DOI: 10.1111/micc.
12545
ABSTR ACT
Highlights from the World Congress of Microcirculation 2018
Donald G. Welsh; Shayn Peirce‐Cottler
Robarts Research Institute and the Department of Physiology & Pharmacology, University of Western Ontario, London, Ontario, Canada; Department of Biomedical
Engineering, University of Virginia, Charlottesville, Virginia
Correspondence
Donald G. Welsh, Robarts Research Institute and the Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada.
Email: dwelsh@robarts.ca
The 11th World Congress for Microcirculation (WCM2018) was held
in Vancouver, BC, Canada from September 9‐13, 2018. It marked the
first return of the Congress to Canada since its inaugural meeting
in Toronto in 1975. The World Congress is held on a rotating 4 year
basis in a unique location that aligns with a regional microcirculatory
society that includes, but is not exclusive to Japan, China, Australia
and New Zealand, North America, Britain and the European Union.
The theme of WCM 2018 was “Microcirculation in health and dis‐
ease” with a focus on emerging technologies. Its objective was to
advance research and education in the microcirculation, aptly de‐
scribed as “an organ within an organ”. As microcirculatory function
is linked to disease progression, its viewed as a rich therapeutic tar‐
get in the treatment of peripheral vascular disease, diabetes, stroke,
dementia, cancer and sepsis. Working with the microcirculatory
societies around the world and our scientific advisory committee,
the local organizing committee developed an exciting meeting with
research presentations concentrated within six themes: (a) tone reg‐
ulation, oxygen transport and blood flow control; (b) angiogenesis
and remodeling; (c) inflammation, injury, oxidative stress and immu‐
nology; (d) lymphatics, permeability, and cell surface interactions; (e)
bioengineering, computational analysis and instrumentation; and (f)
translational microcirculation.
Four hundred people attended the 4‐day conference which
consisted of two plenary lectures, two honorary award lectures,
and 34 designed symposia. The plenary lectures were delivered
by Drs. Sussan Nourshargh and David Kleinfeld. Dr. Geert Schmid‐
Schoenbein received the “Nishumura‐Tsuchiya International Award”
from the Japanese Society for Microcirculation and Dr. Steven S.
Segal received The Microcirculatory Society Benjamin W. Zweifach
Award. Each symposium (2 hour duration) was moderated by a
chair who set the scientific theme and invited three speakers
(30 minutes presentation). Greater than 150 abstracts were pre‐
sented at the three poster sessions and are reprinted in this issue of
Abbreviations: WCM2018, World Congress of Microcirculation 2018.
Microcirculation. 2019;26:e12545. wileyonlinelibrary.com/journal/micc © 2019 John Wiley & Sons Ltd  |  1 of 1
https://doi.org/10.1111/micc.12545
“Microcirculation”. Each poster session was moderated and trainee
posters were judged by three senior investigators with a total of 15
poster prizes offered over the sessions.
In designing the conference, organizers paid careful attention to
career stage, sex and minority status. In this regard, >40% of the
speakers were female, and >40% were early career investigators
(assistant professor or junior). Our commitment to early career in‐
vestigators was further enhanced with the inclusion of 15 young
investigators as symposium chairs or co‐chairs. The local organiz‐
ing committee also added six small group workshops to further en‐
gage early career trainees as well as established investigators. Three
workshops focused on science‐as‐a‐career and topics, including: (a)
Transitioning from trainee‐to‐academic; (b) Interacting with indus‐
try; and (c) Publishing high impact research. Three further workshops
focused on emerging methodology that included: (a) An introduction
to fMRI; (b) An introduction to multi‐photon imaging; and (c) Using
gene‐altering technology. All of the workshops were well attended.
Deep gratitude is extended to the local organizing commit‐
tee chaired by Dr. Shayn Peirce‐Cottler (University of Virginia &
President of the Microcirculatory Society) and Dr. Donald Welsh
(University of Western Ontario). Our conference management team
was comprised of Ms. Suzanne Brett Welsh (Secretariat, University
of Western Ontario), Dr. William Cupples (Professor, Simon Fraser
University, Vancouver), and A111; their dedicated planning efforts
led to a seamless program. Many thanks also to Ms. Brett Welsh and
Ms. Michelle Kim for their efforts in compiling the abstracts for pub‐
lication. The conference was supported by 35 sponsors and special
appreciation is due the National Heart, Lung, and Blood Institute,
the Canadian Institute for Health Research, the Microcirculatory
Society, the Robarts Research Institute, Schulich School of Medicine
(University of Western Ontario), Danish Myotechnology, MBF
Bioscience, the Totman Foundation (University of Vermont), Monash
University, and lastly the journal “Microcirculation”.
DOI: 10.1111/micc.12524

ABSTR ACTS

K E Y N OTE S PE A K E R

Imaging neutrophil-­microvessel interactions in I N V ITE D PR E S E NTATI O N S

sterile inflammation
Sussan Nourshargh
Centre for Microvascular Research, William Harvey Research Institute, Barts and
S O C I E T Y AWA R D S PE A K E R : N I S H I M U R A-­
The London Medical School, Queen Mary University of London
T S U C H I YA I NTE R N ATI O N A L
AWA R D ( JA PA N E S E S O C I E T Y FO R
Post-­capillary venules are the primary site of leukocyte egress in in-
M I C RO C I RC U L ATI O N)
flammatory reactions. Within our laboratory we investigate neutro-
phil trafficking, most notably neutrophil-­venular wall interactions as
analysed by confocal intravital microscopy. The application of this
methodology to quantitative imaging of physiological reactions has
shed much light on our understanding of the mode, mechanisms and Autodigestion: a fundamental mechanism for
dynamics of neutrophil endothelial cell interactions, and the previ- microvascular dysfunctions, diseases and death
ously un-­
explored response of neutrophil-­
pericyte interactions.
Geert Schmid-Schoenbein
Furthermore, analysis of pathological settings, such as ischemia-­
Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
reperfusion injury has identified numerous aberrant neutrophil be-
haviours. This includes neutrophils exhibiting reverse motility at the
Consider the following fundamental question: How is it possible that
level of endothelial cell junctions and moving from the basolateral to
one digests food in the small intestine but not one's own intestine?
apical aspect of the endothelium, a response that we have associated
The answer to this question has become for our team a starting point
with systemic dissemination of a local inflammatory response. Overall
to develop a new analysis of inflammation and the associated cell
our studies have demonstrated that direct and rigorous analysis of
dysfunctions in the microcirculation. During a meal the pancreatic
neutrophil-­vessel wall behaviours and dynamics are likely to identify
digestive enzymes are carried into the lumen of the small intestine
novel and disease-­specific phenomena that could promote a change in
as a cluster of concentrated and fully active enzymes. An important
thinking towards development of new therapeutic strategies.
mechanism that prevents (auto)digestion of one's own intestine is by
compartmentalization of digestive enzymes inside the lumen of the
small intestine by the intestinal barrier which consists of the intes-
K E Y N OTE S PE A K E R tinal epithelium and its mucin layer. An increasing body of evidence
suggests, however, that this barrier can fail. Digestive enzymes are
transported into the intestinal wall and the systemic circulation. They
How blood flows throughout the brain convert inactive into activate degrading proteases (e.g. MMPs), cleave
plasma proteins as well as membrane receptors and in the process
David Kleinfeld1,2 cause microvascular dysfunctions. In hemorrhagic shock, for example,
1
Crick-­Jacobs Center for Theoretical & Computational Biology, Salk Institute, La cleavage of the insulin receptor causes acute “insulin resistance”. The
Jolla, CA, USA 2Department of Physics, University of California San Diego, San
Diego, CA, USA
digestive enzymes generate short peptides by proteolytic degradation
of plasma proteins. The degrading process during autodigestion can
lead to complete microvascular and multi-­organ failure. Our current
The angioarchitecture of the rodent brain was reviewed, and the in-­vivo evidence suggests that enteral blockade of digestive enzymes
consequences of this architecture in terms of ability for the brain to serves to significantly attenuate the autodigestion process. The clini-
control it's own blood flow, as well as the susceptibility of different cal efficacy of such enteral treatment needs to be determined.
brain regions and types of blood vessels to occlusions and minis- Supported by NIH grant GM 85072 and the ShockOmics Project
trokes. The connection of arteriole vasodynamics to the activity of of the European Union.
neighboring neurons during wakefulness and sleep was discussed.
