Journal of the American College of Cardiology Vol. 55, No.

19, 2010
2010 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00
Published by Elsevier Inc. doi:10.1016/j.jacc.2009.08.090

FOCUS ISSUE: BIOMARKERS IN CARDIOVASCULAR DISEASE

Biomarkers of Peripheral Arterial Disease

John P. Cooke, MD, PHD,* Andrew M. Wilson, MBBS, PHD
Stanford, California; and Victoria, Australia

Atherosclerotic arterial occlusive disease affecting the lower extremities is also known as peripheral artery dis-
ease (PAD). This disorder affects 8 to 12 million individuals in the U.S. and is increasingly prevalent in Europe
and Asia. Unfortunately, most patients are not diagnosed and are not optimally treated. A blood test for PAD, if
sufficiently sensitive and specific, would be expected to improve recognition and treatment of these individuals.
Even a biomarker panel of moderate sensitivity and specificity for PAD could refine risk stratification to select
individuals for diagnostic vascular examination. Alternatively, biomarkers for PAD may be useful in determining
prognosis, the risk for progression, or the response to therapy. Finally, the discovery of biomarkers associated
with PAD may provide novel insights into the pathophysiology of PAD and new therapeutic avenues to pursue.
Biomarkers may be derived from studies of the genome, transcriptome, proteome, or metabolome. The focus of
this review is on proteomic biomarkers associated with PAD. (J Am Coll Cardiol 2010;55:201723) 2010 by
the American College of Cardiology Foundation

The prevalence of lower-extremity peripheral artery disease
(PAD), assessed using the ankle-brachial blood pressure
index (ABI), has been estimated to be 10% to 20% in
individuals older than 65 years of age in community-based
studies (1 4). Even greater prevalence is observed in indi-
viduals attending general medicine practices, in which 20%
to 30% of patients age 50 years and older have the disease
(5,6). Peripheral arterial disease causes limb pain with exertion,
reduces functional capacity and quality of life (7), and is
frequently associated with coronary, cerebral, and renal artery
disease (8). Individuals with PAD are at increased risk for acute
cardiovascular events such as myocardial infarction, cerebro-
vascular attack, aortic aneurysm rupture, and vascular death, as
well as ischemic ulceration and amputation (9,10). This in-
creased risk for cardiovascular morbidity and mortality is seen
even in patients without symptoms (11).
Aggressive medical treatment of risk factors can substan-
tially reduce the mortality and morbidity of PAD (12).
Unfortunately, PAD is underdiagnosed and undertreated,
with most patients not receiving optimal management,
including therapies proven to reduce mortality such as
antiplatelet agents, statins, and converting enzyme inhibi-
tors (13). Suboptimal physician recognition and manage-
ment of the condition is in part because of poor public
awareness of PAD (14), inadequate training and tools for
primary physicians, a lack of remuneration for screening
From the *Division of Cardiovascular Medicine, Stanford University, Stanford,
California; and the Department of Medicine, University of Melbourne, Victoria,
Australia. Drs. Cooke and Wilson are inventors on the patent that Stanford
University has filed for biomarkers for peripheral arterial disease. Dr. Cooke has
worked with Vermillion.
Manuscript received May 17, 2009; revised manuscript received July 22, 2009,
accepted August 16, 2009.
(15), and the absence of the classic symptom complex in a
majority of the patients (16). Classical intermittent claudi-
cation (i.e., exertional leg discomfort relieved by rest) is only
noted by 10% to 30% of patients with PAD (7,13).
Musculoskeletal disease or neuropathy commonly coexist
with PAD and confound the clinical picture (7). Accord-
ingly, clinical assessment for PAD has a relatively poor
predictive value (10%) (17). Structured questionnaires
such as the Edinburgh Claudication Questionnaire have
improved sensitivity and specificity compared with clinician
assessment (18), but these questionnaires only identify
patients with classical symptomatology. Because the current
recognition of PAD is suboptimal, and because effective
therapy that improves mortality is available for these indi-
viduals, an efficacious strategy to screen the population for
PAD is highly appealing.
PAD: The Case for Screening
Compared with angiography, the ABI can detect hemody-
namically significant lesions with a sensitivity of 80% to 95%
and a specificity of 95% to 100% (19,20). Furthermore, the
ABI has independent prognostic value beyond the Framing-
ham risk factors (21). The ABI is calculated from Doppler-
derived measurements of the systolic pressure at the brachial
and ankle arteries. By convention, for each lower extremity,
the higher of the 2 ankle artery pressures is used for the ABI
calculation. The ABI for that extremity is the higher ankle
pressure divided by the higher of the 2 brachial artery
pressures.
Targeted screening with ABI is recommended by all
professional vascular societies, including the American Col-
lege of Cardiology (22). The American College of Cardi-
2018 Cooke and Wilson
Biomarkers for PAD?
