are not common in calcified or hyalinized human arterial plaques, suggesting a strong link between neovascularization, inflammation and thrombosis11,13. The phenotype of plaque neovessels could also be important in determining stability of a developing plaque, for example, new vascular networks, and immature vessels with poor integrity and no smooth muscle cell/pericyte coverage would likely act as sites for inflammatory cell infiltration, inflammatory cell leakage and intraplaque haemorrhage respectively9. The correlation of focal collections of inflammatory cells with areas of intraplaque neovascularization and haemorrhage, suggests that release of growth factors and cytokines by macrophages and leukocytes may have a key role in modulating the vascularization process14. Evidence for the existence of hotspots or neovascular milieu was found in lesions from ApoE-/- mice where the density of VV correlated with the presence of inflammatory cells rather than plaque size. Deposition of RBC membranes within the necrotic core of plaques has also been shown to result in an increase in macrophage infiltration and therefore may further potentiate the inflammatory response9,15.

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Figure 6.3. Intima+media and adventitia microvessel angiogenesis. Photomicrographs of microvessel angiogenesis 28 days after coronary angioplasty and VEGF or LacZ gene transfer with the needle injection catheter. Adjacent cross sections of the coronary arteries were immunolabeled with an antibody to vWf showing ECs (endothelial cells) of intima+media (A) and adventitia (B) microvessels from VEGF-transfected artery and ECs of intima+media (C) and adventitia (D) microvessels from LacZ-transfected artery. Note the significant microvessel angiogenesis in the adventitia of VEGF-transfected compared to LacZ-transfected arteries and the similar angiogenic microvessel response in the intima+media area after (peri)adventitial VEGF and LacZ plasmid/liposome transfer, respectively (bar = 200 m). Reproduced with permission from Oxford Journals and taken from the article by Pels K et al. Cardiovasc Res 2003;60:664-672.

Perhaps surprisingly, hypercholesterolaemia may also be a key factor associated with proliferation of VV in coronary and carotid vessels at early stages of plaque development. Williams JK16 first demonstrated that the presence of atherosclerosis in hypercholesterolemic monkeys induced an increase in blood flow through the VV and plaque regression caused by removal of the high lipid diet reduced the VV concentration and blood flow to the coronary media and intima. High cholesterol levels are associated with increased serum VEGF expression and may cause up-regulation of growth factor receptors on both endothelial and smooth muscle cells17. Furthermore, oxidized LDL (ox-LDL) generated in response to pro-oxidative cellular changes can exacerbate the inflammatory response, since engulfment of intact apoptotic cells was reduced in the presence of ox-LDL and in its absence, rapid phagocytosis suppressed macrophage inflammatory cytokine release, suggesting a link between high lipid levels, inflammation and possibly angiogenesis17. In vitro studies have demonstrated that stimulation of HUVEC with ox-LDL up-regulates adhesion molecules (including intracellular adhesion molecule -1 (ICAM-1), E-selectin and P-selectin), inflammatory proteins including interleukin-6 (Il-6), thrombotic factors including tissue factor and remodeling proteins such as matrix metalloproteinase (MMP)-2 and MMP-9, many of which are also stimulators of angiogenesis18.

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103

Medial and intimal thickening induced by hypercholesterolemia may result in a limited supply of oxygen and nutrients reaching these areas from either the lumen and/or VV, resulting in a hypoxic environment. Since the major outcome of hypoxia is increased vascularization, intraplaque vessels may proliferate in association with this potent stimulus. Hypoxia-inducible factor (HIF-1) is expressed in hypoxic regions of expanding and developing plaques, and directs migration of endothelial cells (EC) towards the hypoxic environment via direct HIF-1 binding the regulatory gene of Vascular Endothelial Cell Growth Factor (VEGF) and subsequent induction of VEGF transcription19. VEGF is a potent angiogenic growth factor, stimulating EC mitogenesis and blood vessel formation via activation of intracellular signaling intermediates including mitogen-activated protein kinase 1/2 (MAPK1/2) and src. Increased expression of VEGF and its receptors in hypoxic areas, in association with interaction with cell membrane integrins including 53, is one of the main causes of vessel leakiness20. Leaky plaque VV have been identified by ultrastructural visualization of defects between endothelial tight, gap and adherens junctions, and VEGF is known to affect junctional adhesion molecule expression, block gap junctional communication between adjacent endothelial cells and disrupt tight junctional communication through a src-dependent pathway21. Oxidized phospholipids such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), also prevalent in atherosclerotic lesions, can also up-regulate VEGF expression. In addition, they can regulate leukocyte-endothelial cell interactions and induce expression of inflammatory cytokines from local endothelial cells, monocytes and macrophages22. Hence, a large number of signaling intermediates could be operating within the micro-environment of rapidly remodeling restenotic lesions, but due to the complexity of this process, can we really expect to produce a solution to prevent re-formation of hyperplasia and stent thrombosis by blockade of individual signaling pathways, or do we need an approach using combinational therapy?

THE INFLAMMATORY RESPONSE AND VASCULAR REMODELLING DURING RESTENOSIS


As mentioned briefly above, one of the earliest signs of damage following removal of existing plaque by balloon catheters or implantation of a stent is initiation of the inflammatory reaction. Both methods of treatment result in the induction of a systemic inflammatory response, the extent of which can be measured using the marker C-reactive protein, higher levels generally suggesting a worse clinical outcome23. Insertion of stents/drug eluting stents may exacerbate endothelial dysfunction and delay vascular healing, a critically important process for protection of the intima against subsequent immune cell infiltration and hyperplasia24,25. In fact, endothelial dysfunction and incomplete neointimal coverage of the stent struts are the main cause of subsequent late stent thrombosis26. Present conventional research is therefore directed to delivery of therapeutics to attenuate the inflammatory response, alleviate endothelial dysfunction, and produce and maintain a protective and coherent endothelial cell barrier. Several generalized studies have attempted to provide an overview of transcriptional changes occurring following arterial balloon injury. The most useful example, carried out by Zhang27, employed a rat carotid artery balloon-injury model and measured changes in gene transcription from 1-28 days after injury. They found rapid induction of a large number of pro-inflammatory genes including tumor necrosis factor-alpha (TNF-) followed by an increase in expression of important angiogenic molecules including CD44 and Cxcl12. They concluded that many of the genes for de-regulated via altered production in mesenchymal stromal cells which could therefore be a potential target for clinical intervention.

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INFLAMMATION, ENDOTHELIAL CELL ACTIVATION AND PROLIFERATION. THE KEY TO PREVENT RESTENOSIS?
Attempts to inhibit inflammation have met with some success using both animal models of restenosis and in humans. Although some of these studies have been performed using a carotid model of restenosis, they should be translatable to coronary disease and hence are relevant to this review. Since leukocyte recruitment mediated by up-regulation of adhesion molecules on the endothelial cell surface is a key instigator of the early stages of this process, Qu28 applied a lentiviral construct containing siRNA directed against vascular endothelial cell adhesion molecule-1 (VCAM-1) and applied it to rats prior to carotid surgical mechanical de-endothelialization (CSMDE). The importance of inhibiting VCAM-1 was shown by a significant reduction in stenosis/increase in blood velocity, and a decrease in the intima/media ratio compared with control, untreated animals. A similar study employed adenoviral delivery of A20 protein (a zinc finger protein which is a negative regulator of tumor necrosis factor-induced signaling pathways and therefore inflammation) to rat carotid arteries prior to balloon angioplasty29. A20 was able to down-regulate adhesion markers and chemokine production (e.g. ICAM-1; monocyte chemotactic protein-1) as well as adventitial neovascularisation. These are all key requisites for macrophage trafficking and their results demonstrated that administration of A20 significantly reduced intimal hyperplasia and macrophage infiltration making it a potential drug candidate for inhibition of restenosis. Other methods that have been used to reduce inflammation include the use of simvastatin coated liposomes delivered intravenously (single injection). Fourteen days after balloon carotid artery injury, a significant reduction in proliferation of monocytes and macrophages was noticed concomitant with suppression of neointimal formation30. These results suggest that there may be alternative approaches to delivery of drugs for control of restenosis and these will be described in greater detail later in the chapter. Since the introduction of metal stents became the most popular method of treatment, experiments have been performed to examine the effects of concomitant anti-inflammatory treatments with effects determined by direct measurements of intimal growth and hyperplasia by coronary angiography as well as histological approaches and measurements of systemic circulating immune cells to examine the anti- inflammatory efficacy. Pesarini et al.31 treated a group of patients who had received coronary bare metal stents with high dose oral prednisone (a synthetic corticosteroid which acts as an immunosuppressant and is highly anti-inflammatory) over a period of 40 days following the implant. Some of the major pro-inflammatory cytokines including TNF-, interleukin-6 and NF-B were significantly reduced as measured in patients serum as was late luminal loss and this was associated with reduction in their synthesis by circulating monocytes. In another study, inhibition of p-38 mitogen activated protein kinase signaling (which is associated with the stress response) by treatment with the pharmacological inhibitor SB-681323 (28 days), significantly reduced inflammation as measured by decreased circulating levels of C-reactive protein suggesting that direct inhibition of intermediate signaling proteins could also limit post-stent restenosis after percutaneous coronary intervention32. Although many other studies have examined individual components of the endothelial-inflammatory complex in an attempt to minimize immune cell infiltration and cellular activation following stent the most effective treatment based on a mono- or duel-therapeutic strategy might be to inhibit key adhesion molecules associated with transmission and/or activation of membrane bound receptor-linked primary signaling effectors associated with down-stream pro-inflammatory cascades. An example of this might be inhibition or knockdown of platelet derived endothelial cell adhesion molecule-1 (PECAM-1) together with NFB and or TNF- or its receptors. This strategy has shown promise with

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several published studies, one demonstrating notable reductions in inflammation and restenosis in PECAM-1 knockout mice via a reduction in activation of the NFB/AKt pathway33. Endothelial cell activation and angiogenesis in medium-long-term restenosis: In the early phase response, the composition of neointima covering the metal struts is associated with the healing response and consists mainly of ECM material and vascular smooth muscle cells. However, since in-stent restenosis is associated with longer-term incidents of thrombosis and myocardial infarction, usually 5-10 years after implant, it is important to understand if and how the composition of plaque like material changes to become destabilized over this period of time? Takano et al.34 used intracoronary Optical Coherence Tomography (OCT) to characterize the tissue components in patients approximately 6 months and 5 years after stent implant. They found after 6 months that approximately 60% of the patients demonstrated with neovascularized areas in the persistent zone. In the late phase group, intra-intimal neovascularisation was found in approximately 86% of patients studied. Interestingly they found no direct significant correlation between intraintimal neovascularisation and thrombus formation. However, neovessel development, which was highest in lipid rich areas (associated with intimal disruption and thrombosis), may have a role in promotion of intimal expansion, recruitment of inflammatory cells to the region, subsequent haemorrhage and contribute to plaque instability as occurs during normal atherosclerotic plaque formation. Knowledge of the pathobiological mechanisms which are responsible for promotion of angiogenesis in restenotic plaques could help in preventing their de-stabilization. It is well known that many of the chemokines and growth factors produced by infiltrating leukocytes/monocytes/macrophages are pro-angiogenic, and that these may also have activating effects on other cells of vascular origin35. Vogt36 showed that down-regulation of angio-associated migratory cell protein (AAMP) inhibited smooth muscle cell migration and neointima formation in a porcine model of coronary restenosis, whilst increase in the concentration of more conventional factors such as VEGF and fibroblast growth factor-2 (FGF-2) also leads to peri-adventitial angiogenesis and intimal thickening in animal models of restenosis37. Endothelial progenitor cells and re-endothelialization. Although a discussion of the importance of endothelial progenitor cells in the pathobiology of restenosis has been mentioned in other chapters of this book, their importance in re-initiation of and maintenance of proper EC function should be discussed as they could potentially help to re-form the critical luminal barrier to prevent accumulation of inflammatory cells within susceptible hyperplastic regions liable to undergo thrombosis. There is substantial evidence to show that bone marrow-derived endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularisation in the atherosclerotic environment, perpetuated by signals emanating from local inflammatory cells38. Also, in patients who have impaired production and lower circulating levels, such as those with risk factors for coronary artery disease such as diabetes, hypercholesterolaemia and smoking, statistics demonstrate an increased risk of cardiovascular events. However, Pelliccia39, showed that higher numbers of circulating EPCs were associated with increased restenosis in patients with stable angina, similarly, significant increases in stem cell mobilization were observed following coronary bare metal stent implantation in patients, and this was associated with activation of MAC-1 (a leukocyte specific integrin) and subsequent production of MMP-940. So here we have a complex situation where increased EPCs can help to repair dysfunctional endothelium and reduce late stage thrombosis, whilst on the other hand, influx of large numbers of EPCs during the inflammatory acute phase following angioplasty/stent implant, could contribute to more rapid development of restenosis. To combat the deleterious effects of an over influx of EPCs, Fukuda41 showed that sirolimus (otherwise known as rapamycin-an immunosuppressant) coated stents although reducing inflammation following implant, also resulted in increased late-stage thrombosis due to a lack of re-endothelialization. When they treated patients

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with fluvastatin, which is known to increase the number of circulating EPCs, re-endothelialization was stimulated and the effects reversed. These results suggest that a combinational treatment such as this may be beneficial for patients in receipt of modified coronary/carotids stents.

NOVEL AND EMERGING STRATEGIES/CONCLUSIONS


Integration of nanotechnology into stent design could improve efficiency and time-course of drug delivery. As we have mentioned previously, coating of stents with anti-proliferative, anti-inflammatory drugs such as paclitaxel and sirolimus demonstrates lower rates of restenosis over a 6 month period, but also prohibits normal vessel remodeling, improper integration of the stent into the vessel wall, endothelial dysfunction and incomplete coverage, all of which could result in late-stage thrombosis42. Nanoporous stent surfaces such as those containing aluminium oxide or carbon nanoparticle matrices have been used to deliver anti-inflammatory compounds, whist nano-texturing may be able to enhance the interaction between endothelial cells and stent surfaces by increasing cell adhesion43. Similarly, Epstein44 formulated gadolinium nanosuspensions of aldendronate (a liposomal biphosphonate) inhibited macrophage growth in vitro and also IL-1- and TNF- following a single IV dose in a rat model of carotid artery vascular injury (balloon catheter endothelial denudation).

Figure 6.4. Hypothetical representation of a micro-area of a major artery showing vasa vasorum and potential hypoxic micro-environment responsible for cellular activation, endothelial cell proliferation and ultimately plaque development in restenosis. Phase 1 treatment may involve prevention of vasa vasorum proliferation by administration of targeted siRNA or lentiviral vectors to block relevant gene expression. Phase 2 treatments would be designed to induce stabilization of vessels liable to rupture and bleed by administrating a mixture of factors to induce maturation of fragile vessels. Vascularization in atherosclerosis/restenosis represents a complex interaction between multiple molecules, manipulation of which could benefit patients by slowing down the process of its development and stabilizing developing stenotic regions.

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The ultimate aim would be to employ nanotechnology to combine the integration of non-porous stent surfaces which can deliver drugs on a time release basis45 whilst promoting endothelialization thus reducing restenosis and late-stage mortality due to thrombosis (Figure 6.4).

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Cutlip DE, Chabra AG, Baim DS, Chauhan MS, Marulkar S, Massaro J, Bakhai A, Cohen DJ, Kuntz RE, Ho KK. Beyond restenosis: five year clinical outcomes from second generation coronary stent clinical trials. Circulation. 2004;110(10):1226-30. Gupta S, Gupta MM. Stent thrombosis. J Assoc Physcians India. 2008;56:969-79. Tkachuk VA, Plekhanova OS, Parfyonova YV. Regulation of arterial remodeling and angiogenesis by urokinase type plasminogen activator. Can J Physiol Pharmacol. 2009;87(4):231-51. Pauletto P, Puato M, Faggin E, Sartore S. Low dose cerivastatin inhibits spontaneous atherogenesis in heterozygous watanable hyper lipidemic rabbits. J Vasc Res. 2000;37(3)189-94. Katsargyris A, Klonaris C, Bastounis E, Theocharis S. Toll-like receptor modulation : a novel therapeutic strategy in cardiovascular disease ? Expert Opin Ther Targets. 2008;12:1329-1346. Frantz S, Vincent KA, Feron O, Kelly RA. Innate immunity and angiogenesis. Circ Res. 2005;96:15-26. Hansson GK, Robertson AK, Sderberg-Nauclr C. Inflammation and atherosclerosis. Annu Rev Pathol. 2006;1:297-329. Hayden MR, Tyagi SC. Vasa vasorum in plaque angiogenesis, metabolic syndrome, type 2 diabetes mellitus, and atheroscleropathy: a malignant transformation. Cardiovasc Diabetol .2004;4:1. Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C, Sylvin E, Lo KM, Gillies S, Javaherian K, Folkman J. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci. 2003;100:4736-4741. Zhao Q, Egashira K, Hiasa K, Ishibashi M, Inoue S, Ohtani K, Tan C, Shibuya M, Takeshita A, Sunagawa K. Essential role of vascular endothelial growth factor and Flt-1 signals in neointimal formation after peri-adventitial injury. Arterioscler Thromb Vasc Biol. 2004;24:2284-2289. OBrien ER, Garvin MR, Dev R, Stewart DK, Hinohara T, Simpson JB, Schwartz SM. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol 1994, 145:883-894. Langheinrich AC, Sedding DG, Kampschulte M, Moritz R, Wilhelm J, Haberbosch WG, Ritman EL, Bohle RM. 3-Deazaadenosine inhibits vasa vasorum neovascularization in aortas of ApoE (-/-)/LDL(-/-) double knockout mice. Atherosclerosis 2008, [Epub ahead of print]. Moreno PR, Purushothaman KR, Fuster V, Echeverri D, Truszczynska H, Sharma SK, Badimon JJ, OConnor WN. Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation 2004, 110:2032-2038. Herrmann J, Lerman LO, Mukhopadhyay D, Napoli C, Lerman A. Angiogenesis in atherogenesis. Arterioscler Thromb Vasc Biol. 2006;26:1948-1957. Kolodgie FD, Gold HK, Burke AP, Fowler DR, Kruth HS, Weber DK, Farb A, Guerrero LJ, Hayase M, Kutys R, Narula J, Finn AV, Virmani R. Interplaque haemorrhage and progression of coronary atheroma. N Eng J Med. 2003;349:2316-2325. Williams JK, Armstrong ML, Heistad DD: Vasa vasorum in atherosclerotic coronary arteries: response to vasoactive stimuli and regression of atherosclerosis. Circ Res. 1988;62:515-523. Trape J, Morales C, Molina R, Filella X, Marcos JM, Salinas R, Franquesa J. Vascular endothelial growth factor serum concentration in hypercholesterolemic patients. Scand J Clin Invest. 2006;66:261-267. Garbin U, Fratta Pasini A, Stranieri C, Manfro S, Mozzini C, Boccioletti V, Pasini A, Cominacini M, Evangelista S, Cominacini L. Effects of nebivolol on endothelial gene expression during oxidative stress in human umbilical vein endothelial cells. Mediators Inflamm. 2008;367590. Ziello JE, Jovin IO, Huang Y. Hypoxia-inducible factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischaemia. J Biol Med. 2007;80:51-60. Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature. 2005;437:497-504. Suarez S, Ballmer-Hofer K. VEGF transiently disrupts gap junctional communication in endothelial cells. J Cell Sci. 2001;114:1229-1235. Khan M, Pelengaris S, Cooper M, Smith C, Evan G, Betteridge J. Oxidised lipoproteins may promote inflammation through the selective delay of engulfment but not binding of apoptotic cells by macrophages. Atherosclerosis. 2003;171:21-29. Montone RA, Ferrante G, Baca M, Niccoli G. Predictive value of C-reastive protein after drug eluting stent implantation. Future Crdiol. 2010; 6(2):167-79.

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24. Ertas G, van Beusekom HM, van der Giessen WJ. Late stent thrombosis, endothelialisation and drug-eluting stent. Neth Heart J. 2009;17(4):177-80. 25. Versari D, Lerman LO, Lerman A. The importance of reendothelialization after arterial injury. Curr Pharm Des. 2007;13(17):1811-24. 26. Gupta S, Gupta MM. Stent thrombosis. J Assoc Physcians India. 2008;56:969-79. 27. Li JM, Zhang X, Nelson PR, Odgren PR, Nelson JD, Vasiliu C, Park J, Morris M, Lian J, Cutler BS, Newburger PE. Temporal evolution of gene expression in rat carotid artery following balloon angioplasty. J Cell Biochem. 2007;101(2):399-410. 28. Qu Y, Shi X, Zhang H, Sun w, Han S, Yu C, Li j. VCAM-1 si-RNA reduces neointimal formation after surgical mechanical injury of the rat carotid artery. J Vasc Surg. 2009;50(6):1452-8. 29. Damrauer SM, Fisher MD, Wada H, Siracuse JJ, Da Silva CG, Moon K, Csizmadia E, Maccariello ER, Patel VI, Studer P, Essayagh S, Aird WC, Daniel S, Ferran C. A20 inhibits post-angioplasty restenosis by blocking macrophage trafficking and decreasing adventitial neovascularization. Atherosclerosis. 2010;211(2):404-8. 30. Afergan E, Ben David M, Epstein H, Koroukhov N, Gilhar D, Rohekar K, Danenberg HD, Golomb G. Liposomal simvastatin attenuates neointimal hyperplasia in rats. AAPS J. 2010;12(2):181-7. 31. Pesarini G, Amoruso A, Ferrero V, Bardello C, Fresu LG, Perobelli L, Scappini P, De Luca G, Brunelleschi S, VassanelliC, Ribichini F. Cytokines release inhibition from activated monocytes, and reduction of in-stent neointimal growth in humans. Atherosclerosis. 2010;211(1):242-8. 32. Sarov-Blat L, Morgan JM, Fernandez P, James r, Fang z, Hurle MR, Baidoo C, Willette RN, Lepore JJ, Jensen SE, Sprecher DL. Inhibition of p38 mitogen-activated protein kinase reduces inflammation after coronary vascular injury in humans. Aeterioscler Thromb Vasc Biol. 2010 (Epub ahead of print). 33. Chen Z, Tzima E. PECAM-1 is necessary for flow-induced vascular remodelling. Arteriorscler Thromb Vasc Biol. 2009;29(7):1067-73. 34. Takano M, Yamamoto M, Inami S, Murakami D, Ohba T, Seino Y, Mizuno K. Appearance of lipid-laden intima and neovascularization after implantation of bare-metal stents. JACC. 2010;55(1):26-32. 35. Slevin M, Krupinski J, Badimon L. Controlling the angiogenic switch in developing atherosclerotic plaques: possible targets for therapeutic intervention. J Angiogenesis Res. 2009;1:4. 36. Vogt F, Zernecke A, Beckner M, Krott N, Bosserhoff AK, Hoffmann R, Zandvoort MA, Jahnke T, Kelm M, Wel R. Blockade of angio-associated migratory cell protein inhibits smooth muscle cell migration and neointima formation in accelerated atherosclerosis. J Am Coll Cardiol. 2008;52(4):302-11. 37. Khurana R, Zhuang Z, Bhardwaj S, Murakami M, De Muinck E, Yia-Herttuala S, Ferrara N, Martin JF, Zachar M. Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation. 2004; 110(16): 2436-43. 38. Besler C, Doerries C, Giannotti g, Luscher TF, Landmesser U. Pharmacological approaches to imrove endothelial repair mechanisms. Expert Rev Cardiovasc Ther. 2008;6(8):1071-82. 39. Pelleccia F, Cianfrocca C, Rosano G, Mercuro G, Ppeciale G, Pasceri V. Role of endothelial progenitor cells in restenosis and of progression of coronary atherosclerosis after precutaneous coronary intervention: a prospective study. JACC Cardiovasc Interv. 2010; 3(1):87-9. 40. Inoue T, Taguchi I, Toyoda S, Nakajima K, Sakuma M, Node K. Activation of matrix metalloproteinase -9 in associated with mobilization of bone marrow-derived cells after coronary stent implantation. Int J Cardiol. 2010 (Epub ahead of print). 41. Fukuda D, Enomoto S, Shirakawa I, Nagai R, Sata M. Fluvastatin accelerates re-endothelialization impaired by local sirolimus treatment. Eur J Pharmacol. 2009; 612(1-3):87-92. 42. Godin B, Sakamoto JH, Serda RE, Grattoni A, Bouamrani A, Ferrari M. Emerging applications of nanomedicine for the diagnosis and treatment of cardiovascular diseases. Cell Press. 2010;32(5):199-205. 43. Caves JM, Chaikof EL. The evolving impact of microfabrication and nanotechnology on stent design. J Vasc Surg. 2006;44:1363-1368. 44. Epstein H, Berger V, Eisenberg G, Koroukhov N, Gao J, Golomb G. Nanosuspensions of alendronate with gallium or gadolinium attenuate neointimal hyperplasia in rats. J Control Release. 2007;117(3):322-32. 45. Nakano K, Egashira K, Masuda S, Funakoshi K, Zhao G, Kimura S, Matoba T, Sueishi K, Endo Y, Kawashim Tsujimoto H, Tominaga R, Sunagawa K. Formulation of nanoparticle-eluting stentd by a cationic electrodeposition coating technology: efficient nano-drug delivery via bioabsorbable polymetric nanoparticle-eluting in porcine coronary arteries. JACC Cardiovasc Interv. 2009;2(4):277-83.

CHAPTER 7

SHEAR STRESS AND STENT RESTENOSIS


MICHAIL J. PAPAFAKLIS, KATERINA K. NAKA and LAMPROS K. MICHALIS
Introduction ............................................................................................................................... Biology of the Shear Stress Effect on Atherosclerosis and Restenosis...................................... In vivo Assessment of Shear Stress............................................................................................ Impact of Stent Deployment and Design on Local Flow Dynamics.......................................... Shear Stress and Neointimal Hyperplasia in Bare Metal Stents ................................................ Effect of Flow Dynamics on Drug Deposition and Neointima Distribution in Drug-Eluting Stents ............................................................................................................................... Conclusions ............................................................................................................................... References ................................................................................................................................. 109 110 110 111 115 116 117 117

INTRODUCTION
Coronary stenting has proven to be beneficial in blocking vascular contraction and to provide good long-term clinical results. However, in-stent restenotic lesions following bare metal stent (BMS) implantation occur in relatively high rates of 20 to 30% underscoring the major problem of restenosis which hampers the procedures efficacy1. Localized delivery of immunosuppressive agents using drug-eluting stents (DES) emerged as an alternative in order to inhibit the pathogenic path of restenosis occurring in the early phase of 6 months after stent implantation. DES implantation has the advantage of reducing neointimal growth, but is also associated with some side-effects such as delayed healing and late incomplete stent apposition which may cause stent thrombosis2,3. Neointimal hyperplasia, which is the major contributing pathophysiologic mechanism of in-stent restenosis, is influenced by clinical characteristics, such as diabetes4, and procedural and lesion-related parameters, such as residual stenosis, number of stents, stent length and plaque burden5,6. Recent studies have shown that altered geometry and associated blood flow disturbances established after stenting can also influence restenosis, while stent design, material and configuration are critical in determining neointimal hyperplasia7,8. Local haemodynamic factors, shear stress (SS) in particular, have been implicated in both the atherosclerotic disease and the pathobiology of in-stent restenosis and stent thrombosis9-11. The purpose of this chapter is to introduce the concept of SS and its effects on vascular pathobiology, and summarize current knowledge on the influence of stent deployment on local flow dynamics and the involvement of SS in neointimal hyperplasia following bare metal and drug-eluting stent implantation.

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BIOLOGY OF THE SHEAR STRESS EFFECT ON ATHEROSCLEROSIS AND RESTENOSIS


Despite the systemic nature of risk factors (i.e., hypertension, smoking, hyperlipidemia, diabetes mellitus, social stress) associated with atherosclerotic disease, atherosclerosis is a site-specific disease, which affects primarily the carotid bifurcation, the coronary arteries, the infrarenal aorta and the arteries of the lower limbs having a propensity to involve the outer edges of blood vessel bifurcations12 and the inner walls of curved arteries13. Furthermore, neointima formation causing in-stent restenosis is often observed at specific locations in the stented segment14. Local haemodynamic factors that create a unique environment seem to be a major determinant of the behavior of atherosclerosis and restenosis in a susceptible individual. Flowing blood imposes on the wall of the arterial lumen (endothelial surface) a haemodynamic stress that has two components: a normal (i.e., perpendicular) component, pressure, and a tangential component, SS, which is equal to the shear force (frictional force) per unit area and is due to the viscous nature of blood. The magnitude of SS is proportional to the velocity gradient (shear rate) near the arterial wall, that is, the difference in flow velocity when moving from a point on the arterial wall to an immediately adjacent point in a direction normal to the wall towards the centerline of the artery. Consequently, low local flow velocity gives rise to low shear rate and SS15. It has been observed that regions of moderate to high SS, where flow remains unidirectional and axially aligned, are spared of intimal thickening, while focal lesions develop only in areas of low and recirculating flow, and thus, low SS values16. Low SS exerts a multifactorial influence on the arterial wall, which involves the conversion of biomechanical stimuli to biochemical responses by endothelial cells leading to the activation of inflammatory pathways (e.g., nuclear factor kappa B) and muting of atheroprotective transcriptional pathways (e.g., Kruppel-like factor-2)10,17. The result of this process is: 1) the increased expression of adhesion molecules (e.g., ICAM-1, VCAM-1)16,18 and chemokines (e.g., MCP-1, fraktalkine)19, which mediate the recruitment of inflammatory cells in the intima, and 2) the decrease of atheroprotective molecules, such as nitric oxide and prostacyclin20. Conversely, normal or high SS exerts protective functions on the endothelial layer by increasing nitric oxide synthase21 and reducing endothelial cell death and proliferation22. The pathophysiology of in-stent restenosis involves accumulation of new tissue within the arterial wall, and neointima formation is caused by a cascade mechanism initiated by platelet aggregation due to vascular injury and followed by leukocyte (mainly macrophages) intimal infiltration. The interplay of growth factors (e.g. PDGF, VEGF) released during vessel injury ultimately controls smooth muscle cell migration and proliferation in the intima through intracellular positive and negative regulators of cell cycle events23. Smooth muscle cells migration to the intima is also followed by their phenotypic switch from a contractile (differentiated) phenotype to a synthetic (de-differentiated) one, which leads to increased extracellular matrix production. SS has been demonstrated in vitro to act either through the endothelium or directly, as in cases of a damaged endothelial layer after stenting, and modulate smooth muscle cell gene expression, proliferation and phenotypic modulation24-26.

IN VIVO ASSESSMENT OF SHEAR STRESS


To investigate the role of SS in atherosclerosis and in-stent restenosis in animals and humans, in vivo assessment of local SS distribution is required. Direct measurement of the local velocity gradient and thus, SS in coronary arteries is very difficult due to their small size and

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complex time-dependent three-dimensional (3D) geometry. Therefore, in vivo SS assessment necessitates a mathematical approach, and an approximate solution can be derived from the simple Hagen-Poiseuille formula (SS=32 Q/d3, : viscosity, Q: flow, d: lumen diameter), which is valid in idealized cases of steady (time unvarying), fully-developed flow of a Newtonian fluid through a straight tube of constant circular cross-section. An alternative way, which provides a more detailed approach compared to the simplistic Poiseuille flow, involves the use of the differential equations of continuity and momentum (Navier-Stokes equations). The solution of these equations for complex flow fields like those in coronary arteries is feasible by applying computational fluid dynamics (CFD) techniques in 3D reconstructions of coronary arteries. Realistic 3D coronary artery reconstruction can be obtained either by using stand-alone biplane angiographic data or by using fusion methodologies of biplane angiographic and intravascular ultrasound (IVUS) data27-31. The incorporation of IVUS data in the 3D reconstruction methodology is technically more demanding, but increases the accuracy and also has the advantage of providing both 3D lumen and outer vessel wall (media-adventitia border) reconstructions (Figure 7.1). Latest advancements in medical imaging have also enabled the use of computed tomography angiography for 3D reconstruction of the coronary arterial tree32,33. In CFD modeling, the complex geometry of the 3D artery reconstruction is then separated into a large number of smaller finite elements, creating a grid on which the equations describing the phenomenon are discretised. By assuming the velocity field within those elements, it is possible to solve the equations of fluid motion at the nodes connecting these elements, provided that certain boundary conditions for velocity (e.g., patient-specific flow) are imposed and material properties (i.e., blood density and viscosity) are defined. This procedure finally provides invaluable knowledge of the detailed flow characteristics (e.g., velocity profile) and spatio-temporal variations that SS may exhibit in coronary arteries (Figure 7.2), and can be used for investigating in vivo the relationship of SS with wall characteristics in native arteries and neointimal hyperplasia in stents.

IMPACT OF STENT DEPLOYMENT AND DESIGN ON LOCAL FLOW DYNAMICS


Arterial geometry is an important factor that influences local flow dynamics and SS distributions. Stent deployment is known to alter coronary artery haemodynamics due to the effects of the metal scaffolding on vascular resistance and local arterial geometry according to animal studies or simulations in 3D theoretical vessels reflecting the in vivo environment. Measurement of indices of flow dynamics and SS in the left anterior descending coronary artery of anesthetized dogs demonstrated that stent implantation produces a modest increase in coronary blood flow related to a reduction in coronary vascular resistance and leads to increases in regional SS. Importantly, the same study demonstrated that stent deployment attenuated alterations in fluid dynamics (i.e., reductions in coronary vascular resistance and increases in Reynolds number [Re] and SS indices) during maximal vasodilation34. Stent implantation has also been shown in vivo to cause prominent increments in curvature (increase by 121% at the entrance and by 100% at the exit of the stent) near the stent edges resulting in local SS maxima and minima35. The inherent curvature of a coronary artery due to its course on the epicardial surface cannot be fully preserved after stent deployment and the stent-induced axial arterial straightening leads to skewing of the velocity profile at the inlet and outlet of the stented region, and consequently, causes large alterations in spatial SS distributions. Conversely, implantation of a stent that conforms to native vessel curvature causes minimal changes in SS distribution and may be beneficial from a haemodynamic perspective36.

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Figure 7.1 A. Angiographic views of a right coronary artery including the guidewire and intravascular ultrasound (IVUS) catheter (arrows indicate the IVUS probe). B. Corresponding three-dimensional reconstructed surfaces of the lumen (red) and outer vessel wall (light blue) of the right coronary artery using a methodology based on the fusion of biplane angiographic and IVUS data. LAO, left anterior oblique; RAO, right anterior oblique.

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Figure 7.2 A. Color-coded velocity vectors indicating the flow velocity profile in various cross-sections along the axial direction of a right coronary artery. B. Detailed view of the mid-RCA demonstrating skewing of the velocity profile towards the outer wall of this curved arterial segment. C. Color-coded map of the shear stress distribution on the arterial wall (endothelial surface); different views are provided.

Simplified representations of normal coronary arteries modeled as cylindrical tubes, but also including the individual stent struts, have provided detailed knowledge on the alterations in SS caused by stent implantation. LaDisa37 showed that a slotted-tube stent design has a profound influence on near-wall velocity and wall SS patterns compared to a non-stented vessel

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in a three-dimensional CFD model of an epicardial coronary artery using in vivo canine data as boundary conditions. Minimum wall SS is decreased by approximately 80% within the stent and at the outlet of the stent in stented compared to non-stented vessels, and regions of low SS are localized around stent struts and are associated with stagnation flow and boundary layer separation immediately upstream and downstream of the struts. Similar results were presented by another study which compared four different types of bare metal stents and demonstrated that the zones with low SS values are found near the stent struts at the diastolic peak of the cardiac cycle, while the highest SS values are localized over the stent struts, which are the regions most exposed to blood flow38. Stent design components (e.g., strut number, thickness, angle) also significantly influence local flow dynamics. Increasing the number of struts (four to eight) has been shown to produce a 2.75-fold increase in exposure to low SS39. A reduction in strut thickness (0.096 to 0.056 mm) has been reported to cause regions subjected to low SS to decrease by approximately 87% according to a steady-state simulation39, whereas another study using pulsatile flow and more realistic arterial geometry incorporating the deformed shape of the inner surface of the arterial wall reported an increase in the overall percentage area with low SS values in the deceleration phase of the cardiac cycle with thinner struts (0.05 versus 0.15 mm)38. Of note, larger stent struts have also been associated with less endothelial coverage, underscoring the importance of stent characteristics on both flow dynamics and the vascular response40. Furthermore, the stent strut profile has a significant impact on the wall SS both on the struts and in between struts. Streamlined profiles (elliptical and tear-drop) exhibit better haemodynamic performance compared to the standard square and circular profiles since the former have been shown to have smaller recirculation zones and a lower percentage of inter-strut area where the SS level is decreased41,42. Areas exposed to low SS and reverse flow are also proportional to the connector length in the cross-flow direction and the lengths of flow recirculation depend on the overall strut-connector-strut configuration, an essential component of a stent design as the presence of strut-connectors makes the stent flexible, which in turn improves stent deployment43. In addition to stent design, procedural parameters (e.g., stent-to-artery ratio, over-expansion, overlapping stents) related to stent deployment vary among patients and can affect SS distribution. An increase in the deployment ratio (1.1:1 to 1.2:1) has been demonstrated to increased exposure to low SS by 12-fold39. Latest data also indicate that over-expanding the stent with a second balloon affects the alignment of the stent geometry, and leads to higher SS at the inlet and lower values at mid-stent, while overlapping stents show disturbed flow and a SS deficit region downstream of the overlapped region; thus, the use of overlapping stents in place of a single longer stent appears to disrupt the flow within the stented region44. Stent implantation with or without balloon post-dilatation can also influence the cross-sectional vascular shape of the stented segment since an implanted stent especially one with a small number of stent struts in the circumference may lose its circular profile. Polygonal computational vessels due to circumferential straightening are subjected to greater areas of low SS and elevated spatial SS gradients than those with circular geometry and may predispose to subsequent neointimal hyperplasia45. Further, stent foreshortening (the difference between the original and actual stent length after stent deployment) may occur and influences the angle created between axially aligned stent struts and the principal direction of blood flow increasing the intra-strut area of the luminal surface exposed to low SS and elevated spatial SS gradients46. Overall, stent design characteristics and stent implantation exert significant influence on local arterial geometry and flow features causing alterations in SS distribution and thus, may contribute to restenosis risk. It is noteworthy from a pathobiological standpoint that the

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afore-mentioned simulations incorporating individual stent struts illustrate changes in acute SS induced by stent implantation. However, it is highly likely that progressive endothelialization and smooth muscle cell proliferation around a chronically implanted stent would ultimately affect the SS distribution in vivo.

SHEAR STRESS AND NEOINTIMAL HYPERPLASIA IN BARE METAL STENTS


During the last decade, a number of groups studied in vivo the effect of flow disturbances and SS on the development of neointimal hyperplasia following bare metal stent implantation. Animal data have shown that neointimal hyperplasia occurs in arterial regions of low SS and elevated spatial SS gradients47. In the ilio-femoral bifurcation of a porcine model, stenting of the main branch has been found to lead to highly eccentric restenotic lesions with a maximum at the lateral wall of the main branch, which were in concert with the extent, shape and location of an area of boundary layer separation, flow stagnation and thus, decreased SS magnitude. Regions at the lateral wall had a 3.5-fold higher neointimal thickness compared to high SS regions at the flow divider and were fibrin-rich both around the stent struts and in regions between and away the stent struts showing dense macrophage infiltration of the intima in the acute phase48. In contrast to the adverse effect of low SS on stent patency, an artificial 2-fold augmentation of SS after placing a prototype flow divider in stented segments of rabbit iliac arteries has been demonstrated to result in significant lower (>50%) mean late luminal loss, reduction (>40%) in neointimal thickness and a reduced inflammation and injury score, as assessed by macrophage infiltration and internal elastic lamina disruption49. This study also indicates that it is possible to artificially modify the in-stent SS distribution; stenting devices of non-conventional shape could be presumably manufactured for increasing SS and thus, reducing in-stent restenosis50. The first results based on patient data were reported by Wentzel, et al.,51 who demonstrated a significant negative correlation (r range: -0.65 to -0.04, p<0.05) between SS and neointimal thickness in 9 out of the 14 patients studied 6 months after self-expandable Wallstent (Schneider, AG) implantation. SS and neointimal thickness were shown to be geometrically interrelated such that maximal thickness is preferentially located near the inner curve of the coronary artery, where the minimal SS is also observed, whereas the minimal thickness is more frequently detected in the outer curve, which is primarily where the maximal SS is also located. Similarly, a negative correlation of in-stent neointimal thickness to baseline SS was also reported in 5 out of the 7 patients studied in a later investigation with balloon-expandable bare metal stents52. Conversely, Stone, et al.53 reported a limited role of SS in in-stent restenosis since neointimal hyperplasia appeared to occur to some degree in all categories of SS in the six stented coronary segments they studied. Latest patient data from our group support the role of SS in neointimal proliferation, but also indicate that SS cannot predict the exact locations of neointimal hyperplasia in all cases, since the thickening does not necessarily occur in all regions where SS is low and not all regions where SS is high are spared from neointimal hyperplasia. In-stent restenosis is a multi-factorial process and regions with low SS should thus be considered as regions with a higher probability for neointimal hyperplasia to occur and be more profound. Of note, in the same study, vascular brachytherapy following bare metal stent implantation, a therapeutic modality used in the recent past for reducing in-stent restenosis, was found to diminish the inverse relationship between neointimal thickness and SS54.

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EFFECT OF FLOW DYNAMICS ON DRUG DEPOSITION AND NEOINTIMA DISTRIBUTION IN DRUG-ELUTING STENTS
Although drug-eluting stents have been proven to exert a potent anti-proliferative effect, neointimal hyperplasia or even in-stent restenosis may still occur especially when DES are used in complex clinical and anatomic settings55. Further, drug-eluting stent implantation has been associated with poor endothelialization of stent struts56, delayed healing57, incomplete stent apposition and tissue regression58. The degree of drug penetration into the tissue dictates drug efficacy, but may also contribute to the side-effects of drug-eluting stent implantation. Drug deposition is determined by a complex interplay between strut-wall contact, amount and location of drug release, and flow profiles, which depend on stent design and strut position, as we previously mentioned. Simulations using coupled computational fluid dynamics and mass transfer models have revealed that direct strut contact accounts for only 38% of peak and 11% of total arterial drug, whereas non-contacting strut surfaces can contribute up to 90% of arterial drug deposition, and the adluminal surface provides half of this amount59. Evidently, flow patterns around stent struts determine the quantity of blood-solubilized drug, which is washed away by the free flow stream or trapped in flow stagnation zones and deposited not only under or adjacent to struts but also in distal tissue segments or in inter-strut zones60. Overlapping drug-eluting stents dramatically affect drug distribution not only by adding to the amount of local drug and area of contact with the arterial wall, but also by influencing the degree of strut protrusion into the lumen, and consequently, flow disruption59. In cases of bifurcational stenting, drug deposition is determined not only by stent strut configuration, but also by flow disturbances imposed by the flow divider; the presence of the side branch affects drug distribution in the stented main vessel, thereby creating zones of excessive drug deposition or areas of drug depletion, which could ultimately lead to vascular toxicity or restenosis, respectively61. Latest in vivo studies investigating the haemodynamic characteristics and the relationship of SS with neointima distribution following drug-eluting stent implantation have provided early data on the role of SS in a clinical setting. In-stent SS has been reported to remain in the normal range 6 months after sirolimus-eluting stent implantation compared to bare metal stents, which showed elevated SS values at follow-up (1.60.7 versus 3.93.1 Pa, respectively, p=0.003) 62. The predictive value of SS just after stent implantation on neointima distribution (i.e., neointimal hyperplasia and tissue regression) at follow-up has been assessed mostly after sirolimus-eluting stent implantation and has provided unclear results. Gijsen, et al.63 first reported that neointimal thickness 6 months after sirolimus-eluting stent implantation was inversely related to SS in 4 out of the 6 patients studied by angiography and intravascular ultrasound for 3D coronary artery reconstruction; tissue regression was observed in the form of inter-strut shallow pits and was attributed to intima regression induced by high SS. Both neointimal hyperplasia and tissue regression were also present at 6-month follow-up in a small series of 16 patients after either sirolimus- or paclitaxel-eluting stent implantation, but an overall significant negative relationship was found only between neointimal hyperplasia thickness and SS, whereas the role of SS in tissue regression was limited64. Similarly, only neointimal growth was inversely correlated to SS, while no association was found between tissue regression depth and SS, in a case of lumen dilatation shortly (10 weeks) after paclitaxel-eluting stent implantation65. Post-procedural stent malapposition and thrombus dissolution were potential mechanisms for the lumen enlargement in this case. Furthermore, prediction of in-stent restenosis after sirolimus-eluting stent implantation on the basis of low flow velocity and SS was recently reported in 21 patients with restenosis compared to a

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non-restenotic group (n=246)66. However, in this study TIMI frame count was used for assessing gross flow velocity and Re, which were used as indirect measures of SS, and may have influenced the results. In contrast, another study investigated the correlation between neointimal hyperplasia volume and SS in a diabetic population (n=9) at 9-month follow-up after sirolimus-eluting stent implantation and found no association between the two variables67.

CONCLUSIONS
The role of SS in vascular pathobiology has been lately highlighted by both molecular investigations and in vivo studies, which demonstrated that SS distribution is critical in explaining the spatial heterogeneity of atherosclerotic lesions and may also contribute to restenosis following stent implantation. Modern imaging methodologies allow the in vivo serial assessment of the natural history of coronary artery disease, as well as that of the restenotic process following endovascular interventions in relation to haemodynamic factors. Stent implantation and various design characteristics have a major impact on local flow dynamics since they change native arterial geometry and create zones of flow separation and recirculation around the stent struts, thereby altering SS distribution. Low SS values are associated with an increased thickness of neointimal hyperplasia and consequently, an augmented restenosis risk after bare metal stent implantation, while regions with high SS are protected from biological sequelae following stent-induced vascular injury. In the era of drug-eluting stents, sophisticated simulations provide insight into the role of flow patterns in determining drug deposition and distribution in the inter-strut areas, while the first patient studies indicate that further investigations are needed for reaching concrete conclusions. Newly acquired knowledge regarding the importance of blood flow characteristics in percutaneous coronary interventions is invaluable in guiding the construction of haemodynamically driven next-generation stenting devices and thus, improving success rates and clinical outcomes.

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11. Wentzel JJ, Gijsen FJ, Schuurbiers JC, van der Steen AF, Serruys PW. The influence of shear stress on in-stent restenosis and thrombosis. EuroIntervention 2008;4 Suppl C:C27-32. 12. Papafaklis MI, Bourantas CV, Theodorakis PE, Katsouras CS, Fotiadis DI, Michalis LK. Association of endothelial shear stress with plaque thickness in a real three-dimensional left main coronary artery bifurcation model. Int J Cardiol 2007; 115:276-8. 13. Asakura T, Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res 1990; 66:1045-66. 14. Thury A, Wentzel JJ, Vinke RV, Gijsen FJ, Schuurbiers JC, Krams R, et al. Images in cardiovascular medicine. Focal in-stent restenosis near step-up: roles of low and oscillating shear stress? Circulation 2002;105:e185-7. 15. Katritsis D, Kaiktsis L, Chaniotis A, Pantos J, Efstathopoulos EP, Marmarelis V. Wall shear stress: theoretical considerations and methods of measurement. 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Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming. Am J Physiol Cell Physiol 2007; 293:C1824-33. 27. Wahle A, Prause PM, DeJong SC, Sonka M. Geometrically correct 3-D reconstruction of intravascular ultrasound images by fusion with biplane angiography--methods and validation. IEEE Trans Med Imaging 1999; 18:686-99. 28. Slager CJ, Wentzel JJ, Schuurbiers JC, Oomen JA, Kloet J, Krams R, et al. True 3-dimensional reconstruction of coronary arteries in patients by fusion of angiography and IVUS (ANGUS) and its quantitative validation. Circulation 2000; 102:511-6. 29. Bourantas CV, Kalatzis FG, Papafaklis MI, Fotiadis DI, Tweddel AC, Kourtis IC, et al. ANGIOCARE: an automated system for fast three-dimensional coronary reconstruction by integrating angiographic and intracoronary ultrasound data. Catheter Cardiovasc Interv 2008; 72:166-75. 30. Giannoglou GD, Chatzizisis YS, Sianos G, Tsikaderis D, Matakos A, Koutkias V, et al. In-vivo validation of spatially correct three-dimensional reconstruction of human coronary arteries by integrating intravascular ultrasound and biplane angiography. Coron Artery Dis 2006; 17:533-43. 31. Stone PH, Coskun AU, Yeghiazarians Y, Kinlay S, Popma JJ, Kuntz RE, et al. Prediction of sites of coronary atherosclerosis progression: In vivo profiling of endothelial shear stress, lumen, and outer vessel wall characteristics to predict vascular behavior. Curr Opin Cardiol 2003; 18:458-70. 32. Rybicki FJ, Melchionna S, Mitsouras D, Coskun AU, Whitmore AG, Steigner M, et al. Prediction of coronary artery plaque progression and potential rupture from 320-detector row prospectively ECG-gated single heart beat CT angiography: Lattice Boltzmann evaluation of endothelial shear stress. Int J Cardiovasc Imaging 2009; 25:289-299. 33. van der Giessen AG, Wentzel JJ, Meijboom WB, Mollet NR, van der Steen AF, van de Vosse FN, et al. Plaque and shear stress distribution in human coronary bifurcations: a multislice computed tomography study. EuroIntervention 2009; 4:654-61.