The role of technological advances in addressing longstanding issues
and creating new opportunities for inquiry will be emphasized.

Microcirculation. 2019;26:e12524. wileyonlinelibrary.com/journal/micc © 2019 John Wiley & Sons Ltd  |  1 of 96

 |
2 of 96       ABSTRACTS

S O C I E T Y AWA R D S PE A K E R : TH E CONCURRENT SESSIONS

M I C RO C I RCU L ATO RY S O C I E T Y B E N JA M I N
W. Z W E I FAC H AWA R D

Where provided, abstracts or short descriptions are

included. Presenting authors are in bold text.
How does blood flow know where to go and how
do we learn where to look?
Steven S. Segal SESSION A.1 LESSONS FROM THE MOUSE
Department of Medical Pharmacology and Physiology & Dalton Cardiovascular
MICROCIRCULATION
Research Center, University of Missouri, Columbia USA
CHAIR: DR NAVEED AKBAR
Blood flow to skeletal muscle can increase more than 100-­fold when
transitioning from rest to intense aerobic exercise. While sympa-
thetic vasoconstriction restricts blood flow to inactive regions,
the resistance vasculature of contracting muscles undergoes func- A.1.1 | Recapitulation of developmental
tional sympatholysis to direct cardiac output according to local de- mechanisms to revascularise the ischemic heart
mand. Intravital microscopy reveals that dilation of distal arteriolar
Karina Dubé; Tonia Thomas; Nicola Smart
branches increases capillary perfusion, thereby expanding the func-
BHF Centre of Regenerative Medicine, University of Oxford, Oxford, UK
tional surface area for diffusion of oxygen and metabolites between
myofibers and their blood supply. As oxygen demand increases with
Following myocardial infarction (MI), the survival of existing and re-
the intensity of activity, vasodilation ascends the resistance network
generated tissue depends upon formation of new coronary blood
to encompass intermediate arterioles, which distribute blood flow
vessels, yet endogenous neovascularisation is poorly understood
to active myofibers. Ascending vasodilation into feed arteries in-
and attempts to therapeutically enhance coronary angiogenesis
creases the total volume of flow into microvascular networks, with
have met with limited success. In the embryo, coronary vessels form
their effluent converging into venous return. While transmural pres-
through carefully orchestrated signalling and contribution from dis-
sure, luminal shear stress and perivascular nerves affect vasomotor
tinct cellular sources: sinus venosus (SV), endocardium and proepi-
control and thereby contribute to blood flow regulation, vasodila-
cardium. We investigated whether developmental mechanisms of
tion travels along network branches independent of these stimuli.
coronary vessel formation are intrinsically reactivated in the adult
Electrical coupling, dye transfer and connexin expression establish
mouse post-­MI. We demonstrated a significant role for de novo ves-
that the rapid component of conducted vasodilation reflects local
sel formation post-­MI by induced hypertrabeculation-­compaction of
generation of hyperpolarization which spreads from cell to cell along
the redeployed endocardium and sprouting angiogenesis from the
the endothelium through gap junctions. Myoendothelial coupling
coronary sinus, the adult SV derivative. The quiescent epicardium
enables direct transmission of hyperpolarization into surrounding
is rapidly reactivated in response to injury and, while direct cellular
smooth muscle cells, which relax through electromechanical signal-
contribution to new vessels is minimal, it appears to play a key role
ing. In turn, the slow component of conducted vasodilation reflects
in supporting the directional expansion of the neovessel network
coordinated intercellular calcium waves along the endothelium that
towards the ischaemic myocardium. Reactivation of vascular cell
elicit nitric oxide release to relax smooth muscle cells via pharmaco-
sources in the adult heart utilises some of the molecular mechanisms
mechanical signaling. Complementary studies of brain, heart, intes-
that are key in embryonic vessel growth, however, there are also im-
tine, kidney and lung demonstrate that vascular conduction directs
portant differences. Thymosin β4, a peptide with established roles
blood flow in tissues and organs throughout the body. (Supported by
in vascular development, was required for endocardial compaction
NIH R37-­HL041026).
and endocardial-­mesenchymal transformation, for epicardial vessel
expansion and smooth muscle cell recruitment. Insight into path-
ways that regulate endogenous vascular repair, drawing on compari-
sons with development, may reveal tractable molecular targets to
therapeutically enhance neovascularisation and regeneration of the
infarcted heart.
ABSTRACTS
A.1.2 | Different endothelial cell capacity to
respond to angiogenic stimuli in male and female
mice
Omid Rezvan; Martina Rudnicki; Tara Haas
School of Kinesiology and Health Science, York University, Toronto, Canada
Data from our lab indicate that female mice have higher adipose
angiogenesis than males in response to a high-­f at diet, which leads
us to hypothesize that sex-­related differences exist in the response
of endothelial cells to angiogenic stimuli. To address this question,
visceral adipose tissue was harvested from 10 weeks-­old male and
female C57BL/6J mice (ethics approved) and used for RNA analy-
sis, ex vivo angiogenesis assays and endothelial cell isolation. Gene
expression analysis showed similar mRNA levels of the endothelial
cell markers (Pecam1 and von Willebrand Factor) and angiogenic
factor (Vascular Endothelial Growth Factor A-­VEGFA) in whole
adipose tissue from male and female mice. VEGFA-­s timulated en-
dothelial cell outgrowth from 3D collagen adipose explant cultures
was observed in 85% of female explants compared to only 50% of
male explants. However, the sprouting area and cell density was
greater in male compared to female explants, suggesting a higher
number of endothelial cells. In a serum-­s timulated cell proliferation
assay, male endothelial cell growth rate was 1.5-­fold greater than
that of female cells. These data unexpectedly indicate that male
endothelial cells have higher capacity to proliferate compared to
female cells, which rules out the hypothesis that the higher adipose
tissue angiogenesis previously observed in high-­
f at-­
fed female
mice results from sex-­differences in the proliferative capacity of
endothelial cells. Further experiments will examine contributions
of signalling pathways such as estrogen receptor 1 to the observed
sexual dimorphism in the angiogenic capacity of adipose tissue.
Funding: CIHR.
A.1.3 | Balancing arteriolargenesis and
angiogenesis to achieve functional control of
blood flow
1 1 1
Maria J. C. Machado ; Rachel Boardman ; Federica Riu ;
Andrew V. Benest1,2; David O. Bates1,2
1
Tumour Vascular Biology Laboratories, Cancer Biology, Division of Cancer
and Stem Cells, School of Medicine, Queen's Medical Centre, University of
Nottingham, Nottingham, UK; 2COMPARE University of Birmingham and
University of Nottingham, UK
Arteriolargenesis, the muscularization of pre-­existing capillaries,
can be stimulated by concomitant NO-­T ie signaling in a model of
physiological angiogenesis (Stone et al, 2017). We hypothesized
that stimulation of NO-­T ie signaling would have a positive impact on
the recovery of blood flow in a model of murine hindlimb ischemia.