Abbreviations ology/American Heart Asso-
and Acronyms ciation guidelines support ABI
screening in high-risk patients
ABI ankle-brachial blood
pressure index (defined as individuals age 50
2M 2 microglobulin
years with diabetes and 1 other
atherosclerosis risk factor, those
CAD coronary artery
disease
age 50 to 69 years with a history
of smoking or diabetes, individ-
CRP C-reactive protein
uals age 70 years, those with
MD mass spectroscopy
leg symptoms with exertion or
PAD peripheral artery
ischemic rest pain, and those with
disease
an abnormal lower-extremity pulse
examination) (22). Also, the
American Diabetes Association recommends annual screening
for PAD in diabetics (23).
Despite the abundant evidence supporting the value of
the ABI, and despite careful studies that have revealed
suboptimal recognition of individuals with PAD and inad-
equate utilization of therapies that reduce mortality, there is
resistance to adopting the ABI as a screening tool. The U.S.
Preventive Services Task Force has given the practice a D
level recommendation (i.e., in their opinion, routinely pro-
viding the service to asymptomatic patients is ineffective or
harm from the test may outweigh benefits). Also, the
American Academy of Family Physicians recommends
against the use of the test in asymptomatic persons (24).
These opinions are contrary to the recommendations of
vascular specialty societies and have been convincingly
rebutted (15). In brief, these unfortunate recommendations
are driven by the concern that screening may lead to
unnecessary tests and increased risk from subsequent inva-
sive studies or procedures. However, of much greater
concern is the very real cost to the health care system and to
the patient of not identifying individuals with PAD. The
primary purpose in screening for PAD is to identify indi-
viduals at high risk of vascular events (8,9,2527) to target
them for aggressive risk reduction interventions (28 33).
Unfortunately, most patients with PAD are currently not
diagnosed and are not receiving therapies that can improve
their prognosis (13,34).
Beyond the ABI
Among vascular specialists, there is widespread recognition
of the value of the ABI and evidence-based documentation
of its sensitivity and specificity. However, a practical con-
cern is that most primary practitioners lack the specialized
equipment and trained personnel to perform ABI measure-
ments in the office setting. In the absence of an effective
screening strategy in the primary practitioners office, all
individuals at risk could be referred for a formal vascular
laboratory evaluation. This would be a costly screening
strategy.
The number of individuals that should be screened for
PAD (i.e., all smokers who are 50 years of age, all patients
with diabetes who are 50 years of age, and all individuals
JACC Vol. 55, No. 19, 2010
May 11, 2010:201723
who are 70 years of age) represents approximately 60
million individuals in the U.S. An alternative screening
approach would be to develop a blood biomarker, or panel
of biomarkers, that could stratify the risk for individuals in
the primary practitioners office. Such a panel could be
assessed by a blood draw in the office and would optimally
identify a smaller subset of patients for vascular evaluation.
Such an approach could reduce the overall cost of screening
while improving recognition and proper management. This
alternative diagnostic paradigm requires progress toward
developing novel biomarkers of PAD.
Challenges in Discovering New Biomarkers
There are hurdles to the discovery of any new blood protein
biomarkers. The most daunting problem is the great diver-
sity of the proteome (i.e., plasma contains approximately
10,000 plasma proteins and even more protein fragments)
and its dynamic range (approximately 10 orders of magni-
tude difference between the least and most abundant pro-
teins) (35). The discovery process is complicated by the fact
that the 22 most abundant proteins, such as albumin and the
immunoglobulins, constitute approximately 99% of the total
proteome mass (36). However, it is the low-abundance
proteins that are often of the greatest interest as novel
disease markers. Any technology to profile the plasma
proteome in an informative manner must be able to delve
deeply into the proteome and to discriminate differences in
the levels of low-abundance proteins. For example, cardiac
markers such as troponin are found in the nanomolar range,
whereas cytokines are in the femtomolar range.
Another important issue is confounding by medications
or associated diseases. Careful phenotyping of the subjects is
critical for proteomic discovery, and the control group
should be matched for variables already known to influence
disease risk and outcome. Renal or hepatic disease may
influence the excretion or metabolism of a biomarker. Other
disorders may influence the level of a biomarker by patho-
physiologic processes unrelated to the disease of interest
(e.g., infection increases the plasma level of the cardiovas-
cular biomarker C-reactive protein [CRP]). Technical de-
tails such as how the blood is drawn, processed, and stored
can substantially affect the findings and lead to spurious
results if the samples from different patient groups are not
treated similarly. For example, multiple freeze-thaws while
samples are studied cause protein degradation, introducing
artifactual peaks in mass spectroscopic analyses.
Despite these challenges, the field of cardiovascular pro-
teomics continues to develop rapidly, and a range of
collaborative initiatives have been undertaken. The National
Institutes of Health/National Heart, Lung and Blood In-
stitute has funded several centers for cardiovascular pro-
teomics (37). The Human Proteome Organization has re-
cently initiated a plasma proteome project (38). The early
phase of the project has reported the identification of approx-
imately 345 cardiovascular disease-related proteins in human
JACC Vol. 55, No. 19, 2010
May 11, 2010:201723
plasma (39). However, until recently, there have been few
attempts to detect biomarkers associated with PAD.
Do Biomarkers of PAD Exist?