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34. LaDisa JF, Jr., Hettrick DA, Olson LE, Guler I, Gross ER, Kress TT, et al. Stent implantation alters coronary artery hemodynamics and wall shear stress during maximal vasodilation. J Appl Physiol 2002;93:1939-46. 35. Wentzel JJ, Whelan DM, van der Giessen WJ, van Beusekom HM, Andhyiswara I, Serruys PW, et al. Coronary stent implantation changes 3-D vessel geometry and 3-D shear stress distribution. J Biomech 2000;33:1287-95. 36. LaDisa JF, Jr., Olson LE, Douglas HA, Warltier DC, Kersten JR, Pagel PS. Alterations in regional vascular geometry produced by theoretical stent implantation influence distributions of wall shear stress: analysis of a curved coronary artery using 3D computational fluid dynamics modeling. Biomed Eng Online 2006;5:40. 37. LaDisa JF, Jr., Guler I, Olson LE, Hettrick DA, Kersten JR, Warltier DC, et al. Three-dimensional computational fluid dynamics modeling of alterations in coronary wall shear stress produced by stent implantation. Ann Biomed Eng 2003;31:972-80. 38. Balossino R, Gervaso F, Migliavacca F, Dubini G. Effects of different stent designs on local hemodynamics in stented arteries. J Biomech 2008;41:1053-61. 39. LaDisa JF, Jr., Olson LE, Guler I, Hettrick DA, Audi SH, Kersten JR, et al. Stent design properties and deployment ratio influence indexes of wall shear stress: a three-dimensional computational fluid dynamics investigation within a normal artery. J Appl Physiol 2004;97:424-30; discussion 416. 40. Charonko J, Karri S, Schmieg J, Prabhu S, Vlachos P. In vitro, time-resolved PIV comparison of the effect of stent design on wall shear stress. Ann Biomed Eng 2009;37:1310-21. 41. Mejia J, Ruzzeh B, Mongrain R, Leask R, Bertrand OF. Evaluation of the effect of stent strut profile on shear stress distribution using statistical moments. Biomed Eng Online 2009;8:8. 42. Jimenez JM, Davies PF. Hemodynamically driven stent strut design. Ann Biomed Eng 2009;37:1483-94. 43. Pant S, Bressloff NW, Forrester AI, Curzen N. The influence of strut-connectors in stented vessels: a comparison of pulsatile flow through five coronary stents. Ann Biomed Eng 2010;38:1893-907. 44. Charonko J, Karri S, Schmieg J, Prabhu S, Vlachos P. In vitro comparison of the effect of stent configuration on wall shear stress using time-resolved particle image velocimetry. Ann Biomed Eng 2010;38:889-902. 45. LaDisa JF, Jr., Olson LE, Guler I, Hettrick DA, Kersten JR, Warltier DC, et al. Circumferential vascular deformation after stent implantation alters wall shear stress evaluated with time-dependent 3D computational fluid dynamics models. J Appl Physiol 2005;98:947-57. 46. LaDisa JF, Jr., Olson LE, Hettrick DA, Warltier DC, Kersten JR, Pagel PS. Axial stent strut angle influences wall shear stress after stent implantation: analysis using 3D computational fluid dynamics models of stent foreshortening. Biomed Eng Online 2005;4:59. 47. LaDisa JF, Jr., Olson LE, Molthen RC, Hettrick DA, Pratt PF, Hardel MD, et al. Alterations in wall shear stress predict sites of neointimal hyperplasia after stent implantation in rabbit iliac arteries. Am J Physiol Heart Circ Physiol 2005;288:H2465-75. 48. Richter Y, Groothuis A, Seifert P, Edelman ER. Dynamic flow alterations dictate leukocyte adhesion and response to endovascular interventions. J Clin Invest 2004;113:1607-14. 49. Carlier SG, van Damme LC, Blommerde CP, Wentzel JJ, van Langehove G, Verheye S, et al. Augmentation of wall shear stress inhibits neointimal hyperplasia after stent implantation: inhibition through reduction of inflammation? Circulation 2003;107:2741-6. 50. Papaioannou TG, Christofidis C, Mathioulakis DS, Stefanadis CI. A novel design of a noncylindric stent with beneficial effects on flow characteristics: an experimental and numerical flow study in an axisymmetric arterial model with sequential mild stenoses. Artif Organs 2007;31:627-38. 51. Wentzel JJ, Krams R, Schuurbiers JC, Oomen JA, Kloet J, van Der Giessen WJ, et al. Relationship between neointimal thickness and shear stress after Wallstent implantation in human coronary arteries. Circulation 2001;103:1740-5. 52. Sanmartin M, Goicolea J, Garcia C, Garcia J, Crespo A, Rodriguez J, et al. [Influence of shear stress on in-stent restenosis: in vivo study using 3D reconstruction and computational fluid dynamics]. Rev Esp Cardiol 2006;59:20-7. 53. Stone PH, Coskun AU, Kinlay S, Clark ME, Sonka M, Wahle A, et al. Effect of endothelial shear stress on the progression of coronary artery disease, vascular remodeling, and in-stent restenosis in humans: in vivo 6-month follow-up study. Circulation 2003;108:438-44. 54. Papafaklis MI, Bourantas CV, Theodorakis PE, Katsouras CS, Fotiadis DI, Michalis LK. Relationship of shear stress with in-stent restenosis: bare metal stenting and the effect of brachytherapy. Int J Cardiol 2009;134:25-32. 55. Alfonso F. Treatment of drug-eluting stent restenosis the new pilgrimage: quo vadis? J Am Coll Cardiol 2010;55:2717-20.

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56. Jimenez-Valero S, Moreno R, Sanchez-Recalde A. Very late drug-eluting stent thrombosis related to incomplete stent endothelialization: in-vivo demonstration by optical coherence tomography. J Invasive Cardiol 2009;21:488-90. 57. Farb A, Heller PF, Shroff S, Cheng L, Kolodgie FD, Carter AJ, et al. Pathological analysis of local delivery of paclitaxel via a polymer-coated stent. Circulation 2001;104:473-9. 58. Hong MK, Mintz GS, Lee CW, Park DW, Park KM, Lee BK, et al. Late stent malapposition after drug-eluting stent implantation: an intravascular ultrasound analysis with long-term follow-up. Circulation 2006; 113:414-9. 59. Balakrishnan B, Tzafriri AR, Seifert P, Groothuis A, Rogers C, Edelman ER. Strut position, blood flow, and drug deposition: implications for single and overlapping drug-eluting stents. Circulation 2005;111:2958-65. 60. Kolachalama VB, Tzafriri AR, Arifin DY, Edelman ER. Luminal flow patterns dictate arterial drug deposition in stent-based delivery. J Control Release 2009;133:24-30. 61. Kolachalama VB, Levine EG, Edelman ER. Luminal flow amplifies stent-based drug deposition in arterial bifurcations. PLoS One 2009;4:e8105. 62. van't Veer M, Pijls NH, Aarnoudse W, Koolen JJ, van de Vosse FN. Evaluation of the haemodynamic characteristics of drug-eluting stents at implantation and at follow-up. Eur Heart J 2006;27:1811-7. 63. Gijsen FJ, Oortman RM, Wentzel JJ, Schuurbiers JC, Tanabe K, Degertekin M, et al. Usefulness of shear stress pattern in predicting neointima distribution in sirolimus-eluting stents in coronary arteries. Am J Cardiol 2003;92:1325-8. 64. Papafaklis MI, Bourantas CV, Theodorakis PE, Katsouras CS, Fotiadis DI, Michalis LK. Role of shear stress in predicting neointima distribution: drug-eluting stents versus bare metal stents [Abstract]. J Am Coll Cardiol 2008;51:A300. 65. Papafaklis MI, Katsouras CS, Theodorakis PE, Bourantas CV, Fotiadis DI, Michalis LK. Coronary dilatation 10 weeks after paclitaxel-eluting stent implantation. No role of shear stress in lumen enlargement? Heart Vessels 2007;22:268-73. 66. Hikita H, Sato A, Nozato T, Kawashima T, Takahashi Y, Kuwahara T, et al. Low coronary flow velocity and shear stress predict restenosis after sirolimus-eluting stent implantation. Scand Cardiovasc J 2009; 43:298-303. 67. Suzuki N, Nanda H, Angiolillo DJ, Bezerra H, Sabate M, Jimenez-Quevedo P, et al. Assessment of potential relationship between wall shear stress and arterial wall response after bare metal stent and sirolimus-eluting stent implantation in patients with diabetes mellitus. Int J Cardiovasc Imaging 2008;24:357-64.

CHAPTER 8

HYSTOLOGICAL PREDICTORS AND MYOCARDIAL REGENERATION


JANG-HO BAE and TAEK-GEUN KWON
Introduction ............................................................................................................................... Hystologic Predictors ............................................................................................................... Conclusion................................................................................................................................. Myocardial Regeneration .......................................................................................................... References ................................................................................................................................. 121 122 124 124 125

INTRODUCTION
In-stent restenosis (ISR) is an unsolved problem even in percutaneous coronary intervention (PCI) with drug eluting stent (DES). Various predictors of ISR among clinical, angiographic, and procedural parameters, including diabetes mellitus, long lesion and stent length, small stent diameters, and smaller post-stenting minimal luminal diameter and area were frequently reported (Table 8.1)1. ISR is also known as an inflammatory process like atherosclerosis, since pathological findings in ISR was disclosed such as platelet deposition, leukocyte recruitment, smooth muscle cell deposition/proliferation and matrix deposition. In this chapter, the association between histologic findings including plaque characteristics and ISR and myocardial regeneration will be discussed.
Table 8.1. Mechanisms of ISR after Drug-Eluting Stent
Biological Diabetes Mellitus Increased high-sensitivity C-reactive protein Long lesion Mechanical Stent fracture Polymer peeling Technical Gap/geographical miss Stent underexpansion/inapposition Peri-stent injury

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HYSTOLOGIC PREDICTORS Calcified Plaque


Calcified lesion is relatively easy to detect by conventional coronary angiogram and/or intravascular ultrasound (IVUS) than the other pathology such as fibrous and/or lipid component in coronary trees. PCI of calcified and complex lesion has been associated with lower success rates and higher restenosis rate than PCI of simple lesions2. In heavily calcified lesions, it is hard to deliver and expand a stent properly, resulting in lower post-stenting minimal stent area which is a crucial factor of ISR3. There are several reports that DES and plaque modification can reduce ISR in heavy calcified lesions. TAXUS-IV substudy showed that ISR rate was non-significantly lower in patients with paclitaxel-eluting stent (PES) than bare metal stent (BMS) (7.5% versus 18.3%, p=0.10) in calcified lesion and in-segment restenosis was similar in patients with calcified and noncalcified lesions after PES implantation (7.5% versus 8.0%, p=1.0). However, the rate of failure to deliver with PES was higher than BMS (5.8% versus 2.4%, p=0.21)4. In moderate to severe calcified lesion, PCI with sirolimus-eluting stent (SES) was high procedural success rate that similar with noncalcified lesion. However, binary restenosis (9.2% versus 3.6%, p<0.05) and target lesion revascularization (TLR) (7.3% versus 2.8%, p<0.05) were significantly higher in calcified lesion. TLR was increased proportionally with severity of calcification5. DES implantation was associated with lower incidence of ISR in calcified lesion. However, restenosis rate after BMS implantation in calcified lesion was lower (18.3% versus 29%, p=0.13) than noncalcified lesion and TLR was similar regardless the severity of calcification4. These data is consistent with IVUS observation that neointimal proliferation is less after BMS implantation in calcified compared with noncalcified lesions.6 However, mechanism of restenosis after DES implantation is unclear. PCI with ablation of rotational atherectomy (RA) in calcified lesion showed high procedural success rate, but showed a very high rate of f TLR also7,8. Meta-analysis of plaque modification revealed that routine ablation is not beneficial during PCI9. However, recent data suggest that DES implantation after selective RA is effective in patients with calcified lesion. The failure of DES deployment and full expansion is known as main mechanism of procedural failure and high ISR in calcified lesion, although mild degree calcified lesion can be managed with high pressure balloon dilation before stent delivery. In small and non-randomized studies, RA followed by DES implantation has high rate of procedural success (95 ~ 95.8%) and low-incidence of TLR (3.4% ~ 9.5%)10,11. ISR in calcified lesion is related with an inadequate stent expansion and minimal stent area rather than neointimal proliferation and inflammation process. Thus, it is very important to make good lesion preparation before stenting in order to make easy stent delivery and well expansion of the stent. DES implantation after conservative RA appears to be feasible in selective patients with heavily calcified lesion.

Soft Plaque
It is well known that neointimal hyperplasia is main pathophysiology of ISR. Recent reports suggest that inflammation induced by vascular injury including deendothelialization and thrombus deposition after stent implantation plays a key role to smooth muscle cell proliferation/migration and extracellular matrix deposition12.

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Hher M et al. reported that smooth and yellow plaques detected by angioscopy before coronary angioplasty was associated with restenosis and suggested that a higher proliferative response after a mechanical trauma of such lesions may responsible for restenosis. Yellow plaque consisted of mainly lipid.13 This study suggests that lipid component is responsible for increased ISR. Soft, echo-lucent, plaque detected on IVUS is associated with increased ISR. After single BMS implantation with diameter over 3 mm, ISR was more frequently developed in soft plaques than non-soft plaques14,15. Soft plaques on IVUS represent loose fibrous plaque with intra- and extra-cellular abundant lipid, and/or fibromuscular lesions composed of smooth muscle cells and loose connective tissue. Sahara et al. suggested that soft plaque lesion can be considered a proliferative in the process of atherosclerosis and associated with ISR. Another reports suggest that soft plaques with increased high-sensitivity C-reactive protein (hs-CRP) level that represent an inflammation are associated with a higher incidence of ISR15. Spectral analysis of IVUS radiofrequency data may be a useful tool because it allows detailed assessment of plaque composition, with a high predictive accuracy of 89.5% to 92.8% in fibrous, fibro-fatty, calcified and necrotic core regions with tissue mapping16,17. Our data with virtual histology-IVUS revealed that percent necrotic core component was associated with increased ISR in patients with coronary artery disease and percent fibro-fatty volume was associated with ISR in patients with acute coronary syndrome as well as elevated hs-CRP in both groups. In detail, study population consisted of 241 consecutive patients who underwent. PCI with DES, VH-IVUS examination and 9-months follow up coronary angiogram. ISR was developed in 27 patients (8.2%). Our study also suggests that lipid component and high inflammatory status reflected as increased hs-CRP level were associated with ISR. In conclusion, soft plaque on IVUS and high inflammatory status are responsible for ISR after BMS implantation. However, incidence of ISR is not higher in patients with acute coronary syndrome that frequent related with soft plaque and inflammation. Further evaluation is needed to evaluate the association between plaque tissue characteristics and ISR, especially in DES era.

Role of Inflammation in Restenosis


Out of histologic findings except heavily calcified and high lipid component, high inflammatory status manifested as increased hs-CRP is known as increased ISR. Inflammatory cells, mainly monocytes, are recruited in stented intima just 3 days after stenting in rabbit aorta and macrophages can be seen in the neointima 14 days after stenting18. In human pathologic study of 116 stents from 87 patients >90 days after stenting, there was strong link between plaque component, medial damage, inflammation and neointima area19. This study suggests that lesion characteristics where stent is deployed are important determinant for subsequent inflammation and degree of neointima formation as well as deployed stent can induce inflammation at sites of vascular injury where denuded endothelium and platelet and fibrin have been attached. Necrotic core than fibrous plaque is significantly associated with subsequent high inflammation thus high neointima formation, which is related with ISR. Damaged media also induced severe inflammation like lipid core than fibrotic plaque. Hs-CRP, inflammatory marker, is significantly increased 24 hours after stenting only and to peak level 48 hours after stenting20. It showed stenting itself cause inflammatory reaction, which is similar findings to the human pathology study. Patients with normal (0.5 mg/dl) plasma level of hs-CRP 72 hours after coronary artery stenting had significantly lower adverse events, especially target vessel revascularization, which represent lower ISR, than those with hs-CRP >0.5 mg/dl20. Other study also showed that the higher pre-procedural hs-CRP

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(hs-CRP>1.4 mg/dl versus hs-CRP<0.5 mg/dl), the higher late loss (0.970.8 mm versus 0.660.7 mm, p=0.02) and higher ISR (45.1% versus 19%, p=0.002) in 276 patients after stenting21. These studies suggest that inflammation, which is related with stenting procedure itself and with the site where the stent is deployed in the coronary tree, is clearly associated with ISR beyond lesion characteristics such as calcium and lipid content in the coronary plaque. Thus, it is inevitable to use statins in order to reduce inflammation and/or ISR in patients underwent stenting22. The studies using statins will be described in other chapters.

Role of Endothelialization in Restenosis


Endothelial dysfunction is described in the previous chapter. However, it is important to review about endothelium in histologic predictors for ISR, because incomplete stent endothelialization is closely related with ISR and/or stent thrombosis23. Normal endothelium has anti-atherosclerotic effect such as inhibition of platelet adhesion and aggregation, inhibition of smooth muscle cell proliferation and migration, anti-thrombosis and control of vessel wall permeability and tone24. Delayed re-endothelialization after DES is major concern compared to the bare metal stent (BMS), because polymer may increase local inflammation and thus delay re-endothelialization over the stent strut25. Although animal model study showed similar degree of endothelial cell coverage between DES and BMS and pathological study showed almost complete re-endothelialization of DES late after stenting26,27. However, other pathological study25 in rabbit model revealed that endothelial coverage at 14 days after various DES above strut remained poor in SES, PES, and zotarolimus-eluting stents (30%) relative to everolimus-eluting stents and multi-link vision stents (70%), whereas no significant differences were observed at 28 days. This means that types of DES can show disparities in arterial healing and recovery. In turns, poor endothelial coverage is associated with poor endothelial function, which is related with atherosclerosis and ISR. DES, especially 1st generation DES, is associated with poor re-endothelialization early after stenting compared to BMS or new generation DES. This is known to have an association with stent thrombosis and possibly ISR.

CONCLUSION
Calcium and lipid component of coronary artery plaque are known as important histologic predictors for ISR by current available data. In addition to the calcium and lipid, poor endothelial coverage or deendothelialization possibly due to DES-related problems such as used drug and/or polymer is also an important predictor for ISR. Other important predictor for ISR is inflammation, which is not single phenomenon, but related with several factors such as lipid component of coronary plaque, vessel wall injury during stenting, and polymer in DES. But, the exact pathogenesis of ISR is still need to be disclosed.

MYOCARDIAL REGENERATION
Myocardial regeneration using bone marrow derived stem cell in patients with acute myocardial infarction (AMI) seems promising. There are several clinical trials to evaluate the efficacy and safety of mobilization of stem cells using granulocyte-colony-stimulating factor (G-CSF). We will not discuss about the additional effect of G-CSF treatment to recovery of the left ventricular function or perfusion after primary PCI treatment of acute STEMI. We will focus to restenosis after G-CSF treatment.

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The first randomized trial reported the restenosis after G-CSF treatment is the MAGIC cell randomized clinical trial28 that suggested that stem-cell therapy might accelerate neointimal growth, maybe by mobilization of smooth muscle progenitor cells and increased inflammation. However, another randomized control studies, including the FIRSTLINE-AMI trial29, the STEMMI trial30,31 and the REVIVAL-2 trial32, showed identical restenosis rates in G-CSF-treated and control groups (Table 8.2).
Table 8.2. Myocardial regeneration with stem cell treatment in patients with acute myocardial infarction Study Kang et al.,28 Magic Design Randomized, open-label Cytokine dose and duration G-CSF 10 mg/kg/day 4 days before PCI G-CSF 10 mg/kg/day 6 days after primary PCI G-CSF 10 mg/kg/day 6 days after primary PCI G-CSF 10 mg/kg/day 5 days after PCI Follow-up (months) 6 n Cell infusion = 7 G-SCF = 3 Control = 1 G-CSF = 25 Control = 25 G-CSF = 39 Control = 39 G-CSF = 56 Control = 58 6 Cell infusion = 25 Control = 25 Restenosis 5/7 2/3 16% 20% 3/32 (10%) 3/30 (10%) 18/50 (36.0%) 17/47 (36.2%) 0/23 2/22

Ince et al.,29 Randomized, FIRSTLINE-AMI open-label, blinded evaluation 30,31 Ripa et al., Randomized, STEMMI double-blinded, placebo-controlled Zohlnhfer et al.,32 Randomized, REVIVAL-2 double-blinded, placebo-controlled

4-6

Kang et al.,35 Randomized, open-label G-CSF 10 mg/kg/day Magic Cell-3-DES 3 days after PCI

This discrepancy in restenosis rates can be explained with timing of G-CSF treatment. In the MAGIC cell randomized clinical trial, G-CSF was administrated for 4 successive days prior to PCI and stent was implanted when the G-CSF induced increase of leukocytes was at its maximum, which could increase the inflammatory response33. In recently reported meta-analysis, intracoronary bone marrow stem cell (BMSC) transfer at 4 to 7 days post-AMI decreased the incidence of revascularization (odd ratio: 0.06, 95% CI: 0.37-0.97, p=0.04) compare to emergent transfer (within 24 hour post-AMI).34 The possible mechanism is that BMSC transfer at 4 to 7 days post-AMI reconstitute the damaged endothelium and promote neovascularization, which increase coronary flow reserve in the infarct-related artery and release nitric oxide having antiatherosclerotic effects, in turns. Another possible mechanism of ISR after G-CSF treatment is mobilization of smooth muscle progenitor cell and enhancement of re-endothelization. Neointimal hyperplasia aggravated by G-CSF can be reduced by DES. Kang HJ et al. reported that intracoronary infusion of mobilized peripheral blood stem cells by G-CSF after successful DES implantation in patients with AMI did not increase the restenosis rate35.

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PART I. RESTENOSIS NEW DISEASE Moussa I, Ellis SG, Jones M, Kereiakes DJ, McMartin D, Rutherford B, et al. Impact of coronary culprit lesion calcium in patients undergoing paclitaxel-eluting stent implantation (a TAXUS-IV sub study). Am J Cardiol 2005; 96:12427. Kawaguchi R, Tsurugaya H, Hoshizaki H, Toyama T, Oshima S, Taniguchi K. Impact of lesion calcification on clinical and angiographic outcome after sirolimus-eluting stent implantation in real-world patients. Cardiovasc Revasc Med 2008;9:28. Shimada Y, Ako J, Courtney BK, Morino Y, Bonneau HN, Yock PG, et al. Influence of vessel wall calcium on neointimal growth following bare metal and sirolimus-eluting stent implantation: a 3-D intravascular ultrasound substudy of the SIRIUS trial. J Am Coll Cardiol 2004;43:45A. Dill T, Dietz U, Hamm CW, Kchler R, Rupprecht HJ, Haude M, et al. A randomized comparison of balloon angioplasty versus rotational atherectomy in complex coronary lesions (COBRA study). Eur Heart J 2000; 21:175966. Reifart N, Vandormael M, Krajcar M, Ghring S, Preusler W, Schwarz F, et al. Randomized comparison of angioplasty of complex coronary lesions at a single center. Excimer Laser, Rotational Atherectomy, and Balloon Angioplasty Comparison (ERBAC) Study. Circulation 1997;96:918. Bittl JA,Chew DP, Topol EJ, Kong DF, Califf RM. Meta-analysis of randomized trials of percutaneous transluminal coronary angioplasty versus atherectomy, cutting balloon atherotomy, or laser angioplasty. J Am Coll Cardiol 2004;43:93642. Vaquerizo B, Serra A, Miranda F, Triano JL, Sierra G, Delgado G, et al. Aggressive plaque modification with rotational atherectomy and/or cutting balloon before drug-eluting stent implantation for the treatment of calcified coronary lesions. J Interv Cardiol 2010;23:2408. Tamekiyo H, Hayashi Y, Toyofuku M, Ueda H, Sakuma T, Okimoto T, et al. Clinical outcomes of sirolimus-eluting stenting after rotational atherectomy. Circ J 2009;73:2042-9. Gaspardone A, Versaci F. Coronary stenting and inflammation. Am J Cardiol 2005;96(Suppl.):65L70L. Hher M, Hombach V, Whrle J. Angioscopic predictors of restenosis following coronary angioplasty--the impact of yellow smooth plaques. Z Kardiol 2001;90:1119. Sahara M, Kirigaya H, Oikawa Y, Yajima J, Nagashima K, Hara H, et al. Soft plaque detected on intravascular ultrasound is the strongest predictor of in-stent restenosis: an intravascular ultrasound study. Eur Heart J 2004; 25:202633. Hong YJ, Jeong MH, Lim SY, Lee SR, Hong SN, Kim KH, et al. Relation of soft plaque and elevated preprocedural high-sensitivity C-reactive protein levels to incidence of in-stent restenosis after successful coronary artery stenting. Am J Cardiol 2006;98:3415. Nair A, Kuban BD, Tuzcu EM, Schoenhagen P, Nissen SE, Vince DG. Coronary plaque classification with intravascular ultrasound radiofrequency data analysis. Circulation 2002;106:22006. Bae JH, Kwon TG, Hyun DW, Rihal CS, Lerman A. Predictors of slow flow during primary percutaneous coronary intervention: an intravascular ultrasound-virtual histology study. Heart 2008;94:155964. Rogers C, Welt FGP, Karnovsky MJ, Edelman ER. Monocyte recruitment and neointimal hyperplasia in rabbits: coupled inhibitory effects of heparin. Arterioscler Thromb Vasc Biol 1996;16:13128. Farb A, Weber DK, Kolodgie FD, Burke AP, Virmani R. Morphological predictors of restenosis after coronary stenting in humans. Circulation 2002;105:297480. Gaspardone A, Crea F, Versaci F, Tomai F, Pellegrino A, Chiariello L, et al. Predictive value of C-reactive protein after successful coronary-artery stenting in patients with stable angina. Am J Cardiol 1998;82:515-8. Walter DH, Fichtlscherer S, Sellwig M, Auch-Schwelk W, Schchinger V, Zeiher AM. Preprocedural C-reactive protein levels and cardiovascular events after coronary stent implantation. J Am Coll Cardiol 2001;37:83946. Bae JH, Bassenge E, Kim KY, Synn YC, Park KR, Schwemmer M. Effects of low-dose atorvastatin on vascular responses in patients undergoing percutaneous coronary intervention with stenting. J Cardiovasc Pharmacol Ther 2004;9:18592. Joner M, Finn AV, Farb A, Mont EK, Kolodgie FD, Ladich E, et al. Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk. J Am Coll Cardiol 2006;48:193202. Glasser SP, Selwyn AP, Ganz P. Atherosclerosis: risk factors and the vascular endothelium. Am Heart J 1996;131:37984. Joner M, Nakazawa G, Finn AV, Quee SC, Coleman L, Acampado E, et al. Endothelial cell recovery between comparator polymer-based drug-eluting stents. J Am Coll Cardiol 2008;52:33342. Suzuki T, Kopia G, Hayashi S, Bailey L, Llanos G, Wilensky R, et al. Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation 2001; 104: 118893.

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27. Sousa JE, Costa MA, Farb A, Abizaid A, Sousa A, Seixas AC, et al. Vascular healing 4 years after the implantation of sirolimus-eluting stent in humans: a histopathological examination. Circulation 2004; 110: e5e6. 28. Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, et al. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocytecolony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet 2004;363:7516. 29. Ince H, Petzsch M, Kleine HD, Schmidt H, Rehders T, Krber T, et al. Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI). Circulation 2005; 112:3097106. 30. Ripa RS, Jrgensen E, Wang Y, Thune JJ, Nilsson JC, Sndergaard L, et al. Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction: result of the double-blind, randomized, placebo-controlled stem cells in myocardial infarction (STEMMI) trial. Circulation 2006;113:198392. 31. Jrgensen E, Baldazzi F, Ripa RS, Friis T, Wang Y, Helqvist S, et al. Instent neointimal hyperplasia after percutaneous intervention for ST-elevation myocardial infarction and treatment with granulocyte-colony stimulating factor. Results from the stem cells in myocardial infarction (STEMMI) trial. Int J Cardiol 2008 Dec 2. [Epub ahead of print] 32. Zohlnhfer D, Ott I, Mehilli J, Schmig K, Michalk F, Ibrahim T, et al. Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA 2006; 295:100310. 33. Kastrup J, Ripa RS, Wang Y, Jrgensen E. Myocardial regeneration induced by granulocyte-colonystimulating factor mobilization of stem cells in patients with acute or chronic ischaemic heart disease: a non-invasive alternative for clinical stem cell therapy? Eur Heart J 2006;27:2748-54 34. Zhang S, Sun A, Xu D, Yao K, Huang Z, Jin H, et al. Impact of timing on efficacy and safety of intracoronary autologous bone marrow stem cells transplantation in acute myocardial infarction: a pooled subgroup analysis of randomized controlled trials. Clin Cardiol 2009;32:458-66. 35. Kang HJ, Lee HY, Na SH, Chang SA, Park KW, Kim HK, et al. Differential effect of intracoronary infusion of mobilized peripheral blood stem cells by granulocyte colony-stimulating factor on left ventricular function and remodeling in patients with acute myocardial infarction versus old myocardial infarction: the MAGIC Cell-3-DES randomized, controlled trial. Circulation 2006; 114(1 Suppl):I145-51.

CHAPTER 9

THE ANATOMICAL ORIGIN OF CELLS IN ARTERIAL RESTENOSIS


LUIS RODRIGUEZ-MENOCAL and ROBERTO I. VAZQUEZ-PADRN
Introduction .............................................................................................................................. Morphological and Functional Characteristic of Neointimal Cells .......................................... The Role of Local Contractile SMC in Post-Injury Neointimal Formation (NF).................. The Role of Circulating Progenitors Cells in Post-Injury Neointimal Formation..................... The Role of Adventitial Myofibroblasts and Progenitors Cells in Post Injury Neointimal Formation ................................................................................................................................ The potential role of endothelial-mesenchymal transitions in post-injury neointimal formation .... Conclusions .............................................................................................................................. References ................................................................................................................................ 129 129 130 132 134 135 136 136

INTRODUCTION
Despite dramatic technological advances in coronary intervention, restenosis following percutaneous coronary intervention (PCI) remains an important cause of morbidity with major financial implications1-3. Restenosis can be seen as the pathological healing response of dilated atherosclerotic arteries to PCI or virtually any other type of vascular injury that eventually leads to vascular re-narrowing. Neointima formation due to accumulation of new cells and extracellular matrix components (ECM) within the innermost layer of the artery is the prominent characteristic of restenosis4-7. Neointima formation is not only a salient characteristic of restenosis, it also a key event during the closure of the ductus arteriosus after birth8,9, the development of atherosclerosis10-12, and the failure of homograft transplanted organs13-15, arteriovenous fistulas16-18 and arterial bypass conduits19-21. This chapter gives an overview of novel developments in one of the most controversial issues in the field of restenosis, the origin of the cells forming the post injury neointima. We assimilate, criticize and re-conciliate experimental and clinical evidence suggesting that neointimal cells within an injured blood vessel may derive from variety of anatomical sources that include the contractile Smooth Muscle Cells (SMC)22-27, circulating bone marrow progenitor cells28-30, and adventitial (myo)fibroblasts31. Of note, we limit the overview to the origin of neointimal cells of restenotic lesions and avoid doing inferred conclusion from irrelevant animal models or dissimilar vascular pathologies.

MORPHOLOGICAL AND FUNCTIONAL CHARACTERISTIC OF NEOINTIMAL CELLS


A brief overview of the basic characteristics of post injury neointimal cells is necessary to fully understand the cellular mechanisms underlying the vascular remodeling process.

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These cells are quite distinct from medial contractile SMCs. They are characterized by a large cell body that contains synthetic and secretary organelles32-34. These synthetic SMCs secrete extracellular matrix components35, 36 and express low levels of the smooth muscle specific contractile proteins37, 38. In contrast with the contractile SMCs that are quiescent in the cell cycle, these synthetic SMCs proliferate and migrate in response to growth factors39, 40. Neointimal cells have profound alterations in calcium handling and contractility41,42. Unfortunately, there not exist unique markers for neointimal cells.

THE ROLE OF LOCAL CONTRACTILE SMC IN POST-INJURY NEOINTIMAL FORMATION


For many years the long-standing dogma in the field has been that the majority of SMC in post injury neointimal lesions are derived from preexisting SMC that undergo a phenotypic modulation from contractile to synthetic phenotype and migrate and proliferate into the intima. This dogma has been supported by many years for an extensive amount of indirect evidences. However, more recently solid experimental facts have validated this dogma in animal models and demonstrated that SMC phenotypic modulation is the sufficient condition for neointimal formation. Initially, this hypothesis was supported for the observed extraordinary plasticity of vascular SMC in culture. Dating back to the 70s, pioneer studies noted that human SMC did not retain their morphological and functional characteristics in vitro for many generations. Cultures of those cells spontaneously lose contractility and gained the capacity of synthesizing significant amounts of extracellular matrix43,44. Moreover, this type of SMCs showed an unusual capacity to proliferate in response to environmental factors (e.g. serum, hemodynamic forces, etc.) and was rich in rough endoplasmic reticulum and free ribosomes45-47. Cells with this phenotype expressed low levels of the contractile SMC markers. These undifferentiated cells that undoubtedly resemble those of the injured vessels were called synthetic SMC. Nowadays, the simple two-state model resulted totally inadequate to explain the diverse range of phenotypes that can be exhibited by the SMC during post injury arterial remodeling. New developments have clearly established that SMC plasticity goes beyond two simple cellular stages and that SMC may acquire more complex phenotypes48. In fact, the SMC phenotype is influenced by species, methods of isolation, culture conditions, pre-existing conditions and donor age49-51. Several seminal articles have recently revised the molecular mechanisms controlling VSMC phenotypic modulation26,39,40. An independent line of evidence also supported the hypothesis that neointimal cells come from contractile SMC were originated from the direct pathological inspection of human and animals restenotic lesions. A remarkable study of Schwartz et al.52 showed morphologically identifiable SMC in the process of migrating through the internal elastic lamina upon vascular injury. On the other hand, Clowes and coworkers53,54 found that thymidine-labeled SMC (proliferative) appeared quickly after injury which suggested that the tunica media was the source of the proliferating cells found in the intima. Moreover, SMC in arterial neointimal lesions as synthetic SMC in cultures, expressed low levels of the contractile markers such as, smooth muscle -actin (SMA), myosin heavy chains (SM-MHC), myosin light chains, calponin, SM22 , h-caldesmon, vinculin, and the intermediate filaments proteins vimentin and desmin, and smoothelin37,55,56. It was illustrated by a series of studies published by Aikawa and coworkers37, 55 using human and rabbit restenotic lesions. They found that SM-1 and SM-2 the SM-MHC isoforms were significantly down-regulated after injury in neointimal cells but not in medial SMC at 16 and 20 days after Percutaneous Transluminal Coronary Angioplasty (PTCA). They interpreted that those cells have entered in a more undifferentiated and immature state

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similar to that seen in cultures of synthetic SMC. Lately, transcriptomics studies provide another set of indirect evidences for the SMC phenotypic modulation after arterial injury. Li and coworkers57, characterize the global transcription profile along arterial remodeling and neointima formation in the rat balloon-injury model. They found that neointimal formation was characterized by an increase in expression of genes functionally related to angiogenesis and production of ECM such as Spp1, CD44, and SCF-1. Interestingly, similar signatures were found in a preceding study by Geary et al.35. In this case, microarray-based profiling analysis was performed in the neointima of primates formed 4 weeks after aortic grafting and were compared with those from the normal aorta. A total of 147 genes were differentially expressed in neointimal SMCs versus normal aorta SMCs, most of up-regulated genes underscored the importance of matrix production during neointimal formation. However, the most significant studies supporting the phenotypic modulation theory have been recently published. A group of independent reports have demonstrated that the phenotypic modulation of medial SMC regulated by microRNAs (miR) is sufficient for neointimal formation58-60. MicroRNAs are post-transcriptional regulators that bind to complementary sequences in the 3 untranslated regions of target mRNAs to usually cause gene silencing. Neointimal development after vascular injury induces a rapid perturbation in multiple miR levels61. In particularly, two remarkable studies that we discuss further in this section intended to reveal the role of miR-143/145 cluster in the acquisition and/or maintenance of the contractile phenotype in SMC. In both cases the expression of those miRs was confined to SMC in transgenic mice and the suppression of this locus in knockout mice locked arterial SMC in an irreversible synthetic and state. Surprisely, the 18 month-old miR 143/145 mutant mouse developed spontaneous neointimal lesions in the femoral arteries in the absence of injury or any other type of vascular insult60. Paradoxically in the study published by Xin et al.58, carotid arteries of miR-143/145 mutant mice did not develop neointima after blood restriction injury (carotid artery ligation). The discrepancy between those studies is conciliated if we assumed that mechanisms for aging-related neointimal formation differ from those dictating the vascular remodeling after arterial ligation. Nonetheless, outcomes from these investigations clearly indicate that the presence of synthetic SMC in arteries is sufficient for the development of a restenotic lesion. It must be borne in mind, that most of animal models used to support this and any other hypothesis do not recapitulate all biological events observed in humans after PCI and therefore, have limitations62, 63. Despite all these evidences supporting the dogma that repair of vascular injury is carried out principally (or exclusively) by reversible phenotypic modulation of preexisting SMCs, two observations still challenge this model. The first is the absence of proliferation markers in human restenotic. Low proliferation index suggests that the neointima is formed by accumulation of infiltrating cells and not by proliferation of arterial SMCs. The second observation is that neointima formation occurs after a massive post injury onset of apoptosis that significantly reduce the number of medial SMC. The low proliferation index found in human atherosclerotic and restenotic lesion has been matter of debate for many years. Initially, the SMC proliferation was considered the sine qua non of restenosis. This assumption was made from studies using the rat balloon injury model. In this experimental system during the first 48 to 72 hours following balloon angioplasty, 10% to over 50% of medial SMC undergo DNA replication and incorporate significant amounts of radio-labeled thymidine53,54. The controversy arose at the time of confirming that SMC multiplication after PCTA in humans. Initially and in agreement with the rat model, Pickering et al.64 found that restenotic coronary specimens had high percentages of cells positive for the proliferating cell nuclear antigen (PCNA), a surrogate marker for cell proliferation, by

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either immunocytochemistry (15.213.6%) or in situ hybridization (20.618.2%). However, OBrien et al.65 were not able to reproduce those results and found PCNA- positive cells in only 26 out of a 100 of human restenotic lesions. Those specimens showing positivity had only a modest number of PCNA-positive cells per slide (typically <50 cells per slide). PCNA labeling was detected over a wide time interval after the initial procedure (e.g., 1 to 390 days), with no obvious proliferative peak. Chung and collaborators also found few PCNA positive cells in restenotic lesions of coronary arteries after stent deployment66. Furthermore, cell culture experiments have shown that human smooth muscle cells derived from atherosclerotic and normal arteries grow slowly and appear to undergo a finite number of population doublings67. The discrepancies among studies may reflect methodological flaws e.g controls for PCNA labeling and perhaps, the failure of the rat injury model to mimics restenosis in humans. The low proliferation index of human restenotic lesions would fit within the phenotypic modulation theory if we considered that that only an small portion of cell duplications are necessary to reach the critical cell mass in the neointimal lesion. The second observation challenging the phenotypic modulation hypothesis comes from arterial injury models that demonstrate a rapid onset of apoptosis among cells in the media after injury68,69. These models have shown that up to 70% of medial SMCs died within 30 minutes after the trauma68. The medial SMCs lying closest to the arterial lumen were most likely to undergo apoptosis. The rapid onset of post injury apoptosis occurs through a Fas ligand independent mechanism70 and is ameliorated by locally delivering ZVAD-fmk, a caspace inhitor71. Undoubtedly, still neointimal cells could arise from small groups of surviving contractile SMCs that remain in the artery after injury.

THE ROLE OF CIRCULATING PROGENITORS CELLS IN POST-INJURY NEOINTIMAL FORMATION


The long-standing paradigm that neointimal cells are originated of phenotypically modulated medial cells that migrate and proliferate into the intima in response to injury has been recently challenged by several alternative hypothesis. For instance, Han et al. in 200172 injured arteries of chimeric female C57Bl/6 mice carrying BM cells from congenic (Ly5.1) male donors by either scratching the femoral artery with prove or inserting a silk suture along the axis of the left common carotid artery to produce an organized arterial thrombus. They later found with a Y-chromosome-specific probe and in situ hybridization that a significant proportion of BM circulating cells (Y-chromosome positive, 56%) populated the neointima. Two years later Sata et al.73 published a notorious study that truly sparked controversy. These authors wire injured the femoral artery of chimeric mice carrying ROSA26 BM cells that constitutively express the bacterial -galactosidase (LacZ) gene. Four weeks after injury, they found a significant fraction of neointimal (63.0%) and medial cells (45.9%) staining positively for both LacZ and SMA, the marker for SMC. It was interpreted as a strong evidence for the contribution of BM-derived cells to the neointima cell population. Since then, numerous reports have shown wide diversity in the homing of BM-derived cells on mechanically injured vessels ranging from negligible74 to substantial numbers of vascular SMCs75-77. For example, Tanaka et al.75 demonstrated that the contribution of BM cells to post injury neointimal hyperplasia is diverse and depends on mechanical vascular injury. They found a significant contribution of BM circulating cells to the neointima of wire injured (38%) but not in those perivascularly insulted arteries (7%). Of notice, it is the study of Soda et al. that explored the origin of neointimal cells in stented porcine coronary arteries.

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In this case, cells around struts staining positive for CD34+, c-kit and AC 133 were considered of hematopoietic origin and therefore, coming from the BM. Interestingly, recent studies have found sub-population of cells isolated of blood mononuclear cells and named as smooth muscle progenitor cells (SPC) that are capable of acquiring the SMC phenotype under culture conditions. For instance, Simper et al.78 isolated mononuclear cells from human blood and cultured them on collagen type I matrix in the presence of PDGF-BB. Cells that grew in these cultures stained positively for SMA, SM-MHC and calponin79. The circulating progenitor cells isolated by these authors also expressed the endothelial markers CD34, Flt-1, and Flk-1. Other studies have isolated SMC-like cells from blood mononuclear cells using a variety of collagen-coated plates or media supplemented with growth factors such as transforming growth factor (TGF-), fibroblast growth factor (FGF) and insulin-like growth factor (IGF)79,80. These circulating smooth muscle cell progenitors have been associated in different disease states. The number of collagen-and SMA- expressing SPC is increased in patients with type 1 diabetes indicating a possible role in adverse tissue remodeling81. In peripheral blood of patients with coronary artery disease (CAD), circulating cells expressing CD14, CD105 and SMA were increased compared to non- CAD patients79. Of note, the studies from Weber and co-workers demonstrating that outsourced Lin-/c-kit-/Sca1+/PDGF R + cells outgrowth from blood mononuclear cells migrated and populated the mouse post injury neointima82. Whether those cells were truly vascular progenitor or only a culture-induced phenotype remains unclear; however, it is apparent that cells with smooth muscle potential reside within the mononuclear fraction of blood. Alternatively, others and we have shown strong experimental evidences indicating that differentiation of BM-derived cells into SMC is a very rare event74. For instance, we find no contribution of BM cells to the neointima of balloon injured arteries. In this study, we used chimeric rats generated by rescuing lethal irradiated animals with GFP+ BM cells from transgenic animals. We found BM derived cells (mainly CD68+ monocytes/macrophages) in the media and adventitia of injured vessels at 7 days but only few GFP+ and SMA+ (2.34 % 1.61) among neointimal cells at four weeks later. The vascular origin of cells in the neointima was further confirmed by transplanting injured GFP arteries into wild type recipients. In those grafts 94.23 0.44 % of medial and 92.95 19.34 % of neointimal cells were GFP+ SMA+. Accordantly we our results, previous study using chimeric mice carrying BM from SM22 promoter-LacZtransgenic mice had shown that donor BM cells do not give rise to SM22 expressing neointimal cells83. Most recently two new studies have fueled controversy providing evidences that the differentiation of BM-derived progenitor cells into SMC or endothelial cell lineages seems to be an exceedingly rare event if even happened. In one of these studies, the femoral artery of chimeric C57BL/6 mice transplanted with GFP BM cells were wire injured and harvested at 3 days, 1, 2, 3, 4, 6, and 16-weeks after procedure84. Using high-resolution microscopy, Daniel et al.84 found that the expression of SMC or endothelial cell markers in GFP cells was a very rare event. Indeed, most of the GFP+ cells found infiltrate in the vascular wall were monocytes/macrophages, and their number declined at later time points. Independently, Groenewegen et al.85 had determined the contribution of BM-derived cells in a rat model of in-stent restenosis. They found few transgenic cells expressing the human placental alkaline phosphate (hPAP) in the neointima of wild type F344 rats that had received stent implantation 6 weeks after lethal total body irradiation and infusion of BM from a R26-hPAP transgenic rat. A critical question is whether neointimal SMC in human restenotic lesions are derived from circulating BM cells. Unfortunately, relatively few studies have been published in this area due to inherent limitations underlying the use of human subjects and none have inspected

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human restenotic lesions. The origin of vascular cells in cardiac allographs severely affected by transplant arteriosclerosis was assessed by Quaini et al.86 These authors estimated that as many as 60% of cells in newly developed arterioles in the collateral circulation of male patients who had received a female heart were Y-chromosome/SMA+ and therefore, were from the recipient. In contrast, Glaser et al.87 showed that only 6% of the cells were Y-chromosome/SMA+ in medium and small arteries. Consistent with the latter findings, Hruban et al. found no evidence of BM-derived SMCs within the coronary lesions88. More recently Caplice et al.89 used 13 sex-mismatched BM transplant human subjects to examine the contribution of BM-derived cells to the atherosclerosis in coronary arteries. Of interest, they observed 5.9 to 10.4% of BM-derived SMC in those coronary lesions. Additionally, they demonstrated that the co-expression of SMC markers and BM markers was not the result of cell fusion by performing chromosomal ploidy analyses using markers for chromosome 18. In contrast, Yokote et al.30 examined coronary artery autopsy specimens of two individuals who had undergone allogenic BM transplantation to determine the origin of SMC in the vessel wall. In this study none of the intimal SMCs were of BM origin. The disease state, treatments and medications may be the cause of contradictory results. For instance, the specimens in Caplice et al. study were collected from patients exposed to variables like immunosuppression, chemotherapy, and graft-versus-host disease that are not present during the normal healing of coronary vessels after PCI. Why is so controversial the participation of circulating BM cells in the post injury neointimal formation? Most of the controversy arises from the methodological limitations frequently observed in the studies claiming the participation of BM-derived cells in post-injury neointimal formation that, in fact, have significantly dimmed the enthusiasm for this novel hypothesis. The most commons flaws found in those experimental reports that may lead to the detection of false-positive cells are: 1) detection of labeling cells in unfixed tissues where the tracer marker could potentially diffuse from sectioned cells; 2) lack of resolution to discriminate GFP or LacZ cells from arterial background (tissue autofluorescence or senescence-associated beta-galactosidase activity); 3) lack of high-resolution confocal microscopy to showing definitive co-localization of BM and SMC lineage markers in single cells, 4) lack of appropriate experimental controls to detect cell fusion events, 5) presence of confounding (e.g. irradiation) factors that may modify normal vascular repair process, 6) failure to provide compelling evidence regarding expression of definitive markers of SMC lineage such as SM-MHC; and 7) severe damage of the vessel including complete loss of endothelium and extensive medial SMC apoptosis. In summary, whereas it is certainly plausible that bone marrow-derived SMCs play a major role in development of atherosclerosis, further extensive studies will be necessary to prove this is the case.