Mice injected with Ad.eNOS+Ad.Ang1 (NO-­Tie) had a faster rate
of blood flow recovery compared to control mice, as assessed by
laser speckle imaging. After 21 days of ischemia, blood flow in the
|
      3 of 96
control virus injected mice had recovered to that of the NO-­Tie mice.
There was no change in capillary density, but the ischemic muscle had
higher arteriole density than control (assessed by α-­SMA staining).
Given the previously documented beneficial effect of VEGF signal-
ing, we tested whether mice injected with Ad.eNOS+Ad.Ang1 + Ad.
VEGF would show further improvement. Surprisingly, these mice
recovered no differently than control. There was no difference in
arteriole density and capillary density was lower than control.
Because Dll4 is a driver of arterial specification, we hypothesized
that Notch1 expression would be involved in this process. There was
a significant upregulation of Notch1 transcripts in NO-­Tie-­VEGF
treated compared with NO-­Tie treated mice. Using the Notch acti-
vator, soluble Dll4 (sDll4), we stimulated Notch signaling in the isch-
emic muscles of mice. NO-­Tie-­sDll4 mice had significantly increased
capillary and arteriole density, while presenting a worst outcome in
blood flow recovery.
We conclude that a local balance between angiogenesis and ar-
teriolargenesis needs to occur for efficient recovery of blood flow
after ischemia.
A.1.4 | Signalling pathway investigations of
microvascular endothelial function: from bench
to bedside
Faisel Khan; Naveed Akbar
Division of Systems Medicine, Ninewells Hospital and Medical School, University
of Dundee, Dundee, UK
Chronic inflammation is strongly associated with the develop-
ment of cardiovascular disease (CVD) via its effect on endothelial
dysfunction and atherosclerosis. Previous work has shown that
mitogen and stress kinase 1/2 (MSK 1/2) knock-­o ut mice are
prone to hyper-­i nflammatory responses, but the role of this path-
way in vascular dysfunction is unknown. MSK 1/2 are activated
downstream of toll-­like receptors (TLRs) which engage with the
adaptor protein MyD88. We have shown that MyD88 knock-­o ut
mice are unable to activate TLRs and show preservation of en-
dothelial function and reduced cytokine expression, despite the
onset of hypercholesterolaemia. The MSK 1/2 kinases also inter-
act with MAPKAP 2/3, which limit cytokine synthesis, similarly
to MyD88 KO mice. MAPKAP 2/3 KO mice fed a cholesterol diet
show reduced cytokine expression and preservation of endothe-
lial function in the presence of hypercholesterolemia. Deficiency
of MSK 1/2 in knock-­o ut mice show increased plasma levels of
pro-­i nflammatory cytokines (IL-­1α, IL-­6 and TNF-­α) and a loss of
anti-­i nflammatory IL-­10, with subsequent endothelial dysfunction
through attenuated production of nitric oxide (NO), a modulator
of vascular tone. Cholesterol feeding to MSK 1/ 2 deficient mice
induced significantly greater hypercholesterolemia than in wild-­
type cholesterol fed mice, subsequently this exacerbated inflam-
matory cytokine expression and induced endothelial dysfunction.
These data show that MSK 1/2 may provide a novel target for
 |
4 of 96       ABSTRACTS
intervention to target vascular inflammation. Selective activation
of MSK 1/2 could reduce pro-­inflammatory responses and pre-
serve endothelial dysfunction.
SESSION B.1 GASEOUS TRANSMITTERS: CARBON
MONOXIDE AS MODULATOR OF INFLAMMATION
CHAIR: DR GEDIMINAS CEPINSKAS
B.1.1 | Design and therapeutic applicability of
co-­releasing molecules
Roberto Motterlini; Roberta Foresti
Inserm U955, Equipe 12, Faculty of Medicine, University Paris Est, Creteil, France
Mammalian cells continuously generate carbon monoxide (CO)
during the degradation of heme by the enzyme heme oxyge-
nase. CO serves as a ubiquitous signaling molecule that has also
been shown to possess vasodilatory, anti-­
ischemic and anti-­
inflammatory properties. From a mere biochemical view point, CO
selectively binds with high affinity to transition metals in a specific
redox state leading to the formation of stable metal carbonyl com-
plexes. Based on this peculiar feature, we have designed and de-
veloped metal carbonyls that function as CO-­releasing molecules
(CO-­R Ms) and may have unique therapeutic potentials. Substantial
data from our group revealed that CO-­R Ms are pharmacologi-
cally active by exerting a variety of protective and beneficial ef-
fects both in vitro and in animal models of disease. More recently
we have focussed our research on CORM-­4 01, a water-­soluble
manganese-­b ased carbonyl that has the ability to deliver CO ef-
ficiently to cells as well as to tissues when orally administered in
vivo. Our studies provide evidence for the anti-­inflammatory ef-
fects of CORM-­4 01 in mice challenged with lipopolysaccharide
and highlight an important role of CO in counteracting insulin re-
sistance in high fat diet-­induced obesity. The use of CORM-­4 01
has also helped us to identify mitochondria as a preferential target
of CO in the control of processes as diverse as aerobic metabolism,
inflammation and mitochondrial bioenergetics.
B.1.2 | Therapeutic benefits of carbon
monoxide in vascular-­proliferative disease
Leo E. Otterbein
Harvard Medical School
An overview of the effects of carbon monoxide in preclinical models
of acute vascular injury and its role in modulation of innate immune
responses.
B.1.3 | Carbon monoxide-­releasing molecule
(CORM)-­3 modulates protease-­induced damage
to the endothelium
Eric K. Patterson1; Douglas D. Fraser1,2; Gediminas
Cepinskas1,3
1
Centre for Critical Illness Research, Lawson Health Research Institute, London,
Canada; 2Department of Pediatrics, Western University, London, Canada;
3
Department of Medical Biophysics, Western University, London, Canada
Severe inflammation is accompanied by an overwhelming ac-
cumulation of neutrophils in affected tissues. Neutrophil serine
proteinases, e.g. leukocyte elastase (HLE), proteinase 3 (PR3)
contribute to neutrophil-­induced injury to the microvasculature
during inflammation, resulting in increased vascular perme-
ability and edema. Our previous work showed CORM-­3 inhibits
myeloperoxidase (MPO) activity in a dose-­d ependent manner,
preventing MPO-­induced endothelial injury. The aim of the cur-
rent study was to assess the effect of CORM-­3 on PR3 and HLE-­
induced endothelial injury. We hypothesized that CORM-­3 could
benefit the microvasculature by interfering with PR3 and HLE's
detrimental effects.
We used real-­time monitoring of HUVEC monolayer resistance
employing an ECIS Zθ system (Applied Biophysics). HUVEC were
treated with 2 μg/mL of either PR3 or HLE in the presence or absence
of 100 μM CORM-­3 for up to 2.5 hours in a cell culture incubator. The
data obtained indicate CORM-­3 inhibited both PR3 and HLE-­induced
damage to HUVEC monolayer integrity. Our preliminary results also
indicate that CORM-­3 has a minor effect on HLE enzymatic activity;
however, it is effective in reducing HLE binding to the HUVEC surface.
These findings suggest that CORM-­3 may limit neutrophil-­induced
damage to tissues by inhibiting MPO activity and reducing serine
protease binding to the vascular endothelium.
Funding Support: Heart and Stroke Foundation of Ontario
G-­17-­0 018622.