It could be argued that proteomic profiling is unlikely to
yield plasma biomarkers specific for peripheral, as opposed
to coronary, artery disease. This is because PAD and
coronary artery disease (CAD) share common cardiovascu-
lar risk factors and have many commonalities in pathophys-
iologic processes. A number of biomarkers have been
associated with PAD in population-based studies (Table 1)
(40 61), such as inflammatory cytokines or chemokines,
markers of endothelial dysfunction, mediators of angiogen-
esis or vascular regeneration, lipoproteins or lipid-associated
proteins, indicators of oxidative stress or ischemia-
reperfusion, metabolic modulators, and coagulation factors
(40,50,52,53,62 64).
However, none of these biomarkers are specific for PAD
because they are elevated in CAD and other vascular
disorders. This is not surprising because these biomarkers
were derived from candidate protein- based investigations
that focused on proteins known to be associated with other
vascular diseases or with pathophysiologic mechanisms.
Nevertheless, it is quite likely that pathologic processes in
the peripheral circulation will lead to the release of PAD-
specific biomarkers. This is because significant differences
exist throughout the vasculature in endothelial and vascular
smooth muscle gene expression (65,66). Preclinical studies
have revealed functional differences as well; endothelium-
dependent vasodilation and smooth muscle vasoconstric-
tion, in response to a variety of agonists, differ in coronary
and limb arteries (67). Similarly, in humans, endothelium-
dependent responses in the peripheral arteries are quite
different than those in central arteries (68). These differ-
ences in vascular reactivity between the peripheral and
coronary arteries reflect differences in the expression of cell
surface receptors and signaling pathways. There are also
well-established differences between vascular beds in the
expression of endothelial chemokines and adhesion mole-
cules. These geographic differences are known to play a
physiologic role in lymphocyte homing and other cellular
Biomarkers
Table 1 Associated
Biomarkers With PAD With PAD
Associated
Inflammatory cytokines: CRP (40), interleukin-6 (41,42)
Markers of endothelial dysfunction: ADMA (4346);
soluble cell adhesion molecules (47,48), Von Willebrand factor (49)
Modulators of angiogenesis: soluble Tie 2, VEGF (50);
hepatocyte growth factor (51)
Lipoproteins: oxidized LDL (5254), lipoprotein(a) (55)
Indicators of oxidative stress: homocysteine (56),
8-hydroxy-2-deoxy-2-deoxyguanosine (57), isoprostanes (58)
Coagulation factors: thrombomodulin (59), D-dimer, TPA, PAI-1,
fibrinogen (60,61)
ADMA asymmetric dimethylarginine; CRP C-reactive protein; LDL low-density lipoprotein;
PAD peripheral artery disease; PAI plasminogen activator inhibitor; TPA tissue plasminogen
activator; VEGF vascular endothelial growth factor.
Cooke and Wilson 2019
Biomarkers for PAD?
trafficking in immune surveillance (69). The phenotypic
variation among vascular cells is a manifestation of their
exposure to disparate milieus, their developmental diver-
gence, and their persistent epigenetic differences (70,71).
We hypothesized that known and undiscovered pheno-
typic differences between peripheral and coronary vascular
cells may affect their response to ischemia. Physiologists
have long known that the stimulus of ischemia, followed by
reperfusion, causes characteristic changes in a microcircula-
tory bed, including the release of adhesion molecules and
production of inflammatory cytokines (72). We reasoned
that chronic bouts of ischemia-reperfusion in the lower ex-
tremity of the claudicant might induce the expression and
release of proteins characteristic of the peripheral circulation.
Approach to Discovery
of Novel Biomarkers in PAD
Accordingly, we developed a discovery program for more
specific biomarkers of PAD (73). Previous investigations of
biomarkers for PAD have used a candidate protein-based
approach. By contrast, to discover PAD-specific biomark-
ers, we employed an agnostic approach, using plasma
proteomic profiling by mass spectroscopy (MS). We used a
version of MS known as surface-enhanced laser desorption
time-of-flight MS. The advantage of this version is that it
provides for a rapid chip-based segregation of peptides
based on charge and lipophilicity. When combined with pH
fractionation, the technique enhances MS resolution of the
thousands of plasma proteins. Proteins in the sample are
bound to the chip. An energy-absorbing matrix is applied,
redissolving the proteins on the chip, allowing the proteins
to cocrystallize with the matrix as it dries. Laser excitation
ionizes the matrix, causing it to release the proteins into the
flight tube of a mass spectrometer; the proteins subsequently
strike a detector at the end of the tube. The resulting signal
provides data from which the molecular weight and amount
of the peptide can be assessed. The comparison of the
spectra from cases and controls can lead to the identification
of a peak that is different between the groups. This peak
represents a protein that may be a new biomarker. Subse-
quent studies using complementary methods (MS-MS,
immunoaffinity columns, enzyme-linked immunosorbent
assay, Western analysis) are completed to confirm the
identity of the protein.
We performed comprehensive proteomic profiling using
this method in 88 patients with or without PAD. Our
studies revealed several MS peaks that were increased in
PAD cases. Based upon its molecular weight, one of these
peaks was thought to be 2 microglobulin (2M). We chose
this protein to study further as a proof of concept (73). In a
confirmatory study using other proteomic approaches (i.e.,
Western analysis and immunoaffinity studies), we found
2M to be elevated in subjects with PAD (and its plasma
levels were inversely correlated with treadmill exercise ca-
pacity). Finally, we validated this biomarker using a neph-
2020 Cooke and Wilson
Biomarkers for PAD?