THE ROLE OF ADVENTITIAL MYOFIBROBLASTS AND PROGENITORS CELLS IN POST-INJURY NEOINTIMAL FORMATION
Evidences coming from in vitro cultures and animal models indicate that adventitial myofibroblast have the extraordinary capacity as do SMC of migrate toward the intima in response to trauma and contribute in part to the neointimal formation. Interestingly, adventitial myofibroblast expresses, albeit less strongly, the SMC markers SMA, SM-22 and vimentin90 and display most of the ultrastructural characteristics of cultured SMC91. In vivo, neointimal

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myofibroblasts identical to fibroblasts cultured from the adventitia can be isolated from the surface of injured arteries. The migration of myofibroblasts through the media in response to injury has been documented in several occasions92-94. For instance, Slomp et al.95 using whole vessel wall organ cultures demonstrated that adventitial myofibroblasts can migrate to the luminal surface, where they acquired endothelial and SMC-like phenotypes. Shi et al.93 using the porcine model of coronary artery balloon injury also showed the migration of intima-bound myofibroblasts along medial arterial fissures. Other independent studies have demonstrated that activation of myofibroblasts by laceration of the adventitia is sufficient to cause neointimal lesions even in the absence of de-endothelialization. More recently, Li et al.94 elegantly showed that in response to moderate luminal injury, transfected carotid adventitial fibroblasts expressing -galactosidase migrate to the neointima. Although these studies do not clarify the relative contribution of medial versus adventitial cells, they do support the concept that adventitial fibroblasts contribute to neointima formation. Finally, a novel concept for adventitial progenitor cells has emerged from a study published by Hu and collaborators96. These authors showed a Sca-1+ population of adventitial cells that populated the neointima of vein grafts in ApoE-deficient mice after perivascular cell transplantation. They concluded that those cells were vascular progenitor cells from the adventitia that have the potential to differentiate into SMCs and contribute to the neointima formation. Unfortunately, these results have not been confirmed in any model of vascular injury or restenosis.

THE POTENTIAL ROLE OF ENDOTHELIAL-MESENCHYMAL TRANSITIONS IN POST-INJURY NEOINTIMAL FORMATION


New developments have revealed that the sources for neointimal cells are more diverse than those traditionally considered to contribute to the neointima of vascular lesions which are the resident SMC, the circulating progenitor cells and adventitial fibroblasts. Recently, it has been demonstrated the possibility that endothelial cells have the capability of transitioning into a mesenchymal or SM-like phenotype in a process known as endothelial to mesenchymal transition (EnMT). Once again first evidences come from in vitro studies. Endothelial cells derived from the adult bovine aorta convert to spindle-shaped SMA actin-expressing cells when treated with TGF-97. Certain murine endothelial-like cell lines irreversibly transform into mesenchymal cells upon overgrowth in culture98. One concern raised regarding the early reports describing EnMT was the possibility that the primary endothelial cell cultures were simply contaminated with small numbers of mesenchymal cells. Recent studies have indicated that the endothelial-to-mesenchymal transition (EnMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis99. No study so far has demonstrated that EnMT contributes to the neointima formation after vascular injury. While EnMT could be a possible contributor to the neointima in other multiple vascular diseases, it occurrence in restenosis is unlikely as most of the luminal endothelial cells are severely damage after injury.

CONCLUSIONS
The intent of this review was to present the current state of knowledge regarding to the anatomical sources contributing to the neointimal formation, which occurs not only during the healing of injured vasculature but also in the pathogenesis of various vascular diseases. We intended to reveal the complexity of the controversy about the origin of neointimal cells.

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Unfortunately, many catastrophic studies plagued by methodological flaws have promoted confusion rather than clearness. Additional studies are required for a better understanding of the anatomical source(s) of post injury neointimal cells while the conciliatory theory for the origin of pathological cells in restenotic lesions remains unformulated.

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72. Han CI, Campbell GR, Campbell JH. Circulating Bone Marrow Cells Can Contribute to Neointimal Formation. J Vasc Res. 2001; 38(2):113-119. 73. Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med. 2002; 8(4):403-409. 74. Rodriguez-Menocal L, St-Pierre M, Wei Y, Khan S, Mateu D, Calfa M, Rahnemai-Azar AA, Striker G, Pham SM, Vazquez-Padron RI. The origin of post-injury neointimal cells in the rat balloon injury model. Cardiovasc Res 2009; 81(1):46-53. 75. Tanaka K, Sata M, Hirata Y, Nagai R. Diverse Contribution of Bone Marrow Cells to Neointimal Hyperplasia After Mechanical Vascular Injuries. Circ Res 2003; 93(8):783-790. 76. Schober A, Knarren S, Lietz M, Lin EA, Weber C. Crucial Role of Stromal Cell-Derived Factor-1{alpha} in Neointima Formation After Vascular Injury in Apolipoprotein E-Deficient Mice. Circulation 2003; 108(20):2491-2497. 77. Karshovska E, Zagorac D, Zernecke A, Weber C, Schober A. A small molecule CXCR4 antagonist inhibits neointima formation and smooth muscle progenitor cell mobilization after arterial injury. J Thromb Haemost. 2008; 6(10):1812-1815. 78. Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth Muscle Progenitor Cells in Human Blood. Circulation 2002; 106(10):1199-1204. 79. Sugiyama S, Kugiyama K, Nakamura S, Kataoka K, Aikawa M, Shimizu K, Koide S, Mitchell RN, Ogawa H, Libby P. Characterization of smooth muscle-like cells in circulating human peripheral blood. Atherosclerosis. 2006; 187(2):351-362. 80. Le Ricousse-Roussanne S, Barateau V, Contreres JO, Boval B, Kraus-Berthier L, Tobelem G. Ex vivo differentiated endothelial and smooth muscle cells from human cord blood progenitors home to the angiogenic tumor vasculature. Cardiovasc Res. 2004; 62(1):176-184. 81. Nguyen T, Chon H, van Nieuwenhoven F, Braam B, Verhaar M, Goldschmeding R. Myofibroblast progenitor cells are increased in number in patients with type 1 diabetes and express less bone morphogenetic protein 6: a novel clue to adverse tissue remodelling? Diabetologia. 2006; 49(5):1039-1048. 82. Subramanian P, Karshovska E, Reinhard P, Megens RTA, Zhou Z, Akhtar S, Schumann U, Li X, van Zandvoort M, Ludin C, Weber C, Schober A. Lysophosphatidic Acid Receptors LPA1 and LPA3 Promote CXCL12-Mediated Smooth Muscle Progenitor Cell Recruitment in Neointima Formation. Circ Res 2010; 107(1):96-105. 83. Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, Dietrich H, Xu Q. Smooth Muscle Cells in Transplant Atherosclerotic Lesions Are Originated From Recipients, but Not Bone Marrow Progenitor Cells. Circulation 2002; 106(14):1834-1839. 84. Daniel J-M, Bielenberg W, Stieger P, Weinert S, Tillmanns H, Sedding DG. Time-Course Analysis on the Differentiation of Bone Marrow-Derived Progenitor Cells Into Smooth Muscle Cells During Neointima Formation.ATVBAHA.2010, 110.209692. 85. Groenewegen HC, Onuta G, Goris M, Zandvoort A, Zijlstra F, van Gilst WH, Rozing J, de Smet BJ, Roks AJ, Hillebrands JL. Non-bone marrow origin of neointimal smooth muscle cells in experimental in-stent restenosis in rats. J Vasc Res. 2008; 45(6):493-502. 86. Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, Kajstura J, Leri A, Anversa P. Chimerism of the Transplanted Heart. N Engl J Med 2002; 346(1):5-15. 87. Glaser R, Lu MM, Narula N, Epstein JA. Smooth Muscle Cells, But Not Myocytes, of Host Origin in Transplanted Human Hearts. Circulation 2002; 106(1):17-19. 88. Hruban RH, Long PP, Perlman EJ, Hutchins GM, Baumgartner WA, Baughman KL, Griffin CA. Fluorescence in situ hybridization for the Y-chromosome can be used to detect cells of recipient origin in allografted hearts following cardiac transplantation. Am J Pathol. 1993; 142(4):975-980. 89. Caplice NM, Bunch TJ, Stalboerger PG, Wang S, Simper D, Miller DV, Russell SJ, Litzow MR, Edwards WD. Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation. Proc Natl Acad Sci 2003; 100(8):4754-4759. 90. Skalli O, Schurch W, Seemayer T, Lagace R, Montandon D, Pittet B, Gabbiani G. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest 1989; 60(2):275-285. 91. Yoshida T, Owens GK. Molecular Determinants of Vascular Smooth Muscle Cell Diversit y. Circ Res 2005; 96(3):280-291. 92. Siow RC, Mallawaarachchi CM, Weissberg PL. Migration of adventitial myofibroblasts following vascular balloon injury: insights from in vivo gene transfer to rat carotid arteries. Cardiovasc Res. 2003; 59(1):212-221.

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93. Shi Y, Pieni ek M, Fard A, O'Brien J, Mannion JD, Zalewski A. Adventitial Remodeling After Coronary Arterial Injury. Circulation 1996; 93(2):340-348. 94. Li G, Chen S-J, Oparil S, Chen Y-F, Thompson JA. Direct In Vivo Evidence Demonstrating Neointimal Migration of Adventitial Fibroblasts After Balloon Injury of Rat Carotid Arteries.Circulation 2000; 101(12):1362-1365. 95. Slomp J, Gittenberger-deGroot AC, van Munsteren JC, Huysmans HA, van Bockel JH, van Hinsbergh VW, Poelmann RE. Nature and origin of the neointima in whole vessel wall organ culture of the human saphenous vein. Virchows Arch. 1996; 428(1):59-67. 96. Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, Xu Q. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 2004; 113(9):1258-1265. 97. Arciniegas E, Sutton AB, Allen TD, Schor AM. Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 1992 103(Pt2):521-529. 98. Kohler A, Jostarndt-Fgen K, Rottner K, Alliegro MC, Draeger A. Intima-like smooth muscle cells: developmental link between endothelium and media? Anat Embryol (Berl). 1999; 200(3):313-323. 99. Li J, Bertram JF. Review Article: Review: Endothelial-myofibroblast transition, a new player in diabetic renal fibrosis. Nephrology 2010; 15(5):507-512.

CHAPTER 10

LESION COMPLEXITY, QUANTIFICATION, PREDICTIVE FACTORS


KWEN CHUL SHIN and WOONG CHOL KANG
Introduction.............................................................................................................................. Aorto-Ostial Lesion ................................................................................................................. Bifurcation ............................................................................................................................... Chronic Total Occlusion........................................................................................................... Small Reference Vessel ........................................................................................................... Long Lesion ............................................................................................................................. Saphenous Vein Graft............................................................................................................... Conclusions.............................................................................................................................. References................................................................................................................................ 141 142 142 143 144 145 147 147 148

INTRODUCTION
In-stent restenosis is the major limitation of coronary stenting. Several studies have evaluated the impact of baseline and procedural characteristics on the risk of restenosis after stent implantation, with a number of high-risk factors, such as diabetes, lesion length, vessel size and lesion characteristics1,2 (Table 10.1).
Table 10.1. Predictors of restenosis after coronary stenting Clinical factors Diabetes Mellitus Chronic renal failure Procedural factors Minimal lumen area Lesion factors Aorto-ostial lesion Bifurcation Chronic total occlusion Small vessel Long lesion Saphenous vein graft

Unfortunately, these characteristics are commonly found in the daily practice, where treatment of complex patients frequently appears as a challenging therapeutic dilemma. Despite the dramatic reduction in restenosis conferred by drug-eluting stents (DES), restenosis remains

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a significant problem for real-world patients. Restenosis is a complex phenomenon, and a variety of stent-, drug-, patient- and lesion-related factors have been studied as the determinants of restenosis after stent implantation. In this chapter, we discuss the lesion related predictors for restenosis after stent implantation, and this may help guide the practice in real world.

AORTO-OSTIAL LESION
True aorto-ostial stenting poses a number of potential problems. It requires a high level of experience as it can often be technically demanding and requires precision and skill in guiding catheter manipulation and stent placement. It is also uniquely challenging because of their high rate of lesion rigidity and elastic recoil3,4. Although the limitation of study data for the use of stents in aorto-ostial lesions make it difficult to evaluate the aorto-ostial lesion as a predictor factor of restenosis, aorto-ostial lesion had an especially higher restenosis rate than non-ostial location after bare-metal stent (BMS) implantation5. Topol et al. reported a 38% restenosis rate (48% clinical recurrence rate) in ostial coronary artery lesions after BMS implantation4. DES has a low incidence of restenosis after treatment of noncomplex restenotic lesions, but its efficacy in treating more complicated restenotic lesions remains to be established. Compared with the BMS group, several reports using DES have shown encouraging angiographic and clinical results in coronary ostial lesions, including aorto-ostial and left anterior descending ostial lesions6. In Lemos et al. study of patients with complex lesions and off-label indications, ostial lesions had a relatively high restenosis rate (14.7%) and were identified as independent predictors of restenosis after DES implantation7. The higher incidence of restenosis observed after treating aorto-ostial lesions may be due to its unique histologic characters showing frequently heavily calcified, fibrotic, and sclerotic lesion. It has also been suggested that there may be more elastic recoil even after stent implantation at ostial sites because of the highly elastic tissue in the adjacent aortic wall8. Because aorto-ostial lesions usually have a very large reference vessel size despite the small intraluminal lesion diameter, more appropriate or larger stents may be needed to ensure more complete apposition of the stent strut to the vessel wall and more uniform drug distribution. This issue therefore deserves further investigation.

BIFURCATION
Bifurcation stenosis is one of the most complex coronary lesions for coronary intervention because lumen of both the main vessel and the side branch needs to be restored. Balloon angioplasty alone to treat bifurcation lesions has resulted in relatively low angiographic success and high restenosis rates. With balloon angioplasty alone, the risk of side-branch occlusion due to plaque shift (snowplough effect) while dilating the main branch may occur in up to 14% of patients9,10. In earlier studies with balloon angioplasty, there was usually less than satisfactory immediate lumen restoration, a high complication rate, and an unacceptable restenosis rate11,12. Although the introduction of BMS resulted in more predictable results and higher success rates, angiographic restenosis rates still remained high 13. Various stenting techniques for bifurcation lesions have been described. The kissing balloon technique resulted in improved, though suboptimal, lumen restoration12. In a retrospective reports, the technical success rate in opening of the main as well as the side branch, was over 87% in the majority of cases, but the clinical outcome data remained variable. The restenosis rate was 2562% in the two stents group versus 12.548% in single stent group, and the target lesion revascularization rate (TLR) was 2443% in the two stents group versus 836% in the

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single stent group13. Al Suwaidi et al. reviewed 131 patients with bifurcation lesions treated with one or more stents. One group had BMS in the parent vessel and balloon angioplasty in the side branch. Another group included patients who underwent Y-stenting or T-stenting. At 1-year follow-up, there were no significant differences in death, myocardial infarction, or TLR between two groups14. Colombos group reported 92 patients with bifurcation lesions, 39 had stenting with BMS in the parent vessel with balloon angioplasty of the side branch, and 53 had stenting of both vessels. In-hospital major adverse cardiac events (MACEs) occurred only in the group with two stents (13% versus 0%, P < 0.05). At 6 months, angiographic restenosis rates and TLR rates were similar in the two groups15. For treatment of true bifurcation lesions, a complex strategy of stenting both vessels has no advantage in late outcomes over a strategy of stenting one branch and performing balloon angioplasty of the other16. There are no large prospective randomized trials addressing long-term clinical outcome after placement of BMS versus balloon dilatation or surgery and of different stenting techniques. The majority of the information is based on reports from registries and retrospective data. The introduction of DES has substantially improved the outcome in bifurcation lesions compared with BMS, resulting in lower adverse events and parent vessel restenosis rates. In the Arterial Revascularization Therapies Study II (ARTS-II), the subgroup of patients with bifurcation lesions treated with sirolimus-eluting stents (SES) had the same incidence of 1-year MACEs as patients without bifurcation lesions. The analysis of the Stenting Coronary Arteries in Non-Stress/Benestent Disease Trial (SCANDSTENT) was a randomized controlled study comparing implantation of SES with BMS in patients with complex coronary artery disease. Thuesen et al. investigated SCANDSTENT subgroup analysis of patients with a bifurcation lesion. Compared with BMS, SES reduced the restenosis rate from 28.8% to 4.8% in the main branch and from 43.4% to 14.8% in the side branches (both P <0.001). MACEs occurred in 9% with SES versus 28% with BMS (P = 0.01), and stent thrombosis was observed in 0% versus 9% (P = 0.02)18. Steigen et al. compared the strategy of stenting both the main vessel and the side branch with stenting of the main vessel only, with optional stenting of the side branch, with SES. In this study, there were no significant differences in cardiac death, myocardial infarction, stent thrombosis, target-vessel revascularization (TVR) or composites after 6 months between both groups19. Overall, the data on the use of DES suggest a tendency toward less restenosis rates at long-term follow-up in comparison with BMS. Stenting both main vessel and side branch offers a better visual satisfaction but a slightly increased propensity towards subacute stent thrombosis in early follow-up period13. Single stent technique has advantage over stenting of both main vessel and side branch with regard to procedural success, fluoroscopic time, and contrast volume with overall decrease in TLR.13

CHRONIC TOTAL OCCLUSION


Recanalization of chronic total occlusion (CTO) is one of the most challenging coronary intervention. Procedural success is hampered by the difficulties associated with crossing the occluded segment with guidewires and recanalization devices, and long-term results are threatened by a high restenosis rate20. Rubartilli et al. have published a series of 110 patients Gruppo Italiano di Studio sullo Stent nelle Occlusioni Coronariche (GISSOC) trial with CTO randomized to receive either Palmaz-Schatz stent or balloon angioplasty alone. At 9-month angiographic follow-up, the stented group (56 patients) had lower restenosis rate (32% versus 68%; P <0.001) and lower reocclusion rate (8% versus 34%; P = 0.003). There were also fewer recurrent ischemic events (14% versus 46%, P = 0.002) and lower TLR rates (5.3% versus 22%; P = 0.038)21. The Stenting in Chronic Coronary Occlusions (SICCO) trial compared 117 patients who were randomized either to receive Palmaz-Schatz stent or balloon angioplasty

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alone. At 6-month follow-up the angiographic restenosis was reduced in the stented group (32% versus 74%; P < 0.001). Furthermore, on long-term follow-up at 336 months, MACE were significantly less in the stented group (24.1% versus 59.3%; P = 0.0002); TLR was also reduced from 53% in the balloon angioplasty group vs. 24% in the stented group (P = 0.002)22. In the Primary Stenting of Occluded Native Coronary Arteries (PRISON) study, in patients with CTO, use of the SES was superior to the BMS. Two hundred patients with CTO were randomly assigned to receive bare-metal BxVelocity stents or Cypher stents. After 3 years, the Cypher stent group showed a significantly lower rate of TLR (7% versus 27%, P <0.01) and lower rate of TVR (11% versus 34%, P <0.02)23. In the Approaches to Chronic Occlusions With Sirolimus-Eluting Stents/Total Occlusion Study of Coronary Arteries-4 (ACROSS/TOSCA-4) trial, 200 patients with complex CTO were treated with the SES after recanalization. Using logistic regression modeling with propensity score adjustment, the absolute reduction in binary restenosis with SES compared with a historical BMS control was 37.7%24. Also, most published DES studies in CTO sreported short-term outcomes (< 12 months in most studies), and longer follow-up is needed to ascertain both the efficacy and safety of DES. Most studies included not only CTO, but also total occlusions of < 3 months duration25. While DES may significantly reduce the risk for in-stent restenosis and the incidence of repeat revascularization following CTO recanalization, it is need to better assess clinical safety and efficacy of DES in this complex lesion subset beyond angiographic outcomes 26.

SMALL REFERENCE VESSEL BMS


In the early studies of BMS, some studies failed to demonstrate a significant independent role for vessel size in the restenosis process. However, although it was part of the analysis, the relation between vessel size and restenosis was not the principal focus of these studies, and the limited number of patients in these studies may have imparted insufficient power in this regard. Most of these studies entered postprocedural minimal lumen diameter (MLD) into the multivariate models, which may have blunted the independent role of vessel size in outcome1. Meanwhile several studies demonstrated a significant independent role for vessel size in the restenosis process. A worse clinical outcome was reported for subsets of patients with small vessel size from the BENESTENT and STRESS trials, especially when small vessel size was combined with greater lesion length27. Elezi et al. investigated the influence of vessel size on clinical and angiographic outcome after successful stent implantation in a large patient population. In this study, restenosis rate demonstrated a steady decrease as vessel size increased: 38.6% in the first group (< 2.8 mm), 28.4% in the second (2.83.2 mm), and only 20.4% in the third group (> 3.2 mm)1. This study indicates that patients with smaller vessel size have a less-favorable clinical outcome at 1 year after coronary stent placement than patients with larger vessels1. Although it is evident from this study that a higher incidence of restenosis is responsible for the more adverse clinical outcome of patients with smaller vessels, the reason smaller vessels carry a higher risk for restenosis was not clear. The multivariate model of restenosis demonstrated that the adjusted risk for restenosis decreases if a greater balloon-to-vessel ratio is used during the intervention. The influence of balloon-to-vessel ratio on the risk for early adverse events must be specifically assessed before the strategy of using oversized balloons is recommended as a remedy for the excessive restenosis verified in the group with small vessels1. Of these variables, the relationship between the final lumen size and restenosis has been well validated, leading to the principle of bigger is better27.

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DES
Although angiographic and clinical restenosis rates with DES are markedly lower compared with BMS, the reported benefit has not been homogeneous across various clinical and angiographic subgroups and with different drug-eluting stents. Consequently, investigators have suggested that several factors may confer a higher risk of restenosis after DES implantation. One of these factors is the reference diameter of the treated vessel, which has been long been regarded as an important predictor of restenosis1. In the early reports on the use of DES, which included small numbers of very carefully selected patients, there was an almost complete inhibition of neointimal hyperplasia, and therefore late loss was almost absent28. Based on these results, investigators concluded that vessel size plays no role on the development restenosis after DES implantation28. Meanwhile, other studies that included larger number of patients with less restrictive criteria treated with DES showed that late lumen loss occurs, albeit not to the same degree with various DES, and that restenosis rates were lower in vessels with bigger reference diameters7,29. In the Sirolimus-Coated BxVelocity Balloon-Expandable Stent in the Treatment of Patients with De Novo Coronary Artery Lesions (SIRIUS) trial, rates of angiographic restenosis were 17.6% in small vessels and 1.9% in large vessels29. With respect to TLR rates, they were 6.3% in the subgroup with a vessel size < 2.75 mm compared with 1.9% in the subgroup with vessels size 2.75 mm30. In treatment of De Novo Coronary Disease Using a Single Paclitaxel-Eluting stent (TAXUS V) trial, the subgroup with small vessels had an angiographic restenosis rate of 31.2%, and the subgroup with large vessels had a restenosis rate of 3.5%. TLR rates in the respective vessel size groups were 10.4% and 0%31. Elezi et al. cohort study is focus on the influence of vessel size on the outcomes of patients after DES implantation. In this study, reference vessel size does not influence the risk of death or myocardial infarction but it is an important determinant of the risk of restenosis was identified as a predictor of restenosis. Although in-stent late lumen loss was not statistically different between the small vessel (< 2.41 mm) and the large vessel group (> 2.84 mm), the incidence of the in-segment binary restenosis was significantly different between groups. Incidence of TLR was 12.1% patients in the small vessel group compared with 8.0% patients in the large vessel group, was significantly different. Considering the similar degree of in-stent late luminal loss regardless of the reference diameter of the target vessel, the same extent of late loss that was easily accommodated in larger vessels was great enough to lead to an increased incidence of restenosis and need of repeat revascularization procedures in smaller vessels32. In Kastrati et al. study, the subgroup of vessels with reference diameter 2.6 mm had a significant reduction in the odds of angiographic restenosis and clinical restenosis compared with the subgroup with reference diameter < 2.6 mm. These results provide strong support for the role of vessel size as an important predictor of restenosis in the DES era33.

LONG LESION BMS


The length of a coronary lesion is a significant predictor of restenosis after balloon angioplasty. The influence of lesion length has not comprehensively been assessed after coronary stent placement34. Schomig et al.35 showed that lesion length was an independent correlate of the amount of late lumen loss. In contrast, other studies36,37 did not demonstrate the relation between lesion length and restenosis.

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Astrati et al. found that the binary restenosis rate was higher for patients with long lesion (15 mm) stenting. Similarly, patients with long lesions presented more late lumen loss than those with short lesions (1.29 0.89 versus 1.07 0.77 mm, p <0.001). Multivariate models for both binary restenosis and late lumen loss demonstrated that lesion length was an independent risk factor for restenosis34. Although the length of the BMS has been shown to correlate with risk of restenosis, it is not known whether this is just a reflection of underlying lesion length or if there is an independent effect of stent length38. In Kobayashi et al. observational study, he showed that persistently higher restenosis rate for the lesions treated with a single long stent as compared with a single short stent.The results of multivariate analysis demonstrate that the length of the stented segment is an independent predictor of restenosis, although multiple stents are not predictive. The longer the segment stented, regardless of whether a single long stent or multiple stents were used, the higher the restenosis rate39. A compelling strategy proposed by Colombo et al. is spot stenting, whereby short stents are deployed using intravascular ultrasound study (IVUS) guidance at only the most diseased segments in a coronary artery with diffuse disease. The stented lesion in the spot stenting group is 10.4 13 mm and 32.4 13 mm in traditional stenting group. The spot stenting group had significantly better long-term outcome with lower restenosis rates (26% versus 38%, P <0.05), less TLR (20% versus 32%, P<0.05) compare with traditional stenting group. Given that total stented length is predictive of increased restenosis rates, this approach might prove to be the most reasonable in this difficult group of patients40. Serruys et al. investigated the clinical benefit of additional stent implantation after achieving an optimal result of balloon angioplasty in long coronary lesions (>20 mm). The TVR rate was significantly lower in those with stents <32 mm (13.1% versus 23.8%; p = 0.13), and use of multiple stents was associated with an almost doubling of the need for TVR (29.6% versus 16.7%; p = 0.17)41. Mauri et al. examined the independent contribution of BMS coverage strategies to the risk of restenosis. In conclusion, stent length exceeded lesion length in most stented lesions, and the amount of excess stent length increased the risk of restenosis independent of the stented lesion length. This analysis supports a conservative approach of matching stent length to lesion length to reduce the risk of restenosis with BMS. These findings in BMS suggest that there is no advantage of excess stent length in terms of restenosis38.

DES
Although the use of DES has decreased the effect of lesion length on restenosis, long coronary lesions remain at a higher risk of unfavorable outcomes after coronary intervention33,42. The efficacy of the SES implantation has been evaluated in the context of 2 large randomized trials. The Randomized study with the sirolimus-eluting BxVELocity balloon-expandable stent in the treatment of patients with de novo native coronary artery Lesions (RAVEL) trial43 included only single lesions covered by an 18-mm-long stent and had a zero restenosis rate. In the US Multicenter, Randomized, Double-Blind Study of the SIRolImUS-eluting Bxvelocity balloon expandable stent (SIRIUS) trial relatively long stent placement was allowed (maximum of 2 overlapping 18-mm-long SESs) and the restenosis rate was 9.2%30. Degertekin et al. evaluate the outcomes of patients receiving overlapping stents implanted over a length >36 mm to treat native de novo coronary lesions. Although it implanted markedly long length SES (61 mm on average), the incidence of TVR are relatively low (6.2%)42. An investigation to identify a differential outcome between the 2 leading DES in the treatment of long coronary lesions is clinically important to the physicians choice of stent during PCI. Several previous reports of randomized studies, registry data, and a meta-analysis

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showed that SES was more effective than paclitaxel-eluting stent (PES) in reducing the restenosis rate44, 45. Those studies assumed that the lower late loss associated with SES might contribute to lower incidences of angiographic restenosis and repeat revascularization than PES. Not all studies, however, found SES to be superior46. Kim et al. reported the results of the Long-DES II trial which compared the use of Cypher and Taxus stents in patients with long (25 mm) native coronary lesions. In patients treated with Cypher stents, the lesion length (odds ratio [OR] 1.025, 95% confidence interval [CI] 1.005 to 1.046, p = 0.014) and postintervention minimal lumen diameter (OR 0.215, 95% CI 0.098 to 0.472, p = 0.001) were significant predictors of restenosis. In patients implanted with Taxus stents, the lesion length (OR 1.022, 95% CI 1.001 to 1.043, p = 0.036) and postintervention minimal lumen diameter (OR 0.213, 95% CI 0.104 to 0.437, p = 0.001) were also significant predictors of restenosis47. Recent reports evaluating follow-up outcomes across various clinical and angiographic subgroups, however, showed that several factors conferred a higher risk of restenosis even after DES use. A long diseased segment is a key predictor of worse prognostic outcome in terms of restenosis33,48. Long stenting is frequently associated with prolonged intracoronary manipulation due to multiple and overlapping stent placement, which may lead to injury to the vessel wall integrity42.

SAPHENOUS VEIN GRAFT


PCI of saphenous vein grafts (SVG) is associated with worse outcomes and a high incidence of in-stent restenosis49. The higher local prothrombotic conditions in SVG and the expected delay in endothelial healing after DES are claimed as possible drawbacks because both can lead to higher rates of acute, subacute, and late thrombosis17. The Saphenous vein de Novo (SAVED) trial is a prospective randomized controlled trial comparing stenting and balloon angioplasty alone in the management of graft disease. In this study, there was only a trend toward reduced angiographic restenosis and the difference between the two groups did not reach statistical significance (37% versus 46%; P = 0.24)49. The advantage of DES versus BMS has been well documented for native coronary artery disease, data are limited for the systematic evaluation of DES and BMS in SVG disease50. The two randomized trials evaluating DES and BMS in SVG disease provided very different results. The Stenting Of Saphenous vein grafts (SOS) trial reported that the quantitative in segment, in-stent, and binary angiographic restenosis rates at 12 months were significantly superior in the paclitaxel eluting stent group (p < 0.0001)51. This was accompanied by a lower rate of TVR in the PES group compared to the BMS group (15% versus 31%, P = 0.08). However, in the DELAYED RRISC (Reduction of Restenosis In Saphenous Vein Grafts With Cypher Sirolimus-Eluting Stent) trial, no difference in TVR (SES group vs BMS group, 34% versus 38%, respectively; p = 0.74) was observed52. Now, larger randomized trials are needed to evaluate the impact of DES and BMS on restenosis.

CONCLUSIONS
Despite the advantages of coronary intervention over surgical revascularization from both initial patient morbidity and mortality, the need for reintervention due to restenosis has limited the longer-term benefits of coronary intervention, particularly among high-risk lesions. Aorto-ostial lesion, bifurcation, chronic total occlusion, small vessel disease, long lesion and saphenous vein graft have been shown to be predictive variables for restenosis after coronary intervention. Recently, DES has been widely used as a revascularization strategy for a broad

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variety of clinical and anatomic situations, and restenosis remains a major clinical issue. Further studies are required in order to identify the factors that can be used to reliably predict whether an individual patient will experience restenosis after DES implantation. The knowledge of predictive factors for restenosis after coronary intervention may help to optimize indications and to guide strategies against restenosis.

REFERENCES
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19. Steigen TK, Maeng M, Wiseth R, Erglis A, Kumsars I, Narbute I, Gunnes P, Mannsverk J, Meyerdierks O, Rotevatn S, Niemela M, Kervinen K, Jensen JS, Galloe A, Nikus K, Vikman S, Ravkilde J, James S, Aaroe J, Ylitalo A, Helqvist S, Sjogren I, Thayssen P, Virtanen K, Puhakka M, Airaksinen J, Lassen JF, Thuesen L. Randomized study on simple versus complex stenting of coronary artery bifurcation lesions: The nordic bifurcation study. Circulation. 2006; 114:1955-1961. 20. Grantham JA, Marso SP, Spertus J, House J, Holmes DR, Jr., Rutherford BD. Chronic total occlusion angioplasty in the united states. JACC Cardiovasc Interv. 2009; 2:479-486. 21. Rubartelli P, Niccoli L, Verna E, Giachero C, Zimarino M, Fontanelli A, Vassanelli C, Campolo L, Martuscelli E, Tommasini G. Stent implantation versus balloon angioplasty in chronic coronary occlusions: Results from the gissoc trial. Gruppo italiano di studio sullo stent nelle occlusioni coronariche. J Am Coll Cardiol. 1998; 32:90-96. 22. Sirnes PA, Golf S, Myreng Y, Molstad P, Albertsson P, Mangschau A, Endresen K, Kjekshus J. Sustained benefit of stenting chronic coronary occlusion: Long-term clinical follow-up of the stenting in chronic coronary occlusion (sicco) study. J Am Coll Cardiol. 1998; 32:305-310. 23. Rahel BM, Laarman GJ, Kelder JC, Ten Berg JM, Suttorp MJ. Three-year clinical outcome after primary stenting of totally occluded native coronary arteries: A randomized comparison of bare-metal stent implantation with sirolimus-eluting stent implantation for the treatment of total coronary occlusions (primary stenting of totally occluded native coronary arteries [prison] ii study). Am Heart J. 2009; 157:149-155. 24. Kandzari DE, Rao SV, Moses JW, Dzavik V, Strauss BH, Kutryk MJ, Simonton CA, Garg J, Lokhnygina Y, Mancini GB, Yeoh E, Buller CE. Clinical and angiographic outcomes with sirolimus-eluting stents in total coronary occlusions: The across/tosca-4 (approaches to chronic occlusions with sirolimus-eluting stents/total occlusion study of coronary arteries-4) trial. JACC Cardiovasc Interv. 2009; 2:97-106. 25. Saeed B, Kandzari DE, Agostoni P, Lombardi WL, Rangan BV, Banerjee S, Brilakis ES. Use of drug-eluting stents for chronic total occlusions: A systematic review and meta-analysis. Catheter Cardiovasc Interv. 2010 26. Weisz G, Moses JW. Contemporary principles of coronary chronic total occlusion recanalization. Catheter Cardiovasc Interv. 2010; 75 Suppl 1:S21-27. 27. Kuntz RE, Gibson CM, Nobuyoshi M, Baim DS. Generalized model of restenosis after conventional balloon angioplasty, stenting and directional atherectomy. J Am Coll Cardiol. 1993; 21:15-25. 28. Regar E, Serruys PW, Bode C, Holubarsch C, Guermonprez JL, Wijns W, Bartorelli A, Constantini C, Degertekin M, Tanabe K, Disco C, Wuelfert E, Morice MC. Angiographic findings of the multicenter randomized study with the sirolimus-eluting bx velocity balloon-expandable stent (ravel): Sirolimus-eluting stents inhibit restenosis irrespective of the vessel size. Circulation. 2002; 106:1949-1956. 29. Popma JJ, Leon MB, Moses JW, Holmes DR, Jr., Cox N, Fitzpatrick M, Douglas J, Lambert C, Mooney M, Yakubov S, Kuntz RE. Quantitative assessment of angiographic restenosis after sirolimus-eluting stent implantation in native coronary arteries. Circulation. 2004; 110:3773-3780. 30. Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O'Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, Jaeger JL, Kuntz RE. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med. 2003; 349:1315-1323. 31. Stone GW, Ellis SG, Cannon L, Mann JT, Greenberg JD, Spriggs D, O'Shaughnessy CD, DeMaio S, Hall P, Popma JJ, Koglin J, Russell ME. Comparison of a polymer-based paclitaxel-eluting stent with a bare metal stent in patients with complex coronary artery disease: A randomized controlled trial. JAMA. 2005; 294: 1215-1223. 32. Elezi S, Dibra A, Mehilli J, Pache J, Wessely R, Schomig A, Kastrati A. Vessel size and outcome after coronary drug-eluting stent placement: Results from a large cohort of patients treated with sirolimus- or paclitaxel-eluting stents. J Am Coll Cardiol. 2006;48:1304-1309. 33. Kastrati A, Dibra A, Mehilli J, Mayer S, Pinieck S, Pache J, Dirschinger J, Schomig A. Predictive factors of restenosis after coronary implantation of sirolimus- or paclitaxel-eluting stents. Circulation. 2006; 113: 2293-2300. 34. Kastrati A, Elezi S, Dirschinger J, Hadamitzky M, Neumann FJ, Schomig A. Influence of lesion length on restenosis after coronary stent placement. Am J Cardiol. 1999; 83:1617-1622. 35. Schomig A, Kastrati A, Dietz R, Rauch B, Neumann FJ, Katus HH, Busch U. Emergency coronary stenting for dissection during percutaneous transluminal coronary angioplasty: Angiographic follow-up after stenting and after repeat angioplasty of the stented segment. J Am Coll Cardiol. 1994; 23:1053-1060. 36. Ellis SG, Savage M, Fischman D, Baim DS, Leon M, Goldberg S, Hirshfeld JW, Cleman MW, Teirstein PS, Walker C, et al. Restenosis after placement of palmaz-schatz stents in native coronary arteries. Initial results of a multicenter experience. Circulation. 1992; 86:1836-1844.

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37. Strauss BH, Serruys PW, de Scheerder IK, Tijssen JG, Bertrand ME, Puel J, Meier B, Kaufmann U, Stauffer JC, Rickards AF, et al. Relative risk analysis of angiographic predictors of restenosis within the coronary wallstent. Circulation. 1991; 84:1636-1643. 38. Mauri L, O'Malley AJ, Cutlip DE, Ho KK, Popma JJ, Chauhan MS, Baim DS, Cohen DJ, Kuntz RE. Effects of stent length and lesion length on coronary restenosis. Am J Cardiol. 2004; 93:1340-1346, A1345. 39. Kobayashi Y, De Gregorio J, Kobayashi N, Akiyama T, Reimers B, Finci L, Di Mario C, Colombo A. Stented segment length as an independent predictor of restenosis. J Am Coll Cardiol. 1999; 34:651-659. 40. Colombo A, De Gregorio J, Moussa I, Kobayashi Y, Karvouni E, Di Mario C, Albiero R, Finci L, Moses J. Intravascular ultrasound-guided percutaneous transluminal coronary angioplasty with provisional spot stenting for treatment of long coronary lesions. J Am Coll Cardiol. 2001; 38:1427-1433. 41. Serruys PW, Foley DP, Suttorp MJ, Rensing BJ, Suryapranata H, Materne P, van den Bos A, Benit E, Anzuini A, Rutsch W, Legrand V, Dawkins K, Cobaugh M, Bressers M, Backx B, Wijns W, Colombo A. A randomized comparison of the value of additional stenting after optimal balloon angioplasty for long coronary lesions: Final results of the additional value of nir stents for treatment of long coronary lesions (advance) study. J Am Coll Cardiol. 2002; 39:393-399. 42. Degertekin M, Arampatzis CA, Lemos PA, Saia F, Hoye A, Daemen J, Tanabe K, Lee CH, Hofma SJ, Sianos G, McFadden E, van der Giessen W, Smits PC, de Feyter PJ, van Domburg RT, Serruys PW. Very long sirolimus-eluting stent implantation for de novo coronary lesions. Am J Cardiol. 2004; 93:826-829. 43. Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Hayashi E, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, Molnar F, Falotico R. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med. 2002; 346:1773-1780. 44. Windecker S, Remondino A, Eberli FR, Juni P, Raber L, Wenaweser P, Togni M, Billinger M, Tuller D, Seiler C, Roffi M, Corti R, Sutsch G, Maier W, Luscher T, Hess OM, Egger M, Meier B. Sirolimus-eluting and paclitaxel-eluting stents for coronary revascularization. N Engl J Med. 2005; 353:653-662. 45. Kim YH, Park SW, Lee CW, Hong MK, Gwon HC, Jang Y, Lee MM, Koo BK, Oh DJ, Seung KB, Tahk SJ, Yoon J, Park SJ. Comparison of sirolimus-eluting stent, paclitaxel-eluting stent, and bare metal stent in the treatment of long coronary lesions. Catheter Cardiovasc Interv. 2006; 67:181-187. 46. Airoldi F, Briguori C, Iakovou I, Stankovic G, Biondi-Zoccai G, Carlino M, Chieffo A, Montorfano M, Cosgrave J, Michev I, Rogacka R, Sangiorgi GM, Colombo A. Comparison of sirolimus versus paclitaxel eluting stents for treatment of coronary in-stent restenosis. Am J Cardiol. 2006; 97:1182-1187. 47. Kim YH, Park SW, Lee SW, Park DW, Yun SC, Lee CW, Hong MK, Kim HS, Ko JK, Park JH, Lee JH, Choi SW, Seong IW, Cho YH, Lee NH, Kim JH, Chun KJ, Park SJ. Sirolimus-eluting stent versus paclitaxel-eluting stent for patients with long coronary artery disease. Circulation. 2006; 114:2148-2153. 48. Lee CW, Suh J, Lee SW, Park DW, Lee SH, Kim YH, Hong MK, Kim JJ, Park SW, Park SJ. Factors predictive of cardiac events and restenosis after sirolimus-eluting stent implantation in small coronary arteries. Catheter Cardiovasc Interv. 2007; 69:821-825. 49. Savage MP, Douglas JS, Jr., Fischman DL, Pepine CJ, King SB, 3rd, Werner JA, Bailey SR, Overlie PA, Fenton SH, Brinker JA, Leon MB, Goldberg S. Stent placement compared with balloon angioplasty for obstructed coronary bypass grafts. Saphenous vein de novo trial investigators. N Engl J Med. 1997; 337: 740-747. 50. Lee MS, Yang T, Kandzari DE, Tobis JM, Liao H, Mahmud E. Comparison by meta-analysis of drug-eluting stents and bare metal stents for saphenous vein graft intervention. Am J Cardiol. 2010; 105:1076-1082. 51. Brilakis ES, Lichtenwalter C, de Lemos JA, Roesle M, Obel O, Haagen D, Saeed B, Gadiparthi C, Bissett JK, Sachdeva R, Voudris VV, Karyofillis P, Kar B, Rossen J, Fasseas P, Berger P, Banerjee S. A randomized controlled trial of a paclitaxel-eluting stent versus a similar bare-metal stent in saphenous vein graft lesions the sos (stenting of saphenous vein grafts) trial. J Am Coll Cardiol. 2009; 53:919-928. 52. Vermeersch P, Agostoni P, Verheye S, Van den Heuvel P, Convens C, Van den Branden F, Van Langenhove G. Increased late mortality after sirolimus-eluting stents versus bare-metal stents in diseased saphenous vein grafts: Results from the randomized delayed rrisc trial. J Am Coll Cardiol. 2007; 50:261-267.

CHAPTER 11

THROMBUS FORMATION ON DISRUPTED PLAQUES


ATSUSHI YAMASHITA and YUJIRO ASADA
Introduction............................................................................................................................... Pathology of Coronary Atherothrombosis................................................................................. Pathology of Late Drug-Eluting Stent Thrombosis................................................................... Mechanisms of Plaque Rupture ................................................................................................ Mechanisms of Paque Erosion .................................................................................................. Coagulation Factors Involved in Thrombus Growth................................................................. Role of Blood Flow in Thrombus Growth ................................................................................ Platelet Recruitment in Thrombus Growth ............................................................................... Conclusions............................................................................................................................... References................................................................................................................................. 151 151 154 155 156 158 160 161 163 163

INTRODUCTION
Acute cardiovascular events that usually involve thrombus formation at sites of disrupted atherosclerotic plaques are currently described as atherothrombosis. Thrombosis is a major complication of atherosclerosis and also a rare but serious complication after stent implantation. However, it does not always result in complete thrombotic occlusion with subsequent acute symptomatic events1. Therefore, thrombus growth is critical to the onset of clinical events. Thrombus formation is probably modulated by the thrombogenicity of exposed plaque constituents, local hemorheology, systemic thrombogenicity and fibrinolytic activity. Although the mechanisms of thrombus formation have been intensively investigated, little is known about either the mechanisms involved in thrombogenesis or thrombus growth after plaque disruption and stent implantation. This article examines the pathology of atherothrombosis, including late drug-eluting stent (DES) thrombosis, and recent advances in the understanding of thrombogenetic mechanisms and thrombus growth on atherosclerotic lesions, especially coronary atherothrombosis.

PATHOLOGY OF CORONARY ATHEROTHROMBOSIS


Arterial thrombi were traditionally considered to mainly comprise aggregated platelets because of rapid flow. However, recent evidence indicates that atherothrombi are composed

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of aggregated platelets, fibrin and other types of blood cells2-4. Occlusive thrombi are consistently composed of platelets, fibrin and erythrocytes from the early phase of onset. Glycoprotein (GP) IIb/IIIa, a platelet integrin, intermingles with fibrin, and the surface of thrombi is mainly covered with GPIIb/IIIa and von Willebrand factor (VWF), a blood adhesion molecule that colocalizes with GPIIb/IIIa. Tissue factor (TF), an initiator of the coagulation cascade, is closely associated with fibrin3. Tissue factor and/or VWF might contribute to thrombus growth and obstructive thrombus formation on disrupted atherosclerotic plaques. Two major morphological features of plaque disruption are rupture and erosion (Figure 11.1).

Figure 11.1. Microphotographs of human coronary plaque rupture and erosion. Upper: Ruptured plaque has a large necrotic core and disrupted thin fibrous cap associated with thrombus formation. Lower: Eroded plaque has SMC- and proteoglycan-rich lesion with thrombus. (From Sato et al.2, with permission).

Plaque rupture is caused by disruption of the fibrous cap, which allows blood to come into contact with the thrombogenic necrotized core, resulting in thrombus formation. Plaque rupture likely occurs in plaques with a large necrotized lipid core and fibrous caps that are usually thinner than 65 m and heavily infiltrated by macrophages, lymphocytes and occasional smooth muscle cells (SMC)s5. Plaque erosion is relatively superficial and probably occurs in plaques with thick fibrous caps with an abundance of SMCs and a proteoglycan matrix, especially when composed of versican and hyaluronan, but the necrotic core is small and often absent. Inflammatory cell infiltration is relatively low compared with plaque rupture5. Angiographically, the diseased artery is less narrowed and irregular in erosion. Thrombi that develop on ruptured and on eroded plaques are fibrin- and platelet-rich, respectively. Both TF and C-reactive protein are abundant in ruptured, compared with eroded plaques2. These distinct morphological features suggest that different mechanisms are involved in plaque rupture and erosion. On the other hand, the disruption of atherosclerotic plaques does not always result in complete thrombotic occlusion with subsequent acute symptomatic events (Figure 11.2).

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Figure 11.2. Microphotographs of human coronary thrombi in patients with non-cardiac death (A) and acute myocardial infarction. Atherosclerotic plaque rupture (arrows) does not result in thrombotic occlusion. Fibrous cap is disrupted and thrombus has formed in both arteries. However, thrombus size significantly differs. Thrombus from patients with non-cardiac death (A) is smaller and ruptured plaque has a smaller lipid core and mild stenosis, compared with those from patients with acute myocardial infarction (B).

Clinical studies using angiography and intravascular ultrasound have revealed that multiple plaque rupture is a frequent complication in patients with coronary atherothrombosis6. Healed stages of plaque disruption are also occasionally observed in autopsy cases with or without coronary atherothrombosis7. To evaluate the incidence and morphological characteristics of thrombi and plaque disruption in patients with non-cardiac death, Sato et al.8 examined 102 hearts from non-cardiac death autopsy cases and 19 from patients who died of acute myocardial infarction (AMI). Surprisingly, they found coronary thrombi in 16% of the cases with non-cardiac death, and most of them developed on eroded plaques. However, the thrombi were too small to affect the coronary lumen (Table 11.1).
Table 11.1. Incidence of thrombosis in NCD and AMI NCD (n = 102) Fresh thrombus Erosion Rupture Old thrombus 10 case (10%) 7 case (7%) 3 case (3%) 6 case (6%) AMI (n = 19) 14 case (75%) 4 case (21%) 10 cases (54%) 5 cases (26%) p value <0.001 0.07 <0.001 <0.05

NCD, non-cardiac death; AMI, acute myocardial infarction (From Sato et al.8, with permission).

The disrupted plaques from the patients who died of non-cardiac causes had smaller lipid areas, thicker fibrous caps and more modest luminal narrowing than those who died of AMI. These findings suggest that the onset of acute coronary events represents the tip of the atherothrombosis iceberg and that regional factors influence coronary thrombus growth after plaque disruption.

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PATHOLOGY OF LATE DRUG-ELUTING STENT THROMBOSIS


Coronary drug-eluting stent (DES) implantation has become the treatment of choice for patients with symptomatic coronary artery disease undergoing percutaneous coronary revascularization. Although DES have dramatically reduced rates of restenosis compared with bare metal stents, late thrombosis has emerged as a major concern9. Premature discontinuation of antiplatelet therapy, renal failure and diabetes mellitus are clinical predictors of DES thrombosis. However, the pathogenesis of late/very late DES thrombosis remains obscure. Figure 11.3 shows immunofluorescent and immunohistochemical images of coronary late DES thrombus10.