B.1.4 | CO-­RMs and inflammatory vascular
perfusion in compartment syndrome
Abdel-Rahman Lawendy1,2,3; Aurelia Bihari1; Gediminas
Cepinskas2
1
Div. Orthopaedic Surgery, Department of Surgery, London Health Sciences
Centre, London, Canada; 2Centre for Critical Illness Research, Lawson Health
Research Institute, London, Canada; 3Department of Medical Biophysics,
Western University, London, Canada
Acute limb compartment syndrome (CS), a devastating complica-
tion of musculoskeletal trauma, develops in response to elevation
of the pressure within a closed osteofascial compartment, produc-
ing muscle-­and limb-­threatening ischemia. Full decompression of all
involved compartments by fasciotomy is the current gold-­standard
therapy, but it must be performed within a surgical window of 6-­8 h,
before tissue damage becomes permanent.
ABSTRACTS
Carbon monoxide (CO), a byproduct of heme metabolism, has
been shown protective in ischemia. While inhalation of CO leads to
elevation of carboxyhemoglobin (COHb), recent development of tran-
sitional metal carbonyls, CO-­releasing molecules (CO-­RMs), particu-
larly the water-­soluble CORM-­3, delivers CO in a controlled manner
without COHb formation, making it well suited to clinical applications.
The purpose of our studies was to examine the effects of CORM-­
3-­derived CO on microvascular dysfunction due to elevated com-
partment pressure within skeletal muscle, using clinically relevant
models of CS. The efficacy of both CO and CORM-­3 was tested in
the rat, demonstrating that CORM-­3 was able to prevent the CS-­
associated microvascular perfusion deficits, tissue injury and inflam-
mation, all without the CO-­generated elevation of COHb.
The effects of CORM-­3 were then tested in a preclinical large
animal model of CS (pig), demonstrating the abolition of CS-­induced
systemic leukocyte activation correlated with the inhibition of sys-
temic TNF-­α release, improved tissue microvascular perfusion, di-
minished tissue injury and apoptosis.
The data suggest that CO-­RMs may have an enormous clinical
potential, capable of at least prolonging the surgical window, if not
significantly reducing the need for fasciotomy.
SESSION C.1 EMERGING TECHNOLOGIES IN
MICROVASCULAR IMAGING
CHAIRS: DR BOJANA STEFANOVIC AND DR JOHN G
SLED
C.1.1 | Quantitative imaging of cerebral
microvasculature
Bojana Stefanovic1; John G. Sled2
1
Sunnybrook Health Sciences Centre, Toronto, Canada; 2Mouse Imaging Centre,
The Hospital for Sick Children, Toronto, Canada
Fundamental to the brain's health over the life span is its ability to
adapt to changing environmental conditions. This capacity is, to a
significant part, reflected in the milieu-­driven reorganization of the
cerebral microvasculature. The study of cerebrovascular remodeling
is however, challenging due to the high complexity of brain's micro-
vascular network and the many spatial and temporal scales over
which its adjustments occur. Non-­invasive imaging via two-­photon
fluorescence microscopy, when combined with computational analy-
sis, offers an invaluable platform for measuring these changes in situ
and relating them to changes in the cerebrovascular function, thus
elucidating the link between structural organization of the vascu-
lature and metabolite delivery to the brain tissue. Deployment of
this approach to the models of brain disease has been particularly
fruitful in shedding new light onto the cerebrovascular pathology
and its contribution to disease progression. This talk will review our
use of this technology, and its combination with mesoscale brain
|
      5 of 96
perfusion imaging via arterial spin labeling MRI and neuronal activa-
tion via channel-­rhodopsin photostimulation, to study fundamental
questions regarding cerebrovascular demise in early Alzheimer's
Disease and with repeated, mild traumatic brain injury. Beyond fur-
thering our understanding of the cerebrovascular pathology evolu-
tion, quantitative in situ imaging confers unprecedented sensitivity,
with specificity buttressed either by the transgenic manipulations or
post mortem histopathological assessments. Strengths and limita-
tions of this approach will be discussed in the context of competing
techniques, along with identification of promising future directions.
C.1.2 | Advancement of in vivo MRS imaging
technology for studying brain energy metabolism
and neuroenergetics at ultrahigh field
Wei Chen; Xiao-Hong Zhu
Center for Magnetic Resonance Research (CMRR), Departments of Radiology and
Biomedical Engineering, University of Minnesota, Minnesota, USA
Brain predominantly relies on glucose and oxygen metabolisms to
produce biochemical energy molecules in the form of ATP in the mito-
chondria for supporting electrophysiological activities, neural signal-
ing and brain function under resting and working states. The figure
on the right side shows a schematic diagram illustrating the complex
relations between brain energy metabolism, neuroenergetics, vas-
cular activity and brain function. Impaired energy metabolism and
vascular integrity as well as declined mitochondrial functionality have
been hallmarks for many brain disorders including stroke, brain tumor
and neurodegenerative diseases. However, there is an unmet need
in developing novel neuroimaging tools and providing sensitive bio-
markers to study brain physiology and function or dysfunction in the
early stage of brain diseases. Recent progresses in developing in vivo
X-­nuclear (e.g., 2H, 17O and 31P) magnetic resonance spectroscopic im-
aging (MRSI) techniques at ultrahigh field with significantly improved
sensitivity and spectral quality have shown great promise for quan-
titative and non-­invasive assessment of fundamental cerebral meta-
bolic rates of glucose (CMRGlc) and oxygen (CMRO2) consumption,
ATP production (CMR ATP), TCA cycle rate (V TCA) as well as NAD redox
ratio (RXNAD) in preclinical animal and human brains (1). In this talk, I
will briefly describe the new technical development and advancement
enabling to measure an array of physiological parameters of interest:
1. Quantitative in vivo
2
H MRS/MRSI technique for simultaneous measurement of
CMRGlc and V TCA (2);
2. Quantitative in vivo
17
O MRS/MRSI technique for simultaneous measurement of
CMRO2, cerebral blood flow (CBF) and oxygen extraction fraction
(OEF) (3-5);
3. Quantitative in vivo
31
P MRS/MRSI techniques for measurement of CMR ATP and
RXNAD (6,7).
 |
6 of 96       ABSTRACTS
The new utilities of the metabolic neuroimaging techniques have
been demonstrated for studying resting and stimulus-­evoked brain
(8-­10), stroke (4) and brain tumor (11,12), and mitochondrial function
decline associated with aging process (6), which provide new insights
into brain function and dysfunction.
Acknowledgements: The work reported herein was partly supported
by NIH Grants U01 EB026978, R24 MH106049, RO1 NS070839,
RO1 MH111413, P41 EB015894, P30 NS076408; and the W.M.
Keck Foundation.
References: 1. Zhu XH, Lu M, Chen W. J Magn Reson
2018;292:155-­170.
2. Lu M, Zhu XH, Zhang Y, Mateescu G, Chen W. J Cereb Blood Flow
Metab 2017;37:3518-­3530.
3. Zhu XH, Zhang Y, Tian RX, Lei H, Zhang N, Zhang X, Merkle H,
Ugurbil K, Chen W. Proc Natl Acad Sci U S A 2002;99:13194-­13199.
4. Zhu XH, Chen JM, Tu TW, Chen W, Song SK. Neuroimage
2013;64:437-­4 47.
5. Zhu XH, Wiesner HM, Lee BY, Lu M, Chen W. Proc Intl Soc Mag
Reson Med 2015; 23: p. 895.
6. Zhu XH, Lu M, Lee BY, Ugurbil K, Chen W. Proc Natl Acad Sci U S
A 2015;112:2876-­2881.
7. Lu M, Zhu XH, Chen W. NMR Biomed 2016;29:1010-­1017.
8. Zhu XH, Zhang N, Zhang Y, Ugurbil K, Chen W. J Cereb Blood Flow
Metab 2009;29:10-­18.
9. Zhu XH, Lee BY, Chen W. J Cereb Blood Flow Metab
2018;38:959-­972.