Odds Ratio of CAD PAD Status by
Figure 1
AHA Risk Score and by Biomarker Panel Score
There is a positive interaction between the 2 assessments of disease risk. Individ-
uals were assigned an AHA risk score using the traditional cardiovascular risk fac-
tors as described (19). AHA risk scores of 5 (low), 5 to 10 (medium), and 10
(high) were associated with increasing risk of PAD (p 0.006 for men and p
0.001 for women using the score from the linear regression by analysis of vari-
ance). The tertile cutoffs of the biomarker panel score were used to determine the
risk level: low (0.991), medium (0.991 to 1.033), and high (1.033). AHA
American Heart Association; CAD cardiac artery disease; n.s. not significant;
PAD peripheral artery disease.
elometric assay in a cross-sectional case-control study of
patients undergoing elective coronary angiography for a
diagnosis of CAD. We chose our cases and controls so that
confounding clinical covariates would contribute minimally
ROC Analysis Using Risk Factors
Figure 2
Biomarkers for PAD Diagnosis
Receiver-operating characteristic (ROC) analysis of conventional risk factors, the
biomarker panel score, and a combination of conventional risk factors and the
biomarker panel score. DM diabetes mellitus; PAD peripheral artery disease.
JACC Vol. 55, No. 19, 2010
May 11, 2010:201723
to the difference between the 2 phenotypes. Cases had
hemodynamically significant PAD as documented by re-
duced ABIs, whereas controls had normal ABIs. This study
validated 2M as a biomarker associated with PAD, inde-
pendently of other cardiovascular risk factors.
Biomarker Panels
For any single biomarker (such as CRP), a certain percent-
age of subjects with abnormal levels will not have disease
(false positives), whereas those with disease may have
normal levels (false negatives). One approach to addressing
this problem is to use a panel, in which each of the
biomarkers contributes independent diagnostic information.
Biomarker panels and index scores are beginning to be
used in medicine to refine diagnosis and to aid in prognos-
tication. For example, such index scores incorporating novel
biomarkers have been used to predict clinical outcomes in
hepatocellular and breast malignancies (74,75). Recently,
Wang et al. (76) combined multiple biomarkers from the
Framingham study to predict cardiovascular outcomes and
death.
In our discovery study, an additional biomarker candidate
identified was cystatin C. A subsequent study of 540
high-risk individuals revealed that 2M, cystatin C, high-
sensitivity CRP, and glucose were associated with PAD
independently of the traditional risk factors of age, diabetes
mellitus, hyperlipidemia, hypertension, and tobacco use.
Among the plasma markers tested, 2M and cystatin C had
the highest correlation with ABI, higher than any of the
Figure 3 Biomarker Panel for PAD
The optimal PAD blood test is likely to be composed of a panel of biomarkers
that circulate systemically but which reflect the activity of local pathophysio-
logic processes contributing to inflammation, oxidative stress, matrix remodel-
ing, endothelial dysfunction, coagulation, metabolic perturbations, and
ischemia-reperfusion. Unique characteristics of the peripheral circulation may
provide for specificity of the biomarkers that are released. In brackets are
biomarkers that are elevated in PAD but which are not specific. The identifica-
tion of novel and specific biomarkers is underway (48 51,54 61,79 89).
ADMA asymmetric dimethylarginine; B2M 2 microglobulin; EPC endothe-
lial progenitor cell; glu glucose; IL interleukin; I/R ischemia-reperfusion;
Lp(a) lipoprotein (a); MCP monocyte chemotactic protein; MMP matrix met-
alloproteinase; PAD peripheral artery disease; sRAGE soluble receptor for
advanced glycosylation end products; sVCAM soluble vascular cell adhesion
molecule; TF tissue factor; VEGF vascular endothelial growth factor.
JACC Vol. 55, No. 19, 2010
May 11, 2010:201723
Features
Table 2 of an IdealofPAD
Features Biomarker
an Ideal PAD Biomarker
Sensitive for the presence of disease
Specific, good negative predictive value
Correlates with prognosis
Measurable with high-throughput techniques
Correlates with disease-specific features: ABI, walking time
Levels minimally or predictably affected by confounding factors
Reproducible
Cost effective
Acceptable to patients and clinicians
Complementary to current strategies
ABI ankle-brachial blood pressure index; PAD peripheral artery disease.
conventional risk factors of age, smoking status, and diabe-
tes status. A biomarker panel score derived from 2M,
cystatin C, high-sensitivity CRP, and glucose had an
increased association with PAD status (77), independently
of the traditional risk factors (Fig. 1).