Figure 11.3. Representative immunofluorescent and immunohistochemical images of very late drug-eluting stent (DES) thrombus. Upper and middle: Images stained with fluorescein isothiocyanate-labeled GPIIb/IIIa (green), Cy3-labeled fibrin or P2Y12 (red), and merged immunofluorescent images. Areas with colocalized factors are stained yellow. Lower: Oval CD34-positive cells in clusters (left) and flat CD34-positive cells covering DES thrombus at organizing stage (left). (From Nishihira et al. 10, with permission).

All DES thrombi are composed of aggregated platelets and fibrin (Figure 11.3 upper), together with erythrocytes and white blood cells, and they were all constitutively immunopositive for GPIIb/IIIa, fibrin, glycophorin A (a membrane protein expressed on erythrocytes), P2Y12 receptor (a pyrinergic receptor targeted by thienopyridine), VWF and CD16 (a marker of neutrophils). The proportions of these factors and cells did not significantly differ from those of de novo coronary thrombi associated with AMI (Table 11.2).

CHAPTER 11. THROMBUS FORMATION ON DISRUPTED PLAQUES Table 11.2. Immunopositive area or cell counts in late/very late DES thrombus and de novo AMI thrombus de novo AMI (n = 29) median; range GPIIb/IIIa (%) Fibrin (%) Glycophorin A (%) P2Y12 (%) VWF (%) CD16 (/mm ) CD34 (/mm ) Eosinophil (/mm )
2 2 2

155

late/very late DES (n = 9) median; range 17.0; 6.630.3 36.8; 8.277.2 25.6; 0.243.3 16.4; 8.932.2 12.2; 1.338 487; 2172887 18.2; 0530 0.6; 03.6

p value 0.46 0.16 0.62 0.57 0.68 0.37 0.34 0.28

17.2; 7.231.4 30.1; 4.250.2 22.3; 0.251.5 20.5; 0.848.1 11.9; 1.125.2 877; 1332428 4; 044.3 1.2; 028.2

DES, drug-eluting stent, AMI, acute myocardial infarction, GP, glycoprotein, VWF, von Willebrand factor.

The P2Y12 receptor colocalized with GPIIb/IIIa (Figure 11.3 middle), which supports the clinical fact that intensive anti-platelet therapy with thienopyridine prevents DES thrombosis. Because cytochrome p-450 influences the response to clopidogrel, the novel thienopyridine prasugrel might be more suitable for preventing DES thrombosis, as well as de novo AMI. Interestingly, the shape of the CD34-positive cells in thrombi is heterogeneous (Figure 11.3 lower), which could reflect differentiation or a different origin, and might participate in the organizing process of thrombi. Because CD34-positive endothelial progenitor cells can bind and inhibit platelet function and thrombus formation11, capturing the cells on struts or in thrombi might protect thrombus growth12. The mechanisms of late/very late DES thrombosis are poorly understood. Angioscopic and pathological studies have revealed that delayed neointimal covering is associated with DES thrombosis13,14. In contrast, a serial analysis with optical coherence tomography demonstrated neointimal coverage between 6 and 12 months after DES implantation15. Drug-eluting stents might induce a local hypersensitivity reaction with positive coronary remodelling, resulting in incomplete stent apposition16. Vascular remodelling positively correlates with eosinophil counts in coronary thrombi. However, the role of eosinophils in late DES thrombosis is controversial, because eosinophils were found in only half of the late/very late DES thrombi as a minor component (Table 11.2).

MECHANISMS OF PLAQUE RUPTURE


Thinning and disruption of the fibrous cap caused by metalloproteases is likely to be involved together with local rheological forces and emotional status. Accumulating evidence supports the notion that inflammation plays a key role in the pathogenesis of plaque rupture. The numerous inflammatory cells in rupture-prone atherosclerotic plaques can produce enzymes that degrade the extracellular matrix of the fibrous cap. Macrophages in human atheromas overexpress interstitial collagenases, gelatinases and elastolytic enzymes. Activated T lymphocytes and macrophages can secrete interferon (INF-), which inhibits collagen synthesis and induces the apoptotic death of SMC17. Moreover, INF- can induce interleukin (IL)-18, which accelerates inflammation. Interleukin-18 colocalizes with

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INF- in macrophages located at the shoulder region, but not at the necrotic core, and it is associated with coronary thrombus formation in patients with ischemic heart disease18. Interestingly, the important anti-inflammatory cytokine IL-10 is also upregulated in macrophages in atherosclerotic lesions from patients with unstable, compared with stable angina19. The heterogeneity of macrophages in atherosclerotic plaque could explain these paradoxical findings20. This evidence indicates that an imbalance of the inflammatory pathway appears to participate in the plaque destabilization that triggers thrombosis in fibrous cap rupture. Intraplaque hemorrhage might also trigger plaque rupture. The frequency of previous hemorrhages is greater in coronary atherosclerotic lesions with late necrosis and with a thin fibrous cap than in those with early necrosis or intimal thickening21. Intraplaque hemorrhage and iron deposition are more prominent in coronary culprit lesions obtained by directional coronary atherectomy from patients with unstable, than with stable angina pectoris. The iron deposition correlated with oxidized low density lipoprotein and thioredoxin, an anti-oxidant protein, and was also associated with thrombus formation22. The pathological findings imply relationships among intraplaque hemorrhage, oxidative stress and plaque instability. However, direct evidence linking intraplaque hemorrhage and plaque instability remains elusive.

MECHANISMS OF PLAQUE EROSION


Some authors have postulated that erosions result from vasospasm, but the mechanisms of plaque erosion remain poorly understood. About 80% thrombi of plaque erosions are non-occlusive despite sudden coronary death5. Platelet-rich emboli are found in 74% of patients with plaque erosion who suddenly die, which is an event that is more frequently associated with plaque rupture (40%). Because activated platelets release vasoconstrictive agents such as 5-hydroxytriptamine (5-HT, serotonin) and thromboxane A2, these emboli might increase peripheral resistance leading to altered coronary blood flow. Furthermore, 5-HT can induce the hypervasoconstriction of SMC-rich atherosclerotic vessels via 5-HT2A receptors and reduce blood flow during thrombogenesis23,24. Endothelial cells preferentially undergo apoptosis at areas of atherosclerotic plaques downstream of a blood flow disturbance and shear stress is lower than at upstream areas25. Experimental aortic stenosis can induce acute endothelial change or damage the normal aorta26. Therefore, hemodynamic forces, particularly blood flow disturbed by stenosis or vasoconstriction, could be a crucial factor in generating surface vascular damage and thrombosis. Actually, blood blow disturbed by acute vascular narrowing induces superficial erosive damage to the SMC-rich neointima at post stenotic regions in rabbit femoral arteries. Figure 11.4 shows microscope images of a longitudinal section of the neointima at the post-stenotic region at 15 min after vascular narrowing. Endothelial cells and SMCs at this region were broadly detached, and associated with platelet adhesion to the sub-endothelium. The superficial erosive damage also induced the apoptosis of endothelial cells and superficial SMCs within 15 minutes27. Thus, disturbed blood flow can induce superficial erosive damage to SMC-rich plaque and thrombus formation at post stenotic regions. Computational fluid simulation analysis has indicated that oscillatory shear stress contributes to the development of erosive damage to the neointima27. Although direct clinical evidence has not yet supported the notion that coronary artery vasospasm plays a significant role in plaque erosion, the histological features of this erosive damage caused by disturbed blood flow is similar to those of human plaque erosion2,5. Also, platelet and blood coagulation in the coronary circulation is activated after vasospastic angina28.

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Figure 11.4A. Representative longitudinal microphotographs of rabbit femoral artery with smooth muscle cell-rich neointima 15 minutes after vascular narrowing. Broad superficial neointimal damage at post stenotic region. Neointimal cell detachment (small arrows) at high magnification. N, neointima; M, media; Ad, adventitia.

Figure 11.4B. Representative longitudinal microphotographs of rabbit femoral artery with smooth muscle cell-rich neointima 15 minutes after vascular narrowing. Immunohistochemistry to detect von Willebrand factor (VWF: endothelial marker) and smooth muscle actin (SMA: smooth muscle marker) confirmed detachment of endothelial and smooth muscle cells at post stenotic region. HE (hematoxylin eosin) (From Sumi et al.27).

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Therefore, this evidence suggests that an acute-onset disturbed blood flow due to vasoconstriction could trigger plaque erosion. Hemodynamic factors could play an important role in the development of plaque erosion.

COAGULATION FACTORS INVOLVED IN THROMBUS GROWTH


The most fundamental difference between normal and atherosclerotic arteries is that active TF is abundant in atherosclerotic lesions29. Therefore, vascular wall TF might contribute to thrombus formation/growth on atherosclerotic lesions. However, recent studies have detected low levels of TF in the blood that are nevertheless sufficient to support clot formation in vitro. Plasma TF levels are elevated in patients with unstable angina and AMI, and they correlate with adverse outcomes30. Therefore, whether or not vascular wall and/or blood-derived TF supports thrombus propagation remains controversial. Hematopoietic cell-derived, TF-positive microparticles contribute to laser injury-induced thrombosis in the microvasculature of the mouse cremaster muscle31. In contrast, hematopoietic cell-derived TF does not contribute to photochemically-induced thrombosis in the mouse carotid artery32. Yamashita et al.33,34 demonstrated quantitative and qualitative differences between thrombi on atherosclerotic and normal arteries, and they found that TF derived from the vascular wall rather than from blood significantly contributes to thrombus formation/growth on atherosclerotic lesions. Atherosclerotic lesions express TF and have more procoagulant activity compared with the normal femoral artery (Figure 11.5A). Balloon injury to the neointima induced large platelet-fibrin thrombi, whereas such injury to normal femoral arteries induced small platelet thrombi (Figure 11.5B)33,34. Thrombin generation mediated by neointimal TF contributed to the enhanced platelet aggregation and fibrin formation in atherosclerotic, but not in normal arteries. Moreover, whole blood coagulation in the model was not affected by inhibiting blood TF using a TF antibody even under hyperlipidemic conditions34. Therefore, atherosclerotic plaque-derived TF might contribute to activation of the intravascular coagulation cascade and thrombus growth on atherosclerotic lesions. Recent in vitro studies have shown that various types of blood cells, including monocytes, neutrophils, eosinophils and even platelets, can synthesize TF. Although TF expression in these blood cells is a matter of debate, monocytes are probably the only blood cells that synthesize and express TF, and activated platelets play a role in decrypting monocyte TF activity in a process entailing TF transfer to activated platelets35. A related topic is the contribution of microparticles (MPs) to thrombus formation. Microparticles are small fragments of membrane-bound cytoplasm that are shed from the surface of activated or apoptotic cells. The procoagulant activity of MPs increases with exposure to phosphatidylserine and the presence of TF. Leroyer et al.36 showed that MPs are more abundant and more thrombogenic in human atherosclerotic plaques than in plasma. Most MPs from plaques originate from macrophages, erythrocytes, SMCs and lymphocytes. Both plaque and plasma MPs express TF and generate thrombin, but their coagulation activity is twice as high when isolated from plaques. Thus, MPs might play an important role in thrombus growth after plaque disruption. In fact, MPs are significantly elevated in acute coronary syndrome and ischemic strokes37,38. However, whether elevated levels of MPs are a cause or a consequence of atherothrombosis remains unclear. Future studies are required to clarify the contribution of blood-derived TF and/or MPs to thrombus propagation on atherosclerotic lesions.

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Figure 11.5A. Immunohistochemical and immunofluorescent images of rabbit femoral arteries. Representative immunohistochemical microphotographs of normal femoral artery and of femoral artery at 3 weeks after balloon injury under 0.5% cholesterol diet. Both 0.5% cholesterol diet and balloon injury induced atherosclerotic neointima, which is composed of smooth muscle cells (SMC) and macrophages. Neointimal cells express tissue factor (TF). I, intima; M, media; Ad, adventitia. HE/VB, hematoxylin eosin/Victoria blue stain.

Figure 11.5B. Immunohistochemical and immunofluorescent images of rabbit femoral arteries. Representative immunofluorescent microphotographs of thrombi 15 minutes after balloon injury of normal femoral artery and of atherosclerotic neointima under 0.5% cholesterol diet. Rows show differential interference contrast images, fluorescein isothiocyanate-labeled GPIIb/IIIa (green) and Cy3-labeled fibrin (red) images, as well as merged immunofluorescent images. Areas with colocalized factors are stained yellow. Thrombi on normal intima are composed of small aggregated platelets ( a), while those on atherosclerotic neointima are large, and composed of platelet and fibrin (b). I, intima; M, media; Ad, IEL, internal elastic lamina. (From Yamashita et al.34, with permission).

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Another mechanism that might contribute to thrombus propagation in vivo is the intrinsic pathway of coagulation, which is initiated when coagulation factor XII (FXII) comes into contact with negatively charged surfaces in a reaction involving the plasma proteins, high molecular mass kininogen and plasma kallikrein. In contrast to TF, FXII is likely to become activated on the negatively charged surfaces of activated platelets. Factor XI (XI) is activated by activated FXII, thrombin, and activated XI. Feedback activation of FXI by thrombin promotes further thrombin generation in vitro 39. Studies in vivo have revealed the significance of the intrinsic coagulation pathway in thrombus formation. Initial platelet adhesion and aggregation are normal in mice lacking FXII and FXI, but thrombus propagation is defective in the damaged aorta, carotid artery and mesenteric arterioles40. Mice deficient in FXII are more resistant to thrombosis than those deficient in FXI or Factor IX, indicating that FXII and FXI function via indistinct pathways41. FXI is present in platelet-fibrin thrombus-induced balloon injury of the rabbit atherosclerotic neointima, and anti-FXI antibody reduces thrombus growth without prolonging bleeding time42. Intrinsic coagulation factors play an important role in thrombus growth via further thrombin generation.

ROLE OF BLOOD FLOW IN THROMBUS GROWTH


Blood flow is surely a key modulator of thrombus growth. Clinical studies have shown that blood flow is altered during coronary atherothrombosis and intervention. Marzilli et al.43 reported an approximate 80% reduction in coronary blood flow during ischemia in patients with unstable angina. Hearts obtained from patients after sudden coronary death contained a mean of 4.5 intramyocardial microemboli, which are more common in plaque erosion than rupture and are associated with myocardial necrosis. Most (89%) affected microvessels were < 120 m in diameter44. Not only spontaneous plaque rupture and ulceration, but also coronary intervention can induce microembolization that leads to a reduction in coronary flow reserve45. The mechanisms are considered to be largely responsible for the rapid elevation of distal vascular resistance due to microvascular embolism and vasoconstriction46. However, how blood flow affects thrombus growth on atherosclerotic lesions remains obscure. Yamashita et al.33 demonstrated the effect of blood flow on thrombus propagation in diseased arteries of rabbits. Platelet-fibrin mural thrombi on atherosclerotic neointima became occlusive in the setting of a blood flow reduction of > 75%. In contrast, small platelet thrombi on normal arteries did not grow even under a 90% reduction in blood flow. Increased thrombogenicity of the vascular wall would be essential to platelet-fibrin thrombus formation, and changes in blood flow could be crucial for thrombus growth. In addition to distal vascular resistance, disturbed blood flow induced by acute vascular narrowing promotes thrombus growth at post-stenotic regions. As described above, vascular narrowing of the rabbit femoral artery caused a superficial erosive injury at post stenotic regions of SMC-rich neointima (Figure 11.4A). Mural thrombi became occlusive in 3 of 5 arteries after 3 hours (Figure 11.6) and detached SMCs were involved in the thrombus. The intimal injury significantly progressed 3 h after vascular narrowing. The thrombi that developed on the neointima consisted of a mixture of aggregated platelets and a considerable amount of fibrin. In contrast, the identical vascular narrowing of normal femoral arteries also induced endothelial detachment with small platelet thrombi at post stenotic regions, but fibrin and occlusive thrombi did not develop. Neither vascular narrowing nor anti-rabbit TF antibody affected whole blood hemostatic parameters in the rabbits, and no fibrin was generated in thrombi on eroded normal intima27. The evidence indicates that TF derived from eroded neointima plays a crucial role in fibrin formation and thrombus growth rather than circulating TF. Therefore, flow that becomes disordered after plaque disruption might contribute to thrombus growth in both distal resistance and proximal stenosis.

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Figure 11.6. Thrombus growth at post stenotic region of rabbit femoral artery. Mural thrombus formation (small arrows; A) and occlusive thrombus formation (asterisk; B) on smooth muscle cell rich neointima 30 minutes after vascular narrowing. Large arrows indicate flow direction (From Sumi et al.27) .

The rheological effect on thrombus growth might be partly explained by a shear gradient-dependent platelet aggregation mechanism. Generally, soluble agonists generated at sites of vascular injury are thought to initiate platelet aggregation and thrombus growth. However, platelets can form aggregates in vivo without detectably increasing cytosolic calcium or undergoing a shape change and without substantial -granule secretion during the early phases of thrombus development47,48. These facts have raised the notion that additional mechanisms are involved. Using stenotic microvessels in vitro and in vivo along with imaging systems, Nesbitt et al.49 revealed a shear gradient-dependent platelet aggregation process that is preceded by soluble agonist-dependent aggregation. A shear microgradient at post stenotic regions or at the downstream face of thrombi induces stable discoid platelets aggregates with restricted tethers, and the magnitude and spatial distribution of shear microgradients directly influence the size of platelet aggregation. This process required ligand binding to integrin IIb3 and an inositol triphosphate- and extracellular Ca2+- dependent transient Ca2+ flux, but not global platelet shape, degranulation or soluble agonists. Stabilized discoid platelet aggregates were consolidated within the thrombus base, and depended on soluble agonists. These findings suggest that platelets principally use a biomechanical platelet aggregation mechanism to promote the accumulation and stabilization of discoid platelets at sites of vascular injury. Vessel and/or thrombus geometry itself might promote thrombus formation.

PLATELET RECRUITMENT IN THROMBUS GROWTH


Adhesion molecules and their receptors on platelets are essential for thrombus formation because these molecules support platelet tethering, tight adhesion, aggregation and platelet

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recruitment to the thrombus surface. The crucial importance of platelet adhesion receptors such as GPIb or the GPIIb/IIIa has been confirmed in gene-targeted mice. The absence of these receptors leads to highly impaired thrombus formation, regardless of the model in which vascular injury is induced50. The large, multimeric, plasma protein VWF undergoes a conformational change when bound to a matrix, a process that allow VWF to bind GPIb. Recent experimental evidence in vitro and in vivo has shown that platelet recruitment on the thrombus surface is primarily mediated by VWF and GPIb on flowing platelets51,52. The VWF-GPIb interaction is crucial in both the initial step of platelet adhesion to the injured vessel wall and in the recruitment of new platelets to growing thrombus. However, the roles of VWF on fibrin-rich atherothrombus are limited. Its presence in human coronary thrombi suggests that VWF plays an important role in this process3,4. A monoclonal antibody against the VWF A1 domain, which interacts with platelet GPIb significantly inhibited the formation of fibrin-rich mural thrombus induced by balloon injury of rabbit atherosclerotic neointima53. Moreover, the antibody completely inhibited occlusive thrombus formation on rabbit atherosclerotic vessels even when blood flow was disturbed33. Thus, VWF plays an important role in thrombus propagation on atherosclerotic lesions via platelet recruitment. The size of the VWF multimer can affect thrombus size and it is regulated by a plasma protease, a disintegrin and metalloprotease with a thrombospondin type 1 motif 13 (ADAMTS-13). Hepatic satellite cells synthesize and secrete ADAMTS-13, and its deficiency leads to an increased level of massive circulating VWF multimers, and correlates with the onset of the general thrombotic disease, thrombotic thrombocytopenic purpura (TTP). Surprisingly, mice deficient in ADAMTS-13 are viable with no apparent signs of TTP, but they are susceptible to thrombus formation in small vessels and in the pulmonary circulation54,55. These findings suggest that ADAMTS-13 modulates thrombus formation in various vascular beds and regulates thrombus growth in atherosclerotic arteries. Clinical evidence suggests that the size of the VWF multimer is dysregulated in patients with AMI. The ratio of VWF/ADAMTS-13 antigen is higher in patients with AMI than in those with stable angina pectoris, and the correlation between plasma VWF antigen and ADAMTS-13 activity is inverse in AMI patients56. ADAMTS-13 closely localizes with VWF in fresh coronary thrombi from patients with AMI57. Reducing ADAMTS-13 activity using a monoclonal antibody against the disintegrin-like domain enhances platelet thrombus growth on immobilized type I collagen at a high shear rate (1500S-1) and platelet-fibrin thrombus formation on the injured neointima of rabbit femoral arteries57. That study also showed that massive VWF multimers are cleaved during platelet thrombus formation under a high shear rate. The site of VWF-cleavage by ADAMTS-13 is localized to the surface of platelet thrombus, and ADAMTS-13 activity is shear-dependent58. Thus, ADAMTS-13 may work at the site of ongoing thrombus generation and limit thrombus growth. Other possible factors that enhance thrombus growth on atherosclerotic vessels are molecules that affect thrombus stabilization and platelet potentiation. Activated platelets release adenosine diphosphate (ADP), 5-HT, and thromboxane A2, which further promotes activation. This self-amplifying process will lead to thrombus stabilization. ADP-receptor mediated cyclic calcium signaling requires sustained GPIIb/IIIa activation and thrombus stabilization in vitro59. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) 1 is a cell membrane protein that rapidly hydrolyzes both ADP and adenosine triphosphate to adenosine monophosphate and thereby inhibits platelet aggregation on endothelial cells and SMCs60. The expression of E-NTPDase in atherosclerotic lesions can be down-regulated in patients with unstable angina61 and vascular wall E-NTPDase modulates thrombus formation and vascular contraction in rat carotid arteries60,62. These findings suggest that reduced

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E-NTPDase activity in atherosclerotic lesions affects thrombus stabilization and promotes thrombus propagation on atherosclerotic lesions. However, direct evidence of a correlation between E-NTPDase activity and thrombus size remains elusive.

CONCLUSIONS
Recent evidence indicates that inflammation or altered blood flow plays a key role in the pathogenesis of plaque rupture and erosion, respectively. Occlusive atherothrombus is consistently composed of aggregated platelets, fibrin, erythrocytes, and neutrophils. The enhanced platelet aggregation and fibrin formation in human coronary artery and rabbit atherosclerotic artery indicates excess thrombin generation mediated primarily by plaque TF and a subsequent intrinsic coagulation pathway. Oscillation of shear stress and the shear gradient by vessel and/or thrombus geometry can initiate superficial erosive injury and the early phase of platelet aggregation. The role of blood cell-derived TF and MPs in thrombus formation after plaque disruption is currently unclear, and further studies are required to determine whether or not these factors support thrombus growth. Appropriate models of atherothrombus, but not of thrombus in the normal artery, can provide valuable information about their pathophysiological roles. Moreover, the mechanisms of late DES thrombosis remain unknown. The similar content between late/very late DES thrombus and AMI thrombus de novo implies excess thrombin generation and altered blood flow in thrombus formation after DES implantation.

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11. Abou-Saleh H, Yacoub D, Thort JF, Gillis MA, Neagoe PE, Labarthe B, et al. Endothelial progenitor cells bind and inhibit platelet function and thrombus formation. Circulation. 2009;120:2230-9. 12. Aoki J, Serruys PW, van Beusekom H, Ong AT, McFadden EP, Sianos G, et al. Endothelial progenitor cell capture by stents coated with antibody against CD34: the HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man) Registry. J Am Coll Cardiol. 2005;45:1574-9. 13. Awata M, Kotani J, Uematsu M, Morozumi T, Watanabe T, Onishi T, et al. Serial angioscopic evidence of incomplete neointimal coverage after sirolimus-eluting stent implantation: comparison with bare-metal stents. Circulation. 2007;116:910-6. 14. Joner M, Finn AV, Farb A, Mont EK, Kolodgie FD, Ladich E, et al. Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk. J Am Coll Cardiol. 2006;48:193-202. 15. Katoh H, Shite J, Shinke T, Matsumoto D, Tanino Y, Ogasawara D, et al. Delayed neointimalization on sirolimus-eluting stents: 6-month and 12-month follow up by optical coherence tomography. Circ J. 2009;73:1033-7. 16. Cook S, Ladich E, Nakazawa G, Eshtehardi P, Neidhart M, Vogel R, et al. Correlation of intravascular ultrasound findings with histopathological analysis of thrombus aspirates in patients with very late drug-eluting stent thrombosis.Circulation. 2009;120:391-9. 17. Shah PK. Mechanisms of plaque vulnerability and rupture. J Am Coll Cardiol. 2003;41:15S-22S. 18. Nishihira K, Imamura T, Hatakeyama K, Yamashita, A, Shibata Y, Date H, et al. Expression of interleukin-18 in coronary plaque obtained by atherectomy from stable and unstable angina. Thromb Res 2007;121:275-9. 19. Nishihira K, Imamura T, Yamashita A, Hatakeyama K, Shibata Y, Nagatomo Y, et al. Increased expression of interleukin-10 in unstable plaque obtained by directional coronary atherectomy. Eur Heart J. 2006;27:1685-9 20. Waldo SW, Li Y, Buono C, Zhao B, Billings EM, Chang J, et al. Heterogeneity of human macrophages in culture and in atherosclerotic plaques. Am J Pathol. 2008;172:1112-26. 21. Kolodgie FD, Gold HK, Burke AP, Fowler DR, Kruth HS, Weber DK, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003;349:2316-25. 22. Nishihira K, Yamashita A, Imamura T, Hatakeyama K, Sato Y, Nakamura H, et al. Thioredoxin in coronary culprit lesions: possible relationship to oxidative stress and intraplaque hemorrhage. Atherosclerosis. 2008;201:360-7. 23. Nishihira K, Yamashita A, Tanaka N, Kawamoto R, Imamura T, Yamamoto R, et al. Inhibition of 5-hydroxytryptamine receptor prevents occlusive thrombus formation on neointima of the rabbit femoral artery. J Thromb Haemost. 2006;4:247-55. 24. Nishihira K, Yamashita A, Tanaka N, Moriguchi-Goto S, Imamura T, Ishida T, et al. Serotonin induces vasoconstriction of smooth muscle cell-rich neointima through 5-hydroxytryptamine2A receptor in rabbit femoral arteries. J Thromb Haemost. 2008;6:1207-14. 25. Tricot O, Mallat Z, Heymes C, Belmin J, Lesche G, Tedgui A. Relation between endothelial cell apoptosis and blood flow direction in human atherosclerotic plaques. Circulation. 2000;101:2450-3. 26. Fry DL. Acute vascular endothelial changes associated with increased blood velocity gradients. Circ Res. 1968;22:165-97. 27. Sumi T, Yamashita A, Matsuda S, Goto S, Nishihira K, Furukoji E, et al. Disturbed blood flow induces erosive injury to smooth muscle cell-rich neointima and promotes thrombus formation in rabbit femoral artery. J Thromb Haemost. 2010;8:1394-1402. 28. Oshima S, Yasue H, Ogawa H, Okumura K, Matsuyama K. Fibrinopeptide A is released into the coronary circulation after coronary spasm. Circulation. 1990;82:2222-5. 29. Hatakeyama K, Asada Y, Marutsuka K, Sato Y, Kamikubo Y, Sumiyoshi A. Localization and activity of tissue factor in human aortic atherosclerotic lesions. Atherosclerosis. 1997;133:213-9. 30. Mackman N. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol. 2004;241015-22. 31. Chou J, Mackman N, Merrill-Skoloff G, Pedersen B, Furie BC, Furie B. Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation. Blood. 2004; 104: 3190-7. 32. Day SM, Reeve JL, Pedersen B, Farris DM, Myers DD, Im M, et al. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood. 2005;105:192-8. 33. Yamashita A, Furukoji E, Marutsuka K, Hatakeyama K, Yamamoto H, Tamura S, et al. Increased vascular wall thrombogenicity combined with reduced blood flow promotes occlusive thrombus formation in rabbit femoral artery. Arterioscler Thromb Vasc Biol. 2004;24:2420-2424.

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34. Yamashita A, Matsuda S, Matsumoto T, Moriguchi-Goto S, Takahashi M, Sugita C, et al. Thrombin generation by intimal tissue factor contributes to thrombus formation on macrophage-rich neointima but not normal intima of hyperlipidemic rabbits. Atherosclerosis. 2009;206:418-26. 35. sterud B. Tissue factor expression in blood cells. Thromb Res. 2010;125:S31-4. 36. Leroyer AS, Isobe H, Leseche G, Castier Y, Wassef M, Mallat Z, et al. Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol. 2007;49:772-7. 37. Singh N, Gemmell CH, Daly PA, Yeo EL. Elevated platelet-derived microparticle levels during unstable angina.Can J Cardiol. 1995;11:1015-21. 38. Geiser T, Sturzenegger M, Genewein U, Haeberli A, Beer JH. Mechanisms of cerebrovascular events as assessed by procoagulant activity, cerebral microemboli, and platelet microparticles in patients with prosthetic heart valves.Stroke. 1998;29:1770-7. 39. Gailani D, Broze GJ Jr. Factor XI activation in a revised model of blood coagulation. Science. 1991; 253:909-12. 40. Renne T, Pozgajova M, Gruner S, Schuh K, Pauer HU, Burfeind P, et al. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med. 2005;202:271-81. 41. Cheng Q, Tucker EI, Pine MS, Sisler I, Matafonov A, Sun MF, et al. A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo. Blood in press. 42. Yamashita A, Nishihira K, Kitazawa T, Yoshihashi K, Soeda T, Esaki K, et al. Factor XI contributes to thrombus propagation on injured neointima of the rabbit iliac artery. J Thromb Haemost. 2006;4:1496-501. 43. Marzilli M, Sambuceti G, Fedele S, L'Abbate A. Coronary microcirculatory vasoconstriction during ischemia in patients with unstable angina. Am J Coll Cardiol 2000;35:327-334. 44. Schwartz RS, Burke A, Farb A, Kaye D, Lesser JR, Henry TD, et al. Microemboli and microvascular obstruction in acute coronary thrombosis and sudden coronary death: relation to epicardial plaque histopathology. J Am Coll Cardiol. 2009;54:2167-73. 45. Erbel R, Heusch G. Coronary microembolization. J Am Coll Cardiol. 2000;36:22-4. 46. Taylor AJ, Bobik A, Berndt MC, Ramsay D, Jennings G. Experimental rupture of atherosclerotic lesions increases distal vascular resistance: a limiting factor to the success of infarct angioplasty. Arterioscler Thromb Vasc Biol.2002;22:153-60. 47. van Gestel MA, Heemskerk JW, Slaaf DW, Heijnen VV, Sage SO, Reneman RS, et al. Real-time detection of activation patterns in individual platelets during thromboembolism in vivo: differences between thrombus growth and embolus formation. J Vasc Res. 2002;39:534-43. 48. Furie B, Furie BC. Thrombus formation in vivo. J Clin Invest. 2005;115:3355-62. 49. Nesbitt WS, Westein E, Tovar-Lopez FJ, Tolouei E, Mitchell A, Fu J, et al. A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat Med. 2009;15:665-73. 50. Denis CV, Wagner DD. Platelet adhesion receptors and their ligands in mouse models of thrombosis. Arterioscler Thromb Vasc Biol. 2007;27:728-39. 51. Kulkarni S, Dopheide SM, Yap CL, Ravanat C, Freund M, Mangin P, et al. A revised model of platelet aggregation. J Clin Invest. 2000;105:783-91. 52. Bergmeier W, Piffath CL, Goerge T, Cifuni SM, Ruggeri ZM, Ware J, et al. The role of platelet adhesion receptor GPIbalpha far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis. Proc Natl Acad Sci U S A. 2006;103:16900-5. 53. Yamashita A, Asada Y, Sugimura H, Yamamoto H, Marutsuka K, Hatakeyama K, et al. Contribution of von Willebrand factor to thrombus formation on neointima of rabbit stenotic iliac artery under high blood-flow velocity. Arterioscler Thromb Vasc Biol. 2003;23:1105-10. 54. Chauhan AK, Motto DG, Lamb CB, Bergmeier W, Dockal M, Plaimauer B, et al. Systemic antithrombotic effects of ADAMTS13. J Exp Med. 2006;203:767-76. 55. Banno F, Kokame K, Okuda T, Honda S, Miyata S, Kato H, et al. Complete deficiency in ADAMTS13 is prothrombotic, but it alone is not sufficient to cause thrombotic thrombocytopenic purpura. Blood. 2006;107:3161-6. 56. Kaikita K, Soejima K, Matsukawa M, Nakagaki T, Ogawa H. Reduced von Willebrand factor-cleaving protease (ADAMTS13) activity in acute myocardial infarction. J Thromb Haemost. 2006;4:2490-3. 57. Moriguchi-Goto S, Yamashita A, Tamura N, Soejima K, Takahashi M, Nakagaki T, et al. ADAMTS-13 attenuates thrombus formation on type I collagen surface and disrupted plaques under flow conditions. Atherosclerosis. 2009;203:409-16. 58. Shida Y, Nishio K, Sugimoto M, Mizuno T, Hamada M, Kato S, et al. Functional imaging of shear-dependent activity of ADAMTS13 in regulating mural thrombus growth under whole blood flow conditions. Blood. 2008;111:1295-8.

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59. Goto S, Tamura N, Ishida H, Ruggeri ZM. Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5'-diphosphate receptor stimulation and cyclic calcium signaling. J Am Coll Cardiol. 2006;47:155-62. 60. Furukoji E, Matsumoto M, Yamashita A, Yagi H, Sakurai Y, Marutsuka K, et al. Adenovirus-mediated transfer of human placental ectonucleoside triphosphate diphosphohydrolase to vascular smooth muscle cells suppresses platelet aggregation in vitro and arterial thrombus formation in vivo. Circulation. 2005; 111: 808-15. 61. Hatakeyama K, Hao H, Imamura T, Ishikawa T, Shibata Y, Fujimura Y, et al. Relation of CD39 to plaque instability and thrombus formation in directional atherectomy specimens from patients with stable and unstable angina pectoris. Am J Cardiol. 2005;95:632-5. 62. Furukoji E, Tanaka N, Yamashita A, Matsumoto M, Fujimura Y, Yamamoto R, et al. Ecto-nucleoside triphosphate diphosphohydrolase inhibits ATP- and ADP-induced vasoconstriction. Thromb Res. 2008; 121:583-5.

CHAPTER 12

STENT THROMBOSIS
ALFREDO R. GALASSI, SALVATORE D. TOMASELLO, CORRADO TAMBURINO and DOMINICK J. ANGIOLILLO
Introduction ............................................................................................................................. Current Definition of Stent Thrombosis................................................................................... Pathophysiology of Stent Thrombosis ..................................................................................... Risk Factors of Stent Thrombosis ............................................................................................ Outcome of Stent Thrombosis.................................................................................................. Frequency of Stent Thrombosis ............................................................................................... Treatment of Stent Thrombosis ................................................................................................ Prevention of Stent Thrombosis............................................................................................... The role of Antiplatelet Treatment ........................................................................................... Improving Biocompatibility of Polymers and Stent................................................................. Conclusions.............................................................................................................................. References................................................................................................................................ 167 168 169 172 180 181 182 183 183 185 186 186

INTRODUCTION
Post procedural thrombosis was a bane of percutaneous coronary intervention from its beginning. Abrupt vessel closure, complicating 6% to 8% of balloon angioplasty procedures, was associated with 5% mortality, 40% rate of myocardial infarction (MI), and 40% rate of emergency coronary artery bypass grafting1. The initial experiences with bare metal stents (BMS) in the late 1980s observed a stent thrombosis rate of 6 to 12%2,3. The advent of drug eluting stent (DES) has determined a dramatic reduction in clinical restenosis and the need for target lesion revascularization over prior BMS counterparts4,5. However, the broad use of DES has raised concerns regarding the occurrence of late and very late stent thrombosis related to delayed strut endothelialization and potential prothrombotic characteristics of the DES itself6. Currently, with a dual antiplatelet therapy (DAPT) regimen combining aspirin and a thienopyridine, this incidence has fallen to around 1% over the first 12 months, as reported in the latest studies and meta-analyses of BMS versus drug-eluting stents (DES)5,713. The wide range in the incidence of stent thrombosis across trials with both BMS and DES is explained by differences in definitions, length of follow-up, antithrombotic drug regimens, and complexity of patients and lesions.

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CURRENT DEFINITION OF STENT THROMBOSIS


Definitions of stent trhombosis range from angiographically proven to clinically suspected with the inclusion of MI involving the target vessel to unexplained death (within 30 days or anytime, from stent implantation). Although the first definition characterizes a well-defined mechanism limited to a select patient population undergoing angiography at the time of stent thrombosis and hence underestimates the true incidence of stent thrombosis, the others include events related to atherothrombotic disease progression or arrhythmia and therefore overestimate the true incidence of this event. The lack of consensus on the definition of stent thrombosis among clinical trials has led to disparities through the reports and, in particular, has prevented comparison of stent thrombosis rates between studies. To address this issue, the Academic Research Consortium (ACR) established a stent thrombosis definition14 according to: The timing after initial PCI; The evidence of stent thrombosis.

Timing of Stent Thrombosis


According with the timing after initial PCI, stent thrombosis is considered as acute occurring between 0 and 24 h after stent implantation, subacute between 24 h and 30 days, late between 30 days and 1 year and very late after 1 year (Figure 12.1). Acute or subacute stent thrombosis can also be replaced by the term early stent thrombosis.

Figure 12.1. Timing of stent thrombosis.

Evidence of Stent Thrombosis


In agreement with the ACR consensus, stent thrombosis can be categorized in three types: definite, probable and possible. Definite stent thrombosis requires angiographic or postmortem evidence of thrombotic stent occlusion (thrombus that originates in the stent or in the segment 5 mm proximal or distal to the stent). Probable stent thrombosis is defined as the presence of any unexplained death within the first 30 days after stent implantation, or in the presence of any MI related to documented acute ischemia in the territory of the implanted stent without angiographic confirmation of stent thrombosis and in the absence of any other obvious cause, irrespective of the time after the index procedure. Finally, possible stent thrombosis is defined as any unexplained death from 30 days after intracoronary stenting until the end of follow-up.

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PATHOPHYSIOLOGY OF STENT THROMBOSIS


The pathophysiology of stent thrombosis has not been fully elucidated. A combination of factors might be involved, including procedure-related variables, patient-related issues, and lesion characteristics15,16. Human autopsy series have suggested that DES late and very late thrombosis may be partly attributed to impairment of arterial healing characterized by incomplete re-endothelialization, persistent fibrin deposition, and macrophage infiltration when compared with BMS (Figure 11.2)17.

Proximal

Middle overlapping

Distal

Figure 12.2. Scanning electron micrographs from overlapping bare-metal stents (BMS) and drug-eluting stents (DES) implanted in the rabbit iliac artery model for 28 days. Note, significant less endothelialization is seen in Cypher and Taxus DES as compared to Bx Velocity and Express BMS especially at overlapping sites. Arrows indicate the overlapping regions. Adapted from Nakazawa et al.17

In a necropsy comparison of 23 DES with 25 BMS cases, delayed healing manifested by persistent fibrin deposition and incomplete re-endothelialization emerged as an important discriminator between BMS and DES18 (Figure 12.3). In cases of late/very late stent thrombosis, compared with those of patent DES or BMS, re-endothelialization was reduced (27 26% versus 66 25% versus 90 21%), whereas fibrin scores were increased (3.0 0.9 versus 1.9 1.1 versus 0.9 0.8)18. Fourteen of 21 DES patients suffered late/very late stent thrombosis, which was related to delay healing in all cases. Endothelial coverage was nearly complete in BMS specimens examined beyond 6 months, whereas incomplete re-endothelialization in DES specimens persisted beyond 40 months. Kotani and colleagues compared stent strut coverage 3 to 6 months after BMS and sirolimus

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eluting stent (SES) implantation using intracoronary angiography19. Struts of SES were not or minimally covered, whereas BMS showed complete healing in all cases. Additionally, experimental models suggest that inhibition of proliferation and differentiation of human endothelial progenitor cells might further impede delayed or absent vascular endothelial healing in DES patients.

Figure 12.3. Percentage of endothelialization as a function of time after the index procedure. Adapted from Joner et al.18

Platelets exposed to non-endothelialized stent struts can trigger thrombosis through its key phases: adhesion, activation, and aggregation. Initial platelet adhesion results from binding of von Willebrands factor, glycoprotein Ib and IIb/IIIa, and collagen. Next, activation occurs via release of vasoactive factors including thrombin, adenosine diphosphate, thromboxane A2, and serotonin. Subsequently, activated platelets are linked into an aggregate meshwork primarily mediated by the glycoprotein IIb/IIIa receptor and fibrinogen binding20 (Figure 12.4). The resultant platelet plug might acutely progress to thrombotic stent occlusion21,22. Impairment of endothelial recovery may be considered a potential factor which may cause stent thrombosis and negative vascular remodeling. Preliminary data suggest that DESs are associated with endothelial dysfunction23. Physiological evidence of dysfunctional endothelium comes from studies assessing vasomotion 6 months after DES implantation24,25. Through the use of bicycle exercise during coronary angiography, the segment proximal and distal to DES showed paradoxical vasoconstriction, whereas BMS demonstrated normal vasodilatation, suggesting that DES either prevent re-endothelialization or induce vascular damage with subsequent endothelial dysfunction and reduced nitric oxide availability. Using pharmacological evaluation of endothelial function 6 months after the index procedure, another study

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corroborated paradoxical vasoconstriction distal to the stent with a maximal decrease in the mean coronary diameter by 32% compared with baseline in SES-treated patients with an insignificant decrease in the BMS control group (2%; p<0.03)26.

Figure 12.4. Vascular injury results in the exposure of collagen and von Willebrand factor (vWF) in the vessel wall. Circulating platelets adhere and form a monolayer of activated platelets on the collagen matrix, which drives the release of adenosine diphosphate (ADP) and thromboxane (Tx) A2 from the adherent platelets. Secretion of ADP and TxA2 promotes changes in platelet shape and amplification of platelet activation. Thrombin, generated by locally produced tissue factor (TF), is the most potent platelet activator. In the perpetuation phase, platelet contacts promote growth and stabilisation of the platelet plug. Adapted from Brass et al.20

Hypersensitivity might be an under-recognized component of stent thrombosis, especially in delayed cases. Virmani et al. described a case of local hypersensitivity reaction in a patient suffering DES very late stent thrombosis 18 months after DES implantation27. Autopsy findings revealed extensive vasculitis of the intima, media, and adventitia, consisting predominantly of lymphocytes and eosinophils. Aneurysmal coronary dilation was observed within the stented segment, with evidence of stent malapposition and thick fibrin thrombus between the stent and arterial wall. The hypersensitivity reaction could be caused by the metallic stent, polymer, or sirolimus. Recently an elegant in vivo study by Cook et al. demonstrates that very late stent thrombosis after DES implantation is associated with histopathological and serological signs of inflammation28. The authors performed thrombus aspiration and intravascular ultrasound in a series of 10 patients with very late DES thrombosis, showing that the amount of eosinophils was 3 times higher in thrombus aspirates from patients with very late stent thrombosis compared with other causes of MI. Moreover, eosinophilic infiltrates were associated with evidence of vessel remodeling leading to presumably secondary stent malapposition28. Available pathological evidence, however, supports the hypothesis that hypersensitivity to the polymer is the most likely mechanism. Hypersensitivity to metals such as molybdenum, nickel, and chromium has been reported in 10% of patients undergoing stenting29. Moreover,

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delayed hypersensitivity reactions to nickel and molybdenum have been implicated as trigger for initiation of stent thrombosis30. However, the polymers of DES are considered the predominantly target of type IV hypersensitivity reactions, involving low molecular weight haptens. Macrophages, giant cells, tissue damage, and fibrosis are seen during subcutaneous implantation of poly-nbutylmethacrylate, which is a component of the polymer coating of SES31. Farb et al. suggested other pathological mechanisms of late and very late stent thrombosis according to autopsy data32. Indeed, a plaque rupture that occurs in close proximity to the stent can extend to the adjacent stented segment and present as late stent thrombosis. Moreover, stenting of highly necrotic and inflammatory plaques might also lead to late or very late stent thrombosis. In summary, delayed stent healing, dysfunctional endothelium after DES implantation, and progression of atherothrombotic coronary disease, alone or in combination with chronic inflammation and hypersensitivity reactions, could be considered the triggers for the platelet activation and consequently stent thrombosis (Figure 12.5).

Figure 12.5. Many factors, including impaired neointimal formation, technical issues, possible hypersensitivity to drug-eluting stent components, impaired endothelium, patient characteristics, and adjunct pharmacology, all potentially contribute to and modify platelet activity, the major determinant of stent thrombosis. Solid arrows promotional factors. Dashed arrows with question marks possible promotional factors. Crossed arrows inhibitory factors. ADP = adenosine diphosphate; Ig = immunoglobulin. Adapted from Chen et al.22

RISK FACTORS OF STENT THROMBOSIS


Stent thrombosis is a multifactioral problem (Table 12.1). It is possible to categorize the risk factors for stent thrombosis into three groups: patients related;

CHAPTER 12. STENT THROMBOSIS Table 12.1. Predictors of stent thrombosis identified in the literature Clinical related risk factors Diabetes mellitus Renal failure Low LVEF Discontinuation of antiplatelet therapy Antiplatelet therapy resistance Angiographic related risk factors Lesion length Vessel diameter Type C lesion (including also bifurcation lesion, ostial lesion, CTO) Procedural related risk factors Residual dissection Residual thrombus Final TIMI flow < than 3 Residual stenosis Stent length Number of stent implanted Brachitherapy Stent under-expansion Acute and late incomplete stent apposition

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lesions related; procedural related.

Patient Related Risk Factors (Clinical)


Patients clinical presentation (e.g. acute coronary syndrome), renal failure, diabetes mellitus, and low ventricular ejection fraction are common risk factors for stent thrombosis, already identified in pre-DES era33,34. Renal failure has been linked to cardiac disease with microvascular and metabolic abnormalities that may predispose to thrombus formation35. Renal failure also has been associated by numerous studies with an increased mortality rate despite successful coronary intervention36,37. The results of EVASTEN (valuation cot/efficacit du stent actif au sirolimus chez les patients diabtiques et non diabtiques) registry showed in a series of 1731 patients underwent PCI with SES implantation, an increased risk of stent thrombosis in presence of diabetes and multivessel disease (Figure 12.6)38. Moreover, the rate of stent thrombosis was higher in insulin treated diabetic patients (Figure 12.7). These results are concordant with those reported by another large retrospective evaluation in 8146 treated with SES (n=3823) or paclitaxel eluting stent (PES) (n=4323), showing a strong predictive value of diabetes and acute coronary syndrome at presentation for stent thrombosis39.

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Figure 12.6. Comparison of the stent thrombosis-free survival curves between diabetic and non-diabetic patients. Adapted from Machecourt et al.28

Figure 12.7. Comparison of event rates between insulin-requiring diabetic patients (327 patients, 39%) and noninsulin treated diabetic patients (517 patients, 61%). Adapted from Machecourt et al.28

CHAPTER 12. STENT THROMBOSIS Table 12.2. Incidence of stent thrombosis through the literature Study/Author Karillion et al.65 Moussa et al.48 Schhlen et al.66 RISE Registry67 Cutlip et al.32 Serruys et al.68 Heller et al. 69 Orford et al.57 Wang et al. Lee et al.
71 11 42 70 13

175

Year 1996 1997 1998 1999 2001 2001 2001 2002 2002 2005 2005 2005 2005 2006 2006 2006 2006 2007 2008 2008 2010

Stent type BMS BMS BMS BMS BMS BMS BMS BMS BMS BMS BMS SES, PES SES, PES SES, PES SES, PES SES SES, PES SES, PES SES, PES SES, PES SES, PES

Total population, n 2900 1001 2833 939 6186 600 1855 4509 1191 6058 3012 2006 2229 2974 225 15175 1911 8146 23500 3160 1010

Stent thrombosis, % 1.8 1.9 2.3 1.5 0.9 2.8 1.8 0.5 1.7 1.6 0.3 1.6 1.3 1.3 3.1 0.9 0.8 1.9 2 2.1 1.7

Time Early Early Early Early Early Early Early and late Early Early and late Early and late Early Early Early and late Early and late Early, late and very late Early and late Early, late and very late Early, late and very late Early, late and very late Early, late and very late Early, late and very late

Wenaweser et al. Ong et al.