10. Zhu XH, Liu X, Lu M, Wiesner HM, Ugurbil K, Chen W. Proc Intl
Soc Mag Reson Med 2014; 22: p. 3763.
11. Lu M, Zhu XH, Zhang Y, Low W, Chen W. Proc Intl Soc Mag Reson
Med 2016; 24: p. 3962.
12. Lu M, Zhu XH, Zhang Y, Low W, Chen W. Proc Intl Soc Mag Reson
Med 2018; 26: p. 4852.
C.1.3 | Hypoxia-­induced remodeling of
cerebral capillaries and microglia revealed with
repeated longitudinal imaging with two-­photon
microscopy in vivo
Ryota Hachiya1; Hiroya Yuki1; Takuma Sugashi1; Miyuki
Unekawa2; Yutaka Tomita2; Norihiro Suzuki2; Jin Nakahara2;
Iwao Kanno3; Kazuto Masamoto3,4
1
Faculty of Informatics and Engineering, University of Electro-­Communications,
Chofu, Tokyo, Japan; 2Department of Neurology, Keio University School of
Medicine, Shinjuku, Tokyo, Japan; 3Department of Functional Brain Imaging
Research, National Institute of Radiological Sciences, Inage, Chiba, Japan;
4
Brain Science Inspired Life Support Research Center, University of Electro-­
Communications, Chofu, Tokyo, Japan
Chronic exposure to hypoxia induces remodeling of cerebral capillar-
ies, including vasodilation and angiogenesis. Previously we showed
little involvement of astrocytes which cover cerebral microvessels to
form blood-­brain barrier, in remodeling of cerebral capillaries during
early phases of hypoxia adaptation. Here, we focused on the role
of microglia in hypoxia adaptation of cerebral capillaries. The aim
of the present study was to quantitatively characterize morphologi-
cal changes and motility of cortical microglia during adaptation to
hypoxia. We used CX3CR1-­GFP mice (N = 6, 4-­6 m) in which the
cortical microglia expressed green fluorescence protein (GFP) for
visualization with in vivo two-­photon microscopy. The animal use
and experimental protocols were approved by the institutional
Animal Ethics Committee of Keio University, School of Medicine, and
University of Electro-­Communications. The animals were housed in
a hypoxic cage (8%-­9% O2) for 3 weeks, while GFP-­positive microglia
and microvasculature labeled with sulforhodamine 101 were weekly
imaged through a closed cranial window (Tomita-­Seylaz method)
at the same cortical locations longitudinally. During 3-­week expo-
sure to hypoxia, the cell size, number density, and number of fine
processes of the microglia were not significantly varied from pre-­
hypoxic treatment. In contrast, a significant increase in the micro-
glial motility was found after 2 weeks of hypoxia. Pharmacological
treatment for inhibiting microglia revealed that elongation of cap-
illary sprout toward the existing capillaries to be connected is ac-
companied with migration of microglia. In conclusion, an increase in
microglial motility is accompanied with capillary remodeling during
adaptation to cerebral hypoxia.
C.1.4 | Molecular programming of arterial
venus specification in health and disease—2-­
photon imaging and lineage tracing in live mice
Rong Wang
University of California San Francisco
Using 2-­Photon live imaging in combination with arteriovenous (AV)
fluorescent markers to delineate the mechanisms of AV specification
in normal microcirculation and in vascular abnormalities leading to
arteriovenous malformation.
S E S S I O N D.1 LI N K I N G I O N C H A N N E L
DY N A M I C S TO FU N C TI O N A L H Y PE R E M I A
CH A I R : PRO F W I LLI A M JACK S O N
D.1.1 | KIR channel activation and skeletal
muscle hyperemia in humans
Frank Dinenno
Colorado State University, Fort Collins, USA
The local regulation of blood flow to contracting and/or ischemic
skeletal muscle is complex and involves the mechanical effects
of muscle contraction, and various metabolic, red blood cell and
ABSTRACTS
endothelium-­derived substances. The vasodilation under such con-
ditions is achieved through activating signaling pathways in the en-
dothelium and vascular smooth muscle of microvessels responding
to locally contracting or ischemic muscle fibers, then ascends into
upstream feed arteries, redirecting blood and oxygen to the ap-
propriate region of tissue. Recently, our laboratory has performed
several studies in human subjects in vivo to determine the role of
inwardly rectifying potassium (KIR) channel activation in regulating
blood flow to skeletal muscle. To do so, local intra-­arterial (brachial)
infusion of low doses of barium chloride (BaCl2) are used to inhibit
KIR channels during various local conditions: forearm contractions
(exercise), local ischemia, or infusions of putative vasodilators. Our
findings indicate that inhibition of KIR channels significantly reduces
the peak (~40%) and total (~70%) hyperemic response to a single
muscle contraction. If contractions are rhythmic and sustained, KIR
channel inhibition reduces the initial (~50%) and steady-­state (~30%)
vasodilation during exercise. In response to ischemia, KIR channel in-
hibition significantly reduces peak (~50%) and total (~60%) reactive
hyperemia. These collective findings indicate a significant and ob-
ligatory role in functional hyperemia in humans. Finally, intravascular
ATP has been proposed as a key regulator of muscle blood flow and
BaCl2 inhibits ATP-­mediated dilation by ~50%. Recent unpublished
observations and the potential implications of these findings will be
discussed.
D.1.2 | The ability of astrocytes to work under
pressure: a TRPV4-­mediated event
Michael W. Brands; Juan Ramiro Diaz; Jessica A. Filosa; Ki
Jung Kim
Department of Physiology, Augusta University, Augusta, Georgia, USA
The mechanisms underlying cognitive decline are unknown, but
evidence supports hypertension as an important contributor to
this process. Little is known on the effect hypertension has on as-
trocyte function. Astrocytes form a functional unit with all blood
vessels (the neurovascular unit), and thus, hypertension-­evoked
vascular changes may directly affect astrocyte function. We
tested the hypothesis that hypertension-­evoked increases in ar-
teriole tone concomitantly increase astrocyte Ca2+. We measured
parenchymal arteriole (PA) myogenic responses together with as-
trocyte Ca2+ dynamics using cortical brain slices from normoten-
sive and hypertensive mice. A PA was perfused and pressurized
so that it would develop myogenic tone. Chronic hypertension,
induced by 28-­day angiotensin II infusion, significantly increased
PA myogenic responses. In addition, chronic hypertension signifi-
cantly increased spontaneous Ca2+ events within astrocyte micro-
domains (MD). Enhanced MD activity was also observed during
PA myogenic responses supporting greater vessel-­
to-­
astrocyte
signaling. Transient potential receptor vanilloid 4 (TRPV4) chan-
nels, expressed in astrocyte endfeet, respond to hemodynamic
stimuli such as increased pressure/flow. Cortical astrocytes from
|
      7 of 96
hypertensive mice showed augmented TRPV4 channel expres-
sion, currents and Ca2+ responses to the selective channel agonist
GSK1016790A. Both, pharmacological TRPV4 channel blockade
or genetic deletion abrogated enhanced hypertension-­
induced
increases in PA tone. In hypertensive brains, increased TRPV4
channel expression was also associated with increased expres-
sion of the inflammatory astrocyte marker GFAP. Together, these
data suggest that chronic hypertension increases PA tone and Ca2+
events within astrocytes MD. We conclude that aberrant Ca2+
events in astrocyte constitute an early event towards the progres-
sion of cognitive decline.