This biomarker panel is independent of, but only adds
modestly to, the information provided by the Framingham
risk factors (Fig. 2). Although it is an imperfect prototype,
this panel provides proof of concept for the promise of a
multimarker approach. Of note, a recent prospective cohort
study of 1,000 ambulatory elderly Japanese subjects for 8
years revealed that 2M, cystatin C, and CRP were inde-
pendent predictors of mortality (78). The most informative
biomarker was 2M. The area under the receiver operating
characteristic curve for 8-year mortality was greatest for
2M (0.70; 95% confidence interval [CI]: 0.66 to 0.74),
followed by cystatin C (0.66; 95% CI: 0.62 to 0.70) and
CRP (0.57; 95% CI: 0.53 to 0.61). The authors concluded
that serum 2M is an independent predictor of total
mortality in a general population of older adults and may be
a better predictor than cystatin C or CRP.
Conclusions
There is a strong clinical need for more specific biomarkers
for PAD. A blood test for PAD would increase recognition
of the disease and thereby improve clinical care. It is likely
that a biomarker panel with high sensitivity and high
specificity for PAD will be composed of biomarkers that
circulate systemically but reflect the activity of local patho-
physiologic processes (Fig. 3) (79 89). To be clinically
useful, the optimal biomarker(s) should be stable and easily
measured and should provide diagnostic or prognostic
information that is incremental to existing biomarkers
(Table 2). The search for proteomic biomarkers is currently
being driven by rapid advances in technology and bioinfor-
matics that facilitate high-throughput analysis of blood
from clinical studies with sufficient sample sizes. The
application of these technologies to PAD should be encour-
aged because they are likely to yield useful diagnostics for
PAD, as well as novel insights into the pathophysiology of
the disease.
Cooke and Wilson 2021
Biomarkers for PAD?
Reprint requests and correspondence: Dr. John P. Cooke,
Stanford University School of Medicine, Division of Cardiovas-
cular Medicine, 300 Pasteur Drive, Falk Cardiovascular Research
Center, Stanford, California 94305-5406. E-mail: john.cooke@
stanford.edu.
REFERENCES
1. Brevetti G, Oliva G, Silvestro A, Scopacasa F, Chiariello M. Preva-
lence, risk factors and cardiovascular comorbidity of symptomatic
peripheral arterial disease in Italy. Atherosclerosis 2004;175:131 8.
2. Criqui MH, Vargas V, Denenberg JO, et al. Ethnicity and peripheral
arterial disease: the San Diego Population Study. Circulation 2005;
112:27037.
3. Hayoz D, Bounameaux H, Canova CR. Swiss Atherothrombosis
Survey: a field report on the occurrence of symptomatic and asymp-
tomatic peripheral arterial disease. J Intern Med 2005;258:238 43.
4. Hasimu B, Li J, Nakayama T, et al. Ankle brachial index as a marker
of atherosclerosis in Chinese patients with high cardiovascular risk.
Hypertens Res 2006;29:23 8.
5. Criqui MH, Fronek A, Barrett-Connor E, Klauber MR, Gabriel S,
Goodman D. The prevalence of peripheral arterial disease in a defined
population. Circulation 1985;71:510 5.
6. Criqui MH. Peripheral arterial disease epidemiological aspects. Vasc
Med 2001;6:37.
7. McDermott MM, Liu K, Greenland P, et al. Functional decline in
peripheral arterial disease: associations with the ankle brachial index
and leg symptoms. JAMA 2004;292:453 61.
8. Criqui MH, Denenberg JO. The generalized nature of atherosclerosis:
how peripheral arterial disease may predict adverse events from
coronary artery disease. Vasc Med 1998;3:2415.
9. Newman AB, Shemanski L, Manolio TA, et al. Ankle-arm index as a
predictor of cardiovascular disease and mortality in the Cardiovascular
Health Study. The Cardiovascular Health Study Group. Arterioscler
Thromb Vasc Biol 1999;19:538 45.
10. Newman AB, Tyrrell KS, Kuller LH. Mortality over four years in
SHEP participants with a low ankle-arm index. J Am Geriatr Soc
1997;45:1472 8.
11. Criqui MH, Langer RD, Fronek A, et al. Mortality over a period of
10 years in patients with peripheral arterial disease. N Engl J Med
1992;326:381 6.
12. Gornik HL, Creager MA. Contemporary management of peripheral
arterial disease: I. cardiovascular risk-factor modification. Cleve Clin
J Med 2006;73(suppl 4):S30 7.
13. Hirsch AT, Criqui MH, Treat-Jacobson D, et al. Peripheral arterial
disease detection, awareness, and treatment in primary care. JAMA
2001;286:131724.
14. Hirsch AT, Murphy TP, Lovell MB, et al. Gaps in public knowledge
of peripheral arterial disease: the first national PAD public awareness
survey. Circulation 2007;116:2086 94.
15. Beckman JA, Jaff MR, Creager MA. The United States preventive
services task force recommendation statement on screening for periph-
eral arterial disease: more harm than benefit? Circulation 2006;114:
861 6.
16. McDermott MM, Greenland P, Liu K, et al. Leg symptoms in
peripheral arterial disease: associated clinical characteristics and func-
tional impairment. JAMA 2001;286:1599 606.
17. Criqui MH, Fronek A, Klauber MR, Barrett-Connor E, Gabriel S.
The sensitivity, specificity, and predictive value of traditional clinical
evaluation of peripheral arterial disease: results from noninvasive
testing in a defined population. Circulation 1985;71:516 22.