Iakovou et al.

Kuchulakanti et al.9 Rodrigez et al.72 Urban et al.73 Park et al.74 Daemen et al.38 ESTROFA registry75 Park et al.63 DESIRE registry76

The increased risk in diabetic patients might be related to the more diffuse and aggressive nature of atherosclerosis, accompanied by longer lesion lengths, smaller vessel size, and greater plaque burden, which might incur less optimal procedural results40. In addition, patients with diabetes mellitus are more commonly characterized by antiplatelet drug resistance, an emerging factor associated with recurrent ischemic events, including stent thrombosis. Premature discontinuation of antiplatelet therapy represents an important predictor of stent thrombosis after DES implantation. Ferrari and colleagues hypothesized that aspirin withdrawal in coronary patients represents a real risk for the occurrence and recurrence of coronary event, analyzing every hospitalization for acute coronary syndrome in a period of about 30 months in a European centre41. Notably, they showed a higher rate of stent thrombosis in patients who stop aspirin therapy. Iakovou et al. showed, in a retrospective study, that stent thrombosis occurred in about the 30% of patients who prematurely discontinued dual antiplatelet therapy (DAPT), making treatment adherence of paramount importance42. In the Dutch stent thrombosis registry, discontinuation of clopidogrel was strongly associated with the risk of stent thrombosis within 30 days of the index procedure (HR 36.5, 95% CI: 8.0167.8), whereas the risk was increased but attenuated between 30 days and 6 months (HR 4.6, 95% CI:

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1.415.3) and beyond 6 months (HR 5.9, 95% CI 1.7 to 19.8)43. Recently, Kimura et al. reported the impact of DAPT discontinuation analyzing a series of 611 stent thrombosis44. The authors showed that in 216 patients (36%) stent thrombosis happened after the discontinuation of antiplatelet therapy (both thienopyridine and aspirin in 77 patients; discontinuation of thienopyridine only, in 133 patients; and discontinuation of aspirin in 6 patients), with a median interval between discontinuation and stent thrombosis of 121 days (Figure 12.8).

Figure 12.8. Timing of ST after discontinuation of both aspirin and thienopyridine in the RESTAR registry. Adapted from Kimura et al.44

The median interval from discontinuation of antiplatelet therapy to the onset of stent thrombosis was significantly shorter after discontinuation of both aspirin and thienopyridine than after discontinuation of thienopyridine only (13 days versus 314 days p<0.0001)44. It is important to note, that short intervals between discontinuation of DAPT and stent thrombosis suggest a causal relationship between discontinuation and thrombotic event. Many reports found a positive association between DES late/very late stent thrombosis and cessation of DAPT, suggest perhaps that even further prolongation of treatment might be appropriate4548. Ongoing trials are currently addressing the optimal duration of DAPT. Additionally, there is compelling evidence for clopidogrel or aspirin resistance as strong predictors for DES as well as BMS thrombosis. Several studies have shown and association particularly with clopidogrel resistance and recurrent ischemic events, including stent thrombosis (Table 12.3)49. Among these studies, the first and largest with regards to DES thrombosis is from Buonamici et al. investigated clopidogrel-induced platelet inhibition in patients after DES implantation found an higher incidence of subacute and late stent thrombosis in non-responders compared with responders patients (8.6% versus 2.3%, p<0.001)16. Moreover, they identified clopidogrel non-responsiveness as strong predictor of stent thrombosis at multivariable analysis (HR 3.08, 95% CI 1.32 to 7.16; p< 0.009) (Figure 12.9).

CHAPTER 12. STENT THROMBOSIS Table 12.3. Clinical outcomes and inadequate clopidogrel response defined according to various platelet function assays. Adapted with permission by Ferreiro et al.49 N Matetzky et al. Gurbel et al. Gurbel et al. Cuisset et al. Lev et al. Cuisset et al. 60 Clinical setting LTA (light transmittance aggregometry) STEMI undergoing PCI Outcomes

177

Post-primary PCI ischemic events (6 months) Post-PCI ischemic events (6 months) Post-PCI myonecrosis/inflammation Post-PCI ischemic events (30 days) Post-PCI myonecrosis Post-PCI myonecrosis Post-PCI ischemic events (30 days) Post-PCI major cardiovascular events (3 months) Post-PCI ischemic events (12 months) Periprocedural myocardial infarction Ischemic events (24 months) Post-PCI myonecrosis Stent thrombosis Stent thrombosis Post-PCI major adverse cardiac events (6 months) Post-PCI ischemic events (30 days) Stent thrombosis Stent thrombosis Stent thrombosis Major adverse cardiac events and stent thrombosis (6 months) Major adverse cardiac events (30 days) Major adverse cardiac events (12 months) Major adverse cardiac events (12 months)

192 Nonemergent PCI 120 Elective PCI 106 ACS undergoing PCI 120 Elective PCI 150 Elective PCI

Hochholzer et al. 802 Elective PCI Geisler et al. Bliden et al. Cuisset et al. Angiolillo et al. Marcucci et al. Mller et al. Buonamici et al. Bonello et al. Frere et al. Barragan et al. Gurbel et al. Blindt et al. Price et al. Patti et al. Marcucci et al. De Miguel et al. 379 Stable and unstable angina undergoing PCI 100 Chronic clopidogrel therapy undergoing nonemergent PCI 190 NSTEACS undergoing PCI 173 Type 2 diabetes mellitus on chronic dual antiplatelet therapy 367 MI undergoing PCI 105 Elective PCI 804 PCI with drug eluting stent 144 195 46 120 99 380 160 683 179 Stable angina and low-risk NSTEACS undergoing PCI NSTEACS undergoing PCI Subacute stent thrombosis Subacute stent thrombosis PCI with high risk for stent thrombosis PCI with drug eluting stents PCI ACS undergoing PCI NSTEACS undergoing coronary angiography Others Sibbing et al.

VASP-P (vasodilator-stimulated phosphoprotein phosphorilation) assay

VerifyNow P2Y12 assay

1608 49

Elective PCI with drug eluting stent Subacute stent thrombosis

Stent thrombosis Stent thrombosis

Ajzenberg et al.*

STEMI: ST elevation myocardial infarction; PCI: percutaneous coronary intervention; ACS: acute coronary syndrome; NSTEACS: non-ST elevation acute coronary syndrome. Platelet function test: multiple electrode platelet aggregometry (MEA). * Platelet function test: shear-induced platelet aggregation (SIVA).

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Figure 12.9. Kaplan-Meier Analysis of Primary End Point for Responders and Nonresponders to Clopidogrel. Adapted from Buonamici et al.16

Lesion Related Risk Factors (Angiographic)


Similar to previous experience with BMS, DES thrombosis is influenced by several angiographic lesion characteristics50. Among these, lesion length, vessel diameter, lesions complex severity (type C), bifurcation lesions, chronic total occlusion represent the most common factors identified as predictors of stent thrombosis38,4244.

Procedural Related Risk Factors


Many procedural factors have been clearly related to stent thrombosis. Intravascular ultrasonic predictors of early stent thrombosis after BMS implantation were stent under-expansion, residual dissections, and thrombus51,52. In a recent study of 21 patients with documented SES thrombosis, Fujii and colleagues identified stent under-expansion and a residual reference segment stenosis as predictors of early stent thrombosis53. It was recently suggested that stent malapposition as assessed by intra-vascular ultrasonography (IVUS) imaging plays an important role in patients who develop very late stent thrombosis after DES implantation. Stent malapposition (or incomplete stent apposition) represents a separation of at least one stent strut from the intimal surface of the arterial wall with evidence of blood behind the strut54. Stent malapposition can be acute if detected post-procedure or late if detected at follow-up by IVUS imaging55. Acute stent malapposition can resolve or persist during the follow-up period. Late stent malapposition may be persistent if present both immediately after the procedure and at follow-up, or acquired if present only at follow-up56. The hypothesis of incomplete stent apposition proposed and are depicted schematically in Figure 12.10, as follows: (1) positive arterial remodeling with an increase of external elastic membrane out of proportion to the increase in persistent plaque and media; (2) a decrease in plaque and media due to dissolution of jailed thrombus or plaque debris, (i.e. patients undergoing stent implantation during acute myocardial infarction); (3) stent malapposition not

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recognized at implantation and detected at follow-up (persistent malapposition); this may be mediated in part by severely calcified lesions not allowing for homogeneous stent expansion and resulting in stent under-expansion.

Figure 12.10. Mechanisms leading to incomplete stent apposition. I. Positive arterial remodeling with an increase of EEM out of proportion to the increase in persistent plaque and media, which leads to ISA (B) of an initially well-apposed stent (A). II. A decrease in plaque due to dissolution of jailed material, ie, patients undergoing stent implantation during acute myocardial infarction. A, Completely apposed stent with thrombus jailed behind the struts. B, ISA during follow-up due to thrombolysis. III. ISA not recognized at implantation and detected at follow-up (persistent ISA). This may be mediated in part by severely calcified lesions not allowing for homogeneous stent expansion. Thus, a site of calcification (fulcrum) may result in localized underexpansion of the stent and ISA (lever principle) at its vicinity due to either insufficient balloon pressure or inability to overcome the inherent stiffness of the stent structure (B). Adapted from Cook et al.57

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Another theory considers of chronic stent recoil without any change in arterial dimensions in the pathogenesis of late malapposition. In the retrospective POST (Predictors and Outcomes of Stent Thrombosis) registry, stent malapposition was observed in 49% of 53 patients with early stent thrombosis after BMS implantation50. In a case control study Cook and colleagues analyzed IVUS findings in patients with very late stent thrombosis57. The authors found that incomplete stent apposition was more frequent and larger in patients with stent thrombosis in comparison with control group (77% versus 12%; p<0.001, 8.37.5 versus 4.03.8 mm2 p<0.03, respectively) suggesting a role in the pathogenesis of this adverse event55. A recent meta-analysis of 17 trials showed a higher risk of LSM after DES implantation in comparison with BMS use and a strong association with LSM and stent thrombosis58. Moreover, no difference in term of LSM was found among different types of DES57.

OUTCOME OF STENT THROMBOSIS


Patients presenting with stent thrombosis have a poor prognosis; indeed, it results in abrupt closure of the treated artery with the associated risk of MI and death33. The impact of stent thrombosis is influenced by the myocardial area at risk, its viability, the degree of instantly recruitable collaterals, and rapid reperfusion therapy9,11,33. Moreover, stent thrombosis may be responsible for late complications of MI, including heart failure, arrhythmias, or mechanical complications. It was estimated that 10%30% of patients who experienced stent thrombosis die in hospital. Moreover, stent thrombosis can lead to unexplained sudden death. A pooled analysis of multicenter BMS trials33 and a single-center registry of 6058 BMS patients13 both observed a mortality of 7% at 30 days after definite stent thrombosis. Mortality at 30 days after definite stent thrombosis in DES amounted to 9% in a registry of 8146 patients11 and to 19% in a series of 2974 patients9. A recent report of definite or probable stent thrombosis in randomized clinical trials of DES versus BMS revealed similar rates of mortality for both stent types (SES versus BMS, 31% versus 33%; PES versus BMS, 32% versus 28%)10. However, non fatal acute MI is the most frequent clinical presentation of stent thrombosis (7080% of cases)9,11,33. In a recent Japanese large registry MI was the most common clinical sequelae of stent thrombosis, occurring in 89% of patients and cumulative incidence of death after the index stent thrombosis events was significantly lower in patients with very late compared with patients with early or late stent thrombosis43. However, after adjustment for baseline demographics, differences were of borderline significance for early versus very late stent thrombosis (HR 2.09; 95% CI 1.07 to 4.07; p=0.03) and not significant for late versus very late stent thrombosis (HR 1.29; 95% CI 0.61 to 2.74; p=0.5)44 (Figure 12.11). Concerning the long term outcome, Cutlip et al. reported a 1-month mortality rate of 20%33, while Orford et al. recently found a 6-month mortality rate of 48% after definite stent thrombosis59. Furthermore, this rate does not include unexplained deaths, and underestimates the real overall mortality rate. As described in the literature, currently the only predictive factors of death after definite stent thrombosis are the presence of a residual dissection after stent thrombosis treatment, a TIMI flow grade 3 after treatment of stent thrombosis, and the presence of a residual stenosis above 50% after stent thrombosis treatment13. Finally, it is notable that patients suffering from stent thrombosis are at significant risk of recurrent thrombotic stent occlusion13,39.

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Figure 12.11. Cumulative incidence of death after stent thrombosis according to the timing of stent thrombosis in the RESTART registry. Adapted from Kimura et al.44

FREQUENCY OF STENT THROMBOSIS


Daemen and colleagues showed in a large two-institutional cohort study that stent thrombosis is a rare phenomenon which continues at a steady rate per year39 (Figure 12.12).

Figure 12.12. Occurrence and frequency of stent thrombosis over time. Adapted from Daemen et al.39

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Early stent thrombosis is encountered with a similar or even somewhat lower frequency after DES compared with BMS. A 30-day definite stent thrombosis rate of 1.2% with BMS (506 patients), 1.0% with sirolimus-eluting stents (SES; 1017 patients), and 1.0% with paclitaxel-eluting stents (PES; 989 patients) was found in a sequential cohort comparison11. A meta-analysis of 6 studies comparing BMS with SES in 2963 patients reported ST rates at 30 days of 0.5% with SES and 0.6% with BMS, respectively (RR 0.76; 95% CI 0.30 to 1.88; p=0.55)60. A pooled analysis of 5 trials using PES in 3513 patients showed early stent thrombosis rates of 0.5% with PES compared with 0.6% with BMS (RR, 0.74; 95% CI 0.31 to 1.80; p=0.51)61. Although late stent thrombosis had already been observed during the BMS era, it was largely ignored. Four registries comprising 9465 BMS patients reported rates of late stent thrombosis ranging from 0% to 1% (average, 0.5%) and representing nearly one third of overall ST events (average, 1.6%)62. In a meta-analysis, no differences existed in the overall (0.6% versus 0.5%; OR 1.05; 95% CI 0.51 to 2.15; p=1.00) and late (0.2% versus 0.3%; OR, 99; 95% CI 0.35 to 2.84; p=1.00) incidence of stent thrombosis between DES (2602 patients) and BMS (2428 patients)63. Case reports, observational studies, extended follow-up of trials comparing DES with BMS, and meta-analyses of randomized trials have corroborated that very late stent thrombosis is more common with DES than BMS. The predisposition to this atherothrombotic event more than 1 year after DES implantation has been anticipated as design inherent64. It was first documented in 4 patients who discontinued antiplatelet therapy or underwent a surgical procedure46. Pooled analysis of 4 randomized trials (1748 patients) comparing SES and of 5 randomized trials (3513 patients) comparing PES with BMS revealed similar rates of protocol-defined stent thrombosis up to 1 year but significantly more very late stent thrombosis (SES versus BMS: 0.6% versus 0%, p=0.03; PES versus BMS: 0.7% versus 0.2%, p=0.03)5. Another systematic review of 14 trials comparing SES with PES revealed no difference in the overall incidence of protocol-defined stent thrombosis (SES, 1.5% versus BMS, 1.3%; p=0.75), but very late stent thrombosis was more frequent with SES (0.3% versus 0.04%; p=0.02)8. Recently, an observational Korean study evaluated the outcomes in 7221 patients who received DES or BMS implantation65. The authors showed a similar risk of stent thrombosis between DES and BMS despite a higher risk of very late thrombosis with DES use. The same results were obtained by a meta-analysis of 6675 patients with follow-up ranging from 8 to 48 months reported no difference in the overall incidence of ST between BMS and DES (0.10% versus 0.07%; RR 1.03; 95% CI 0.63 to 1.68; p=0.91) but a significantly higher rate of very late ST in disfavor of DES (0.5% versus 0%; RR 5.02; 95% CI 1.29 to 19.52; p=0.02)66. Table 12.2 shows an overview of the incidence of stent thrombosis throughout the years in literature (endnotes of Table 12.26778).

TREATMENT OF STENT THROMBOSIS


The principal objective of the treatment of stent thrombosis must be to obtain an early effective reperfusion with TIMI flow grade 3, as quickly as possible. Primary PCI must be the first choice considering the low rate of reperfusion obtained with thrombolysis. Nevertheless, one important point needs to be highlighted: the thrombus quality in patients with a stent thrombosis differs from that in patients presenting with a usual acute MI as shown by experimental studies and data from human autopsy studies18,28,32. Indeed, in the case of stent thrombosis, the thrombus is almost totally composed of platelets and contains very little fibrin. Thus PCI should be considered the preferred treatment of this event. Results concerning the impact of thrombus aspiration for stent thrombosis treatment are debated in the literature. If this technique seems feasible and safe, its impact in terms of short- and long-term patient

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outcomes is still unknown7981. According to the results of the TAPAS (Thrombus Aspiration during Percutaneous coronary intervention in Acute myocardial infarction Study), use of thrombus aspiration before stenting of the infarcted artery improves the 1-year clinical outcome after usual ST-elevation MI (STEMI) compared to conventional PCI82. In the EXPIRA trial (thrombectomy with export catheter in infarct-related artery during primary percutaneous coronary intervention) Sardella et al. showed that STEMI patients treated with aspiration device resulted in a high rate of myocardial blush grade (MBG) 2, high rate of ECG-changes resolution and a lower incidence of cardiac death at 9-month compared with those treated with standard PCI (88% versus 60%, p< 0.001, 64% versus 39%, p< 0.001, 4.6% versus 0%, log-rank test, p=0.02 respectively)83. However, in this study the 12% of patients treated with aspiration device achieved a final MBG < 2, proving that in some conditions this device is not able to avoid distal embolization. Rich platelet thrombus seems to be a good rationale for the use of GP IIb/IIIa inhibitors. Some authors have shown the efficacy of this treatment, with a reperfusion success rate around 90% when associated with PCI84,85. Moreover, Wenaweser et al. reported that use of GP IIb/IIIa inhibitors is the only factor associated with a decrease in recurrent stent thrombosis after a first successfully treated stent thrombosis13. Although no impact on mortality was found in these studies, GP IIb/IIIa inhibitor use is associated with increased reperfusion success and decreased recurrences in the case of stent thrombosis.

PREVENTION OF STENT THROMBOSIS


A poor procedural result was recognized as the most important factor which may cause early stent thrombosis after both BMS and DES implantation. Therefore, the basis of early stent thrombosis prevention is represented by performing the best PCI possible, including good expansion and apposition of the stent and avoiding residual vessel dissection. In this setting the role of IVUS must be highlighted, and some authors recommended the use of IVUS systematically86,87.

THE ROLE OF ANTIPLATELET TREATMENT


The advent of DAPT consisting of aspirin in combination with a thienopyridine resulted in a significant reduction of stent thrombosis88,89. Recognition of the appropriate pre-treatment and sufficient loading doses of thienopyridines, and the use of glycoprotein IIb/IIIa antagonists in the setting of acute coronary syndromes led to a further decline in stent thrombosis. Although guidelines recommend one-year use of dual antiplatelet therapy, the optimal length of DAPT in these patients is still a matter of debate90. At present, in fact, there are no direct comparative randomized trials investigating the required duration of clopidogrel use after DES implantation. In addition, the lack of a truly compelling explanation for late stent thrombosis still hampers the attempt of defining optimal duration of dual antiplatelet therapy. Current US Food and Drug Administration-approved indications for the use of clopidogrel following drug eluting stent placement call for 3 to 6 months of dual antiplatelet therapy based on the type of stent implanted. A prospective observational study from Airoldi et al. on 3,021 patients (5389 lesions) treated with DES supported this statement15. In this study, the proportion of patients on dual antiplatelet therapy at 30 days, 6 months, 12 months and 18 months was 97.6%, 80.7%, 42.8% and 23.2%, respectively. During the first 6 months, stent thrombosis was more common among patients not on dual antiplatelet therapy compared with those compliant to the prescribed regimen (4.2% versus 0.9% at 30 days and 0.8% versus 0.3% between 30 and 180 days). Conversely, after 6 months, there was a numerical increase in the

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proportion of patients experiencing stent thrombosis while on dual antiplatelet therapy (0.2% versus 0.4% between 180 and 360 days and 0.1% versus 0.4% between 360 and 540 days). The median time from discontinuation of clopidogrel and stent thrombosis was 13.5 days during the first 6 months and 90 days thereafter, suggesting different existing mechanisms of subacute and late thrombosis15. Indeed, similar findings were observed in other small scale registries91,92. However, despite these provocative results, several evidences have shown over time that DES use is associated with a continuum late increased risk of stent thrombosis than the use of BMS, leading to the argument that clopidogrel intake should be protracted beyond 6 months, ideally up to 12 months45,9395. The most recent 2009 focused updated US guidelines have modified this class I, level of evidence B recommendation introducing prasugrel90, a third generation thienopyridine with more potent antiplatelet effects compared to clopidogrel investigated in the pivotal phase III TRITON TIMI-38 (Trial to assess Improvement in Therapeutic Outcomes by optimizing platelet Inhibition with prasugrel Thrombolysis In Myocardial Infarction 38) trial, as a valid alternative to clopidogrel in patients with acute coronary syndromes undergoing PCI96,97. Notably, in this trial, curves of stent thrombosis rates continue to separate over time (up to 15 months). Parallel findings on stent thrombosis (up to 12 months) were observed with ticagrelor, also a novel and more potent antiplatelet agent compared with clopidogrel, but forming part of a different chemical class in the large scale phase III PLATO (PLATelet inhibition and patient Outcomes) trial98. Cumulatively, these data support the use of DAPT in PCI patients for at least 12-15 months as indicated in current guidelines. The use of DAPT beyond this time frame remains as a class II B recommendation in selected patients receiving a DES who are not at high risk for bleeding90, somewhat underlying an individualized use of this strategy based on patient risk profile and supporting the need for more evidence to prolong dual antiplatelet therapy use for more than one year. Many studies explored the impact of DAPT duration on stent thrombosis after DES implantation (Table 12.4).
Table 12.4. Overview of studies on duration of dual antiplatelet therapy after stent implantation Study/author Airoldi et al. Park et al. Schulz et al. Roy et al. Kimura et al. van Werum et al. Eisenstain et al. Ellis et al. Barr et al. REAL LATE and ZEST LATE N patients 3021 2873 6813 2889 10778 21009 4666 21000 749 2701 Follow-up 18 months 3 years 4 years 1 year 2 years 31 month 2 years 31 month 2 years 2 years Stent type DES DES DES DES SES DES and BMS DES SES DES and BMS DES Results DAPT reduced ST until 6 months after DES implantation No benefits of DAPT after 1 year from DES implantation DAPT reduced ST until 6 months after DES implantation DAPT reduced ST until 6 months after DES implantation DAPT reduced ST in each time point DAPT reduced ST until 6 months after DES implantation DAPT predicted death and MI after 2 years from DES implantation DAPT continued to reduce ST after 6 months from DES implantation DAPT continued to reduce ST after 6 months from DES implantation No benefits of DAPT after 1 year from DES implantation

DES= drug eluting stent; BMS= bare metal stent; ST= stent thrombosis; DAPT= dual antiplatelet therapy.

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185

Recently, Park and colleagues performed a combined analysis of two multicenter trials specifically designed to evaluate the efficacy of long DAPT on clinical outcome [REAL-LATE (Correlation of Clopidogrel Therapy Discontinuation in REAL-world Patients Treated with Drug-Eluting Stent Implantation and Late Coronary Arterial Thrombotic Events, NCT00484926) and the ZEST-LATE (Evaluation of the Long-term Safety After Zotarolimus-Eluting Stent, Sirolimus-Eluting Stent, or PacliTaxel-Eluting Stent Implantation for Coronary Lesions- Late Coronary Arterial Thrombotic Events, NCT00590174)]99. However, these trials present several limitations including the low number of patients enrolled (which determined statistical underpowered), the suspension of clopidogrel in 17% of patients and the large use of cilostazol through the study population. Further investigation will shed more light on the benefit of prolonging DAPT beyond 12 months, in addition to the evolving data on the bleeding risk with long-term clopidogrel and prasugrel use100.

IMPROVING BIOCOMPATIBILITY OF POLYMERS AND STENT


Current evidence suggest that both antiproliferative agents and stent polymers are likely candidates as causes of late and very late stent thrombosis associated with DES implantation. Thus, there is a need of developments aimed to improve biocompatibility of DES polymers which could reduce the risk of stent thrombosis. A number of non-degradable and biodegradable polymers have been proposed and tested for stent manufacturing and coating101104. To prevent stent thrombosis, BMS were initially coated with natural polymers such as the polysaccharide heparin and phosphorylcholine-based polymers105. Polyzene-F [bis(trifluoroethoxy)-phosphazene] (CeloNova Bio-Sciences, Newnan, Georgia) is an inorganic, high molecular weight, ultrapure polymer that can be used to coat multiple substrates and can be applied on stents as an 40-nm surface modification. Recent animal studies have shown that stents treated with a nanothin surface of Polyzene-F are effective in reducing in-stent restenosis, and when compared with uncoated stents, these coated stents have an optimal profile in terms of thromboresistance and inflammation and promote normal endothelial cell growth, reducing perioperative and post-procedural platelet activation and agglomeration106. The results of the first-in-man study with the use of new Catania Polyzene-F coated stents showed an excellent early and mid-term safety profile and high-level efficacy in 55 patients, without any case of stent thrombosis during a follow up of 12 month107. The good safety profile in term of late stent thrombosis achieved with this stent was confirmed by a single registry executed in 254 patients108. Recently, the existence of circulating endothelial progenitor cells (EPCs) has been identified as a key factor for re-endothelialization109. The EPC capture stent (Genous stent, OrbusNeich, Fort Lauderdale, FL) is a stent coated with murine monoclonal antihuman CD34 antibodies designed to attract circulating EPCs to rapidly establish a functional endothelial layer and promote healing. Its safety use in de novo coronary lesion and during acute myocardial infarction PCI has been proven over the years110. Currently the development of the fully bioabsorbable DES systems based on polymer materials or bioabsorbable metals are under clinical evaluation. Potential advantages of having the stent disappear from the treated site include reduced or abolished late stent thrombosis, improved lesion imaging with computed tomography or magnetic resonance, facilitation of repeat treatments (surgical or percutaneous) to the same site, restoration of vasomotion, and freedom from side-branch obstruction by struts and from strut fracture-induced thrombosis and restenosis. The first clinical experience with a biodegradable polymer stent was presented by Tamai et al., who reported on the immediate and 6-month results of the Igaki-Tamai stent from

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biodegradable poly-L-lactic acid (PLLA) in 15 patients (25 stents in 19 lesions)111. The zigzag helical coil design of this stent is balloon-expandable at 50C and self-expanding. The results were encouraging with a restenosis rate and target lesion revascularization rate of 10.5% after 6 months111. The fully absorbable everolimus-euting BVS scaffold (Abbott Vascular, Santa Clara CA, USA) was tested in the first-in-man ABSORB trial enrolling 30 patients with a single de novo coronary artery lesion112. Two year follow up showed: 1) complete absorption of polymeric struts (documented by IVUS and intra-coronary optical coherence tomography); 2) late enlargement of vessel lumen; 3) restoration of vasomotion and endhotelial function in some patients; 4) low two-year adverse cardiac events (3.4%) without any case of stent thrombosis112. However, these findings need to be confirmed in larger trials with more complex coronary lesions.

CONCLUSIONS
Although rare, stent thrombosis is a serious adverse event after PCI which is associated with high risk of MI and death. By far, late and very late stent thrombosis has emerged as a distinct clinical entity more germane to (at least the first-generation) DES than BMS. Delayed healing and impaired endothelialization together with chronic inflammation induced by the drug-polymer are the prevailing mechanisms of late and very late stent thrombosis. Immediate reperfusion, preferably by primary PCI, is the therapy of choice of stent thrombosis. Preventive measures such as careful attention to implantation details and uninterrupted dual antiplatelet therapy for at least 12 months may hamper this adverse event. Moreover, the fully bioabsorbable DES systems based on polymer materials or bioabsorbable metals currently test in early clinical trials could represent an alternative to permanent coronary stent implants and potentially could fully solve the problems associated with long-term biocompatibility of stents.

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First-in-Man 1-Year Clinical Outcomes of the Catania Coronary Stent System With Nanothin Polyzene-F in De Novo Native Coronary Artery Lesions: The ATLANTA (Assessment of The LAtest Non-Thrombogenic Angioplasty stent) Trial. JACC Carciovasc Intv 2009; 2: 197204. 108. Capodanno D, La Manna A, Di Salvo ME, Sanfilippo A, Corcos T, Tamburino C. Early and mid-term clinical outcomes with the CATANIA coronary stent system vs. bare metal stents in patients with coronary artery disease. Cardiovasc Revasc Med. 2009; 10: 216-20. 109. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964 7. 110. Aoki J, Serruys P, van Beusekom H, et al. Endothelial progenitor cell capture by stents coated with antibody against CD34: the HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal GrowthFirst In Man) Registry. J Am Coll Cardiol 2005; 45:1574-9. 111. Tamai K, Igaki E, Kyo K, et al. Initial and 6-month results of biodegradable poly-l-lactic acid coronary stents in humans. Circulation 2000;102: 399404. 112. Ormiston JA, Serruys PW, Regar E, et al. A bioabsorbable everolimus-eluting coronary stent system for patients with single de-novo coronary artery lesions (ABSORB): a prospective open-label trial. Lancet 2007; 371:899-907.

CHAPTER 13

RESTENOSIS IN CHRONIC TOTAL OCCLUSION PERCUTANEOUS CORONARY INTERVENTION


ALBERT E. ALAHMAR, KUSHAL PUJARA and ANTHONY H. GERSHLICK
Introduction .............................................................................................................................. PCI and Stenting in CTO.......................................................................................................... DES versus BMS in CTO......................................................................................................... Molecular Mechanisms of ISR ................................................................................................. Conclusions .............................................................................................................................. References ................................................................................................................................ 191 192 192 193 197 198

INTRODUCTION
Chronic total occlusion (CTO) of a coronary artery is present in almost one-third of patients with coronary artery disease (CAD)1. Furthermore, CTO accounts for 7.2% to 15.6% of all percutaneous coronary intervention (PCI) attempts2. In a consensus document, Stone et al., defined CTO as an estimated > 3-month-old obstruction of a native coronary artery without any luminal continuity and thrombolysis in myocardial infarction (TIMI) grade 0 flow or with minimal contrast penetration though the lesion without distal vessel opacification (TIMI grade 1 flow)35.

Figure 13.1. Right: Occluded right coronary artery (RCA) after sizable right ventricle branch. Left: Septal collaterals from left Cx and left anterior descending (LAD) artery to distal RCA.

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The majority of patients undergoing PCI for CTO have stable or progressive angina, whereas many asymptomatic patients with CTO are managed medically. Outcomes in patients with successful recanalization of CTO with PCI are more favorable in terms of alleviating anginal symptoms, improving left ventricular ejection fraction, reducing predisposition to ventricular arrhythmias, decreasing the need for coronary bypass graft surgery, and may prolong life6,7.

PCI AND STENTING IN CTO


Recently, CTO recanalization has received increased attention as one of the major frontiers of PCIs8,9. Some characteristics for stenting in CTO include the absence of endothelial cells and the exposure of deep plaque components at the site of stenting, the almost invariable presence of well-developed coronary collaterals which may explain why almost 5% of CTO are asymptomatic10, and the frequent need to stent longer than normal coronary segments11. Some of these issues were proposed during the BMS era to explain the high restenosis rates associated with stent implantation in CTO. Some of these features may remain unfavorable in the DES era in terms of achieving controlled strut endothelialization and adequate but not excessive coverage by neointima, an issue of major concern regarding late stent thrombosis (ST)12. Total occlusion recanalization has been shown to have time independent benefit in the British Columbia Cardiac Registry, in which attempted revascularization of CTO lesions accounted for >15% of all PCI procedures13. Among 1458 patients with CTOs, successful percutaneous revascularization was associated with increased survival and a reduced need for surgical revascularization over a 7-year follow-up period (both P<0.001). CTO success was associated with a 56% relative reduction in late mortality (hazard ratio 0.44; 95% CI, 0.30 to 0.64); It should be noted that this was a registry however. In the prospective Total Occlusion Angioplasty StudySociet Italiana di Cardiologia Invasiva (TOAST-GISE), successful PCI of a CTO (attempted in 390 lesions in 369 patients) was associated with a reduced 12-month incidence of cardiac death or MI (1.1% versus 7.2%; P=0.005), a reduced need for coronary artery bypass surgery (2.5% versus 15.7%; P<0.0001), and greater freedom from angina (88.7% versus 75.0%; P=0.008)14. In the overall study population, the only factor associated with enhanced 1-year event-free survival was successful CTO recanalization (odds ratio 0.24; P=0.018).

DES versus BMS IN CTO


It has been suggested that the opening of total coronary occlusions can be of benefit by restoring blood flow to an under-perfused, hibernating myocardium and thus improve symptoms and left ventricular function15,16. Successfully revascularized chronic total occlusions confer a survival advantage compared with failed revascularization17. In 1991, Meier18 stated that compared with other lesion types, total coronary occlusions, albeit a different animal, are of the same species and deserve improved percutaneous revascularization techniques. Since its introduction in 1977 by Andreas Grntzig19, percutaneous transluminal coronary angioplasty (PTCA) has become a popular procedure used to dilate atherosclerotic vessels to alleviate angina pectoris, eventually preventing myocardial infarction, or to revascularize the infarcted myocardium20. The major limitation of the long-term success of PTCA is restenosis, a pathological process provoking recurrent arterial narrowing at the site of the intervention. Restenotic lesions typically lack lipid deposits and grow during 4-6 months post-PTCA, unlike native atheromas, which normally accumulate high content of lipids and develop over longer time periods, typically years or decades21.

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Excessive restenosis leads to hemodynamically relevant vessel obstruction and eventually recurrence of clinical symptoms thus forcing target-vessel revascularization. Restenosis after conventional PTCA, which is mainly due to a combination of recoil, negative arterial remodeling, intimal hyperplasia, affected 2550% of patients. Currently, more than 90% of the percutaneous coronary interventions (PCI) are performed using metallic stents, which increase the safety of interventional revascularization and, by attenuating recoil and negative arterial remodeling, reduce the rates of restenosis to 1530%. By attacking the intimal over-proliferative repair process, In-stent restenosis is further reduced using drug-eluting stents22,23.

MOLECULAR MECHANISMS OF ISR


Neointimal hyperplasia is regarded as the main cause of ISR. This proliferative process can be considered as the arterial walls healing response to the acute mechanical injury provoked by stent deployment (e.g., injury of the endothelial cell lining, denudation, disruption of the lamina elastica, etc.). The acute early phase of ISR is characterized by the activation of platelets and ensuing thrombosis accompanied by the recruitment into the intimal area of blood-borne monocytes, neutrophils and lymphocytes. These cells engage in the production of a plethora of mitogenic and chemotactic factors which trigger a chronic inflammatory response leading to the activation of the smooth muscle cells (SMCs) residing in the tunica media, which then undergo aberrant cell proliferation and migration toward the growing neointimal lesion. Moreover, activated SMCs exhibit a less (un)differentiated, so called synthetic phenotype featuring broader and flatter shape, expression of embryonic isoforms of contractile proteins, and abundant synthesis of extracellular matrix (ECM) components, unlike medial SMCs in normal adult arteries, which are fusiform and exhibit a differentiated, contractile phenotype characterized by the expression of contractile proteins and reduced proliferative and migratory activity. Although it is becoming increasingly evident that recruitment of bone marrow-derived and adventitial SMC progenitors and adventitial myofibroblasts also contribute to the accumulation of neointimal SMCs, their relative contribution to restenosis remains undefined. Putative regulators of neointimal hyperplasia include thrombogenic factors (e.g., thrombin receptor, tissue factor) cell adhesion molecules (e.g., VCAM, ICAM, LFA-1, Mac-1), transcription factors (e.g., NF-kB, E2F, p53, AP-1, c-myc, c-myb, YY1, Gax, IRF-1), signal transduction molecules (e.g., MEK/ERK, PI 3-kinase/Akt), inflammatory cytokines (e.g., TNFa), chemotactic factors (e.g., CCR2, MCP-1), growth factors (e.g., PDGF-BB, TGFb, FGF, IGF, EGF, VEGF), cell cycle regulators (e.g., CDK2, CDC2, cyclin B1, PCNA, p21, p27, pRb), and metalloproteases (e.g., MMP-2, MMP-9). At later stages post-PCI characterized by the resolution of inflammation and wound healing, neointimal SMCs return to a contractile phenotype characterized by low proliferative and migratory activity and production of ECM components to more closely resemble the undamaged arterial wall2429. When compared with conventional bare metal stents (BMS), the introduction of drug-eluting stents (DES) has resulted in a substantial reduction in the incidence of in-stent restenosis30. DES systems eluting either sirolimus31 or paclitaxel32 from a polymer stent coating have been shown in randomized trials to effectively inhibit the process of neointimal proliferation resulting in restenosis reduction. Indeed, nonrandomized studies suggest a benefit of drug-eluting stent implantation over bare metal stent implantation in the treatment of total coronary occlusions, leading to more patients treated with PCIs rather than coronary artery bypass graft surgery.

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Figure 13.2. Molecular structure of sirolimus, everolimus, biolimus, zotarolimus, tacrolimus, and pimecrolimus [Alahmar, Gershlick U S Cardiology Vol 6 :2009].

Werner et al.,33 investigated the effect of paclitaxel-eluting stents in chronic total occlusions in 48 patients and compared them with 48 matched patients with chronic total occlusions treated with a bare metal stent. The angiographic restenosis rate in this study was 8.3% with paclitaxel versus 51.1% with bare metal stents (P<0.001), with a higher survival free of major adverse cardiac events in the paclitaxel group due to a reduced target lesion revascularization rate (12.4% versus 47.9%; P<0.001). In a nonrandomized trial, Nakamura et al.,34 found lower rates of restenosis, showing 2% in 60 patients who underwent sirolimus stent implantation versus 32% in 120 patients who received a bare metal stent (P=0.001), with a target lesion revascularization rate of 2% versus 23% (P=0.001) and a target vessel revascularization rate of 3% versus 30% (P=0.001), respectively. In another nonrandomized study, Ge et al.,35 found restenosis rates of 9.2% after sirolimus stent implantation and 33.3% after bare metal stent implantation (P<0.001), with a target lesion revascularization rate of 7.4% versus 26.3% (<0.001) and a target vessel revascularization rate of 9.0% versus 29.0% (P<0.001), respectively. In the Sirolimus-Eluting Stent in Chronic Total Occlusion (SICTO) study, an in-lesion late luminal loss of 0.150.39 mm was found in 25 patients with chronic total occlusions >3 months old treated with a sirolimus-eluting stent and showed a target lesion revascularization rate of 4% and a target vessel revascularization rate of 12% at 12-month follow-up36. PRISON II randomized trial (2005)37, essentially addressed a primary end point of angiographic binary restenosis at six months and secondary end points of MACE, target vessel failure (TVF), in-stent and in-segment mean luminal diameter (MLD), late lumen loss, late-loss

CHAPTER 13. CHRONIC TOTAL OCCLUSION PERCUTANEOUS CORONARY INTERVENTION 195

index, and percent diameter stenosis at six months. A total of 200 patients were randomized to a bare BX Velocity stent or to the Cypher sirolimus-eluting stent. The clinical event rate was higher in the bare-metal-stent group, driven primarily by target lesion revascularization (TLR) and target vessel failure. Six-month angiographic follow-up also pointed to significant differences between the two stents. Notably, in-segment and in-stent restenosis, as well as reocclusions, were significantly higher in the bare-metal-stent group (Table 13.1.)
Table 13.1 End point MACE* (%) TVF** (%) % diameter stenosis, in-stent % diameter stenosis, in-segment Late loss, in-stent (mm) Late loss, in-segment (mm) Binary restenosis (in-stent) (%) Binary restenosis (in-segment) (%) Reocclusions (%) Bare-metal stent 20 24 48.75 53.32 1.09 0.64 41 36 13 Cypher 4 8 22.01 31.85 0.05 -0.07 11 7 4 p <0.001 0.003 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.001 <0.04

* MACE=cardiac death, MI, ischemia-driven TLR.

Although PRISON II trial was criticized as it recruited relatively low risk CTO patients (lower diabetic, lower renal impairment), the authors conclusion was that as compared with bare-metal stents, the Cypher sirolimus-eluting stent implantation in CTO is superior, with a significant reduction in binary in-segment and in-stent restenosis, resulting in a significant reduction in TLR and [target vessel revascularization] TVR. At 3 years38, DES in PRISON II continued to be superior to BMS in terms of target vessel revascularization (TVR 7% versus 27% p<0.001 respectively). There were no statistically significant differences in death, myocardial infarction, and stent thrombosis according to the Academic Research Consortium criteria between the 2 groups. In recent meta-analysis April 201039, Humberto et al. compared the efficacy and safety of DES and BMS implantation in 4,394 patients underwent CTO PCI in 14 comparative studies. Major cardiac events were reduced significantly in the DES arm compared to the BMS arm (relative risk [RR]: 0.45, 95% confidence interval [CI]: 0.34 to 0.60, p < 0.001), (Figure 13.3). This was primarily driven by the revascularization rate TVR (RR: 0.40, 95% CI: 0.28 to 0.58, p < 0.001), (Figure 13.4). Although there was strong trend toward a higher rate of stent thrombosis was documented in DES-treated patients (RR: 2.79, 95% CI: 0.98 to 7.97, p = 0.06), there was no significant difference in death (RR: 0.87, 95% CI: 0.66 to 1.16, p = 0.88) or myocardial infarction (RR: 0.89, 95% CI: 0.54 to 1.46, p = 0.80), (Figure 13.5). This benefit was sustained at 3 years of follow-up.

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Figure 13.3. RR of a MACE and Target Vessel Revascularization Associated With DES and BMS Use [Humberto et al. J Am Coll Cardiol 2010].

Figure 13.4. RR of a MACE and TVR Associated With DES and BMS Use: Stratified Analysis According to Follow-Up Length [Humberto et al. J Am Coll Cardiol 2010].

CHAPTER 13. CHRONIC TOTAL OCCLUSION PERCUTANEOUS CORONARY INTERVENTION 197

Figure 13.5. RR of Myocardial Infarction and Death Associated With DES and BMS Use [Humberto et al. J Am Coll Cardiol 2010].

It is interesting to note that the observed 11.71% TVR rate associated with DES is virtually identical to that reported by other authors in other off-label DES indications40. Because TVR is a clinical end point, driven mainly by the reappearance of angina, the similarity with non-CTO patients treated with DES might seem paradoxical, given that occluded vessels usually have more collateral support that might attenuate or even abolish anginal symptoms. Potential explanations include the reported regression of collateral support after CTO recanalization41 and the inclusion of a significant number of studies performing angiographic follow-up, which has been shown repeatedly to promote reinterventions42.

CONCLUSIONS
Chronic total occlusion PCI has been underused due to perceived poor success rates. Recent promotion of the value of the procedure (courses, meetings, educational websites) and major changes in the development of techniques and equipments have lead to CTO treatment becoming an achievable challenge for most operators, although techniques such as retrograde approach should remain in the hands of the most experienced. Most studies to date have focused on the need to achieve acute success and have highlighted how much better patients do compared to those in whom CTO has not been acutely successful. However, with increasing likelihood of acute success attention somewhat have turned to the longer term outcomes. Furthermore, in addition to the fact that the use of DES appears to be mandatory, the late stent thrombosis, albeit without excess mortality highlighted in recent meta-analysis needs watching. The real issue is that there have been no randomized trials in patients with CTO who have or have not been treated invasively. The EuroCTO club have for some two years been attempting to secure funding for such a trial. This trial is closed to being initiated.

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Hopefully we will see the results of this trial in the coming years, in the meantime from the available data it would appear that the DES are effective without any significant increase of the risk of death or myocardial infarction and should all other things being equal (e.g. patients ability to take long term dual antiplatelets) be the stent of choice. Good acute results will always lead to improved outcomes in all PCIs. With its coming frontiers (devices, techniques, expertise) has shown significant improvement in success rate and current figure reaches almost 85%43. Drug-eluting stents in this rather different animal PCI have shown similar rates of restenosis to the non CTO rates indicating that DES are effective without any significant increase in the risk of death or myocardial infarction.

REFERENCES
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Colmenarez, Javier Escaned, Cristina Fernndez, Liliana Lobo, Sara Cano, Juan G. del Angel, Fernando Alfonso, Pilar Jimenez, Camino Bauelos, Nieves Gonzalo, Eulogio Garca, Rosana Hernndez, and Carlos Macaya.Efficacy and Safety of Drug-Eluting Stents in Chronic Total Coronary Occlusion Recanalization: A Systematic Review and Meta-Analysis .J Am Coll Cardiol 2010 55: 1854-1866.

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40. Ko DT, Chiu M, Guo H, et al. Safety and effectiveness of drug-eluting and bare-metal stents for patients with off- and on-label indications J Am Coll Cardiol 2009; 53:1773-1782. 41. Werner GS, Bahrmann P, Mutschke O, et al. Determinants of target vessel failure in chronic total coronary occlusions after stent implantation. The influence of collateral function and coronary hemodynamics. J Am Coll Cardiol 2003;16; 42219-25. 42. Ten Berg JM, Kelder JC, Suttorp MJ, Verheugt FW, Thijs Plokker HW. Influence of planned six-month follow-up angiography on late outcome after percutaneous coronary intervention: a randomized study J Am Coll Cardiol 2001; 38:1061-1069. 43. Rathore S, Katoh O, Matsuo H, Terashima M, Tanaka N, Kinoshita Y, Kimura M, Tsuchikane E, Nasu K, Ehara M, Asakura K, Asakura Y, Suzuki T. Retrograde percutaneous recanalization of chronic total occlusion of the coronary arteries: procedural outcomes and predictors of success in contemporary practice. Circ Cardiovasc Interv. 2009 Apr.; 2(2):124-32.

CHAPTER 14

RELATION BETWEEN CORONARY BIFURCATION AND RESTENOSIS


A. STENT RESTENOSIS IN BIFURCATION LESIONS FROM CLINICAL TRIALS SHINICHIRO YAMADA

Introduction............................................................................................................................... Bifurcation Lesion Classifications ............................................................................................ Drug Eluting Stent versus Bare Metal Stent.............................................................................. Classification of Bifurcation Stenting Techniques in DES Era ................................................. Single Main Branch DES versus 2 DES Approach ................................................................... 2-DES Approach ....................................................................................................................... Left Main Trunk Distal Bifurcation Lesions ............................................................................. How to Evaluate Clinical Significance of Side Branch Stenosis............................................... Conclusions...............................................................................................................................

201 202 202 203 204 205 205 206 208

INTRODUCTION
Atherosclerosis is frequently developed at the lateral area of the bifurcation where turbulence of coronary flow favors to occur with low endothelial shear stress. Since coronary arteries have numerous bifurcations, bifurcation lesions accounts for 15% to 20% of all coronary artery disease treated by percutaneous coronary intervention (PCI). Initial clinical results with plain old balloon angioplasty were miserable with a high risk of acute occlusion of the treated branches and a high restenosis rate1. Introduction of coronary stents brought more predictable results with higher success rate, however, angiographic restenosis rates remained high2. Despite of the introduction of drug-eluting stent (DES), bifurcation lesions have remained a significant challenge in the aspect of high frequencies in restenosis and stent thrombosis. However, changing in the strategy of bifurcation PCI in the last several years has improved its clinical outcome considerably3.

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BIFURCATION LESION CLASSIFICATIONS


Currently, 5 major bifurcation lesion classifications have been published47. In the first 4 classifications, capital letters or Roman numerals are used for categorizing various bifurcation lesion types. These classifications are not relevant in the present days because it is lack of association between the numbers or letters and various anatomic abnormalities of bifurcation lesions. Medina classification proposed in 20067 is simpler and easier to remember and understand. It classified by the presence or absence of disease in the proximal main branch, distal main branch and side branch. It was slightly modified in 2008 corresponding to the pathological finding that there was little atherosclerotic lesion in the carina (Figure 14.1). The European Bifurcation Club recommends the Medina classification because of its simplicity8.

1,1,1

1,1,0

1,0,1

0,1,1

1,0,0

0,1,0
Figure 13.1. Medina Classification.