D.1.3 | Neuronal activity driven TRPV4
channel-­and IP3 receptor-­mediated capillary
Ca2+ signaling generates nitric oxide which tunes
local brain blood flow
Thomas Longden1; Osama Harraz1; Grant Hennig1; Evi
Kostensis2; Gabriela Konig3; Michael Kotlikoff4; David Hill-
Eubanks1; Mark Nelson1,5
1
Department of Pharmacology, University of Vermont, Burlington, VT, USA;
2
Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical
Biology, University of Bonn, Bonn, Germany; 3Institute of Pharmaceutical
Biology, University of Bonn, Bonn, Germany; 4Department of Biomedical
Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA;
5
Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK
Brain capillaries constitute a vast, arborescent network of micro-
scopic tubes composed of end-­to-­end endothelial cells (ECs) that
are in close apposition with all neurons and are responsible for pro-
viding a barrier between the brain and periphery while enabling the
exchange of gas and nutrients with neural tissue. We demonstrate
that in vivo capillary ECs exhibit high amplitude, long lasting and fre-
quent Ca2+ signals that are driven by neural activity. Activation of EC
GqPCRs drives these signals through Ca2+ release through IP3 re-
ceptors in the endoplasmic reticulum and Ca2+ entry through TRPV4
channels. Furthermore, capillary Ca2+ elevations increase local nitric
oxide (NO) production, which in turn can tune blood flow. As NO
is fundamental to many cellular and brain processes, these findings
provide a means through which capillaries can not only precisely
control blood flow, but also influence intra-­and intercellular signal-
ing to support brain function.
Supported by American Heart Association postdoctoral fel-
lowships and a Scientist Development Grant (14POST20480144;
17SDG33670237 to T.A.L.; 17POST33650030 to O.F.H), the Totman
Medical Research Trust (to M.T.N.), Fondation Leducq (to M.T.N.),
EC Horizon 2020 (to M.T.N.), and National Institutes of Health (NIH
4P20 GM103644/4-­
5 to the Vermont Centre on Behavior and
Health (T.A.L); P30-­GM-­103498 to the COBRE imaging facility at
the University of Vermont College of Medicine; R24-­HL-­120847 to
M.I.K; P01-­HL-­095488, R01-­HL-­121706, R37-­DK-­053832 and R01-­
HL-­131181 to M.T.N., and FOR2372 to G.M.K and E.K).
 |
8 of 96       ABSTRACTS
D.1.4 | Conducted vasodilation initiated
by capillary TRPA1 channels in the cerebral
microcirculation
Paulo Pires1; Harry Pritchard1; Pratish Thakore1; Thomas
Longden2; Mark Nelson2,3; Scott Earley1
1
Department of Pharmacology, Center for Cardiovascular Research, University
of Nevada, Reno School of Medicine, Reno, NV, USA; 2Department of
Pharmacology, College of Medicine, University of Vermont, Burlington, VT, USA;
3
Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK
A recent study showed that cerebral capillary endothelial cells (ECs)
can initiate functional hyperemia in the brain through a mechanism
that requires the activity of inwardly-­rectifying K+ channels. Here
we tested the hypothesis that additional molecular sensors are pre-
sent in brain capillary ECs that can also orchestrate dilation of up-
stream parenchymal arterioles (PAs). We found that Ca2+-­permeable
transient receptor potential ankyrin 1 (TRPA1) cation channels are
present and functional in native cerebral capillary ECs. Using a re-
cently developed ex vivo capillary-­parenchymal arteriole microvas-
cular preparation, we observed that picospritzing capillaries with
the TRPA1 activator allyl isothiocyanate (AITC) induced dilation of
upstream PAs. This effect was inhibited by the selective TRPA1 an-
tagonist HC-­030031 and was absent in microvascular preparations
obtained from EC-­specific TRPA1 knockout (eTRPA1−/−) mice. The
vasodilatory response was insensitive to block of the nitric oxide
synthase and cyclooxygenase pathways but was abolished by inhibi-
tion of small-­and intermediate-­conductance Ca2+-­activated K+ chan-
nels with TRAM34 and apamin. Using EC-­specific Tek:GCaMP6f
Ca2+ biosensor mice, we also found that picospritzing capillaries with
AITC initiated intercellular Ca2+ waves that propagated from capil-
laries to upstream PAs. Ca2+ wave propagation was inhibited by HC-­
030031 and the P2X receptor blocker PPADS. Using laser Doppler
flowmetry, we found that functional hyperemic responses induced
by somatosensory stimulation were smaller in eTRPA1−/− mice com-
pared with TRPA1 fl/fl littermates. We conclude that stimulation of
TRPA1 channels on capillary ECs initiates retrograde propagating
Ca2+ waves that induce dilation of upstream PAs and contribute to
functional hyperemia in the brain. R01HL091905; R01HL137852;
R01HL139585; K99HL140106; 15POST2472002.
S E S S I O N A . 2 M EC H A N I S M S O F
A RTE R I O L A R DYS FU N C TI O N I N
C A R D I OVA S C U L A R D I S E A S E
CH A I R : D R K A R EN S TO K E S
A.2.1 | Exercise uncovers novel vasodilator
mechanisms in human adipose arterioles
Ibra S. Fancher1,2; Abeer M. Mahmoud1,3,4; Shane A.
Phillips1,4,5; Austin T. Robinson6; Richard Severin1,4
1
Department of Physical Therapy, University of Illinois at Chicago, Chicago, IL,
USA; 2Division of Pulmonary, Critical Care, Sleep, and Allergy, Department of
Medicine, University of Illinois at Chicago, Chicago, IL, USA; 3Department of
Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL, USA;
4
Integrative Physiology Laboratory, University of Illinois at Chicago, Chicago,
IL, USA; 5Division of Endocrinology, Diabetes, and Metabolism, Department of
Medicine, University of Illinois at Chicago, Chicago, IL, USA; 6Department of
Kinesiology and Applied Physiology, University of Delaware, USA
Impaired endothelium occurs early in the development of CVD and
occurs in the microcirculation where changes in function may pro-
mote the development of hypertension and insulin resistance. We
have found that vasodilator responses to endothelium dependent
stimuli such as flow and acetylcholine are reduced in adipose mi-
crocirculation following single bouts of acute exercise in sedentary
and obese adults. In contrast, we have found that that post-­acute
exercise -­induced resistance artery dysfunction does not occur in
individuals who exercise regularly or in obese adults following ex-
ercise training. The preserved endothelium-­dependent vasodilator
function in microvessels following acute exercise or isolated high
intraluminal pressure in microvessels obtained from trained adults
is maintained by the action of hydrogen peroxide (H2O2) whereas
resting flow induced dilation is nitric oxide (NO) dependent. This
presentation will discuss the potential mechanisms whereby regular
exercise elicits mechanisms to preserve vasodilation when NO bio-
availability is reduced.
A.2.2 | Uridine adenosine tetraphosphate in
the coronary microvasculature in health and
disease
Daphne Merkus
Erasmus MC, The Netherlands
Up4A induces endothelium-­
dependent coronary microvascular
vasodilation in the coronary microvasculature, through activation
of different purinergic receptors. Cardiovascular disease modulates
expression of these purinergic receptors and thereby alters Up4A-­
induced coronary vasodilation.
ABSTRACTS
A.2.3 | Assessing the renal macro-­and
microcirculation during cardiopulmonary bypass:
a pre-­clinical ovine model
Yugeesh R. Lankadeva1; Roger G. Evans2; Sally Hood1;
Naoya Iguchi1,3; Bruno Marino 4; Andrew D. Cochrane5,6;
Clive N. May1
1
Florey Institute of Neuroscience and Mental Health, Melbourne, VIC,
Australia; 2Cardiovascular Disease Program, Biomedicine Discovery Institute
and Department of Physiology, Monash University, Melbourne, VIC, Australia;
3
Department of Anesthesiology and Intensive Care Medicine, Graduate School of
Medicine, Osaka University, Osaka, Japan; 4Department of Perfusion Services,
Austin Hospital, Heidelberg, VIC, Australia; 5Department of Cardiothoracic
Surgery, Monash Health, Monash University, Melbourne, Vic., Australia;
6
Department of Surgery, School of Clinical Sciences at Monash Health, Monash
University, Melbourne, Vic., Australia
Objectives: Acute kidney injury (AKI) develops in ~30% of patients
following cardiac surgery on cardiopulmonary bypass (CPB). There
are no interventions to prevent post-­
operative AKI, because its
pathophysiology remains unclear. Our aims are (1) to determine the
effects of CPB on the renal macro-­and micro-­circulation and (2) to
determine the effects of altering pump flow on global and regional-­
kidney perfusion and oxygenation during CPB.