18. Leng GC, Fowkes FG. The Edinburgh claudication questionnaire: an
improved version of the WHO/Rose questionnaire for use in epide-
miological surveys. J Clin Epidemiol 1992;45:11019.
19. Lijmer JG, Hunink MG, van den Dungen JJ, Loonstra J, Smit AJ.
ROC analysis of noninvasive tests for peripheral arterial disease.
Ultrasound Med Biol 1996;22:391 8.
20. Fowkes FG. The measurement of atherosclerotic peripheral arterial
disease in epidemiological surveys. Int J Epidemiol 1988;17:248 54.
21. Fowkes FG, Murray GD, Butcher I, et al. Ankle brachial index
combined with Framingham Risk Score to predict cardiovascular
events and mortality: a meta-analysis. JAMA 2008;300:197208.
2022 Cooke and Wilson
Biomarkers for PAD?
22. Hirsch AT, Haskal ZJ, Hertzer NR, et al. ACC/AHA 2005 practice
guidelines for the management of patients with peripheral arterial
disease (lower extremity, renal, mesenteric, and abdominal aortic): a
collaborative report from the American Association for Vascular
Surgery/Society for Vascular Surgery, Society for Cardiovascular An-
giography and Interventions, Society for Vascular Medicine and
Biology, Society of Interventional Radiology, and the ACC/AHA
Task Force on Practice Guidelines (Writing Committee to Develop
Guidelines for the Management of Patients With Peripheral Arterial
Disease): endorsed by the American Association of Cardiovascular and
Pulmonary Rehabilitation; National Heart, Lung, and Blood Institute;
Society for Vascular Nursing; TransAtlantic Inter-Society Consensus;
and Vascular Disease Foundation. Circulation 2006;113:e463 654.
23. American Diabetes Association. Peripheral arterial disease in people
with diabetes. Diabetes Care 2003;26:3333 41.
24. United States Preventive Services Task Force. Recommendation state-
ment: screening for peripheral arterial disease. Washington, DC:
Agency for Healthcare and Quality 2005:1 8.
25. Leng GC, Fowkes FG, Lee AJ, Dunbar J, Housley E, Ruckley CV.
Use of ankle brachial pressure index to predict cardiovascular events
and death: a cohort study. BMJ 1996;313:1440 4.
26. McKenna M, Wolfson S, Kuller L. The ratio of ankle and arm arterial
pressure as an independent predictor of mortality. Atherosclerosis
1991;87:119 28.
27. Ouriel K. Peripheral arterial disease. Lancet 2001;358:1257 64.
28. Hankey GJ, Norman PE, Eikelboom JW. Medical treatment of
peripheral arterial disease. JAMA 2006;295:54753.
29. Hiatt WR. Medical treatment of peripheral arterial disease and
claudication. N Engl J Med 2001;344:1608 21.
30. Daskalopoulou SS, Daskalopoulos ME, Liapis CD, Mikhailidis DP.
Peripheral arterial disease: a missed opportunity to administer statins
so as to reduce cardiac morbidity and mortality. Curr Med Chem
2005;12:44352.
31. Federman DG, Ranani DC, Kirsner RS, Bravata DM. Lipid-lowering
therapy in patients with peripheral arterial disease: are guidelines being
met? Mayo Clin Proc 2005;80:494 8.
32. Hiatt WR, Krantz MJ. Masterclass series in peripheral arterial disease.
Antiplatelet therapy for peripheral arterial disease and claudication.
Vasc Med 2006;11:55 60.
33. Feringa HH, van Waning VH, Bax JJ, et al. Cardioprotective
medication is associated with improved survival in patients with
peripheral arterial disease. J Am Coll Cardiol 2006;47:11827.
34. Okaa RK, Umoh E, Szuba A, Giacomini JC, Cooke JP. Suboptimal
intensity of risk factor modification in PAD. Vasc Med 2005;10:91 6.
35. Gerszten RE, Wang TJ. The search for new cardiovascular biomark-
ers. Nature 2008;451:949 52.
36. Anderson NL, Polanski M, Pieper R, et al. The human plasma
proteome: a nonredundant list developed by combination of four
separate sources. Mol Cell Proteomics 2004;3:31126.
37. Granger CB, Van Eyk JE, Mockrin SC, Anderson NL. National
Heart, Lung, And Blood Institute Clinical Proteomics Working
Group report. Circulation 2004;109:1697703.
38. Muthusamy B, Hanumanthu G, Suresh S, et al. Plasma Proteome
Database as a resource for proteomics research. Proteomics 2005;5:
3531 6.
39. Berhane BT, Zong C, Liem DA, et al. Cardiovascular-related proteins
identified in human plasma by the HUPO Plasma Proteome Project
pilot phase. Proteomics 2005;5:3520 30.
40. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH.
Plasma concentration of C-reactive protein and risk of developing
peripheral vascular disease. Circulation 1998;97:425 8.
41. Tzoulaki I, Murray GD, Lee AJ, Rumley A, Lowe GD, Fowkes FG.
C-reactive protein, interleukin-6, and soluble adhesion molecules as
predictors of progressive peripheral atherosclerosis in the general
population: Edinburgh Artery Study. Circulation 2005;112:976 83.