0,0,1

DRUG-ELUTING STENT VERSUS BARE METAL STENT


The use of DES reduced the rate of restenosis compared with bare metal stents (BMS)9,10. However, there were a few randomized controlled trials comparing DES to BMS which included the bifurcation lesion. In a subanalysis of the SCANDSTENT (Stenting Coronary Arteries in Non-Stress / Benestent Disease) trial, which evaluated 126 bifurcation lesions treated by sirolimus-eluting stents or BMSs11, restenosis rates were dramatically reduced in both main branch (4.9% versus 28.3%, p<0.001) and side branch (14.8% versus 43.8%, p<0.001). Similarly, other non-randomized studies have demonstrated reduction in restenosis rates compared to BMS controls12,13. According to these studies, DES implantation has become

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the first-line strategy for bifurcation lesions except the cases of contraindication of prolonged dual antiplatelet therapy.

CLASSIFICATION OF BIFURCATION STENTING TECHNIQUES IN DES ERA


In 2008, Louvard et al. published the classification of the bifurcation stenting techniques14 (MADS: Main, Across, Distal, Side). In this classification, an accurate definition of each of the numerous stenting techniques used combined with a classification to facilitate their description was proposed. In the first family (M: Main), stent is deployed in the proximal main vessel at first (PMV) and this procedure may be followed by the opening of the stent towards both branches (SKIRT), with subsequent stenting in one or both distal branches (extended Y). In the second family (A: Across), stenting is firstly performed from proximal to distal main branch across the side branch. In this strategy, provisional side branch stenting may be added (Internal crush, Culotte, TAP). The third family (D: Distal) involves distal main branches, and starts with simultaneous stent placement at the ostium of both distal branches (V). Recently variant method which creates new carina by simultaneous 2 stents deployment in the proximal segment (Simultaneous Kissing Stent: SKS). The forth family (S: Side), stent is deployed at side branch first, either at ostium (SB ostial stenting) or sometimes pronounced protrusion into the proximal main vessel. The side branch stent may be crushed by balloon (SB crush, SB mini crush) or additional stent (Crush, Mini crush) in the main vessel. Stankovic et al. reviewed the advantages and disadvantages of each stenting technique in MADS classification8 (Table 14.1).
Table 14.1. Advantage and disadvantage of each stenting technique according to MADS classification Stenting technique M family Advantages Maintains SB access Low risk of proximal dissection Treatment flexibility for complex anatomy Stent number, Easy extension of a provisional one stent techniques to two stents Good SB scaffolding with angles > 70 6 Fr compatible, Angle independent, Allows different diameter SB/MV, Predictable stent scaffolding Easy and quick technique No risk of SB occlusion Less hemodynamic instability Good for emergency situations Procedure is comprehensively simple, Low risk of SB occlusion Disadvantages Stent number Requires high precision stent placement Risk of large metallic carina Gap at SB ostium (need distal MB stent strut access) Stent protrusion in the MV Two layers stent proximally Demanding in terms of time, material, concentration of the operator/team, Risk of losing SB or MB access Guiding catheter > 7 Fr Longitudinal stent overlap and metallic carina (flow disturbance, thrombogeneticity, restenosis) Over dilatation in proximal MV, Gap between the two stents, Difficulty in additional treatment (proximal stenting, restenosis) Difficult SB positioning Difficult SB wiring (3 layers of stents) Difficult to optimize SB ostium dilatation

A family Provisional T-stenting

Provisional Culotte stenting

D family

S family

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SINGLE MAIN BRANCH DES VERSUS 2 DES APPROACH


There had been considerable debate about the priority between single main branch (MB) stenting and 2-stent approach in the true bifurcation lesion. However, recent clinical trials have revealed that provisional strategy is feasible for the majority of bifurcation lesions. There have been 5 large randomized studies1519 that compare a provisional approach of 1-DES stenting in the MB versus approach of 2-DES stenting in both MB and side branch (SB) (Table 14.2).
Table 14.2. Randomized studies comparing provisional stenting to both branch stenting with DES Author, Year SIRIUS Bifurcation; Colombo et al., 2004 NORDIC Steigen et al., 2004 BBK; Ferenc et al., 2008 CACTUS; Colombo et al., 2007 BBC; Hildick-Smith et al., 2010 No. of Patients 1-Stent 2-Stent Angiographic Group Group Follow-up Restenosis, Restenosis, % (%) % MB / SB MB / SB 84 4.8 / 14.2 5.7 / 21.8 Provisional Stenting If

Strategy

Both branches vs. provisional stenting Both branch vs. provisional stenting T stenting vs. provisional stenting Crush vs. provisional stenting Crush, culotte vs. provisional stenting

85

>50% residual stenosis TIMI flow=0 after SB dilation >60% stenosis and/or flow-limiting dissection >50% stenosis and/or flow-limiting dissection TIMI<3 and/or >70% stenosis and/or dissection >type A

91

73

4.6 / 19.2

5.1 / 11.5

202

95

7.3 / 9.4

3.1 / 12.5

350

86

6.7 / 14.7

4.6 / 13.2

500

N/A

N/A

N/A

Among these studies, no advantage of 2-DES strategy was observed over a provisional strategy in the aspect of restenosis. Furthermore, there were no differences between those two strategies in death, myocardial infarction, target lesion revascularization (TLR), or composite major adverse cardiac events (MACE). Furthermore, complex procedure of 2-DES strategy increased contrast, fluoroscopy time and the prevalence of periprocedural myocardial infarction19. Since the criteria to cross-over from 1-DES to 2-DES deployment varied among these trials, the actual rate of cross-over also varied. In Sirius Bifurcation study15, residual stenosis of > 50% in SB was defined suboptimal and it had led to more than 50% of cross-over rate. On the other hand, in Nordic study16, SB had just to remain patency with TIMI flow grade > 0. As a result, only 5% of the cases in each randomized groups crossed over to the unassigned strategy. In CACTUS (Coronary Bifurcation Application of the Crush Technique Using Sirolimus-Eluting Stents) study18, 350 patents were randomized to a single MB stent or the mini-crush technique, and 2-stent approach were arrowed with >50% residual SB stenosis, >grade C dissection, or <TIMI 3 flow, resulting in the 31% of the provisional group received SB stenting. The prevalence was much higher than that in BBK study (19%)17 and BBC (British Bifurcation Coronary) study (3%)19. Although the 2-stent crush approach did not show any advantage to a single MB stent in CACTUS study, there was no increased risk of mid-term MACE including TLR at 6 months18. Therefore 2-DES strategy is acceptable if it is required in the true lesion which diffuse plaque is extended in the SB.

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2-DES APPROACH
When rich plaque burden is extended diffusely (> 5 mm longitudinal) in the large-size (> 2.5 mm diameter) SB, 2-DES approach could be an effective option to obtain optimal initial and long-term result. The large-scale studies in which 2-DES strategy was used for bifurcation lesions are summarized in Table 14.3 20-23.
Table 14.3. Studies Evaluating 2-DES in bifurcation lesions with both MB and SB Author, Year Ge et al. 2004 Hoye et al. 2006 Ge et al. 2007 NORDIC II Erglis et al. 2009 Strategy Crush with FKI vs. without FKI Crush (observational) Crush vs. T Culotte vs. Crush No. of Patients 181 241 182 424 Follow-up, (%) 80 77 78 88 Restenosis, % MB / SB FKI 8.9 / 11.1 FKI 6.4 / 9.6 Crush (n=121) 16.2 / 19.2 Crush (n=209) 4.7 / 9.2 Restenosis, % MB / SB No FKI 15.5 / 37.9 No FKI 10 / 41.3 T stenting (n=61) 13 / 26.1 Cullote (n=215) 2.0 / 4.5

These studies imply that optimal selection and performance of the 2-stenting technique improves clinical outcomes. Ge et al.20 and Hoye et al.21 have reported the importance of final kissing inflation (FKI) in crush stenting technique in reducing the late loss and restenosis especially at the SB. FKI had become the indispensable performance of all 2-DES technique. There are data suggesting that bifurcation angle between MB and SB plays an important role on clinical outcomes8. Dzavik et al. reported that high bifurcation angle (>50 degree) of SB is associated with increased rates of MACE after crush stenting (22.7% versus 6.2%; p=0.0007)24. This group extended these observations to culotte stenting25. Nordic II study is only the large scale randomized trial which compared the 2 different 2-DES techniques (Culotte versus Crush)23. Although there was no significant difference in 6-month clinical outcomes, there was a trend toward lower restenosis rates in the SB in culotte stent group. However, it was limited by less frequent FKI in the crush group. Therefore, still few data have demonstrated the superiority of either type of the 2-DES approaches.

LEFT MAIN TRUNK DISTAL BIFURCATION LESIONS


In PCI for left main trunk (LMT) disease, low rate of restenosis and TLR after DES implantation comparable to bypass surgery was reported in a randomized study26. However, LMT distal bifurcation lesion is an independent predictor of restenosis and MACE. Valgimigli et al. reported that cumulative incidence of target vessel revascularization was significantly higher in patients with LMT distal bifurcation disease compared with those without bifurcation (13% versus 3%, p=0.02)27. The impact of a 2-stent strategy was reported from the distal left main registry of the GISE-SICI (Groppo Italiano Studi EmodinamiciSocieta Italiana di Cardiologia Invasia) study28. In this observational study of 773 patients, the adjusted survival free from MACE during 2-year follow-up was significantly higher in patients treated by 1-DES strategy compared with 2-DES. Comparing patients who received 1-DES (n=456) with those who received 2-DES (n=317), the propensity adjusted hazard ratio for the risk of 2-year MACE was 0.53 (95% confidence interval 0.370.76). Other registry of PCI for unprotected LMT lesions also reported higher incidence of TLR and cardiac death in 2-DES strategy29 (Table 14.4).

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PART I. RESTENOSIS NEW DISEASE Table 14.4. Incidence of MACE during follow-up period. From two large unprotected left main trunk registry

Author, Year GISE-SICI Palmerini et al. 2009 J-Cypher Toyofuku et al. 2009

No. of Patients 773 582

Follow-up (%) 2 years 3 years

1-DES group MACE (%) 13.0 30.9

2-DES group MACE (%) 26.9 11.1

P value < 0.0001 < 0.0001

These studies suggested that systematic 2-DES strategy appeared unacceptable for LMT bifurcation lesions. In contrast, in French Left Main Taxus Registry30, which included 78% patients who had distal LMT disease, provisional SB T-stenting was performed in 92%, and final kissing balloon inflation in 97% of LMT bifurcation lesions. Angiographic binary restenosis was observed in 13%, and 80% of restenosis lesions was focal. Given the potentially serious consequences of restenosis after LM stenting, it is reassuring that 65% of patients with restenosis were asymptomatic. The more benign form of restenosis observed with DES may explain the relatively low rate of cardiac death and MACE (3.1% and 12.2% at 1 year follow-up, respectively). This study concluded that unprotected LMT stenting with paclitaxel-eluting stents, with a strategy of provisional side-branch T-stenting for distal lesions, provides excellent acute angiographic results and good mid-term clinical outcomes, with a 15.8% rate of MACE at 2-year follow-up.

HOW TO EVALUATE CLINICAL SIGNIFICANCE OF SIDE BRANCH STENOSIS


Bifurcation PCI includes some potential risks in the SB (e.g., jailed strut, acute occlusion, dissection and subsequent biomarker elevation indicative of myocardial infarction). It also leads to the risk of lower long-term successful results (Figure 14.2).
%

BMS

DES

Figure 14.2. Angiographic restenosis rates at SB.

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Although the provisional SB stent strategy is preferred in most bifurcation lesions, operators should consider the necessity of additional balloon angioplasty or stenting in the jailed SB narrowing in the angiography. However, accuracy of angiographic evaluation alone is sometimes inadequate and does not reflect the functional severity especially in short ostial lesions. Angiographic overestimation due to various factors (e.g., angulation, branch overlap, imaging artifact) has led to additional procedure on SB lesions without physiologically significant ischemia. Fractional flow reserve (FFR) is an easily obtainable, lesion specific physiological parameter of epicardial coronary artery stenosis31. SB evaluation using FFR is performed by passing a pressure sensor FFR-guidewire through the jailed MB stent strut. Hyperemia is induced by intracoronary bolus or intravenous adenosine infusion and FFR is measured at least 5 mm distal of SB ostium. Wire is then pulled back and FFR is measured again in MB to evaluate the influence of proximal lesion32. Koo et al. reported that FFR evaluation of jailed SB lesions is feasible and angiographic evaluation overestimates the severity of these lesions32,33. In their study, angiographic criteria of 75% stenosis would have resulted in 68, 32, and 48% of the lesions requiring additional SB intervention after MB stent implantation, after kissing balloon dilatation and at follow-up, respectively. However, these were reduced to 31, 8, and 8% with the application of the functional criteria of FFR <0.75 (Figure 14.3)32. Measurement of FFR is therefore an accurate way to decide the indication of ischemia-driven intervention for jailed SB. If, after MB stent deployment, there is a >70% angiographic stenosis in the SB ostium, then FFR plays an important role in further management. If FFR is >0.75, no further treatment is necessary, whereas if FFR is <0.75 the treatment of kissing balloon dilatation with provisional SB stenting is recommended until the establishment of an FFR >0.75. An effort to limit stenting in SB with focal disease using a provisional stenting strategy would appear warranted. The decision of SB intervention according to FFR can avoid unnecessary procedure to reduce PCI-related adverse events.

Figure 14.3. The percentage at each step of provisional side-branch intervention strategy according to angiography and FFR.

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CONCLUSIONS
In the last decade, improvements have occurred in the treatment of bifurcation lesions. Numerous clinical trials for bifurcation lesions have demonstrated: 1) efficacy of drug-eluting stent; 2) provisional approach for the majority, and selective usage of complex 2-stents strategy as intention-to-treat; 3) acceptance of suboptimal results in relatively small side branch; 4) physiological assessment for provisional side branch intervention. As a result, clinical outcomes including restenosis and target lesions revascularization after bifurcation PCI have markedly improved, and currently, approximate those of non-bifurcation PCI. However, PCI for bifurcation remains challenging especially in complex lesions. Bifurcation lesions vary in plaque burden, location of plaque, angulation between branches, and diameter of branches. There are no 2 identical bifurcation lesions. Therefore, the most important issue in bifurcation PCI is the selection of appropriate strategy for an individual lesion. Although numerous techniques have been proposed for complex bifurcation lesions, no method completely resolve the limitations of the current stent platforms. Dedicated bifurcation devices which can simplify the procedure are hopeful in the future technology.

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B. INSIGHT FROM BENCH STUDY

YOSHINOBU MURASATO
Introduction............................................................................................................................... Can 2-Stent Technique Provide Complete DES Coverage of Coronary Bifurcation? ............. Kissing Ballon Inflation Technique (KBT)............................................................................... Jailed Side Branch..................................................................................................................... Polymer Damage....................................................................................................................... Rheological Assessment .......................................................................................................... Limitation of Bench Test .......................................................................................................... Conclusion ................................................................................................................................ 209 210 215 216 216 216 218 218

INTRODUCTION
Since the introduction of drug-eluting stent (DES), restenosis and target lesion revascularization (TLR) have been also reduced in the bifurcation lesion. Although bifurcation is prominent risk factor of restenosis in the bare metal stent (BMS) era34, ARTS II trial demonstrated that there was no significant difference between bifurcation and non-bifurcation lesions in the rate of revascularization at 1-year follow up after sirolimus-eluting stent (SES) implantation3. However, high restenosis rate in the side branch (SB) has been still demonstrated in other randomized clinical trials with high percentage (922%) of follow up angiography1518. In the SYNTAX study which was a randomized study comparing the paclitaxel-eluting stent (PES) treated group and the bypass surgery group in the clinical outcome of the coronary artery disease combined with left main and/or three-vessel disease, the subgroup including any bi- or trifurcation lesion demonstrated worse TLR in the PES-treated group compared to the surgery group35. The bifurcation lesion has been still an Achilles heel of restenosis even in the DES era. Recent randomized studies which compared elective 2-stent with provisional 1-stent techniques revealed that no advantage was found in the aspect of restenosis in each branch15-19. The reason why the DES has not yet overcome the SB restenosis attributes to its unique anatomical and physiological characteristics. The bifurcation lesion has the following unmet needs for the complete revascularization. In the SB-related aspect, differential profile and bifurcation angle should be considered to maintain SB access. In the ostium-related aspect, elliptical shape of SB ostium is more prominent according to the degree of narrowness of bifurcation angle. The compromise of the healthy SB after main vessel (MV) stenting sometimes requires the intervention. The restenosis at the bifurcation is also related to distorted strut due to the complex procedure, excess strut layers in the 2-stent technique, gap in scaffolding, polymer injury and unpredictable drug release due to implant injury36. Using the high quality imaging devices, the previous bench testing studies have clearly revealed these aspects36-45. Real phantom model provides us clinically useful and available information with the reproducibility.

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CAN 2-STENT TECHNIQUE PROVIDE COMPLETE DES COVERAGE OF CORONARY BIFURCATION? T-Stenting
In the first randomized trial using SES for the bifurcation, disappointing result for the SB was revealed in the 2-stenting group in which T-stenting was mainly used. The binary restenosis of SB were 24.0% in the SB stenting group and 18.7% in the 1-stent group15. In addition to the technical difficulty in precise positioning of the stent at the SB ostium, oblique extension of the proximal SB in the narrow-angled bifurcation cannot be covered completely by the stent with this technique45. In order to overcome this potential risk of gap formation in the SB ostium, modified-T stenting was proposed, which the SB stent was deployed firstly with its proximal site minimally protruded in the MV. In the modified-T stenting, the protruded site of the SB stent was compressed by the MV balloon for the successive MV stenting followed by kissing balloon inflation technique (KBT). However, there is a possibility of excessive protruding of the SB stent which results in the same course of mini-crush stenting containing a 3-layer metal overlapping43. The inappropriate re-cross of the guide wire through the outside of the SB stent may cause more deformation of the stent by the KBT36,38. The differentiation whether the position of re-crossed guide wire is proper or not is sometimes difficult in the angiography or even in the intra-vascular ultrasound (IVUS)36,38. T-stenting and protrusion (TAP) technique was a provisional SB stenting with minimal protrusion followed by KBT to diminish the risk of gap formation in the SB ostium46. It was demonstrated that the restenosis was related to the SB ostial expansion47 and neointimal proliferation was more prominent by approximately 150% increase at the SB ostium than at the MV and SB distal stent. The optimal threshold of post-procedural minimal stent area was 4.83 mm2 at the SB ostium to predict minimal lumen area 4 mm2 at the 9-month follow-up period. Bench study revealed the limitation of the expansion of the SB ostium when the closed-cell stent was used in the MV stent36,38 (Figure 14.4). It was also suggested that the excessive protrusion of the SB stent into the MV stent have the risk which might result in the flow disturbance at the bifurcation48.

Crush Stenting
The most beneficial point of this stenting is to secure the patency of the diseased SB. However, the protruded SB stent is crushed to a 3-layer metal overlapping in the proximal MV. The clinical IVUS study49 and animal experiment50 reported the stent malapposition at the SB ostium, thrombogenic metal overlapping and incomplete crush at the proximal MV. The bench testing revealed that bifurcation angle had a great impact on the stent apposition to the SB ostium36-39,41,43,44. Final KBT improved the apposition in the narrow-angled bifurcation, whereas there remained an unstented area at the distal carinal site of the SB even after KBT in the high-angled bifurcation43. In the 3-dimentinal left main coronary artery (LMCA) bifurcation model, which had the bifurcation angle between LAD and LCX of 90 degree with the angles from LMCA to each branch equally divided, there also remained unstented area at the SB ostium36-38,41 (Figures 14.5, 14.6). The cross sectional images at the distal bifurcation demonstrated a wedge-shaped gap between the stents and the absence of the struts36 (Figure 14.5). Clinically, minimizing the crushed site (mini-crush)51, first KBT following the crush of the SB stent by the MV balloon in addition to usual final KBT (double kiss and crush, DK crush)52, and two-step balloon dilation which high-pressure SB ballooning was performed

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A
a b

D LCX LAD G

a b

H E F

Figure 14.4. MFCT images of T-stenting and protrusion (TAP) in the 3-D LMCA bifurcation model. Experiments were
performed using Driver (upper panels) and Bx Velocity stents (lower panels) in the MV. (A) (E) Long axis 3D image. (B) (F) Cross sectional view at the distal LMCA. Blue and red lines indicate the LCX and the LAD stents, respectively. (C) (G) Cross sectional view corresponding to the line a in the 3-D image. The squeezing of the LCX stent at the strut where the LCX stent was protruded into the LMCA was small in the panel C, whereas it was apparent in the panel G (arrows). (D) (H) Cross sectional view corresponding to the line b. There was a gap at the distal carina in the panel H (arrow) (From reference [36]).

abcd

B(1)
a

A B(2)

Figure 14.5 A. Micro focus CT images of crush stenting using Express II stents in the 3-dimentional LMCA bifurcation model. B. Magnified view. A relatively large gap was observed at the epicardial site of the LCX ostium (B2, encircled area). C. Cross sectional views at the indicated lines corresponding to those in the figure B1. The arrows indicate absence of the struts. (From reference [38]).

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before final KBT39 are suggested to acquire good apposition to the SB ostium and improvement of long-term results (Figure 14.7).

a b

C(1)

( 2) B a

d ( 3)

Figure 14.6 A. Micro focus CT images of the worst case of crush stenting with the guide wire recrossed through the distal site
of the LCX ostium. The LCX stent was crushed more distally after kissing balloon inflation (dotted arrows). B. Cross sectional view at the lines corresponding to those in Figure A. White arrows indicate the absence of the strut and the crushed stent was observed at the opposite site of the carina. C. Mechanism of this phenomenon. The guide wire and balloon were advanced outside the proximal segment of the crushed stent (1) and the balloon was inflated (2). Finally, the proximal part of the LCX stent was recrushed more distally (3) (From reference [38]).

Although residual stenosis after two-step ballooning in the SB ostium stenosis was confirmed to be larger in the small cell size stent (52%) compared to larger (>3.5 mm) size cell stent (37%) in a bench study39, TLR frequently occurred in paclitaxel-eluting stent (larger cell size stent) compared to SES (small cell size stent) in a prospective study of classic crush stenting (14.5% versus 6.2%)21 as well as in a prospective study of DK crush stenting (23.3% versus 8.8%)52. Since widely opening of the SB stent and damage of the polymer are the trade-off issue, using large cell stent is not always assured clinical better outcome.

Simultaneous Kissing Stenting (SKS)


The SKS technique deploys stents in the MV and SB simultaneously and this simple and speedy technique obviates the need for re-crossing the stent struts. It has been considered to be favorable for the true bifurcation lesion which includes the stenotic lesions in both branches with large proximal MV. In the bench testing using the 3-D LMCA bifurcation model, stent overlap created a wedge-shape gap beneath the site where the LCX stent crosses over the LAD stent, creating a substrate for restenosis at the LCX ostium36,37,41,42 (Figure 14.8). When kissing site is extended long in the proximal MV, there was a twisting of the two stents. The LCX stents was extended to the opposite side of the LCX ostium at its proximal end36.

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Figure 14.7. The SB ostial dilation after crush stenting followed by kissing balloon inflation. The dotted lines in the right panels indicate maximally dilated cells of the MV stent at the SB ostium. A case with good expansion of the cell (A) and that with poor position of the cell (B) using Bx Velocity stents are shown as well as a case with well-apposed expansion using an Express II stent (C) (From reference [38]).

Figure 14.8. Micro focus CT images after simultaneous kissing stenting in the 3-D LMCA bifurcation model. A. The long axis 3-D image shows the proximal end of the LCX stent crossing the LAD stent in the distal LMCA, rather than being positioned lateral to the LAD stent. B. Proximal to distal (a to c) cross sectional images of the carina. Proximally, the LCX stent was compressed by the LAD stent (a, dotted arrow). Distally, a gap is observed beneath the overlapped stents on the myocardial side (c, solid arrow). C. The plain cross sectional image at level a also shows compression of the LCX stent (From reference [37]).

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There remains a concern about thrombogenic effect of residual metallic carina in the proximal MV. It has been reported that the metallic carina was covered by the intimal tissue in 47% of 30 SKS cases after average of 27-month follow-up53. However, long flow-divider and oval-shape deformation of each stent are not ideal situation for preventing restenosis or future atherosclerotic change in the rheological aspect. Once the restenosis occurs after SKS, complete revascularization is difficult in the following aspects; re-crossing the guide wire in the twisted stents, precise deployment of additional proximal stent without gap and crush of intima-covered metallic carina.

Culotte Stenting
Since this technique induces the jail of both branch ostium by the opposite stent, opening of these jailed struts are necessary. Therefore, open-cell based stent is favorable because the strut can be dilated widely. The 3-D images of Bx Velocity stents, closed cell based stents, demonstrated the napkin ring restriction of the ostium of the MV and the SB, because maximal strut dilation length is 3.0 mm even after the dilation using > 3.0 mm balloon36,38,40 (Figure 14.9A). On the contrary, the images of the Driver (Medtronic, Santa Rosa, California) and Liberte stents, open-cell based stents, seemed to show good appositions to the vessels36 (Figure 14.9 B and C). In most parts of the bifurcation, the cross sectional views also showed good stent apposition. High volume of metal overlapping in the proximal MV is one of the disadvantages in this technique and it would lead to a possible risk of stent thrombosis and excessive drug effect. The modified technique that the proximal SB stent deployed in the MV was minimized to diminish the metal overlapping54. The complicated procedure requiring two times of guide wire exchange and KBT may also include a risk of the polymer damage of DES.

Figure 14.9. Micro focus CT images after culottes tenting. A. Bx Velocity stents in the 2-D bifurcation model at 45 angle between the MV and the SB. The restriction of the stent expansion is observed in both ostium of the daughter branches after kissing balloon inflation using 3.5 mm balloons (arrows). B. Driver stents in the 3-D LMCA bifurcation model. Middle and lower panels show the cross sectional image corresponding to the lines where the star and the circle are located at the upper panel, respectively. C. Liberte stents in the LMCA bifurcation model. The open-cell based stents are ideal for this technique (From reference [36]).

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A prospective study of culotte stenting demonstrated that angiographic restenosis was favored to occur in the SB (0% in the proximal MV, 9.1% in the distal MV, and 16% in the SB)55. There was a numeric trend towards a frequent restenosis with zotarolimus-eluting stent (ZES) and PES but it was not statistically significant (SES 18.0%, ZES 28.6%, PES 34.6%)55. There have been the discrepant results between bench and clinical studies, which cannot be explained only by stent platform.

KISSING BALLOON INFLATION TECHNIQUE (KBT) Impact on 2-Stent Strategy


It has been revealed that final KBT after crush stenting dramatically reduced the restenosis in the SB from 40 to 10%20,21. The KBT improved the underexpansion of the SB stent and the stent apposition as well as the jailed condition by the crushed struts at the SB ostium36-41. In the high-angled bifurcation, a bench test revealed that stent-uncovered area increased close to the carina after crushing the SB stent43 and a clinical study reported that the KBT was more effective for reducing the MACE in the high-angled bifurcation group24. However, bench test clarified some limitations of the KBT. The complete stent apposition was not obtained even after high-pressure ballooning in the 80-degree angle model43 or 3-dimentional LMCA model36,37,41. The point of the SB guide wire crossing also affects on the SB opening. Crossing through the proximal strut results in inadequate stent expansion which leads to stent-uncoverage in the carina. There is a possible risk in crossing through the distal strut, which results in increase of the gap due to the inflation of the balloon located outside of the SB stent36,38,39. The improvement of stent apposition after the KBT was also confirmed after culotte stenting, modified T-stenting, and TAP technique36. However, small closed-cell stent has the limitation in the dilation of the jailed strut, which leads to underexpansion of the stent at the SB ostium36. SB ostial underexpansion has been clarified to be a critical risk factor for the SB restenosis in the IVUS studies of crush stenting49 and TAP technique47. Using the same size and non-compliant balloons for the KBT reduced the deformation and underexpansion of the stents and prevents the deviation of the metallic carina in the SKS and TAP technique36,37,42. The position of two balloons during the KBT also had the influence on the final stent configuration, which led to a possible risk of the deformation of the proximal MV in the high-angled bifurcation and the 3-dimentional LMCA models3638,41,42.

Impact on 1-Stent Strategy


Recently, the inefficacy of final KBT in 1-stent strategy has been reported in the following studies. Korean Coronary Bifurcation Stenting Registry demonstrated adverse effect of the KBT on the MACE (4.7% versus 8.7%)56. Nordic-Baltic III trial which was a randomized comparison between KBT and non-KBT groups also demonstrated similar frequencies of the MACE (2.9% versus 2.9%)57. Koo et al. measured the flow fractional reserve (FFR) in the jailed SB and demonstrated that only 30% of jailed SB whose angiographic stenosis 75% showed physiologically relevant stenosis33. Therefore, Overtreatment derived from the angiographic overestimation of the SB narrowing may cause the adverse effect. Gurin et al. observed in the bench test that the KBT caused elliptic deformation with the eccentricity value of 0.75 in the proximal MV and 30% enlargement was obtained compared to the distal MV58. They also confirmed that metal / artery ratio was decreased as well as drug delivery / surface ratio in the KBT site58. The widely-opened struts tended to have more polymer injury which might impair the DES efficacy of anti-proliferation. The dissection in the SB due to overexpansion and inappropriately proximal position of the SB guide wire, which causes uncoverage of the SB ostium, are possible risk of the restenosis induced by the KBT.

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JAILED SIDE BRANCH


The present concept concerning the mechanism of the SB narrowing after the MV stenting has been shifted from plaque shift to carina shift59. A recent pathological study demonstrated that atherosclerotic change is favored to occur in the lateral area of the bifurcation and carinal involvement was extremely unusual60. The healthy carina shift to the SB ostium, and SB lumen is pinched and narrowed after the MV stenting. Over-size MV stenting and overdilation in the distal MV are the risks of this phenomenon. Although there has been the concern about the deterioration of the SB coronary flow behind the jailed strut and its platelet activation leading to thrombosis61, long-term clinical outcome has not yet been clarified. Koo et al. demonstrated that FFR-guided treatment had not shown the worsening of the FFR value in the jailed SB32. However, the observation using optical coherence tomography showed the growth of neo-intimal tissue on the jailed struts in 12-month follow-up compared to that in 6-month (34.0 17.0 m versus 79.0 26.0 m)50. The change in SB coronary flow pattern due to the pinched ostium may bring newly generated low shear stress area where atherosclerosis favors to occur.

POLYMER DAMAGE
Guieren et al. investigated polymer damage of five DESs (Cypher, Cypher Select, Endeavor, Taxus Express, and Taxus Liberte) after KBT in the bench test58. The polymer breakage due to overlapping strut layers and uneven strut distribution at the SB ostium were observed in the scanning electron microscopy (Figure 14.10). The coating damage on the ostial struts was observed frequently in all DESs, which may reduce drug delivery and increase the restenosis risk at the SB ostium58. Although the Cypher and the Taxus Express stents showed highly damaged polymer coating only in the ostial struts, the Endeavor stent showed subtotal destruction from the proximal to the distal parts of the stent58. The Cypher Select and the Taxus Liberte, which were changed from their old models to obtain more expansion and improvement of stent apposition, showed the increase of the coating damage in the proximal MV as well as in the ostial site58. The balance of the expandability of the stent and polymer damage due to the over expansion should be considered in the newly developed innovation.

RHEOLOGICAL ASSESSMENT
Recent pathological studies demonstrated that atherosclerotic change favored to occur at the lateral area of the bifurcation where low shear stress was induced by low or oscillatory coronary flow without the involvement of the carnal area60,62. Delayed endothelialization in the carinal area after DES deployment has been reported, where high coronary flow was exposed60. Stent deployment in the bifurcation lesion may change the local vessel geometry and bring abnormal flow pattern which promotes atherosclerosis or thrombosis. Williams et al. analyzed wall shear stress after the MV stenting with or without virtual SB angioplasty using computer simulation63. The MV stenting introduced eccentric low shear stress area along the lateral wall of both distal MV and SB. Despite of the SB angioplasty, low shear stress area had been still remained with normal value of FFR. Coisne et al. investigated flow dynamics in LMCA bifurcation model with 60 and 90 degrees in bifurcation angle (Figure 14.11)64. Before stenting, low wall shear stress was observed in the lateral area in both types. The MV stenting promoted the low shear stress behind the jailed struts. Interestingly, after provisional SB stenting, low shear stress area was widely spread to the carinal area in the 90-degree type and more spread even in the distal MV in the 60-degree type.

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B
Figure 14.10. Polymer damage after double stenting. A. Cypher stent. B. Endeavor stent. Courtesy of Dr. Yoshihisa Kinoshita (Toyohashi Heart Center).

Baseline

MV stenting

Provisional S B stenting

60

90
Figure 14.11. Assessment of coronary flow at the baseline (left), after MV stenting (middle), and provisional SB stenting (right) in the phantom model with the bifurcation angle of 60 (upper panels) and 90 degrees (lower panels). The MV stenting promoted the low shear stress behind the jailed struts and provisional SB stenting promoted more in the ostium of both daughter branches more widely. Courtesy of Dr. Damien Coisne (Universit de Poitiers).

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A few clinical pilot studies of assessing the shear stress after DES deployment were reported using 3-dimentional coronary flow simulation by Chen et al. They suggested that the inhomogeneous and lower reduction of shear stress appeared to predict in-stent restenosis after classical crush stenting65. They also reported that contradictory shear stress distribution, which the stress was low in the MV and high in the SB, appeared to prevent the restenosis after provisional stenting66.

LIMITATION OF BENCH TEST


There have been some unexplained phenomena in the clinical studies which were inconsistent with the previous bench studies. Frequent restenosis was observed after crush stenting using the Taxus compared to the Cypher, whereas the Taxus was more ideal for the SB opening21,52. SB compromise frequently occurred in multiple stenting in the Taxus group compared to the Express which had the same platform in the TAXUS V trial (22.1% versus 12.9%)67. Although the opening and access ability of the SB were similar, the Taxus Express group had more SB compromise (5.2% versus 2.8%) and non-Q myocardial infarction (5.0% versus 2.7% at 2 year) compared to the Xience V group in the SPIRIT III trial68. Overall frequency of aggravation of SB was the most in the Endeavor group (Endeavor 11.6%, Cypher 4.7%, Taxus 10.4%) which had a good access ability to the SB, whereas that of the jailed SB was the least (2.8%, 7.7%, 12.8%) in ZEST bifurcation substudy69. Although the bench test is usually performed in the tube or the mold block, the elasticity of the vessel, curvature, 3-dimentional structure and heart beating which are different from human coronary artery affect on the final stent configuration. Physiological condition (i.e. temperature, blood, flow volume, and blood pressure) also affect on the efficacy of DES. Biological reaction (i.e. toxic effect, allergic reaction) cannot be assessed by the bench test. Therefore the results of bench test cannot be simply extrapolated to the clinical practice and an in vivo analysis will be required for the confirmation. However, bench test using high technology in the realistic phantom model will provide useful information for comprehensive analysis of major three components of DES efficacy, platform, polymer and drug.

CONCLUSION
Bench studies have revealed the deformation of the DES after bifurcation intervention that cannot be detected completely by the clinical imaging devices. They have also clarified the crucial factors related to the restenosis, i.e. gap formation, incomplete stent apposition, overexpansion of the strut, and polymer damage. The comprehensive analysis is required for the bench test not only in morphological aspect but also in the assessment of DES efficacy, rheological and physiological aspects.

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C. PATHOLOGY OF BIFURCATION STENTING GAKU NAKAZAWA


Atherosclerosis in Bifurcation .................................................................................................. Predictors of Restenosis in BMS Era ....................................................................................... Delayed Arterial Healing in DES ............................................................................................. Conclusions .............................................................................................................................. References ................................................................................................................................ 219 221 222 228 228

ATHEROSCLEROSIS IN BIFURCATION
It is estimated that coronary bifurcation lesions are involved more than 10% of all percutaneous coronary intervention (PCI)70. Atherosclerotic lesions tend to form at specific regions of the coronary vasculature where flow is disturbed, in particular in areas of low shear stress71-73. Because dramatic hemodynamic alternations occur at branch points within the arterial tree, coronary bifurcation are extraordinarily susceptible to atherosclerosis. Areas of low shear stress are thought to accelerate atherosclerosis through modulation of gene expression including the promotion of endothelial cell dysfunction which causes increased uptake of lipoproteins, up-regulation of leukocyte adhesion molecules, and leukocyte endothelial transmigration, all of which contribute to the development and progression of atherosclerosis74. It is well known that there is a strong correlation between endothelial dysfunction and low shear stress and oscillatory flow which is usually observed in the bifurcation lesions or in areas of abrupt curvatures. Cheng et al. reported that up-regulation of eNOS (endothelial nitric oxide synthase) was observed in a high shear areas utilizing cast-induced increased shear stress model in the mouse carotid artery75. In a later study, Cheng et al. showed elevated gene expressions of interleukins-6, C-reactive protein, ICAM, VCAM, and vascular endothelial growth factor in the low shear area using the same model76. Thus, shear stress appears to play an important role not only in early plaque formation but also its progression. In addition, bifurcation angle variation also plays a central role as shown by Perktold et al. that the larger the angle of bifurcation the greater the turbulence77. Moreover, ratio of the caliber from the proximal to the distal or side branch also influences the flow turbulence78,79. We have recently reported the spatial distribution of coronary atherosclerotic plaques in the bifurcation lesion using autopsy cases with the longitudinal cutting60. From the CVPath registry, 26 bifurcation lesions in 18 sudden cardiac death autopsy cases are selected. A mean age of the population was 60 10 years with a mean body mass index of 29 7. Thirteen patients had known coronary risk factors such as hypertension (n=6), diabetes (n=4), smoking (n=3) and/or pre-existing coronary artery disease (n=4). All coronary bifurcations had atherosclerotic plaques. Plaque types were thin-cap fibroatheroma (n=2), fibroatheroma with or without calcification (n=11, with/without=5/6), and fibrocalcific plaques (n=13). Plaque thickness was the greatest on the lateral wall in the distal main vessel (1.3 0.7 mm) followed by the lateral wall in the proximal main vessel (1.1 0.7 mm). All lateral walls showed significantly greater intimal thickness as compared to those in the flow divider (Figure 14.12).

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Figure 14.12. The bifurcation lesions were divided into 7 regions (Left Panel). The top bar chart shows the thickness of the plaque (mm) in each region, while the bottom graph shows the thickness of the necrotic core (mm). Low shear areas had a higher prevalence of coronary plaque as well as necrotic core. Note, that carinal region was rarely involved (G).

Similarly, necrotic core thickness was also significantly greater in the lateral wall versus the flow divider where the necrotic core was minimal or absent (Figure 14.12). This finding proofs the concept of shear stress being a major role in the progression of disease in human coronary artery disease. Representative cases are shown in Figure 14.13. Not only in the autopsy series, similar findings are reported in living patients using imaging modalities80. Oviedo et al. reviewed 140 left main coronary bifurcation lesions comparing angiography and intravascular ultrasound (IVUS), where they pointed out that atherosclerotic plaques were not seen in the carina but rather in the lateral wall in the bifurcation. Furthermore, most atherosclerotic plaques were diffusely distributed and continuously extending from LMCA to either LAD or LCX whereas those localized to the LAD or LCX ostium were rare. These findings were consistent with our pathologic analysis. In addition, circumferential patterns of plaque distribution was proposed by Oviedo et al. which classifies the axial location of plaques into diffuse, lateral, myocardium, and pericardium. These findings give an important insight into the procedural strategy such as one or two stent technique based on the circumferential plaque burden, which could not be accurately assessed by angiography alone. Another study by Rodriguez-Granillo et al. using multislice computed tomography (MSCT) was reported where they demonstrated that atherosclerotic plaques were commonly located at the opposite site of the flow divider in the bifurcation lesion (i.e. lateral wall)81. Thus, findings in vivo studies with living patients are consistent with our results at autopsy, and both demonstrated the areas of low shear zone tend to show greater atherosclerotic plaques than high shear zone.

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Figure 14.13 A. Longitudinal section of trifurcation (Left main/ LAD/ Ramus intermedicus/ CX). There are atherosclerotic plaques in the lateral wall, while the flow divider regions are spared (B). C. Longitudinal section taken in the region of LCM/ Left obtuse marginal bifurcation. Note, severe luminal narrowing proximal and distal to the bifurcation. Low shear regions show atherosclerotic plaque development including necrotic core formation whereas flow divider regions (the carina) has minimal intimal thickening (D, E).

In these atherosclerotic plaques in the bifurcation lesion, the majority of coronary plaques show complex structures with a large necrotic core and severe calcification. These specific features of bifurcation lesion yield complexity in procedure and vascular response following stent implantation. To better understand the detail of atherosclerotic plaque development and its spatial distribution, invasive and non-invasive multi-modality assessments are likely to be helpful and result in better clinical outcome in PCI involving bifurcation lesions.

PREDICTORS OF RESTENOSIS IN BMS ERA


Following Dr. Gruentzigs groundbreaking performance of coronary balloon angioplasty82, this procedure rapidly spread and became the standard treatment of symptomatic coronary artery disease. In 1990s, coronary artery stents were developed to overcome the high rate of restenosis that resulted from early plaque recoil and late arterial shrinkage after conventional balloon angioplasty83. Although this improvement has been accomplished, one-quarter of all patients still needed repeat revascularization due to restenosis84. To understand this phenomenon, many pathologic studies have been performed. The time course of arterial healing has been well characterized for BMS implantation85. At the early time

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point (<30 days), there is mild luminal thrombus with inflammatory cell infiltrates, consisting of polymorphonuclear leukocyte and macrophages. Platelets aggregation and fibrin deposition are observed for up to 30 days. T lymphocytes begin to infiltrate at 2 to 4 weeks and persist beyond the 6 months period. At the same time, smooth muscle cells begin to appear at 14 to 30 days accompanied by extracellular matrix consisting of proteoglycan and type III collagen. Completion of re-endothelialization occurs by 3 to 4 months following BMS implantation. The neointima peaks at 6 to 12 months and thereafter the neointimal volume decreases as type III collagen is replaced by type I collagen86. These observations are reinforced by clinical studies which showed that the amount of neointimal growth peaks at 69 months and then decreases slowly up to 3 years following BMS implantation87. Not to mention, excessive neointimal formation consisting of smooth muscle cell and proteoglycan matrix proliferation results in restenosis, which occur one-quarter of stented lesions as mentioned above. Mechanisms of restenosis following BMS implantation were reported in early 2000s88. After detailed histological analysis of 116 stents in 87 coronary arteries, Farb et al. have reported that there was a strong correlation between stent area and neointimal area (r2=0.70, P<0.0001). Namely, overexpansion of metal stents followed by arterial medial fracture was associated with significant increase in neointimal thickness as compared to those without. Neointimal thickness, inflammatory cell density, and neointimal vascular channel density were greater arteries with a ruptured arterial media by stent struts than those without. Furthermore, stent strut penetration into a lipid core was associated with increased neointimal thickness and inflammatory cell density. Thus, stent implantation is associated with vessel injury such as endothelial denudation, medial injury and/or fracture, plaque disruption and necrotic core penetration, which resulted in increased risks of in-stent restenosis. The stent induced injury is very similar to that induced by balloon angioplasty alone except that the plaque is tacked back against the arterial wall by the expanding stent88. Early results using bare metal stents (BMS) to treat bifurcation lesions resulted in a high angiographic acute success rate but were limited by a higher rate of restenosis13,34. This could be related to the complex anatomy and underlying plaques in bifurcation lesions. As previously discussed, these lesions frequently contain lipid-rich plaque with large necrotic core, which stent struts can easily penetrate the necrotic core resulting in an excessive vascular response and subsequent restenosis. Furthermore, because of these underlying plaques, bifurcation lesions show infiltration of inflammatory cells, which is one of the important factors in neointimal growth since these cells release many growth factors to promote neointimal formation. In addition, the vessel size changes considerably from the distal to the proximal main vessel due to merging two daughter branches into the main vessel. Therefore, metal stents, which do not have tapering design, can induce the excessive vascular injury especially in the distal portion of the main vessel where restenosis is more frequently seen than the other location in bifurcation. Thus, restenosis in bifurcation lesion with BMS is associated with complex underlying plaques, inflammation, and vascular injury that are essentially common in BMS restenosis regardless of the condition.

DELAYED ARTERIAL HEALING IN DES


Because a restenosis rate was considerably high up to 2030% even after the introduction of BMS, new technologies focused on inhibiting specific cellular targets critical to the restenotic process such as smooth muscle cell proliferation and migration. Although early attempts to reduce restenosis after PCI with the use of systemic pharmaceutical agents proved unsuccessful89,90, local stent based delivery of anti-proliferative agents such as sirolimus, a cytostatic macrocyclic lactone with both anti-inflammatory and anti-proliferative properties, and paclitaxel, a lipophilic diterpenoid known to suppress smooth muscle cell proliferation and migration by disrupting

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microtubule dynamics, reduced neointimal proliferation in animal studies. In these, so-called drug eluting stents (DES), local sustained drug delivery takes advantage of polymers to promote controlled elution of drugs from the stent. Encouraging results in preclinical studies using sirolimus and paclitaxel eluting stents paved the way for human studies. Aggressive suppression of neointimal growth by DES has largely overcome restenosis, the Achilles heel of BMS. Pivotal clinical trials with use of DES have demonstrated a significant reduction in restenosis rates compared to BMS9,10. Because of the potent drug effect on the vascular proliferative response, classical pathologic risk factors for restenosis such as necrotic core penetration, inflammatory reaction, and vascular injury along with medial fracture are no longer listed as primary substrate of stent failure in DES era. However, at a cost of dramatic reduction of restenosis, late stent thrombosis, rare but a life threatening complication, has emerged as a major concern91. Virmani et al. were the first to report incomplete endothelial coverage with focal platelets aggregates and persistent fibrin deposition within the necrotic core at 16 months following Sirolimus eluting stent (SES: Cypher) stent implantation, so-called delayed arterial healing in 200392. In 2006, the same group reported a significant delayed arterial healing in DES using initial series of autopsy cases, as evidenced by persistence of fibrin deposition, incomplete re-endothelialization, and sparse smooth muscle cell growth compared with BMS implanted for similar duration93 (Figure 14.14).

Figure 14.14 A. Section from BMS implantation. There is abundant neointimal tissue consisting of smooth muscle cells in a proteoglycan collagen matrix and an overlying endothelium. B. Lymphocytes are present around the stent strut with minimal fibrin underneath the strut. The luminal surface of the stent is covered by smooth muscle cells in a proteoglycan/collagen rich matrix. C. Histologic section of the Cypher stent showing minimal coverage of the struts by fibrin. D. High-power magnification showing peri-strut fibrin with rare endothelial cells but no luminal thrombus, whereas inflammation and smooth muscle cells are only rarely observed.

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Late stent thrombosis was observed in 14 of 23 patients receiving SES or paclitaxel eluting stents (PES: Taxus). Although neointimal thickening was minimal in DES, poor endothelial cell coverage of the lumen was consistently documented in all DES cases regardless of the duration of implantation, while endothelialization in BMS was complete by 3 to 4 months. Later, we showed a lack of endothelial strut coverage as the single best correlate of late stent thrombosis using the data from a larger number of autopsy cases94. The arterial healing in DES lesions was heterogeneous, especially in thrombosed cases (Figure 14.15), suggesting that the underlying lesion morphologies also contribute to the rate of healing.

Figure 14.15. Heterogeneity of neointimal healing following DES placement: 34 year old female underwent placement of one Cypher (22 3 mm) stent in the proximal left circumflex artery for acute myocardial infarction 2 year antemortem. The patient was admitted to emergency room with ST-elevation myocardial infarction and subsequently expired. Consecutive sections of Cypher DES were cut 3 mm apart, stained with Movat Pentachrome (A) and measurements of neointimal thickness (above each strut) and the number of uncovered stent struts performed (B). There is greater neointimal growth above each strut (x) and fewer uncovered stent struts (red circle) within the proximal and distal stented portion, with absence of luminal thrombus formation. At the site of thrombus formation (sections #5 and 6), neointimal thickness is minimal and the number of uncovered stent struts maximal.

One of the underlying plaque morphologies associated with greater incidence of late stent thrombosis (LST) is ruptured plaques, which are frequently observed in patients with acute myocardial infarction (AMI). In our recent autopsy study, we compared the vascular pathologic responses to DES implantation in patients receiving DES for AMI with that of patients receiving them for stable angina95. In this study, we have observed a significantly higher incidence of LST in AMI patients treated with DES as compared to patients with stable angina (41% versus 11%, p=0.04). Furthermore, greater delayed healing was observed at culprit sites (i.e. site of underlying plaque rupture) in AMI patients as compared to those in stable patients (Figure 14.16), which was evidenced by greater fibrin deposition, inflammation, and higher

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prevalence of uncovered struts. Thus, these findings emphasize the importance of underlying plaque morphology on vascular healing. It should also be noted that plaque rupture often occurs at the site of bifurcation that might render the situation even more complicated.