Methods: We implanted a flow probe on the renal artery and laser
Doppler/oxygen-­sensing probes in the cortex and medulla in sheep
(N = 10). After baseline recordings in conscious sheep, animals were
anesthetized, then placed on CPB with inspired oxygen fraction (FiO2)
maintained at 0.6. From a baseline level of 80 mL/kg/min, pump flow
was altered to 60, 80 and 100 mL/kg/min in random order.
Results: During CPB, renal blood flow (RBF; 287 ±  21 to 109 ±  19 mL/
min), medullary perfusion (720 ± 127 to 222 ± 42 BPU) and med-
ullary oxygenation (48 ± 5 to 22 ± 7 mmHg) were all reduced,
compared with conscious sheep. Cortical oxygenation increased
during CPB (46 ± 3 to 70 ± 17 mmHg), despite reduced perfusion
(1954 ± 378 to 872 ± 112 BPU). Reducing pump flow exacerbated
medullary hypoxia (to 11 ± 3 mmHg), while increasing pump flow
tended to improve medullary oxygenation (to 29 ± 7 mmHg).
Conclusions: CPB had detrimental effects on RBF, medullary and
cortical perfusion and medullary oxygenation. Cortical oxygenation
was maintained by the high FiO2. Medullary hypoxia may be a cru-
cial mediator of post-­CPB AKI. Thus, avoiding medullary hypoxia, by
optimizing perfusion conditions on CPB, may be a feasible strategy
to mitigate post-­operative AKI.
A.2.4 | Novel mechanisms of microvascular
dysfunction in human obesity
Noyan Gokce
Boston University Medical Center, Boston, USA
Background: Pathophysiological mechanisms that connect obesity
to cardiovascular disease are incompletely understood. The ac-
cumulation of visceral adiposity, in particular, is closely associated
|
      9 of 96
with cardiometabolic disease. The current study sought to examine
the role of the non-­canonical WNT5A/PCP signaling pathway in the
control of vascular phenotype in human obesity.
Methods: We collected paired subcutaneous and visceral fat sam-
ples from obese individuals during planned bariatric surgeries and
characterized depot-­specific expression of the pro-­inflammatory
Wnt-­
p athway signaling components in relation to adipose tis-
sue microvascular insulin resistance, endothelial function and
angiogenesis.
Results: We observed remarkable over-­expression of components
of the pro-­inflammatory Wnt-­signaling pathway associated with re-
duced vasodilator and angiogenic capacity of microvessels isolated
from the visceral compared to subcutaneous (SC) adipose depots.
Recombinant Wnt5a reduced endothelial cell eNOS phosphoryla-
tion and NO production, and promoted angiogenic dysfunction,
while pharmacological antagonism of Wnt-­signaling improved vas-
cular function. Moreover, in a subset of obese individuals undergo-
ing coronary artery bypass graft (CABG) surgery, Wnt-­expression
was markedly increased in perivascular/pericardial adipose tissue
overlying atherosclerotic coronary arteries compared to non-­
obstructive left internal mammary artery (LIMA) perivascular fat.
Conclusions: We provide strong evidence that signaling modulators
involving the Wnt pathway may represent unique nodal drivers of
disease that negatively influence the vasculature and promote vaso-
motor and angiogenic dysfunction. Aberrant Wnt signaling may play
an important role in the pathogenesis of cardiometabolic disease
linked to obesity.
SESSION B.2 MICROVESSELS AND LYMPHATICS
IN INFLAMED TISSUES: NEW INSIGHTS FROM
MODELS OF INFLAMMATION
CHAIR: DR JEROME BRESLIN
B.2.1 | Lymphatic adipose crosstalk in alcohol
immunomodulation
Flavia Souza-Smith
Louisiana State University HSC
We will describe how alcohol-­induced lymphatic leakage and de-
viation of dendritic cells into the perilymphatic adipose tissue can
chronically disable the induction of mucosal immunity and lead to
immunometabolic dysregulation.
 |
10 of 96       ABSTRACTS
B.2.2 | Traditional medicine extracts to treat
inflammatory Edema
Michiko Jo
University of Toyama, Toyama, Japan
Water is the most abundant molecule in the body. Membrane water
channels called aquaporins (AQPs) are freely permeated by water but
not by ions or charged solutes. AQPs play a central role in urine con-
centration, and changes in AQPs have a profound influence on water
balance in the whole body, such as dehydration or overhydration to
maintain serum osmolality. Pioglitazone is prescribed to improve Insulin
sensitivity in type 2 diabetes mellitus patients, but it is associated with
fluid retention and edema. Pioglitazone may also exacerbate existing
severe side effects that lead to overexpression of AQPs. There are no
effective diuretics for Pioglitazone-­induced edema, furthermore, long-­
term therapy causes cardiovascular dysfunction and worsens insulin
sensitivity in type 2 diabetes mellitus patients prescribed Pioglitazone
and diuretics. Kampo medicines are traditional herbal formulas used
to treat and prevent various diseases. In particular, a Kampo formula-
tion named Goreisan has been used to promote water metabolism to
relieve symptoms such as thirst, oliguria, and sweating. However, the
influence of Goreisan on AQP expression in the kidney is unknown.
The purpose of this study was to investigate the effect of Goreisan
on urinary concentrating ability and body fluid volume associated
with changes of AQP1-­3 expression in Zucker fatty rats that received
Pioglitazone and Goreisan. Our goal was to clarify the mechanisms of
the effects of Goreisan on the renal function of diabetes mellitus rats
to establish a new combination of Pioglitazone and Goreisan.
B.2.3 | Sepsis dysregulates the microvascular
oxygen transport system in skeletal muscle
Ryon Bateman1; Michael Sharpe2; Justin Jagger3;
Christopher Ellis1,4
1
Department of Medical Biophysics, University of Western Ontario, London,
Canada; 2Department of Medicine, University of Western Ontario, London,
Canada; 3Paediatrics, Thunder Bay Regional Health Sciences Centre, Thunder
Bay, Canada; 4Robarts Research Institute, University of Western Ontario,
London, Canada
Sepsis is defined as “life-­threatening organ dysfunction caused by
a dysregulated host response to infection” [1]. Evidence of maldis-
tribution of capillary blood flow and impaired reactive hyperemia in
skeletal muscle suggest sepsis dysregulates the microvascular oxy-
gen transport system. Using a 6-­hour sepsis rat model, our objective
was to test the hypothesis that sepsis would attenuate the red blood
cell (RBC) signal of tissue hypoxia by reducing RBC O2-­dependent
ATP efflux thus delaying the response time in hypoxic capillaries
(defined below). Experimental protocols were approved by Western
University Council on Animal Care. Sepsis was established using
cecal ligation and perforation [2], and the extensor digitorum longus
skeletal muscle microcirculation was imaged using a dual wavelength
intravital system. Capillary RBC hemoglobin O2 saturation (SO2),
RBC supply rate (SR, RBC/s) and oxygen flow rates (qO2, pL O2/s)
were calculated based on absorption spectroscopy and analysis of
space-­time-­images. Hypoxic capillary and capillary response time
defined as RBC SO2 < 20% and time to increase SO2 > 20%, respec-
tively. Plasma ATP measured using a gas exchanger under normoxic
(N) and hypoxic (H) conditions. Analysis of capillary SR-­SO2-­qO2
time-­series revealed sepsis 1) increased capillary qO2 heterogeneity
(P < 0.05), 2) decreased capillary venular-­end qO2 (P < 0.05) and 3)
delayed capillary response time 3.6-­fold (P < 0.05) in hypoxic capil-
laries. Concurrently, RBC O2-­dependent ATP efflux (H/N) decreased
by 63% (P < 0.05). Accordingly, sepsis impaired RBC function and
capillary response to hypoxia, supporting the concept of a dysregu-
lated microvascular oxygen transport system [2].