42. Murabito JM, Keyes MJ, Guo CY, et al. Cross-sectional relations of
multiple inflammatory biomarkers to peripheral arterial disease: the
Framingham Offspring Study. Atherosclerosis 2008;203:509 14.
43. Mittermayer F, Krzyzanowska K, Exner M, et al. Asymmetric di-
methylarginine predicts major adverse cardiovascular events in patients
with advanced peripheral artery disease. Arterioscler Thromb Vasc
Biol 2006;26:2536 40.
44. Krzyzanowska K, Mittermayer F, Krugluger W, et al. Asymmetric
dimethylarginine is associated with macrovascular disease and total
JACC Vol. 55, No. 19, 2010
May 11, 2010:201723
homocysteine in patients with type 2 diabetes. Atherosclerosis 2006;
189:236 40.
45. Miyazaki H, Matsuoka H, Cooke JP, et al. Endogenous nitric oxide
synthase inhibitor: a novel marker of atherosclerosis. Circulation
1999;99:1141 6.
46. Boger RH, Bode-Boger SM, Thiele W, Junker W, Alexander K,
Frolich JC. Biochemical evidence for impaired nitric oxide synthesis in
patients with peripheral arterial occlusive disease. Circulation 1997;95:
2068 74.
47. Brevetti G, Schiano V, Chiariello M. Cellular adhesion molecules and
peripheral arterial disease. Vasc Med 2006;11:39 47.
48. Kappelmayer J, Nagy B Jr., Miszti-Blasius K, Hevessy Z, Setiadi H.
The emerging value of P-selectin as a disease marker. Clin Chem Lab
Med 2004;42:475 86.
49. Lee AJ, MacGregor AS, Hau CM, et al. The role of haematological
factors in diabetic peripheral arterial disease: the Edinburgh artery
study. Br J Haematol 1999;105:648 54.
50. Findley CM, Mitchell RG, Duscha BD, Annex BH, Kontos CD.
Plasma levels of soluble Tie2 and vascular endothelial growth factor
distinguish critical limb ischemia from intermittent claudication in
patients with peripheral arterial disease. J Am Coll Cardiol 2008;52:
38793.
51. Yoshitomi Y, Kojima S, Umemoto T, et al. Serum hepatocyte growth
factor in patients with peripheral arterial occlusive disease. J Clin
Endocrinol Metab 1999;84:2425 8.
52. Tsimikas S. Oxidized low-density lipoprotein biomarkers in athero-
sclerosis. Curr Atheroscler Rep 2006;8:55 61.
53. Tsimikas S, Kiechl S, Willeit J, et al. Oxidized phospholipids predict
the presence and progression of carotid and femoral atherosclerosis and
symptomatic cardiovascular disease: five-year prospective results from
the Bruneck study. J Am Coll Cardiol 2006;47:2219 28.
54. Fraley AE, Tsimikas S. Clinical applications of circulating oxidized
low-density lipoprotein biomarkers in cardiovascular disease. Curr
Opin Lipidol 2006;17:5029.
55. Sutton-Tyrrell K, Evans RW, Meilahn E, Alcorn HG. Lipoprotein(a)
and peripheral atherosclerosis in older adults. Atherosclerosis 1996;
122:119.
56. Malinow MR. Plasma homocyst(e)ine and arterial occlusive diseases: a
mini-review. Clin Chem 1995;41:173 6.
57. Loffredo L, Marcoccia A, Pignatelli P, et al. Oxidative-stress-
mediated arterial dysfunction in patients with peripheral arterial
disease. Eur Heart J 2007;28:608 12.
58. Mueller T, Dieplinger B, Gegenhuber A, et al. Serum total
8-iso-prostaglandin F2: a new and independent predictor of
peripheral arterial disease. J Vasc Surg 2004;40:768 73.
59. Seigneur M, Dufourcq P, Conri C, et al. Levels of plasma thrombo-
modulin are increased in atheromatous arterial disease. Thromb Res
1993;71:42331.
60. Vidula H, Tian L, Liu K, et al. Biomarkers of inflammation and
thrombosis as predictors of near-term mortality in patients with
peripheral arterial disease: a cohort study. Ann Intern Med 2008;148:
8593.
61. Mustonen P, Lepantalo M, Lassila R. Physical exertion induces
thrombin formation and fibrin degradation in patients with peripheral
atherosclerosis. Arterioscler Thromb Vasc Biol 1998;18:244 9.
62. Vu JD, Vu JB, Pio JR, et al. Impact of C-reactive protein on the
likelihood of peripheral arterial disease in United States adults with the
metabolic syndrome, diabetes mellitus, and preexisting cardiovascular
disease. Am J Cardiol 2005;96:655 8.
63. Vainas T, Stassen FR, de Graaf R, et al. C-reactive protein in
peripheral arterial disease: relation to severity of the disease and to
future cardiovascular events. J Vasc Surg 2005;42:24351.
64. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis.
Circulation 2002;105:1135 43.
65. Aird WC. Endothelial Cells in Health and Disease. Boca Raton, FL:
Taylor & Francis; 2005.