Figure 14.16. Histologic sections in the top row (A, B, and C) are from AMI patients. A 64-year-old female who died from congestive heart failure 9 months after Taxus stent implantation (A), a 49-year-old male who died from non-cardiac cause 13 months after Cypher stent implantation (B), and a 34-year-old female who died from late stent thrombosis 24 months after Cypher stent implantation (C). Struts with necrotic core (NC) were observed with fibrin deposition and absence of endothelial coverage (A). Stents in B and C showed minimal coverage of struts above necrotic core at 13 or 24 months duration. Histologic sections in the bottom row (D, E, and F) are from patients with stable lesion. A 61-year-old male who died from a non-cardiac cause 7 months after Cypher stent implantation (D), a 53-year-old male who died suddenly 18 months after Taxus stent implantation (E), and a 68-year-old male who died from a non-cardiac cause 19 months after Cypher stent implantation (F). All patients had underlying fibroatheroma with thick fibrous cap. High magnification images show underlying necrotic core (NC) and thick fibrous-cap (FC) with varying degree of neointimal formation above stent struts (D, E, and F).

Recently, improvement of imaging modalities such as optical coherence tomography (OCT) and angioscopy has allowed the assessment of the neointimal strut coverage following stent implantation in living patients. Greater stent tissue coverage was confirmed in BMS compared to DES in both angioscopy and OCT96,97. Furthermore, Kubo et al. reported that the lack of neointimal strut coverage was more frequently observed in the patients with unstable angina compared to those with stable angina, which is consistent with the findings at autopsy98. Thus, stent imaging followed by the risk stratification of patients receiving DES may be useful and important rather than angiographic follow-up alone. Bifurcation lesion is also one of the most susceptible sites for stent thrombosis especially in DES era99. In our recent autopsy study, the incidence of stent thrombosis in bifurcation

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lesions with DES was significantly greater than BMS60. Detailed morphometric and pathologic analysis revealed greater delayed arterial healing (i.e. greater incomplete stent coverage and fibrin deposition) in bifurcation lesions stented with DES versus BMS. Furthermore, in DES, the percentage of uncovered struts was significantly greater at flow divider sites as compared to lateral wall [median 40% (IQR 16, 76) versus 0% (0, 15), p=0.001] and fibrin deposition was also frequently seen in flow divider sites as compared to lateral wall [median 60% (IQR 21, 67) versus 17% (0, 55), p=0.01]; (Table 14.5), while these differences were not significant in BMS. Most of the thrombi originated at the flow divider sites where uncovered struts were frequently observed (Figure 14.17).
Table 14.5. Morphmetric Comparison Between Flow divider versus Lateral wall in DES and BMS DES Flow divider Neointimal thickness (mm) Fibrin deposition (% Struts) Uncovered struts (% Struts) 0.07 [0.03, 0.15] 60 [21, 67] 40 [16, 76] P value Lateral 0.17 [0.09, 0.23] 17 [0, 55] 0 [0, 15] 0.001 0.01 0.001 BMS Flow divider Lateral 0.26 [0.16, 0.73] 8 [0, 33] 0 [0, 21] 0.44 [0.17, 0.67] 3 [0, 21] 0 [0, 0] P value 0.25 0.21 0.10

DES=Drug-Eluting Stent, BMS=Bare Metal Stent, Values are expressed as median and interquartile range.

Figure 14.17. A 55-year-old male with history of smoking, hypertension, and dyslipidemia, received 2 Taxus stents in the ostium of left anterior descending coronary artery (LAD) and left circumflex (LCX) with overlapping Taxus stents placed in the LAD. The patient died suddenly 2 year after stent implantation. Radiograph shows mildly calcified coronary artery. Both stents are occluded with platelet-rich thrombus (thr) at the ostium of LAD and LCX (A). High magnification images show adherent thrombus in the region of the uncovered struts at the flow divider (B, C). Uncovered struts and adherent thrombus was also observed in all sections of the LCX stent (D, E, G). The middle to distal portion of LAD stent shows absence of thrombus and healed luminal surface with mild neointimal thickening (D, F, H).

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These findings suggest that shear stress and flow disturbance may play an important role in vascular response following stent implantation especially in DES. In addition, in vitro experimental bifurcation model demonstrated that deployment of stents can alter boundary layer separation of the lateral walls and produce disturbances (vortical structures) at the carina (Figure 14.18).

Figure 14.18 A. A gross image of the in vitro stented bifurcation model. The recorded pressure (B) and flow (C) rate measurements are displayed and were maintained within physiological conditions. D. Boundary layer separation in the non-stented model was observed at peak systolic phase at the lateral walls, particularly at the side branch (white line represents boundary layer). E. After stenting, the boundary layer was increased as compared to the non-stented model, however, (F) after side branch dilation, the boundary layer was decreased. No flow disturbances were observed during the early diastolic phase of the cycle in the non-stented model (G), however, in the stented model, vortical structures (arrows) were present in the carinal region (H).

Regions of boundary layer separation is associated with reduced level of wall shear stress, poor mass transfer of blood flow and vessel wall, and an increase in time residence of circulating blood elements. Moreover, the development of vortical structures can prolong and alter localized areas of low-flow regions, and influence drug deposition, arterial healing post stenting, and local fibrin and platelets deposition100-102. Richter et al have shown that a greater neointimal formation occurs in the lateral as compared to flow divider walls following stent implantation in porcine ilio-femoral bifurcation model79, which is consistent with findings in our recent study. On the other hand, these differences were not significant in BMS which may be due to more rapid healing and more uniform neointimal formation after BMS implantation in humans. Thus, a combination of drug effect and flow disturbance both are likely to accelerate the delayed healing in bifurcation lesion. After the introduction of DES, the treatment of

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bifurcation is more feasible in terms of restenosis, however, clinicians should still be cautious when clinically follow these patients with bifurcation stenting.

CONCLUSIONS
The distribution of plaque formation in coronary bifurcations is highly affected by shear stress. Atherosclerotic plaques in bifurcation lesions show complex structure forming a large necrotic core and calcification which yields complexity in procedure and vascular response following stent implantation. Although restenosis in bifurcation lesion with BMS is associated with complex underlying plaques, inflammation, and vascular injury, DES has largely overcome the problem of restenosis. However, delayed arterial healing following DES implantation, primary substrate of late stent thrombosis, is exaggerated especially in complex lesions such as bifurcation. Therefore, physicians must continue paying attention to the patients who received DES in bifurcation lesions.

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CHAPTER 15

SMALL VESSEL STENTING. THE BEST APPROACH


SEUNG-WOON RHA and DONG-JOO OH
Introduction .............................................................................................................................. Definition of Small Vessel Disease........................................................................................... Clinical Significance of Small Vessel Disease.......................................................................... Management Strategy in Small Vessel Disease ........................................................................ Role of Coronary Image and Physiology in Small Vessel Disease ........................................... Discussion................................................................................................................................. Conclusions .............................................................................................................................. References ................................................................................................................................ 235 236 236 237 241 241 242 242

INTRODUCTION
Coronary artery disease (CAD) still remains a major cause of morbidity and mortality despite significant improvement of optimal medical therapy for primary and secondary prevention. Specifically, coronary atherosclerotic lesions often extend to distal and small-caliber coronaries, such that as much as 2030% of patients undergoing percutaneous coronary intervention (PCI) may have significant coronary lesions whose diameter is relatively small (< 2.75 mm).1 Small vessel CAD has been a challenging subset to manage with PCI due to its association with other risky co-morbidities including diabetes, female gender, and diffuse coronary involvement.2 Even during the PCI, the incidence of acute complications are more common and further, the long term outcomes are more frequently suboptimal.3-4 Despite the development of PCI devices and techniques, still the problems associated with restenosis are profound in this particular subset of disease. The results of balloon angioplasty alone (only POBA, plain old balloon angioplasty) and implantation of base metal stents (BMS) have been disappointing due to higher restenosis rate, thus there have been controversies regarding their benefit in managing small vessel disease.5 It means that POBA and BMS have shown only partially effective, as early and midterm failures could occur in up to half of the patients.6 Basically, higher restenosis rates results compare with larger coronary vessels are originated from a lower post-procedural stent area, with small vessels less able to accommodate neointimal accumulation.7 Anyway, despite of these clinically important problems, adequate management of small vessel disease is very important as 30 to 67% of all PCIs involve small coronaries.8-9

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With the help of recently introduced drug-eluting stent (DES), the PCI patterns for small vessel disease have drastically changed. PCI with DES in small vessel disease have shown their potent anti-restenotic effect and remarkable early and midterm safety.10 Compared to BMS, despite DESs have significantly attenuated target lesion revascularization (TLR), small vessel size continues to be an independent predictor of restenosis and TLR even after DES implantation.11-12 In this chapter, authors will provide a concise and updated review on current and future approaches for PCI in patients with symptomatic small vessel disease.

DEFINITION OF SMALL VESSEL DISEASE


There have been different definitions regarding the small vessel from the past. In these days angiographic reference vessel diameter equal or lower than 2.75 mm is considered as most appropriate cutoff.13 Thus, CAD amenable to PCI with a 2.75 mm or smaller device can be considered as small. Further, after development, market approval and subsequent availability of Small DES which is 2.25 mm diameter, some reports have described as Very small CAD for those coronary segments treated with a 2.25 mm device. We have to emphasize the clinical significance of small CAD. Even after the successful PCI, it is more associated with restenosis, thrombosis and subsequent major adverse cardiac events (MACE) as compared to larger CAD. There is a modeling the impact of different late losses on follow-up binary angiographic restenosis after PCI in small (< 2.75 mm), medium (2.75-3.25 mm) and large (> 3.25 mm) reference vessel diameter. The authors suggested that risk of restenosis after balloon only PCI will be 35-55%, after BMS implantation 25-50% and DES implantation 10-35% (30-35% after DES with relatively high late loss such as EndeavorTM, 20-25% with medium late loss such as TaxusTM, and 10-15% with low late loss such as CypherTM or XienceTM), according to previous data including C-SIRIUS, ENDEAVOR-2, 3, MICROSCOPE, SES-SMART, SIRIUS, SIRTAX, SPIRIT-2, 3, TAXUS-5 and 6. 14

CLINICAL SIGNIFICANCE OF SMALL VESSEL DISEASE


Although there are many varying definitions in the publications, there is a consensus that small vessel CAD is highly prevalent: up to 20-30% of patients with symptomatic CAD.1 It is particularly highly prevalent in patients with diabetes or chronic renal insufficiency.15 We should emphasize that not all the small CAD are born equal. We should consider the lesion location of small CAD in deciding management strategy. For example, an extensive diffuse lesion in proximal left anterior descending artery (LAD) with negative remodeling might appear to have similar reference vessel diameter to that of a distal LAD or a distal secondary branch of a post descending artery (PDA). However, the clinical significance of these two different lesion locations will be quite different. It means that the clinical relevance and management strategy should be different. For example, if there are large amount of myocardium at risk, all efforts should be focused on providing the most appropriate sized device and treatment; the larger the better principle. In this particular situation, to get an optimal result, adequate lesion preparation (appropriate predilation or debulking), proper stent size choice based on the intravascular imaging such as intravascular ultrasound (IVUS) or optical coherence tomography (OCT), and proper DES choice which is associated with low late loss. Conversely, a small CAD which is supplying small proportion of myocardium is often not so worthy to give major therapeutic interventional procedures and can be better served with even conservative optimal medical therapy (OMT).

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MANAGEMENT STRATEGY IN SMALL VESSEL DISEASE Optical Medical Therapy (OMT) in Small CAD
We should emphasize the importance of optimal medical therapy regardless of technical issues in treating small CAD. Many trials of aggressive medical therapy including antiplatelet agents, beta-blockers, angiotensin-converting enzyme inhibitors and statins have shown the benefit and now are recommended in all patients with stable CAD regardless of the PCI results. Beyond the standard OMT for the primary and secondary prevention of small CAD, there have been recently several clinical attempts on systemic drug therapy to prevent restenosis after PCI in both large and small CAD. Promising data have been introduced using oral rapamycin,16 steroids,17 and cilostazol.18 However, further clinical data with larger study populations are needed to make final conclusion.

Coronary Artery Bypass Graft (CABG) Surgery in Small CAD


Significant proportion of small CAD is coming from patients with diabetes and chronic renal insufficiency and their CAD are usually extensive, diffuse, small vessel and multi-vessel disease. In this aspect, we should consider the role of bypass surgery in treating extensive small CAD. Bypass surgery has been traditionally regarded as the first option of revascularization strategy for diabetic patients with multivessel disease or unprotected left main disease and concomitant significant left ventricular systolic dysfunction.19 However, recent development of promising devices including newer generation DESs which are very stable to stent thrombosis and lower incidence of restenosis, now we should consider individual patients risk-benefit balance of surgical versus percutaneous revascularization. We should consider reducing periprocedural complications, systemic general condition and also socioeconomic status of the patient. Even after the bypass surgery in patients with small CAD, it is also associated with an increase in late adverse clinical events since small CAD provide poor distal runoff for the aorto-coronary grafts which can lead graft malfunction or occlusion.

Ballon Angioplasty Alone and Non-Stent Devices in Small CAD


Balloon angioplasty only (Plain Old Balloon Angioplasty, POBA) has been the main PCI strategy for small CAD patients for almost 2 decades. POBA is at least similarly safe and effective with a variety of different atheroablation or debulking devices such as directional atherectomy, rotational atherectomy, cutting balloon atherectomy or laser angioplasty.20 However, binary restenosis rates following POBA in patients with small CAD reach to 50-60%. Nonetheless, a favorable midterm clinical outcomes are predicted once an operator can achieve a satisfactory post POBA angiographic results (residual diameter stenosis <20%). In this point of view, POBA results appears even non-inferior to BMS.6 Although several non-balloon and non-sent devices have been challenged in both usual CAD and small CAD, still the data are inconclusive and not superior. Only very selective usage of cutting balloon, Fx MiniRail, or Angioscore can be used in particular subset of patients with small CAD.21-22

Bare Metal Stent (BMS) in Small CAD


The balloon-expandable bare metal stents (BMS) led significant improvements in acute procedural success of PCI, reduced restenosis rate at least mid to long term when compared with POBA period. Hausleiter et al reported that among the 3156 patients treated with BMS in

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small CAD, acute coronary syndrome (ACS) at admission and LV ejection fraction (EF) were main predictors of early adverse clinical outcomes, whereas binary restenosis at midterm follow-up was between 30% and 55% and was directly dependent on lesion length and total stent length.23 After BMS implantation in patients with small CAD, practical clinical problems are the adverse cardiac events associated with restenosis. It is imperative to know the important factors predicting restenosis for adequate clinical implication, especially for patients with small CAD. Strut thickness and density can be a factor; lower strut thickness yielded less abundant neointimal hyperplasia.24 Kasaoka et al. also demonstrated some important procedural predictors for restenosis after BMS implantation using angiography and IVUS. Total stent length, smaller reference lumen diameter and smaller final minimum lumen diameter were strong predictors of in-stent restenosis. More accurately, IVUS stent lumen cross-sectional area was actually a better independent predictor than angiographic parameters.25 There have been continuing arguments and controversies regarding efficacy and safety between BMS and POBA only for this particular challenging subset. To make conclusion, Agostoni et al. conducted an extensive meta-analysis including 13 studies and 4383 patients, showing that BMS may have an overall beneficial impact on binary restenosis (27.8% versus 35.8% for POBA group, p=0.003) and target lesion revascularization (TLR, 14.9% versus 19.7%, p=0.02). However, the benefit was heterogenous (p=0.001) and the results still have significant limitation. Currently we cannot make definite conclusion whether BMS is absolutely needed and superior to the efficacy of POBA only or not.

Hybrid Stenting Using Bare Metal Stent (BMS) and Drug-Eluting Stent (DES) in Small CAD
Even during the DES era, there were limited sizes in every DES platform. Although many studies in the literature suggested that DESs are far superior to BMS in small vessels, DESs of < 2.5 mm were not approved by the FDA until September 2008 when the Taxus atom 2.25 mm stent was approved. So for the treatment of real very small CAD which reference diameter is 2.5 mm had definite limitation in selecting a stent. Target vessels of 2.0 mm in diameter pose a major challenge in view of higher restenosis rates when stented BMS are used and distal edge dissection when oversized stents are deployed. In this point of view, some cardiologists have tried funneling to optimize small vessel stenting.26-27 This strategy combines stenting the distal part of the lesion with short (812 mm length) 2-mm BMS, while stenting with a larger-diameter ( 2.5 mm) DES with considerable DES-BMS overlap (leaving only the distal 4-6 mm of the BMS not overlapped by a DES). They suggested that the two stents create a funnel that is for the post part drug eluting. Their intention with funneling was to get adequate drug elution and larger diameter, minimizing both distal edge dissection and restenosis. Kronsteiner E et al. reported the superiority of this hybrid BMS/SES (n=41) to BMS alone (n=62) in small vessel disease.27 Despite the use of more stents and longer stent length in the BMS/SES group, there was a significant reduction in the composite of restenosis, myocardial infarction and cardiac death (9% versus 29%, p=0.033). They also suggested that the use of BMS cap distally, telescoped in tandem with SES more proximally, can be a reasonable option in treating long lesions in small CAD with distal reference diameters 2.5 mm. This can be an option even in the DES era; however, the prohibitive factors are the excessive cost of two stents and the potential restenosis in the distal edge. Because already several very small DES which is 2.25 mm diameter have been released, we have more cost-effective strategy and more options for very small CAD.

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Drug Eluting Stent (DES) in Small CAD


The development and introduction of DES in daily clinical practice has revolutionized PCI by providing a device that effectively reducing restenosis even in higher risk complex patients and complex lesions including small CAD, thus DES have lead clinically relevant benefits in terms of TLR and MACE. The Canadian Study of the Sirolimus-Eluting Stent in the Treatment of Patients with Long, De Novo Lesions in Small Native Coronary Arteries (C-SIRIUS) trial showed the sirolimus-eluting stents (SES, CypherTM, Cordis, Miami, FL USA) reduced binary restenosis from 52% of BMS to only 2% with SES (P<0.01).28 The Sirolimus-Eluting Stent and a Standard Stent in the Prevention of Restenosis in Small Coronary Arteries Trial (SES-SMART) is the only randomized trial specifically focused on the comparison of DES vs. BMS in small CAD (2.75 mm inclusion but actually less than 2.25 mm).29 In this study, among the 257 patients binary in-segment restenosis in BMS was 53% but 10% in DES group (p<0.001). Similar results were reported by subanalysis of other trials or registries on SES; SES was associated with very low late loss ranging between 0.05 and 0.20 mm in patients with small CAD.30-31 Paclitaxel-eluting stent (PES, TaxusTM, Boston Scientific, Natick, MA, USA) is another most effective first generation DES. However, the late loss revealed from several pivotal studies was averaging 0.30 mm, which can unfavorably impact on outcomes in very small CAD. The direct comparison data with DESs associated with lower late loss such as SES and everolimus-eluting stent (EES), PES failed to show the superiority in treating small CAD due to higher late loss, either from head to head randomized trials32-34 or observational registries.35 Zotarolimus-eluting stent (ZES, EndeavorTM, Medtronic, Minneapolis, MN, USA) is the 3rd major DES which differ in polymer covering (phosphorylcholine) but showed highest late loss (ranging between 0.50 and 0.70 mm) among the major DESs.36 Thus, it may not appealing in patients with small CAD because even a mild increase in late loss can lead to increase in binary restenosis. However, the newer generation Endeavor ResoluteTM which has BioLinx polymer is known to have very low late loss and 2.25 mm platform is available, and can be a good candidate device for patients with very small CAD. Everlolimus-eluting stents (EES, Xience V from Abbott Vascular, Abott Park, IL, USA or Promus from Boston Scientific) are associated with very low late loss (lower than 0.20 mm), making them another alternatives for patients with very small CAD. From the SPIRIT III randomized trial, the EES reduced late loss, restenosis and MACE as compared with PES.34 Interestingly, newly released Promus ElementTM has excellent radioopacity, flexibility and deliverability compared to 1st generation SES, providing wide adaptation to complex small CAD. In real-world clinical practice, we have studied and reported subanalysis data regarding very small CAD from the Korea University Guro Hospital PCI registry, a real world Asian registry. Rha SW et al. reported that among the 137 patients with 143 de novo small coronary lesions, six-month angiographic outcomes showed that the incidence of binary restenosis and late loss were lower in SES group compared PES group (13.9% versus 27.7%, P=0.041 and 0.52 0.41 versus 0.84 0.65, P=0.032). Despite these angiographic benefits, mid-term clinical outcomes were similar between two groups. At 6-month follow up, the survival and freedom from MACEs was similar between two groups (96.06% versus 97.4%, p=0.419). SES for the treatment of small coronary lesions showed more angiographically favorable results compared with PES but failed to translate its benefits into better clinical outcomes.37 Rha SW et al. reported the efficacy of 2.25 mm PES in diabetic patients with very small CAD.38 PES was selectively implanted in 64 pts with very small, de novo coronary artery

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lesions (less than 2.5 mm). The angiographic and clinical outcomes of DM group (n=30) were compared with those of non-DM group (n=34) at 6 months. At 6-month follow-up, the angiographic and clinical outcomes were similar between two groups but showed higher rate of binary restenosis (20% versus 30.8%, P=0.453). The survival and freedom from MACEs was similar between two groups (96.67% versus 97.06%, P=1.000). DES implantation using 2.25 PES in diabetic pts with very small vessel lesion was safe and showed excellent clinical outcomes although the incidence of binary restenosis was relatively higher. Rha SW et al. also investigated whether there are differences in outcomes in small stenting in main branch versus side branch.39 A total 90 patients with single, small (less than 2.50 mm), denovo coronary artery lesion located either MB or SB were implanted with single DES at the lesion segment. Among them, 6-month angiographic and clinical outcomes of DES implanted in MB (51 lesions) were compared with those of DES in SB (39 lesions). At 6 months, the incidence of binary restenosis was relatively high as 22.7%. However, there were no differences between two groups in angiographic and clinical outcomes. The survival and freedom from MACEs was similar between two groups (98.0% versus 97.4%, P=1.000). DES implantation in MB or SB in patients with small vessel bifurcation lesions was safe and showed similarly excellent clinical outcomes despite the incidence of binary restenosis remained high. Thus, as DES in general appear superior to BMS from a variety of studies, DESs with lower late loss would be preferable strategy in managing small vessel disease. With the technical development, now we have more options of DESs which have not only the anti-restenotic effect of DESs, but also the excellent deliverability, flexibility, conformability and quick inflation/deflation properties.

Drug Eluting Balloon (DEB) in Small CAD


After DES introduction, with the time, the DES fantasy becoming down due to several untoward dark side of DES including stent thrombosis, DES associated neoaneurysm, late stent malapposition, DES associated endothelial dysfunction and clinically significant vasospasm and hypersensitivity reaction. Stent-based local drug delivery might be associated with delayed and incomplete endothelialization and potential risk of subacute and even late stent thrombosis.40 First in-man trials with a paclitaxel-coated balloon catheter have shown beneficial effects in the treatment of coronary in-stent restenosis41 and in peripheral arteries.42 For the safety and efficacy of drug-eluting balloon (DES) application in small vessel disease, PEPCAD I trial was conducted.43 This trial was to investigate the SeQuent Please balloon catheter (B. Braun, Melsungen, Germany), a second generation paclitaxel-coated balloon, in the treatment of small CAD. A total 118 patients with small CAD were treated by a paclitaxel-coated balloon (3 g/mm2). The main inclusion criteria encompass diameter stenosis of 70% and 22 mm in length with a vessel diameter of 2.25-2.8 mm. Seventy percent of the patients treated DEB only and rests of patients were required additional stents. At 6 months, mean in-segment late loss was 0.280.53 mm. In patients with DEB only, the in-segment late loss was 0.160.38 mm. At 12 months, the MACE rate was 15% which primarily due to the need for TLR (14 patients, 12%). They concluded that the DEB was well tolerated and may offer an alternative to DES implantation. The concept of a DES allows for a homogenous drug application to the arterial wall. Contrast to DES, it avoids a long lasting drug exposition of stent struts and the use of polymers. Thus, prolonged administration of dual antiplatelets will not be required. The PEPCAD I trial, the clopidogrel was given only for 1 month following DEB treatment and for 3 months after additional BMS. With more technical development, we will have more promising options for patients with small CAD.

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ROLE OF CORONARY IMAGE AND PHYSIOLOGY IN SMALL VESSEL DISEASE


In patients with small CAD, the IVUS evaluation will be critically important when the lesion location is in proximal segment of 3 major epicardial coronary arteries. In small vessels, IVUS clearly demonstrated that the coronary angiography often mistakenly underestimate real reference vessel diameter, especially in patients with diffuse disease such as diabetes.15 Thus, IVUS should be recommended whenever there is uncertainty on the exact reference vessel diameter of the target segment, as well as to optimize the stenting. However, it is unclear whether IVUS should be used routinely or more selectively in patients with small CAD undergoing DES implantation because currently no pertinent controlled trials are yet available. StentBoost (Philips Cine Angiography) is a novel radiographic technique that increases spatial resolution of the cineangiographic equipment to more accurately appraise stent expansion. Either in large or small vessel PCI, this appears a promising and cost-effective tool to appraise stent expansion in small vessels.44 Choi CU, Oh DJ, Rha SW et al. reported the impact of StentBoost enhancement guided PCI on mid-term angiographic and clinical outcomes.45 We compared midterm angiographic and clinical outcomes between StentBoost group (n=301) and No StentBoost group (n=569). At 6 months, the rate of binary restenosis, late loss, TLR, TLR-MACE and target vessel revascularization (TVR)-MACE were lower in stentboost group. This can be similarly implicated into small CAD. Optical coherence tomography (OCT) has superior spatial resolution in comparison to IVUS. It will play an important role in the future. However, the lower depth of penetration can be a major limitation. OCT can be a complement to IVUS and more studies will be needed to make final conclusion. The role of coronary physiology in PCI is dramatically increasing with the development and introduction of coronary pressure wire and fractional flow reserve (FFR) measurement in several particular subsets of patients. Usual cutoff value for intervention is lower than 0.75-0.80 after induction of maximal hyperemia using adenosine. Currently, FFR is strongly recommended in intermediate lesion, jailed side branch, ambiguous ISR lesion and even multivessel disease. Similarly it can be recommended in patients with angiographically intermediate stenosis in small coronary vessels or whenever there is no definite proof of correlation with symptoms or signs of myocardial ischemia.46

DISCUSSION
Currently, DES implantation became main strategy for treatment of small CAD. Now clinical implication of DEB is increasing in small CAD. The development of bioabsorbable stents may be a help to get another option. But still need more development and data. Despite the great technical evolution of PCI, the optimal treatment of small CAD by PCI is still debated. Although the DES is the mainstay of PCI, still some selective patients cannot easily be treated with DES. Small CAD which is relatively clinically not important, thus not worthy to receive a DES, very small but tortuous and calcified lesion may not be comfortable to receive DES. In these selective lesions or patients can be managed with POBA alone and/or subsequent DEB. The clinical relevance of small CAD should be decided on clinical situation. Patients presenting with typical ischemic symptoms and concomitant signs of definite ischemia are clear candidates for PCI. Different sizes of coronary arteries in different populations also should be considered; diabetics, CRI patients, females, elderly populations, Aisan populations.

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CONCLUSIONS
Over the past decade, the techniques and outcomes of DESs and other devices have substantially advanced. Nevertheless, since majority of the clinical trials comparing the therapeutic strategies using different devices for small vessel disease have been limited to selected patient populations, optimal treatment modalities for high-risk patients with small CAD and multiple co-morbidities remain undetermined. Currently, the ideal stent to treat typical small CAD should be a DES with a low late loss, a stent platform with low strut thickness, and high conformability to the vessel. In 2010, two available and promising candidates (Promus Element 2.25 mm and Endeavor Resolute 2.25 mm) are released in the market need extensive evaluation of their safety and efficacy in patients with small CAD. Thus, more patients with small CAD shall be safely and effectively treated than previous days and more clinical benefits are expected to those patients with typical small CAD as primary target for their disease. In selective cases, the usage of DEB is increasing but more data for small CAD will be needed.

REFERECES
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42. Tepe G, Zeller T, Albrecht T, et al. Local taxane with short exposure for reduction of restenosis in distal arteries. THUNDER trial. N Engl J Med 2008; 358:689-699. 43. Unverdorben M, Kleber FX, Heuer H, et al. Treatment of small coronary arteries with a paclitaxel-coated balloon catheter. Clin Res Cardiol 2010; 99:165-174. 44. Mishell JM, Vakharia KT, Ports TA. Determination of adequate coronary stent expansion using StentBoost, a novel fluoroscopic image processing technique. Catheter Cardiovasc Interv 2007; 69:84-93. 45. Choi CU, Oh DJ, Rha SW, et al. Impact of stentboost enhancement guided percutaneous coronary intervention on mid0term angiographic and clinical outcomes. J Am Coll Cariol 2010; 56:13 (Supple B), B87. 46. Costa MA, Sabate M, Staico R, et al. Anatomical and physiologic assessments in patients with small coronary artery disease: final results of the Physiologic and Anatomical Evaluation Prior to and After Stent Implantation in Small Coronary Vessels (PHANTOM) trial. Am Heart J 2007; 153:296.e1-296.c7.

CHAPTER 16

(LATE) STENT MALAPPOSITION IN THE BARE-METAL STENTS AND DRUG ELUTING STENTS ERA
JEFFREY J.W. VERSCHUREN, TAREK A.H.N. AHMED, IOANNIS KARALIS, PAUL H.A. QUAX and J.WOUTER JUKEMA
Introduction............................................................................................................................... Definition and Classification .................................................................................................... Pathophysiology........................................................................................................................ Risk Factors .............................................................................................................................. BMS versus DES ...................................................................................................................... Diagnostics ............................................................................................................................... Treatment .................................................................................................................................. Future Perspectives ................................................................................................................... Conclusion ................................................................................................................................ References................................................................................................................................. 245 245 246 247 249 249 250 250 251 251

INTRODUCTION
Since the introduction of drug eluting stents (DES), the incidence of coronary stent restenosis has been significantly reduced. However, the safety of DES became a major issue after several studies associated DES with an increased risk of the rare but often devastating development of late and very late stent thrombosis1-3. Several risk factors have emerged as being important in the development of stent thrombosis, including stent malapposition. Although it is still under debate what the exact role of stent malapposition in this respect is, it appears undeniable that stent malapposition is involved in the development of this severe complication after stent implantation in at least a part of the cases4-10. In this chapter several aspects of stent malapposition will be discussed.

DEFINITION AND CLASSIFICATION


Stent malapposition (SM), also known as incomplete stent apposition, is defined by a separation of at least one stent strut from the intimal surface of the arterial wall with evidence of blood behind the strut, without involvement of side branches11. Stent malapposition may increase the thrombotic risk due to the presence of intraluminal stent struts12. Although appropriate apposition of the stent to the vessel wall is an important aspect for all stents, it seems to be critical in the case of DES to ensure antiproliferative drug delivery as well as circumferential vascular support.

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Depending on the time point of detection, the following classification can be made; acute if present immediately after the index procedure and late if detected at follow-up. Furthermore, resolved if present after stent implantation but not at follow up, persistent if present both directly after stent implantation and at follow up and as acquired when the stent is well apposed after the index procedure but SM is detected at follow up. Differentiating between the different forms of SM therefore requires intravascular imaging both at stent implantation and at follow up. Schematically this is shown below as depicted by Hur et al., Cardiovasc Revasc Med 200913 (Figure 16.1).

Figure 16.1. Various types of stent malapposition by Hur et al., Cardiovasc Revasc Med 200913.

It is important to realize that the distinction between acute and late SM also implies different pathogenetic mechanisms for these two entities. Furthermore, each form will require a different therapeutic approach, which will be addressed later in the chapter.

PATHOPHYSIOLOGY
The current thought is that acute SM is mostly technique dependent and may result from inadequately sized stent selection or inadequate stent expansion, whereas late acquired SM may result from vessel wall changes in the stented segments that occur during the follow-up period. Focusing on late SM, several mechanisms have been postulated; (1) positive vascular remodeling of the vessel wall; (2) decrease in plaque volume; (3) chronic stent recoil and (4) a hypersensitivity reaction to one of the stent constituents.

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Positive remodeling is the increase in vascular dimension measured by intravascular imaging using intravascular ultrasound (IVUS) or optical coherence tomography (OCT) by analyzing the change in cross sectional area of the external elastic membrane (EEM) in comparison with the change in plaque volume over time. Several studies reported a greater increase of the EEM compared to the change in plaque volume. Since the size of the stent cannot increase over time, except for the now rarely used self expanding stents, this process will eventually lead to stent malapposition13-19. A recent study of Kang et al. used IVUS immediately after intervention and at the 6-month and 2-year of follow-up to evaluate serial vascular changes after DES implantation 19. Their main conclusion was that the development of malapposition was not limited to the first 6 months after implantation as a result of ongoing vascular remodeling even after this period. Therefore, the reported incidence of acquired late SM in previous studies (between 0% and 25%20) maybe underestimated due to relative short-term follow-up periods19. Besides an increase of the vascular dimensions, another possible mechanism to stent malapposition is a decrease of the plaque volume. This can be caused by dissolution of a thrombus, present at the stent implantation, mainly seen in patients with acute myocardial infarction4,13,14,21,22. Furthermore, regression of the plaque under stringent statin treatment can result in creating space between the stent and the vessel wall; however this seems to be only the case in a minority of lesions12, 21, 23. The pattern of late acquired SM is usually focal and is more often found at the borders of the stents (up to 90%, as reported by Mintz et al.)17. Additionally, acquired SM is more likely to occur in the relatively disease-free side of the vessel wall, probably because the normal vessel gets more injured during DES implantation and the delayed healing due to the drugs loaded on these stents13,24. It is therefore more likely that positive remodeling is the most important mechanism for the development of late SM and that the contribution of plaque change is more limited13. Although chronic stent recoil could theoretically be one of the causes of SM, it has not been detected using IVUS in patients with SM, making it therefore unlikely to be a factor in the development of SM17, 25. A final factor of possible influence is the inflammatory response to these stents. In contrast to bare-metal stents (BMS), DES provokes inflammatory responses in animal models, either by a local hypersensitivity reaction to the non-biodegradable polymers of the stent or to the drug released by the stent. Since in a pig model the hypersensitivity reaction only peaks after complete release of the drug, it appears more likely that the polymer is the cause22. Also in human thrombectomy specimens histopathological signs of inflammation were found, thus supporting the theory that a hypersensitivity reaction underlies the development of SM and stent thrombosis5. Furthermore, a growing number of reports documented the formation of coronary artery aneurysm (CAA) after DES implantation. Although the exact mechanism is still unknown, the available evidence suggest that it may be a hypersensitivity-mediated reaction to the delivery polymer26. Studies of related polymers have demonstrated local and systemic hypersensitivity reactions to intravascular polymers5,27-29. Also, animal studies of DES show that 25% of pigs receiving DES develop eosinophilic infiltrates29. Bare-metal stents have not been demonstrated to elicit such hypersensitivity reactions in human autopsy series of over 400 stents29.

RISK FACTORS
Predictors of acute SM include aneurismal appearance of the target lesion, larger vessels, lesion calcification, higher patient age, longer lesions and lower balloon pressure21,30. This is in line with the above statement that acute SM is most likely technique dependent, since these predictors are contributing to the complexity of the target lesion or to the technique itself. Also for late SM, several factors related to the different proposed mechanisms of SM have been shown to be independent predictors. The highest incidence of 25% of late SM in DES

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stented patients has been reported in the MISSION! Intervention study, including patients with acute myocardial infarction21. Several studies have shown AMI as an independent predictor of SM14,16. The explanation for this can be found in the second proposed mechanism of SM. The presence of a thrombus, particularly a large thrombus load, which is frequently encountered in AMI may predispose to the occurrence of late SM after the dissolution of the thrombus at the site of stent implantation. Diabetes mellitus has been associated with a lower rate of stent malapposition. The diabetic subpopulation is known to have an increased neointimal growth leading to more restenosis in BMS. Poor glycemic control has been associated with diminished efficacy of sirolimus (one of the drugs used on DES) on smooth muscle cell proliferation, which may explain the lower rate of late SM in these patients21,30-32. Opposing these reports however, another study found a significant greater proportion of diabetic patients in DES cohort with late SM compared to the BMS cohort14. They reasoned that the proinflammatory role of diabetes may be responsible for a local enhanced inflammatory reaction after DES implantation eventually increasing the risk of late SM. Another predictor of SM is directional coronary atherectomy (DCA) before stenting. The higher incidence of late SM in DCA before stenting might be explained by the fact that aggressive debulking with DCA is associated with deep vessel injury and promotes more positive remodeling15. Despite angiographic optimization with high pressures and adequately sized balloons, malapposed stent struts are frequently found in complex coronary lesions and more often following the implantation of Cypher Select stents which have a thicker stent strut and closed cell design33. Other factors associated with the lesion complexity, such as longer stent length, C-type lesions and overlapping stents, larger vessel reference diameter, have also been associated with an increased risk of SM14,16,21. Also, chronic total occlusion (CTO), defined by the absence of antegrade flow or only minimal flow of contrast distal to the occlusion during coronary angiography before stent implantation, has been reported as an independent predictor of late SM after DES implantation16. Patient age has been associated with the risk of SM, although not consistently. In a recent report of Steinberg et al., subjects with acute SM are older than those without acute SM, whereas (younger) age was the only independent predictor of late acquired SM. Their explanation for this last finding is that most of the other reported risk factors for SM were excluded from their study30. As will be discussed in the next section, also the stent type is associated with the risk of late stent malapposition. All risk factors are summarized in Table 16.1.
Table 16.1. Risk factors for stent malapposition Clinical factors Acute myocardial infarction (L) Absence of diabetes mellitus (L) Chronic total occlusion (L) Patient age (A,L) Procedure related factors Drug eluting stent (L) Lower maximum balloon pressure (A, L) Larger vessel reference diameter (A, L) Longer stent length (A, L) Overlapping stents (A, L) C-type lesion (A,L) Directional coronary artherectomy (L) (A) Risk factor for acute stent malapposition, (L) Risk factor for late stent malapposition.

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BMS VERSUS DES


Acute SM is frequently observed both after DES and BMS implantation21,30. Considering the mechanism of acute SM, a similar incidence in both DES and BMS is in the line of expectation. Late SM is rare after BMS and seems to be related to stent under-expansion in most patients21. Although not all, several studies have reported that late SM is much more common after DES implantation6,14,21,24,30,34. A recent meta-analysis of Hassan et al. aimed on clarifying this, included 2453 patient after BMS implantation and 2195 patients receiving DES from a total of 17 studies20. The incidence of late acquired SM after DES varied between 0% and 25%, whereas after BMS implantation the highest reported incidence of SM was 6% at 6 months. They concluded that the risk of late acquired SM was 4.4-fold higher in patients after DES implantation compared to those with BMS. A comparison of paclitaxel- with limus-eluting stents did not yield significant findings. The higher risk of SM after DES is likely due to the effects of the drugs on the vessel wall, resulting in positive remodeling20,21. In BMS the lumen changes at the site of SM are more related to plaque burden21. Furthermore, Hassan et al. also conducted a meta-analysis on the risk of (very) late stent thrombosis in patients with late SM. They concluded that the risk of stent thrombosis is 6.5 times higher (95% confidence interval 1.3434.91) in patients with late SM compared with those without late SM20. So late SM appears to be more frequent after DES implantation and is associated with an increased risk of late stent thrombosis. After adequately lowering the incidence of restenosis, the primary complication of balloon dilation and BMS implantation, we may have created a potentially more devastating obstacle with late SM and subsequent stent thrombosis in DES.

DIAGNOSTICS
Although intravascular ultrasound (IVUS) has been usually regarded as the gold standard for in vivo assessment of stent strut apposition to the vessel wall, more accurate evaluation of stent strut apposition cannot be successfully performed in some lesions with minimal stent malapposition due to the limited resolution capacity in IVUS (100-150 um)35. Furthermore, this limited axial resolution of IVUS makes it virtually impossible to discriminate between atherosclerotic plaque and thrombus21. It also has problems with stent-related artifacts12, possibly resulting in underestimation of the evaluation of re-endothelialization. SM is associated with a decreased re-endothelialization after DES implantation22, which is in turn associated with an increased risk of stent thrombosis7. Optical coherence tomography (OCT) is a relatively new imaging modality. In contrast with IVUS, OCT visualizes intra-coronary features using near-infrared light instead of ultrasound, leading to a far better axial resolution, able to resolve detail up to 10 m12. Multiple studies investigating stent malapposition using OCT and comparing it with IVUS findings have already been published. There is evidence that minimal stent malapposition which is not detectable by IVUS may disappear or decrease in follow-up OCT evaluation35 and SM without complete re-endothelialization is associated with presence of OCT-detected thrombus12. More detailed information provided by OCT imaging also led to the conclusion that rate of stent strut coverage and malapposition were significantly different among different DES types and among the type of clinical presentation. More SM was found in sirolimus-eluting stents and paclitaxel-eluting stents compared to zotarolimus-eluting stents and the rate of SM was higher in patients presenting with acute coronary syndrome compared to those with stable angina pectoris36,37.

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Despite the clear advantages of OCT over IVUS, some disadvantages should also be mentioned. The cost of the device is higher than that of IVUS and the device is not available in every center. A major limitation of OCT is the requirement of a bloodfree imaging field because red blood cells scatter light. This can be achieved with a continuous saline flush administration or with a temporary balloon occlusion catheter, leading to transient ischaemia which is not desirable and or tolerated in all cases. Another disadvantage of OCT is poor penetration into non-transparent tissues thus allowing evaluation of only superficial structures including coronary plaques with thin-caps38.

TREATMENT
The most important and potential devastating complication of SM is stent thrombosis. The mechanism by which SM may contribute to stent thrombosis remains unclear. SM may serve as a local nidus for thrombus formation, allowing fibrin and platelet deposition. Delayed re-endothelialization, impaired vasomotion and also involvement of chronic inflammation and delayed healing, leading to tissue necrosis around the stent, all contribute to creating a thrombogenic environment20,23. When performing intravascular imaging directly post stent implantation, immediate action can be untaken after diagnosing acute stent malapposition by re-dilatation of the target lesion, further expanding the stent. Considering dissolution of a jailed thrombus as a possible mechanism of SM, it may be important to effectively remove thrombotic material prior to stent implantation. Unfortunately, it has been shown that immediate post-intervention IVUS showing no malapposition does not guarantee an uneventful course after DES implantation8. After diagnosing stent malapposition during follow-up, defining a proper treatment strategy is difficult, especially when the patient is asymptomatic. The main goal should be off course to prevent stent thrombosis. One option could be pharmacological with long-term double anti-thrombotic treatment11. However, this will also lead to a continuous increased bleeding risk and it is evident that SM may also persist for years without leading to complications, creating a risk of exposing patients to unnecessary side effects of the treatment without having the benefit of it. Another option could be dilation and implementation of a second, larger, stent39.

FUTURE PERSPECTIVES
It goes without saying that all the, until now, unanswered questions about the mechanism of stent malapposition, its association with stent thrombosis and the best treatment of SM are to be clarified in future larger studies. One of the current developments in this field is the development of a bioabsorbable stent. The theoretical advantages of such a stent is that it might have less potential for late stent thrombosis because there will eventually be no foreign material exposed to the bloodstream. Also problems with later surgical revascularization, eliminating vasomotor reactions and interference with imaging techniques could be prevented40. However, this remains to be proven and also partial/unequal stent dissolution may perhaps cause problems. Lately, there have been some ongoing clinical trials addressing the use of self-expandable nitinol stents in the treatment of AMI patients with long-term follow-up IVUS and OCT41-43. The hypothesis generated in these trials was that the self-expandable stents would better accommodate to early changes in the vessel wall (thrombus dissolution and vasodilatation) with better apposition due to its self-expandable properties. Initial results have shown that these stents provided 20% increase in lumen area with perfect apposition on 3 days follow up OCT. However, long-term (safety) results are still lacking.

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CONCLUSIONS
Stent malapposition appears to be a relative common finding after stent implantation, especially diagnosed using OCT which has a superior resolution compared to IVUS. The incidence of late SM is higher after DES implantation, mainly caused by positive vascular remodeling. Although SM is associated with stent thrombosis, this is a relatively rare complication. Since the incidence of SM is vastly greater than that of stent thrombosis, SM is not necessarily sine qua non, but more likely only one factor in a complex system. Nevertheless, stent thrombosis is a devastating complication and must be prevented where possible. Therefore, further research unraveling the exact mechanisms of stent malapposition and stent thrombosis and possible treatments will continue in future clinical trials.

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Iakovou I, Schmidt T, Bonizzoni E et al. Incidence, predictors, and outcome of thrombosis after successful implantation of drug-eluting stents. JAMA 2005 May 4;293(17):2126-30. Jeremias A, Sylvia B, Bridges J et al. Stent thrombosis after successful sirolimus-eluting stent implantation. Circulation 2004 April 27;109(16):1930-2. McFadden EP, Stabile E, Regar E et al. Late thrombosis in drug-eluting coronary stents after discontinuation of antiplatelet therapy. Lancet 2004 October 23;364(9444):1519-21. Cook S, Wenaweser P, Togni M et al. Incomplete stent apposition and very late stent thrombosis after drug-eluting stent implantation. Circulation 2007 May 8;115(18):2426-34. Cook S, Ladich E, Nakazawa G et al. Correlation of intravascular ultrasound findings with histopathological analysis of thrombus aspirates in patients with very late drug-eluting stent thrombosis. Circulation 2009 August 4;120(5):391-9. Lee CW, Kang SJ, Park DW et al. Intravascular ultrasound findings in patients with very late stent thrombosis after either drug-eluting or bare-metal stent implantation. J Am Coll Cardiol 2010 May 4;55(18):1936-42. Luscher TF, Steffel J, Eberli FR et al. Drug-eluting stent and coronary thrombosis: biological mechanisms and clinical implications. Circulation 2007 February 27;115(8):1051-8. Bergheanu SC, Van der Hoeven BL, Hassan AK et al. Post-intervention IVUS is not predictive for very late in-stent thrombosis in drug-eluting stents. Acta Cardiol 2009 October;64(5):611-6. Alfonso F, Suarez A, Angiolillo DJ et al. Findings of intravascular ultrasound during acute stent thrombosis. Heart 2004 December;90(12):1455-9. Alfonso F, Suarez A, Perez-Vizcayno MJ et al. Intravascular ultrasound findings during episodes of drug-eluting stent thrombosis. J Am Coll Cardiol 2007 November 20;50(21):2095-7. Alfonso F, Cruz A, Garcia J. Late stent malapposition: innocent phenomenon or major risk marker? Eur Heart J 2010 January;31(2):260-1. Ozaki Y, Okumura M, Ismail TF et al. The fate of incomplete stent apposition with drug-eluting stents: an optical coherence tomography-based natural history study. Eur Heart J 2010 June;31(12):1470-6. Hur SH, Ako J, Honda Y, Sudhir K, Fitzgerald PJ. Late-acquired incomplete stent apposition: morphologic characterization. Cardiovasc Revasc Med 2009 October;10(4):236-46. Chechi T, Vecchio S, Lilli A et al. Mechanisms of late stent malapposition after primary stenting in ST-elevation myocardial infarction: a subanalysis of the selection trial. J Interv Cardiol 2009 June;22(3):201-6. Hong MK, Mintz GS, Lee CW et al. Incidence, mechanism, predictors, and long-term prognosis of late stent malapposition after bare-metal stent implantation. Circulation 2004 February 24;109(7):881-6. Hong MK, Mintz GS, Lee CW et al. Late stent malapposition after drug-eluting stent implantation: an intravascular ultrasound analysis with long-term follow-up. Circulation 2006 January 24;113(3):414-9. Mintz GS, Shah VM, Weissman NJ. Regional remodeling as the cause of late stent malapposition. Circulation 2003 June 3;107(21):2660-3. Shah VM, Mintz GS, Apple S, Weissman NJ. Background incidence of late malapposition after bare-metal stent implantation. Circulation 2002 October 1;106(14):1753-5. Kang SJ, Mintz GS, Park DW et al. Late and Very Late Drug-Eluting Stent Malapposition: Serial 2-Year Quantitative IVUS Analysis. Circ Cardiovasc Interv 2010 July 6. Hassan AK, Bergheanu SC, Stijnen T et al. Late stent malapposition risk is higher after drug-eluting stent compared with bare-metal stent implantation and associates with late stent thrombosis. Eur Heart J 2010 May; 31(10):1172-80.