66. Ho M, Yang E, Matcuk G, et al. Identification of endothelial cell
genes by combined database mining and microarray analysis. Physiol
Genomics 2003;13:249 62.
67. Vanhoutte PM, Miller VM. Heterogeneity of endothelium-dependent
responses in mammalian blood vessels. J Cardiovasc Pharmacol 1985;7
Suppl 3:S1223.
JACC Vol. 55, No. 19, 2010
May 11, 2010:201723
68. Luscher TF, Cooke JP, Houston DS, Neves RJ, Vanhoutte PM.
Endothelium-dependent relaxations in human arteries. Mayo Clin
Proc 1987;62:601 6.
69. Kunkel EJ, Butcher EC. Chemokines and the tissue-specific migration
of lymphocytes. Immunity 2002;16:1 4.
70. Geary RL, Wong JM, Rossini A, Schwartz SM, Adams LD. Expres-
sion profiling identifies 147 genes contributing to a unique primate
neointimal smooth muscle cell phenotype. Arterioscler Thromb Vasc
Biol 2002;22:2010 6.
71. Deng DX, Tsalenko A, Vailaya A, et al. Differences in vascular bed
disease susceptibility reflect differences in gene expression response to
atherogenic stimuli. Circ Res 2006;98:200 8.
72. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion
injury. J Pathol 2000;190:255 66.
73. Wilson AM, Kimura E, Harada RK, et al. 2-microglobulin as a
biomarker in peripheral arterial disease: proteomic profiling and
clinical studies. Circulation 2007;116:1396 403.
74. Snyder N, Nguyen A, Gajula L, et al. The APRI may be enhanced by
the use of the FIBROSpect II in the estimation of fibrosis in chronic
hepatitis C. Clin Chim Acta 2007;381:119 23.
75. Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence
of tamoxifen-treated, node-negative breast cancer. N Engl J Med
2004;351:281726.
76. Wang TJ, Gona P, Larson MG, et al. Multiple biomarkers for the
prediction of first major cardiovascular events and death. N Engl
J Med 2006;355:26319.
77. Fung ET, Wilson AM, Zhang F, et al. A biomarker panel for
peripheral arterial disease. Vasc Med 2008;13:21724.
78. Shinkai S, Chaves PH, Fujiwara Y, et al. 2-microglobulin for risk
stratification of total mortality in the elderly population: comparison
with cystatin C and C-reactive protein. Arch Intern Med 2008;168:
200 6.
79. McDermott MM, Guralnik JM, Corsi A, et al. Patterns of inflam-
mation associated with peripheral arterial disease: the InCHIANTI
study. Am Heart J 2005;150:276 81.
80. Allison MA, Criqui MH, McClelland RL, et al. The effect of novel
cardiovascular risk factors on the ethnic-specific odds for peripheral
Cooke and Wilson 2023
Biomarkers for PAD?
arterial disease in the Multi-Ethnic Study of Atherosclerosis (MESA).
J Am Coll Cardiol 2006;48:1190 7.
81. Libra M, Signorelli SS, Bevelacqua Y, et al. Analysis of G(-174)C
IL-6 polymorphism and plasma concentrations of inflammatory mark-
ers in patients with type 2 diabetes and peripheral arterial disease.
J Clin Pathol 2006;59:2115.
82. Fiotti N, Giansante C, Ponte E, et al. Atherosclerosis and inflamma-
tion. Patterns of cytokine regulation in patients with peripheral arterial
disease. Atherosclerosis 1999;145:51 60.
83. Danielsson P, Truedsson L, Eriksson KF, Norgren L. Inflammatory
markers and IL-6 polymorphism in peripheral arterial disease with and
without diabetes mellitus. Vasc Med 2005;10:191 8.
84. Ziegler S, Kudlacek S, Luger A, Minar E. Osteoprotegerin plasma
concentrations correlate with severity of peripheral artery disease.
Atherosclerosis 2005;182:175 80.
85. DePalma RG, Hayes VW, Cafferata HT, et al. Cytokine signatures in
atherosclerotic claudicants. J Surg Res 2003;111:21521.
86. Nylaende M, Kroese A, Stranden E, et al. Markers of vascular
inflammation are associated with the extent of atherosclerosis assessed
as angiographic score and treadmill walking distances in patients with
peripheral arterial occlusive disease. Vasc Med 2006;11:21 8.
87. Hoogeveen RC, Morrison A, Boerwinkle E, et al. Plasma MCP-1
level and risk for peripheral arterial disease and incident coronary heart
disease: Atherosclerosis Risk in Communities Study. Atherosclerosis
2005;183:3017.
88. Girn HR, Orsi NM, Homer-Vanniasinkam S. An overview of
cytokine interactions in atherosclerosis and implications for peripheral
arterial disease. Vasc Med 2007;12:299 309.
89. Tayebjee MH, Tan KT, MacFadyen RJ, Lip GY. Abnormal circulat-
ing levels of metalloprotease 9 and its tissue inhibitor 1 in angiographi-
cally proven peripheral arterial disease: relationship to disease severity.
J Intern Med 2005;257:110 6.
Key Words: peripheral arterial disease y 2 microglobulin y C-reactive
peptide y cystatin C y ankle-brachial index.