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21. Van der Hoeven BL, Liem SS, Dijkstra J et al. Stent malapposition after sirolimus-eluting and bare-metal stent implantation in patients with ST-segment elevation myocardial infarction: acute and 9-month intravascular ultrasound results of the MISSION! intervention study. JACC Cardiovasc Interv 2008 April; 1(2):192-201. 22. Finn AV, Nakazawa G, Joner M et al. Vascular responses to drug eluting stents: importance of delayed healing. Arterioscler Thromb Vasc Biol 2007 July; 27(7):1500-10. 23. Shiran A, Weissman NJ, Leiboff B et al. Effect of preintervention plaque burden on subsequent intimal hyperplasia in stented coronary artery lesions. Am J Cardiol 2000 December 15;86(12):1318-21. 24. Ako J, Morino Y, Honda Y et al. Late incomplete stent apposition after sirolimus-eluting stent implantation: a serial intravascular ultrasound analysis. J Am Coll Cardiol 2005 September 20;46(6):1002-5. 25. Costa MA, Sabate M, Kay IP et al. Three-dimensional intravascular ultrasonic volumetric quantification of stent recoil and neointimal formation of two new generation tubular stents. Am J Cardiol 2000 January 15;85(2):135-9. 26. Levisay JP, Roth RM, Schatz RA. Coronary artery aneurysm formation after drug-eluting stent implantation. Cardiovasc Revasc Med 2008 October; 9(4):284-7. 27. van der Giessen WJ, Lincoff AM, Schwartz RS et al. Marked inflammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries. Circulation 1996 October 1;94(7): 1690-7. 28. Nebeker JR, Virmani R, Bennett CL et al. Hypersensitivity cases associated with drug-eluting coronary stents: a review of available cases from the Research on Adverse Drug Events and Reports (RADAR) project. J Am Coll Cardiol 2006 January 3;47(1):175-81. 29. Virmani R, Guagliumi G, Farb A et al. Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious? Circulation 2004 February 17;109(6):701-5. 30. Steinberg DH, Mintz GS, Mandinov L et al. Long-term impact of routinely detected early and late incomplete stent apposition: an integrated intravascular ultrasound analysis of the TAXUS IV, V, and VI and TAXUS ATLAS workhorse, long lesion, and direct stent studies. JACC Cardiovasc Interv 2010 May;3(5):486-94. 31. Patterson C, Mapera S, Li HH et al. Comparative effects of paclitaxel and rapamycin on smooth muscle migration and survival: role of AKT-dependent signaling. Arterioscler Thromb Vasc Biol 2006 July; 26(7): 1473-80. 32. Tanabe K, Serruys PW, Degertekin M et al. Incomplete stent apposition after implantation of paclitaxel-eluting stents or bare metal stents: insights from the randomized TAXUS II trial. Circulation 2005 February 22; 111(7):900-5. 33. Tanigawa J, Barlis P, Dimopoulos K, Dalby M, Moore P, Di MC. The influence of strut thickness and cell design on immediate apposition of drug-eluting stents assessed by optical coherence tomography. Int J Cardiol 2009 May 15;134(2):180-8. 34. Jimenez-Quevedo P, Sabate M, Angiolillo DJ et al. Vascular effects of sirolimus-eluting versus bare-metal stents in diabetic patients: three-dimensional ultrasound results of the Diabetes and Sirolimus-Eluting Stent (DIABETES) Trial. J Am Coll Cardiol 2006 June 6;47(11):2172-9. 35. Kim WH, Lee BK, Lee S et al. Serial changes of minimal stent malapposition not detected by intravascular ultrasound: follow-up optical coherence tomography study. Clin Res Cardiol 2010 April 21. 36. Fan C, Kim JS, Lee JM et al. Different vascular healing patterns with various drug-eluting stents in primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: optical coherence tomographic findings. Am J Cardiol 2010 April 1;105(7):972-6. 37. Kim JS, Fan C, Choi D et al. Different patterns of neointimal coverage between acute coronary syndrome and stable angina after various types of drug-eluting stents implantation; 9-month follow-up optical coherence tomography study. Int J Cardiol 2009 August 24. 38. Low AF, Tearney GJ, Bouma BE, Jang IK. Technology Insight: optical coherence tomography--current status and future development. Nat Clin Pract Cardiovasc Med 2006 March;3(3):154-62. 39. Roura G, Teruel LM, Gomez-Hospital JA. Very late stent thrombosis due to late stent malapposition. Rev Esp Cardiol 2009 December;62(12):1499-501. 40. Serruys PW, Ormiston JA, Onuma Y et al. A bioabsorbable everolimus-eluting coronary stent system (ABSORB): 2-year outcomes and results from multiple imaging methods. Lancet 2009 March 14;373(9667): 897-910. 41. IJsselmuiden A, Verheye S. First report on the use of a novel self-expandable stent for treatment of ST elevation myocardial infarction. Catheter Cardiovasc Interv 2009 November 15;74(6):850-4. 42. Verheye S. Latest results of the STENTYS clinical program. 2010. Oral presentation Euro PCR congress 2010. 43. Koolen J. STENTYS - Innovation in Self-Expanding Stent Technology, 2010. Oral presentation Euro PCR congress 2010.

CHAPTER 17

STENT FRACTURE AND RESTENOSIS


JONG-SEON PARK and YOUNG-JO KIM
Introduction............................................................................................................................... Incidence, Contributing Factors and Classification .................................................................. Diagnostic methods................................................................................................................... Clinical implications ................................................................................................................. Conclusions............................................................................................................................... References................................................................................................................................. 253 253 255 257 257 257

INTRODUCTION
Stent fracture, one of the troublesome complications associated with implantation of metallic stents in the human body, was first demonstrated in 1996 with Wall stents implanted in the subclavian vein which underwent fracture due to compression between the clavicle and rib eventually leading to restenosis.1 The heart is also a continuously moving organ usually beating more than 100,000 times a day, 30 million times per year, and therefore, a coronary stent which is implanted in an epicardial location is rendered prone to fracture by the repetitive cardiac motion in both the longitudinal and circumferential directions. In 2004, Sianos et al. described the first two cases of treatment failure with sirolimus-eluting stents related to stent fracture2, and in the drug-eluting stent era, stent fracture, which can develop and be found during and several years after stent deployment, has been recognized as one of the common complications of metallic coronary stent in association with a high rate of in-stent restenosis and cardiac events.3,4 In this chapter, we summarize the clinical features and contribution to cardiovascular complications of stent fracture.

INCIDENCE, CONTRIBUTING FACTORS AND CLASSIFICATION


The incidence of stent fracture varies among studies depending on the types of stent, definition of stent fracture and diagnostic imaging modalities used. The concern of an interventionist for this complication is also a very important determinant in detecting it because stent fracture is usually faintly seen on fluoroscopy. Generally, stent fracture is defined as interruption of the linear or curvilinear connections of stent struts on angiography and fewer visible stent struts than normal in the stented area on intravascular ultrasound (IVUS).5 Retrospective and prospective clinical studies have revealed an incidence of stent fracture ranging from 0.8% to 7.7%,6 however, the true incidence of stent fracture might be

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underestimated because not all patients undergo angiographic follow-up and intravascular ultrasound studies and because of the limited sensitivity of diagnostic methods. Using high-contrast film-based radiography in an autopsy study, Nakazawa et al. observed stent fracture in 29% of lesions. Multiple studies have reported that several risk factors appear to be associated with stent fracture, namely right coronary artery location, overlapping stents, high angulated lesion and Cypher stent use.7-9 Stenting of a highly angulated lesion straightens out and distorts the natural curvature of the coronary artery, and it creates continuous inertial force to recovery. Furthermore, cardiac movement creates complex vessel movement flexion, stretching, and torsion forces9, leading to mechanical force and fracture at the hinge points.8 The right coronary artery is the common location followed by the left anterior coronary artery, the left circumflex artery and saphenous vein grafts. This is attributed to the vulnerable anatomy of right coronary artery, because it is tortuous, angulated and with large degrees of vessel movement during cardiac contractions.8 Overlapping stents create high axial strength and they also are known as a significant risk factor for stent fracture.6 The first generation Cypher stent was associated with a higher incidence of stent fracture than other drug-eluting stents and it accounts for 95% of reported drug-eluting stent fractures.10 Although the exact mechanism is unknown, factors cited for the predisposition include closed-cell design which increased regidity7,11 and higher radiopacity which makes it easier to be diagnosed by fluoroscopy.6 Other factors, such as longer stent, extensive calcification, total occlusions, ostial location and high inflation pressure, are also considered as predictors of stent fracture.12 Usually, the degree or type of stent fracture is classified according to the severity of strut fracture and displacement. In the clinical studies, the authors used different classification systems and there is no unified classification system (Table 17.1).
Table 17.1. Classification of stent fracture Authors Scheinert et al.13 Classification type Minor Moderate Severe Type 1 Type 2 Type 3 Type 4 Nakazawa et al.7 Grade I Grade II Grade III Grade IV Grade V Disruption Avulsion Displacement Definitions Single strut fracture Fracture >1 strut Complete separation of stent segments Single-strut fracture or gap between struts >2 times 2.5 mm expanded cell diameter Incomplete transverse stent fracture that resulted in V-shaped horizontal separation of stent struts without discontinuity at 1 edge of stent Complete transverse fracture of stent without displacement of 2 components of fractured stent by >1 mm Complete transverse fracture of stent with torsion during cardiac cycle of displacement of 2 stent fragments >1 mm Single-strut fracture Two or more strut fractures without deformation Two or more strut fractures with deformation Multiple strut fractures with acquired transection but without gap Multiple strut fractures with acquired transection with gap in the stent body Both inner and outer struts of the angled stent were separated without displacement on the coronary angiogram. The linear or curvilinear alignment of the stent was maintained The connections of the inner struts of the angled stent were maintained; nonetheless, the outer strut was separated and not covered with stent struts The proximal and distal parts of the fractured stent struts were completely separated and displaced, and thus the linear or curvilinear alignment of the stent was not maintained

Popma et al.12

Chung et al.5

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DIAGNOSTIC METHODS Fluoroscopy


Plane fluoroscopy is the most commonly using diagnostic tool for stent fracture although its sensitivity and specificity is not validated. The need for multiple views is inherent because the fractured segment can be obscured by overlying stent images. Stent fracture in the proximal and mid right coronary artery may be appreciated in the left anterior oblique view, the anterior cranial view and the right anterior oblique views. There is no consensus fluoroscopic definition and classification yet. Usually, stent fracture is defined as a newly developed fluoroscopic discontinuity of stent struts at follow-up.14 (Figure 17.1) StentBoost, an improved fluoroscopic stent visualization technique, creates a high-quality stent image, even of fractured segments that are not apparent on ordinary fluoroscopy.15 The combination of fluoroscopic, angiographic and intravascular ultrasound images provides more information. Also, to maximize the diagnostic yield from a fluoroscopy examination of stent fracture, careful attention by the doctor performing the evaluation is essential.

Figure 17.1 A. Angiographic image showing a totally occluded left anterior descending coronary artery 22 months after Cypher 3.5 23 mm stent implantation. The arrow indicates the proximal edge of the stent. B. The fluoroscopic image demonstrates the discontinuity and misalignment in the middle of the stent (arrow in the box).

Intravascular Ultrasound (IVUS)


IVUS is another important tool for the initial diagnosis or confirmation of a suspicious stent fracture on fluoroscopy. Post-procedural and follow-up IVUS examinations with the lowest automated pullback speed (0.5 mm/s) should be compared side by side. Stent fracture is defined as the significant disappearance of stent struts in the stent at follow-up in comparison with the presence of stent struts immediately after stent implantation (Figure 17.2).14 Yamada et al.16 suggest that IVUS can more reliably detect stent fractures than angiography.

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Figure 17.2. Top: Longitudinal Intravascular ultrasound reconstruction of the left anterior descending coronary artery. The white square box indicates the strut-free segment in the middle of the stented segment. Bottom: Cross-sectional images revealing the absence of stent struts in the middle of the stent (b) as compared to the proximal (a) and distal (c) segments.

Multidetector-row Spiral Computed Tomography (MDCT)


In recent years, with the technical innovation of MDCT, it has become possible to accurately detect a stent and its associated stenosis in coronary arteries. Because of the high roentgen attenuation of the metal, stents can be easily detected without the use of contrast media. Stent artifacts depend on the stent material (stainless steel, gold-coated, tantalum, nitinol, and cobalt-chromium), design and thickness. Multiplanar reconstructions are suited to visualize stent struts. The most prominent advantage of MDCT is its multiplanar capability which provides the ability to determine fracture orientation and to obtain three-dimensional images (Figure 17.3). Pang et al. evaluated the ability of 64-slice MDCT, conventional cine-angiography, and IVUS to detect stent fractures in vitro, and CT showed the best accuracy, sensitivity and specificity.17 Coronary CT angiography also depicts stent fractures that are not clearly seen by conventional angiography.18

Figure 17.3 A. Multiplanar reconstruction CT image reveals a stent gap (arrow). B. Three-dimensional volume rendering of the coronary artery shows a very small discontinuity in the middle of the stent (arrow).

CHAPTER 17. STENT FRACTURE AND RESTENOSIS

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CLINICAL IMPLICATIONS
The clinical presentations associated with stent fracture include stent thrombosis, myocardial infarction and restenosis. The majority of stent fractures are found during angiographic follow-up after stent implantation. Because stent fracture creates a stent-free area in the middle of the fractured segment, the underlying atherosclerotic lesion is exposed to the bloodstream with the potential of plaque rupture.4 Furthermore, occasionally there is strut malapposition around the fractured segments4 and aneurysm formation,19 which have the potential to progress to acute coronary syndrome. More than 30% of the reported stent fracture cases presentas stent thrombosis, acute myocardial infarction or unstable angina,10)with clinical presentations differing among reports. Chung et al.5 conducted a multicenter retrospective study, and found 35 Cypher stent fractures with no acute coronary syndrome, in-stent restenosis developing in 24 lesions (65%), all of which were focal restenosis, and target-lesion revascularization being performed on 11 lesions (30%). In a Cypher stent registry, Popma et al.12 analyzed 39 patients with stent fracture. Patients presenting with fractures had high rates of in-stent restenosis (47.4%) and late target-lesion revascularization (52.6%). Also, stent fractures were associated with total occlusion (7.9%) and aneurysm formation (13.2%). Okumura et al.14 also performed a serial angiographic follow-up study after Cypher stent implantation in 169 lesions of 139 patients. Stent fracture was observed in 4 lesions (31%) of 13 restenosis lesions. Ino et al.20 observed 18 stent fractures in 273 patients (264 lesions) who were treated with the Cypher stent. The rates of restenosis (33%) and target-lesion revascularization (28%) were higher in patients with stent fracture. In a prospective study by Aoki et al.8 rate of in-stent restenosis and target-lesion revascularization was 37.5% and 50.0%, respectively, in 8 patients with stent fracture. Interventional strategies for stent fracture lesions include the use of drug-eluting stents or balloon angioplasty, vary among interventionists; however, another drug-eluting stent with an open cell design is being used preferentially in this lesion. The long-term prognosis associated with stent fracture is unclear. Ino et al.20 conducted a long-term follow-up (mean 24 months) evaluation of 18 patients with Cypher stent fractures including 5 patients who underwent repeat intervention, and there were no cardiac events. Lee et al.21 also performed a follow-up study of 15 patients with stent fracture. Of the 8 patients with restenosis, 5 were treated with heterogeneous stents, 2 were implanted with homogenous stents, and 1 was treated with balloon angioplasty. There were no cardiac deaths.

CONCLUSIONS
Coronary stent fracture is an unusual complication after implantation of any kind of stent, particularly in areasof repetitive mechanical force. Within the coronary artery, it occasionally results in serious cardiac problems such acute myocardial infarction and restenosis. The optimal therapeutic strategy and long-term prognosisremainunknown. New biodegradable stents or more physiologic stents may be needed to prevent this complication.

REFERENCES
1. 2. Meier GH, Pollak JS, Rosenblatt M, Dickey KW, Gusberg RJ. Initial experience with venous stents in exertional axillary-subclavian vein thrombosis. J Vasc Surg 1996;24(6):974-81. Sianos G, Hofma S, Ligthart JM, et al. Stent fracture and restenosis in the drug-eluting stent era. Catheter Cardiovasc Interv 2004;61(1):111-6.

258 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

PART I. RESTENOSIS NEW DISEASE Serikawa T, Kawasaki T, Koga H. Impressive sirolimus-eluting stent fracture immediately after stent implantation: a case report. Int J Cardiol 2009;134(3):e113-6. Park JS, Shin DG, Kim YJ, Hong GR, Cho IH. Acute myocardial infarction as a consequence of stent fracture and plaque rupture after sirolimus-eluting stent implantation. Int J Cardiol 2009;134(2):e79-81. Chung WS, Park CS, Seung KB, et al. The incidence and clinical impact of stent strut fractures developed after drug-eluting stent implantation. Int J Cardiol 2008;125(3):325-31. Canan T, Lee MS. Drug-eluting stent fracture: incidence, contributing factors, and clinical implications. Catheter Cardiovasc Interv 2010;75(2):237-45. Nakazawa G, Finn AV, Vorpahl M, et al. Incidence and predictors of drug-eluting stent fracture in human coronary artery a pathologic analysis. J Am Coll Cardiol 2009;54(21):1924-31. Aoki J, Nakazawa G, Tanabe K, et al. Incidence and clinical impact of coronary stent fracture after sirolimus-eluting stent implantation. Catheter Cardiovasc Interv 2007;69(3):380-6. Lee MS, Jurewitz D, Aragon J, Forrester J, Makkar RR, Kar S. Stent fracture associated with drug-eluting stents: clinical characteristics and implications. Catheter Cardiovasc Interv 2007;69(3):387-94. Chhatriwalla AK, Cam A, Unzek S, et al. Drug-eluting stent fracture and acute coronary syndrome. Cardiovasc Revasc Med 2009;10(3):166-71. Shaikh F, Maddikunta R, Djelmami-Hani M, Solis J, Allaqaband S, Bajwa T. Stent fracture, an incidental finding or a significant marker of clinical in-stent restenosis? Catheter Cardiovasc Interv 2008;71(5):614-8. Popma JJ, Tiroch K, Almonacid A, Cohen S, Kandzari DE, Leon MB. A qualitative and quantitative angiographic analysis of stent fracture late following sirolimus-eluting stent implantation. Am J Cardiol 2009;103(7):923-9. Scheinert D, Scheinert S, Sax J, et al. Prevalence and clinical impact of stent fractures after femoropopliteal stenting. J Am Coll Cardiol 2005;45(2):312-5. Okumura M, Ozaki Y, Ishii J, et al. Restenosis and stent fracture following sirolimus-eluting stent (SES) implantation. Circ J 2007;71(11):1669-77. Shinde RS, Hardas S, Grant PK, Makhale CN, Shinde SN, Durairaj M. Stent fracture detected with a novel fluoroscopic stent visualization technique - StentBoost. Can J Cardiol 2009;25(8):487. Yamada KP, Koizumi T, Yamaguchi H, et al. Serial angiographic and intravascular ultrasound analysis of late stent strut fracture of sirolimus-eluting stents in native coronary arteries. Int J Cardiol 2008; 130(2):255-9. Pang JH, Kim D, Beohar N, Meyers SN, Lloyd-Jones D, Yaghmai V. Detection of stent fractures: a comparison of 64-slice CT, conventional cine-angiography, and intravascular ultrasonography. Acad Radiol 2009; 16(4):412-7. Lim HB, Hur G, Kim SY, et al. Coronary stent fracture: detection with 64-section multidetector CT angiography in patients and in vitro. Radiology 2008;249(3):810-9. Okamura T, Hiro T, Fujii T, et al. Late giant coronary aneurysm associated with a fracture of sirolimus eluting stent: a case report. J Cardiol 2008;51(1):74-9. Ino Y, Toyoda Y, Tanaka A, et al. Predictors and prognosis of stent fracture after sirolimus-eluting stent implantation. Circ J 2009;73(11):2036-41. Lee SE, Jeong MH, Kim IS, et al. Clinical outcomes and optimal treatment for stent fracture after drug-eluting stent implantation. J Cardiol 2009;53(3):422-8.

CHAPTER 18

ATHEROSCLEROSIS, VASCULAR DAMAGE AND RENAL DISEASE


DEBORAH MAZZAFERRO, SERENA DI CELLO, PAOLA NACCARATO, ANTONIO PATA, ELIEZER J. TASSONE, ALESSANDRA PASCALE, LAURA GRECO, GIULIA GALIANO LEONE, ROSAMARIA BRUNI and FRANCESCO PERTICONE
Introduction............................................................................................................................... Atherosclerosis and Inflammation ........................................................................................... Prognostic Significance of Endothelial Dysfunction in CV Disease ........................................ Endothelial Dysfunction and Renal Damage ............................................................................ Conclusions............................................................................................................................... References................................................................................................................................. 259 260 262 266 269 269

INTRODUCTION
It is well known that cardiovascular diseases (CVD) are the major health problem and the leading cause of death worldwide, both for women and for men. In Europe CVD cause about half of all deaths (48%), and represent the main cause of the disease burden (23% illness and death)1, contributing substantially to the increase of healthcare costs2,3. In most of the high-income countries the incidence of CVD has been decreasing over the last decades but the incidence in low- and middle income countries has been rising continuously4. On the other hand, in many high-income countries CVD mortality rates are associated with an older age at presentation, and an increased number of subjects suffer from several chronic CVD, such as heart failure, diabetes, hypertension, dyslipidemia and chronic kidney disease. CVD, in particular coronary artery disease and myocardial infarction, as well as peripheral artery disease and cerebrovascular diseases such as ischemic stroke, are caused by the coexistence of multiple risk factors that interact in a multiplicative manner in the appearance and progression of atherosclerotic disease5. However, there are some other risk factors such as genetic, renal disease, autoimmune diseases, elevated heart rate, hyperhomocysteine, thrombogenic factors and inflammation markers which sometimes are no less important than those mentioned above6. Thus, atherosclerosis results from a complex interaction of pro-atherogenic and pro-inflammatory mediators and the subsequent cellular response. Taken together, atherosclerotic disease is considered an inflammatory chronic and degenerative process associated with the most common cardiovascular risk factors that concur to induce endothelial dysfunction, which is the initial step in the pathogenesis of plaque formation7.

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According with this, an impaired endothelium-dependent vasodilation has been found in the forearm, coronary, and renal vasculature of patients with hypertension, dyslipidemia, diabetes mellitus, and coronary artery diseases.

ATHEROSCLEROSIS AND INFLAMMATION


It is well established that the inflammatory process plays a key role in the genesis and progression of the atherosclerotic disease, and its clinical complications. In fact, inflammatory pathways, substantiated by biological and clinical processes, are implicated in early atherogenesis, in the progression of vascular lesions and in their thrombotic complications. In keeping with this, some results, obtained from observational and/or from interventional trials, demonstrated that inflammatory markers may play a major adjunctive role in the assessment of cardiovascular risk in selected patients. Similarly, cardiovascular risk factors are associated with an increase of oxidative stress and an elevation of some inflammatory markers reflecting the response of vascular wall to injury. It is now well-accepted that atherosclerosis is not merely a simple lipid disorder, but also a chronic inflammatory disease7,8. Inflammation is recognized as a major contributor to atherogenesis through adverse effects on lipoprotein metabolism and arterial wall biology. In addition, both the innate (monocyte-derived macrophages) and the acquired immune system (T-cells) have been implicated in the development of atherogenic process9. Substantial biological data implicate inflammatory pathways activation in early atherogenesis, in the progression of vascular lesions and, finally, in the thrombotic complications of this disease10,11. The chronic inflammatory process, that involves the vascular endothelium, may be caused by a response to the oxidative components of modified low-density lipoprotein (LDL), free radicals, hypertension, diabetes mellitus, genetic alterations, hyperhomocysteine, cigarette smoking, and other factors12. According to the oxidation hypothesis, LDL particles retained in the intima, in part by binding to proteoglycan, undergoes oxidative modifications that promote the early activation of both biological and cellular mechanisms of vascular wall as response to the injury13. Modified lipids induce the expression of adhesion molecules, chemokines, proinflammatory cytokines, and other mediators of inflammation in different vascular wall cells, including macrophages. The apoprotein moieties of the lipoprotein particles can also undergo modification in the artery wall, rendering them antigenic and capable of stimulating T-cell responses, thus activating the antigen-specific adaptive limb of the immune response. In the chronic state of the atherosclerotic disease, atheroma contains immune cells such as T lymphocytes, activated macrophages and mast-cells, which are also present in inflammatory infiltrates. This leads to the notion that the inflammatory response is immune-mediated, and the involvement of immune mechanisms in atherosclerosis are, at least in part, elucidated. Considerable evidences support the early involvement of the monocyte/macrophage, the most prominent cellular component of the innate immune response, during atherogenesis. The receptors involved in these primordial host defense responses include several families of macrophage scavenger receptors, also implicated in uptake of modified lipoproteins, and a family of toll-like receptors (TLR). These receptors trigger a complex intracellular signaling cascade that stimulates the production of proinflammatory cytokines and other inflammatory mediators14,15. As result of chronic injury, several markers of inflammation increase such as cytokines [interleukin-1 (IL-1), 6 (IL-6) and 18 (IL-18), tumor necrosis factor- (TNF- )], adhesion molecules (ICAM-1 and VCAM-1), E-selectin and proteins of acute-phase [e.g. fibrinogen and

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C-reactive protein (CRP)]; in addition, all these substances have clinical relevance because they are considered as independent predictors of cardiovascular events16. IL-6 is a multifunctional cytokine that regulates various aspects of the immune response, acute-phase reaction, and haematopoesis. IL-6 is inducible by IL-1 and, consequently, its serum concentrations often reflect IL-1 activity. Nonetheless, IL-6 has been identified as an independent risk factor for coronary artery disease17. Direct involvement of IL-6 in atherosclerotic lesion development was suggested by experiments in which mice, treated for 6-21 weeks with recombinant IL-6, showed a significantly increase in lesion size in comparison with non-cytokine-treated animals18. However, the relationship between fatty streaks in mice and atheromatous plaques in humans remains controversial. In addition, IL-1 contributes to the development of tissue damage by stimulating cells proliferation and the release of matrix metalloproteases (MMPs), that actively contribute to the plaque instabilization19. In particular, MMP-9 (gelatinase B), secreted by macrophages and other inflammatory cells, has been identified in patients with unstable angina, myocardial injury and other different pathological processes such as general inflammation, neoplastic metastasis, respiratory diseases, vascular aneurysms, and remodeling events. Blankenberg et al. noted a strong association between baseline MMP-9 levels and future risk of cardiovascular death20. IL-18 plays a key role in the inflammation cascade and it is an important regulator of both innate and acquired immunity21. In fact, it induces the production of interferon- (IFN-) and T-lymphocytes and it has been found together with a functional receptor in human atheroma lesions. It also stimulates IL-6, IL-8, ICAM-1, and MMP expression in vascular smooth muscle cells, endothelial cells, and macrophages22. Unexpectedly, IL-18 together with IL-12 also promotes IFN- expression in smooth muscle cells. TNF- participates in the inflammatory response, influences the balance of blood lipid composition and is involved in all steps of atherosclerotic process23,24. By being a potent stimulator of several MMPs25 and of plasminogen activator inhibitor-1 (PAI-1), TNF- has been also identified as a player in several atherosclerotic complications. CRP is an acute-phase protein produced primarily in the liver under the stimulation of adipocyte-derived pro-inflammatory cytokines, including IL-6 and TNF-. CRP is the most commonly measured circulating marker for subclinical inflammation, with widely available, stable and standardized assays for its measurement. Most clinical studies report that CRP is an independent predictor of atherosclerotic cardiovascular events26. Based on multiple epidemiological and interventional studies, minor CRP elevation [high-sensitivity CRP (hsCRP)] has been shown to be associated with future major cardiovascular risk (hsCRP: <1 mg/L = low risk; 1-3 mg/L = intermediate risk; 3-10 mg/L = high risk; >10 mg/L = unspecific elevation). Components of the metabolic syndrome (obesity, hypertension, hypertriglyceridemia, low HDL, abnormal glucose) have also been associated with increased levels of CRP that add prognostic information on further cardiovascular events27. Although in a recent epidemiological study classical risk factors have been once again confirmed to explain over 90% of coronary heart disease28, there is increasing evidence that serum CRP represents a reliable indicator of high cardiovascular and renal risk in population-based studies and in a variety of conditions associated with such elevated risks. What said before can be explained by the fact that high serum CRP level is deemed to be a measure of a systemic inflammation implicated in arterial damage. In keeping with this, we previously demonstrated, in a group of never treated and uncomplicated hypertensive patients, an inverse and significant relationship between serum CRP and endothelium-dependent vasodilation (Figure 18.1); in particular, CRP explains the 16% of its variation29.

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ACh-stimulated FBF % of increase % of increase

ACh-stimulated FBF,

CRP , mg/L CRP , mg/L


Figure 18.1. Significant relationship between C-reactive protein (CRP) forearm and endothelium-dependent vasodilation (ACh-stimulated FBF) in hypertensive patients.

PROGNOSTIC SIGNIFICANCE OF ENDOTHELIAL DYSFUNCTION IN CARDIOVASCULAR DISEASE


Atherosclerosis is a chronic inflammatory and degenerative process7 associated with the most common cardiovascular risk factors that concur to induce endothelial dysfunction, the earlier step in the pathogenesis of plaque formation. According with this, an impaired endothelium-dependent vasodilation has been found in the forearm, coronary, and renal vasculature of patients with traditional30-34 and non traditional35-37 CV risk factors. The endothelium is an autocrine, paracrine and endocrine organ, capable to release several vasoactive substances. Among vasodilating molecules, the most important are nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor38-40, and among those vasoconstricting ones are angiotensin II, endothelin-1 and asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of endothelial NO-synthase (e-NOS). In addition, endothelial cells have the ability to repair and modulate themselves, so that in absence of endothelial layer there is not development of atherosclerotic disease36,38-40. Thus, in physiological conditions, endothelium exerts some vasoprotective effects inhibiting platelet aggregation, suppressing the adhesion of leukocytes and monocytes, and inhibiting the migration and proliferation of vascular smooth muscle cells41,42. Endothelial dysfunction is characterized by a reduced bioavailability of vasodilating substances, in particular NO, and a concomitant increase of endothelium-derived contracting factors. This imbalance leads to an impairment of endothelium-dependent vasodilation, which can be measured in clinical practice, and which represents the functional characteristic of endothelial dysfunction. However, considering the nature of the vascular response to the injury mediated by cardiovascular risk factors, is plausible to define the impaired endothelium-dependent vasodilation as a specific state of endothelial activation characterized by a proinflammatory, proliferative, and procoagulatory effects that promote all stages of

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atherogenesis43. Thus, an activated endothelium plays a key pathophysiologic role in the development and progression of atherosclerosis since it loses the ability to protect the vascular system by the impairment of endothelium-dependent vasodilation and the reduction of its antiatherosclerotic and antithrombotic actions. Several possible mechanisms are involved in the appearance and progression of endothelial dysfunction, characterized by a decreased NO bioavailability. This condition, which occurs early in vascular disease, may be caused by various mechanisms including decreased NO synthesis, increased NO degradation due to oxidative stress, or to reduced sensitivity to NO1,34,40,44,45. With regard to the first mechanism, this alteration has been attributed to reduced NO synthesis due to a specific defect in the phosphoinositide pathway leading to activation of e-NOS. In addition, the activity of e-NOS may be inhibited by endogenous analogues of L-arginine such as ADMA46, which has been shown to be increased in patients with chronic renal diseases47, in uncomplicated essential hypertension36, in familial hypercholesterolemia and in a variety of other clinical situations48-51. An increased NO breakdown, due to an excessive production of superoxide anions and interaction with endothelium-derived factors, may be considered another important mechanism involved in the reduction of NO bioavailability documented in patients with cardiovascular risk factors30,34,44,45. Reactive oxygen species (ROS) are important molecules in signal transmission and regulation of vascular function. ROS are small, structurally simple molecules with high and random reactivity; they appear to be subtle physiologic modulators of biochemical processes involved in intracellular signaling52. The ROS-induced oxidation alters the conformation of proteins, eventually resulting in impaired enzyme activity or DNA binding. Notably, the activation of G protein-coupled receptors, such as -adrenergic and angiotensin II (Ang-II) type 1 (AT1) receptors, by particular agonists results in the production of ROS and/or NO, which act as signaling molecules in the receptor-mediated regulation of gene expression and cell growth53. Thus, it is important to remark the key pathogenetic role of oxidative stress in both promoting the endothelial damage and in reducing vascular NO bioavailability30,34,44,45,54. In fact, ROS, reacting with NO, reduce the bioavailability of endothelium-derived NO and promote the production of peroxynitrite (ONOO-), the true effector of vascular damage55. It is useful to underlie that the reaction between ROS and NO is of equimolar type, confirming the role of NO in promoting the injury of endothelial cells. In keeping with this, most evidences confirm that vascular homeostasis depends by the dynamic balance between vasodilating and vasoconstricting substances. All cardiovascular risk factors are associated with overproduction of ROS; thus, it is plausible to affirm that the increased oxidative stress represents the common pathogenetic mechanism that links the cardiovascular risk factors to endothelial dysfunction55. Obviously, the overproduction of ROS, and the consequent oxidative stress, represents an established promoter of inflammatory process, resulting in the expression of several inflammatory mediators and adhesion molecules that initiate the atherosclerotic disease and increase plaque vulnerability. Interestingly, in addition to the known direct vasoconstricting effect, Ang-II also stimulates the production of ROS by increasing the expression of the NADPH oxidase gene and the activity of NADPH oxidase56. Furthermore, Ang-II increases the production of endothelin in the blood vessel walls, which induces vasoconstriction and the proliferation of vascular smooth muscle cells57. Therefore, there are many evidences about the strong link between vascular function and renin-angiotensin-aldosterone system (RAAS). In fact, the alterations of this system promotes endothelial dysfunction that maintains and amplifies the negative effects of the Ang-II that in turn, through the AT1 receptors stimulation, reduces the NO bioavailability, favours the apoptosis, increases the fibroblastic proliferation and interferes with the condition of insulin resistance by the activation of post receptorial defects58. However, it should be noted

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that the activity of e-NOS increases consensually with the production of ROS, as demonstrated in vitro by Cosentino et al. on endothelial cells exposed to high glucose concentrations59. Similar results were observed by our group regarding the interaction between ADMA and vascular function36. In other words, at least in the early stages of vascular damage, the endothelium tends to defend itself from the injury of cardiovascular risk factors, resulting in increased NO production; this is possible because the L-arginine-NO reaction is characterized by a first degree linear kinetics, meaning that the greater is the substrate, the higher is the enzyme activity. As previously reported, most of cardiovascular risk factors, traditional and non-traditional, are related with endothelial damage and atherosclerosis interfering with the shear stress, the most important endogenous modulator of endothelial homeostasis. However, is useful to remark that also the individual genetic background contribute to endothelial damage as reported by our group in essential hypertension. In particular, ACE-gene polymorphism60 as well as the Arg972 IRS-1 polymorphism61 and the 460Trp allele of adducin-162 were associated with a significant impairment of endothelium-dependent vasodilation irrespective of arterial blood pressure values. Less-invasive or non-invasive techniques for the assessment of endothelial function, including strain-gauge forearm plethysmography in conjunction with intra-arterial infusion of endothelium-dependent vasodilators, such as methacholine or acetylcholine, and high-resolution external vascular ultrasound to measure flow-mediated dilation (FMD) of the brachial artery during reactive hyperemia, have been developed. The rationale of these techniques lies on the fact that endothelial dysfunction is not only confined to the coronary arteries but represents a systemic disorder that affects also peripheral vascular beds, including both conduit arteries and small resistance vessels in the extremities. Given the systemic nature of endothelial dysfunction, it has long been debated whether peripheral vascular function parallels that of the coronary arteries and, thus, may be considered as a surrogate marker to identify subjects with coronary endothelial dysfunction. To elucidate this point, two studies demonstrated a close correlation between endothelial function measured in the human coronary and peripheral circulation63,64. This provides the clinical explanation through which endothelial dysfunction, measured at the forearm by FMD or strain-gauge plethysmography, results to be an independent predictor of cardiovascular events in different settings of patients65-69. These findings strengthen the concept that endothelial dysfunction must be considered as a syndrome with different clinical presentations rather than a localized vascular disorder, and contributes to clarify the pathogenetic mechanisms operating in the appearance of cardiovascular outcomes associated with endothelial dysfunction70. Of clinical relevance may be also considered the evidence that coronary endothelial vasodilator dysfunction predicts long-term atherosclerotic vascular disease progression and cardiovascular events rate71,72. As already reported, the endothelial dysfunction is identified as an independent predictor of cardiovascular events in subjects with one or more cardiovascular risk factors or in individuals with a previous cardiovascular event, so as it may be considered the risk of cardiovascular risk factors67. This association is supported by the Rosss hypothesis that considers the endothelial cells activation as a response to the injury caused by risk factors on vascular wall, that promotes the inflammation and proliferative processes characterizing all steps of atherosclerotic disease. However, differently from this hypothesis, we recently demonstrated that endothelial dysfunction/activation can precede the appearance of new diabetes in never treated hypertensive patients without clinical evidence of vascular damage73.

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In addition, more recently, we also showed that endothelial dysfunction is able to predict the progression of renal damage in hypertensive patients with normal renal function (creatinine <1,4 mg/dl) 74. Therefore, for the first time, we demonstrated that endothelial damage, produced by mild inflammation, represents a key pathogenetic mechanism involved in the appearance of diabetes and progression of the renal damage, all conditions that, in turn, cause further vascular damage. In other words, all these biological modifications establish reverberating circuits which, if not early recognized and interrupted, contribute to the progression of atherosclerotic disease, until the cardiovascular events. Of interest, our data provide evidence for a strong association between baseline endothelial function and subsequent development of type-2 diabetes in essential hypertension. Inflammation results involved in this association, and CRP interacts with endothelial damage, increasing in a multiplicative manner the risk of new diabetes. Since inflammation and endothelial dysfunction are present in hypertensive patients30,34,66,70, it could be interesting to know which alteration precedes the appearance of the other. In keeping with this, we suggest a possible pathophysiological mechanism: hypertension-related vascular inflammation, at first, induces endothelial dysfunction which, in turn, promotes insulin resistance and progression to type-2 diabetes. In fact, essential hypertension is associated with increased oxidative stress, which induces mild vascular inflammation and endothelial dysfunction (Figure 18.2).

OXIDATIVE STRESS

INFLAMMATION

DIABETES

ENDOTHELIAL DYSFUNCTION

INSULIN RESISTANCE

Figure 18.2. In this cartoon we graphically report the possible interactions among some biological phenomenon involved in the atherosclerotic disease.

This hypothesis confirms previously published data demonstrating that proinflammatory molecules, such as TNF-, IL-6, and CRP, are associated with endothelial dysfunction and insulin resistance75,76. Accordingly, we observed a significant relationship between CRP and both endothelial dysfunction and type-2 diabetes, supporting the idea that endothelial dysfunction contributes to the development of type-2 diabetes. Taken all together, these data suggest that endothelial dysfunction may contribute to development of type-2 diabetes, preceding rather than following its onset (Figure 18.3).

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Figure 18.3. Endothelial dysfunction and C-reactive protein are independent risk factors for diabetes in essential hypertension.

ENDOTHELIAL DYSFUNCTION AND RENAL DAMAGE


Chronic kidney disease (CKD) is considered a well established risk factor for CVD, and is associated with a significant increase in all-cause mortality77-79. Even if the etiology of the increased CVD risk in subjects with CKD is not completely clarified, it may be due, at least in part, to shared cardiovascular risk factors such as hypertension, lipid abnormalities, diabetes, obesity, and smoking80,81. However, in CKD there is an extra risk attributable to renal-specific risk factors that include proteinuria, increased RAAS activity, chronic volume overload, altered calcium and phosphorus metabolism, inflammation, infection, anaemia, malnutrition, oxidative stress, hyperhomocysteine, uremic toxins and thrombogenic factors. Of interest, the risk excess for CVD was also demonstrated in subjects with mild to moderate CKD77,79 that, at this time, represents a major public health problem. In fact, population-based studies82 and secondary analyses of intervention studies in hypertensive patients83 and in selected patients at high cardiovascular risk84 have shown coherently that classical risk factors represent major correlates of renal dysfunction so that the diagnosis of renal dysfunction should alert the practitioner to correctly define the total burden of CVD. However, CKD is still not included in the group of well-recognized cardiovascular risk factors in many healthcare settings. Despite the established association between CKD and CVD, the burden of cardiovascular risk is often not optimized for these patients in the community setting because screening for CKD is frequently limited to a measurement of serum creatinine, which does not accurately reflect glomerular filtration rate (GFR), the best indicator of renal function in healthy and diseased subjects85,86. This is despite the growing body of evidence demonstrating increased cardiovascular risk associated with CKD in the general population as well as in patients with diabetes, hypertension, obesity, metabolic syndrome, atherosclerotic coronary disease, postmenopausal women87 and chronic heart failure. Moreover, consistent evidence has now accrued that in hypertensive patients, even minor degrees of renal insufficiency entail a high

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risk. In keeping with this, in uncomplicated essential hypertension, a higher serum creatinine, even if in the normal range, is a strong predictor of cardiovascular morbidity, and its prognostic value also persists after adjustment for several powerful confounders, including average 24-hour blood pressure and left ventricular hypertrophy88. Endothelial dysfunction is another feature of major clinical relevance in hypertensive patients30, because independently of arterial pressure levels and other risk factors, it is associated with left ventricular hypertrophy89 and predicts cardiovascular events66,67. Mild to moderate renal insufficiency in hypertensive patients may be considered the expression of a renal microvasculopathy characterized by preglomerular arteriolar involvement and tubulo-interstitial changes. This microvasculopathy may be triggered by sympathetic overactivity, overstimulation of the RAAS, or any factor that causes renal vasoconstriction90. Afferent vasoconstriction per se, without other noxious factor, is not able to cause overt renal dysfunction. By contrast, if dysfunctional endothelium contributes to renal vasoconstriction, renal impairment may be much more likely to follow. Thus, it appears plausible that microvascular disease in the kidney may be related to an alteration involving the endothelium. In support of this possibility, hemodynamic studies exploring the renal response to the NO precursor L-arginine have consistently demonstrated an impaired renal vascular relaxation in hypertensive subjects91,92. Taken together, we evaluated 500 uncomplicated and never-treated hypertensive patients, with normal serum creatinine, to verify the possible relationship between the systemic endothelium-dependent vasodilation and renal function. Our data demonstrated that an impaired endothelium-dependent vasodilatory response appears to be associated with renal function loss, evaluated by estimated GFR, and serum creatinine levels. This association suggests that systemic endothelial dysfunction is involved in mild to moderate renal insufficiency in patients with uncomplicated essential hypertension. In addition, CRP was related directly to serum creatinine and inversely to GFR and vasodilatory response to ACh, which suggests that endothelial dysfunction is a possible mechanism linking inflammation and impaired renal function in essential hypertension93. These data have particular relevance as studies of endothelial function in the kidney vasculature are laborious to perform, mainly because the stimulation and blockade of e-NOS by these substances have prolonged effects on the renal vasculature94. In addition, the systemic nature of endothelial dysfunction67, the parallelism between endothelial dysfunction in various vascular beds63,64, and the fact that endothelial dysfunction in the forearm and in the coronary circulation is predictive of adverse cardiovascular outcomes plausibly suggest that endothelial dysfunction in the forearm is also paralleled by a similar alteration in renal vasculature. If so, the arterial pressureindependent link between the estimated GFR and the endothelium-dependent vasodilation, found in our study, suggests that systemic endothelial dysfunction may be a mechanism contributing to mild renal dysfunction in human essential hypertension. In the same study we also documented that serum CRP was inversely related with both endothelium-dependent vasodilatory response and estimated GFR. These associations suggest that endothelial dysfunction represents an intermediate mechanism able to mediate the effect of inflammation on renal function. Microalbuminuria is another important early marker of renal dysfunction as well as an independent predictor of cardiovascular complication95,96. Of interest, it is also considered an indicator of a generalized endothelial dysfunction, both at the systemic and renal vasculature. In keeping with this, we demonstrated that microalbuminuria was related to the endothelium-dependent vasodilation but, similar to a recent study in patients with essential hypertension97, it was unrelated to the GFR93. Clinically relevant is the fact that the activation of RAAS plays a crucial role in the pathogenesis of endothelial dysfunction and it is determinant to induce kidney organ damage, both at glomerular and tubulo-interstitial level.

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In fact, Ang-II causes vasoconstriction, neurohumoral activation, production of ROS and parallel reduction of NO bioavailability, oxidative modifications of DNA and proteins, lipid oxidation, enhanced mitogenicity and apoptosis of vascular cells, activation of pathophysiologically important genes, such as the receptor for oxidized LDL, adhesion molecules, chemotaxis factors, pro-inflammatory cytokines, regulators of cell cycle progression, and matrix MMPs. Moreover, hypercholesterolemia, a very frequent condition present in patients with CKD, enhances the RAAS activity, by increasing AT1 receptors density and functional responsiveness98. Another mechanism that contributes to endothelial dysfunction in CKD is the increased ADMA levels that are associated with reduced e-NOS activity99 and increased expression of oxidized LDL lecitine-like receptor-1 of (LOX-1)100, the main receptor for LDL-ox in endothelial cells. In addition, LDL oxidation may contribute to the elevation of circulating ADMA levels, through inhibition of dimethylarginine dimethylaminohydrolase (DDAH) activity, an enzyme which degrades ADMA, prior to the renal excretion101. Elevated ADMA levels were associated with higher intima-media thickness102 and cardiovascular events103. On the other hand, there is a potential interaction between ADMA and the RAAS104: in fact, ADMA, interacting with arteriolar RAAS, activates NAD(P)H oxidase to induce oxidative stress which, interfering with NO release, results in the impairment of endothelial function105. As mentioned above, we recently demonstrated that endothelium-dependent vasodilation and systolic blood pressure were associated with a greater decline in renal function (Figure 18.4) after adjustment for other cardiovascular risk factors and antihypertensive treatment74.

P<0.0001 by linear trend

2 0 -2 -4 -6

Q1
(<150)

Q2
(150-249)

Q3 (250-349)

Q4
(>350) FBF % of increase

e-GFR

ml/min/1.73 m2/yr

Figure 18.4. Annual loss of estimated glomerular filtration rate (e-GFR) in hypertensive patients stratified by quartiles of ACh-stimulated forearm blood flow (FBF).

These data suggest that endothelial dysfunction may exacerbate the decline in renal function in patients with hypertension and that therapies to improve endothelial function may help prevent the development of hypertensive kidney disease. Although not tested in the present study, the prevention of progressive renal disease in hypertensive patients may not only rely on intensive blood pressure control but also on other interventions known to improve endothelial

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function, such as lifestyle modification, treatment of dyslipidemia, and smoking cessation. Another important consideration that emerges from the analysis of these results is represented by a bidirectional mechanism involved in the appearance and progression of organ damage. Indeed, the hypertensive renal damage induces endothelial dysfunction which, in turn, promotes the loss of renal function, supporting the concept that an impairment of vascular function is both a consequence and a cause of CKD.

CONCLUSION
Endothelial dysfunction, that is measured by the hemodynamic response to administration of muscarinic receptor agonists, is a very consistent finding in patients with traditional and emerging cardiovascular risk factors. It is well demonstrated that endothelium-dependent vasodilatation in coronary circulation, peripheral arteries and the kidney is a closely related phenomenon, indicating that endothelial dysfunction represents a systemic disorder involving both conduit arteries and small resistance vessels in the extremities. In addition, some studies have clearly demonstrated that endothelial dysfunction, evaluated in both coronary and forearm vasculature, provides prognostic information for future clinical events. Recent evidences also demonstrate that endothelial dysfunction is an important and independent predictor of new diabetes and renal function decline. All this is of particular clinical relevance since endothelial dysfunction is a consequence of cardiovascular risk factors but also can promote the organ damage. Endothelial dysfunction is a reversible disorder, and strategies aimed at reducing cardiovascular risk factors, such as cholesterol lowering drugs, antihypertensive therapy, smoking cessation, estrogen replacement therapy in postmenopausal women, supplementation with folic acid, and physical exercise, also translate into an improvement in endothelial health, further supporting the association between risk factors and endothelial dysfunction. Moreover, the observation that several pharmacological interventions that improve endothelial function are associated with a decrease in cardiovascular events independent of risk factor modification supports the concept that cardiovascular risk factors share a common pathway that leads to endothelial dysfunction, such as oxidative stress.

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