References: 1. Singer M. JAMA 2016, 315(8):801-­810.
2. Bateman RM. Crit Care 2015, 19:389. Support by CIHR Grant.
B.2.4 | Lymphatic adaptations in models of
intestinal inflammation
Pierre-Yves von der Weid
Inflammation Research Network, Snyder Institute for Chronic Diseases,
Department of Physiology & Pharmacology, Cumming School of Medicine,
University of Calgary, Calgary, Canada
The functions and morphology of the lymphatic system are mark-
edly altered during intestinal inflammation. In order to assess the
role of these changes in disease progression, we examined the
mesenteric lymphatic system in several rodent models of intestinal
inflammation. Using immunofluorescence techniques and confocal
microscopy, we observed a significant lymphangiogenesis, dilation
and leakage of mesenteric collectors, and enlargement of draining
lymph nodes in both the DSS mouse model of acute colitis and the
TNFΔARE mouse model of chronic ileitis. Although consistent in
both models, these features were correlated with opposite change
in lymph drainage. Indeed, lymphatic clearance of a fluorescent
lipid administered by gavage to the animals was significantly de-
creased in the acute inflammation model, while it was increased
in the chronic model. Using the model of TNBS-­induced ileitis
in guinea pigs, an animal displaying strong contractile activity
in their mesenteric vessels, we further demonstrated that acute
inflammation-­induced decrease in lymph flow correlated with inhi-
bition of lymphatic pumping. Like in the mouse models, mesenteric
lymphatics were also massively dilated. Interestingly, we reported
in TNFΔARE mice numerous structures we identified as tertiary
lymphoid organs (TLOs), which appear very similar to those re-
cently reported in the gut wall and mesentery of IBD patients.
Our findings suggest that mesenteric lymphatic dysfunctions
may promote submucosal edema and impair immune response, thus
contributing to the severity or chronicity of inflammatory bowel
disease as illustrated by the development of mesenteric TLOs. The
ABSTRACTS
lymphatic system should thus be recognized as a potential therapeu-
tic target for intestinal inflammation.
SESSION C.2 NOVEL MECHANISMS OF KV
CHANNEL REGULATION
CHAIRS: DR MANUEL NAVEDO AND DR MATTHEW
NYSTORIAK
C.2.1 | KV channels and the regulation of
arteriolar tone
William Jackson
Michigan State University, East Lansing, USA
Arteriolar smooth muscle cells (SMCs) express a diverse array of
voltage-­gated K+ (KV) channels with members of the KV1, KV2
and KV7 families being particularly important in non-­proliferating
cells. This family of K+ channels are formed from tetramers of pore-­
forming α subunits and associated accessory subunits. The α subu-
nits have long amino-­and carboxy terminal sequences that serve
as docking sites for a diverse array of signaling proteins. Members
of the KV channel family: 1) are highly expressed in VSMCs; 2) are
active at the resting membrane potential of VSMCs in vivo (−45
to −30 mV); 3) contribute to the negative feedback regulation of
VSMC membrane potential and myogenic tone; 4) are activated
by membrane depolarization, vasodilators that signal through
Gs-­coupled receptors, nitric oxide, hydrogen sulfide and hydro-
gen peroxide; 5) are inhibited by membrane hyperpolarization,
increases in intracellular Ca2+ and vasoconstrictors that signal
through Gq-­coupled receptors through a variety of signaling path-
ways; 6) are involved in the proliferative phenotype of VSMCs; and
7) their expression and function are modulated by diseases such
as hypertension, obesity, the metabolic syndrome and diabetes.
Thus, KV channels participate in every aspect of the regulation
of VSMC function in both health and disease. Supported by NIH
R01-­HL32469, R01-­HL137694, P01-­HL070687.
C.2.2 | Bridging the gap between myocardial
metabolic demand and vascular KV1 function
Matthew Nystoriak
University of Louisville
This talk will focus on the mechanisms coupling smooth muscle in-
termediary metabolism with Kv1 channel function to control mem-
brane potential and blood flow upon changes in myocardial oxygen
demand.
|
      11 of 96
C.2.3 | The conducted response as a pliant
process. shaping electrical signaling in the vessel
wall
Bjorn Hald1; Maria Sancho2; Donald Welsh2
1
Department of Neuroscience, University of Copenhagen, København N,
Denmark; 2Robarts Research Institute & Department of Physiology and
Pharmacology, Schulich School of Medicine and Dentistry, University of Western
Ontario, London, ON Canada
Local blood flow control requires the production of a stimulus,
where upon diffusion, initiates a vasomotor response that conducts
along arteries and across branch points. Conducted responses arise
when stimuli activate sufficient ion channels to change membrane
potential and initiate charge spreads via gap junctions along the
endothelium and then to the overlying smooth muscle. Functional
hyperemia critically depends on grading the distance a response
conducts, thus it is intriguing to consider whether this biological
process is modifiable or “pliable”. This key question is difficult to
address biologically, as experimental approaches lack the refine-
ment needed to study factors in isolation. In silico approaches do,
however, possess this level of control. Consequently, we developed
a vascular model of cell-­cell communication to explore the conduc-
tion of electrical/vasomotor phenomenon along simple arteries and
complex network structures. Focusing on key electrical properties,
foundational principles were set to illustrate how an electronic sys-
tem of vascular cells, variably connected with gap junctions, yields
conducted responses that are dynamic and pliable. Our model spe-
cifically shows how subtle changes in ionic conductance, induced
by voltage-­dependent or independent ion channels, impact current
dissipation and hence conduction. Channel-­induced alterations to
conduction were distinct from those induced by varying gap junc-
tional resistance. We conclude that resting ionic conductance and
gap junctional resistivity substantively impact conduction and con-
sequently local blood flow control in health and disease. Supported
by HSFC and CIHR.
C.2.4 | Pathogenic mechanisms of small vessel
diseases of the brain: insights from genetic
diseases
Anne Joutel
INSERM, France
Implication of the microvascular extracellular matrix in CADASIL and
COL4A1/2-­diseases.
 |
12 of 96       ABSTRACTS
SESSION D.2 RECOVERY OF SKELETAL MUSCLE
MICROCIRCULATION AND ITS REGULATION
FOLLOWING INJURY
CHAIRS: PROF STEVEN S SEGAL AND DR J GEOFFREY
PICKERING
D.2.1 | (Re)Making a muscle: activity and
interactions of muscle stem cells and other cell
types during muscle regeneration
DDW Cornelison
Biological Sciences, University of Missouri, Columbia, MO, USA
Skeletal comprises nearly half of adult human body mass and is
necessary for not only for locomotion but also for respiration,
posture and metabolism. While the focus of the muscle commu-
nity is primarily on differentiated contractile myofibers and their
dedicated stem cell population (satellite cells), multiple other cell
and tissue types are present in muscle; these cells are molecularly,
mechanically, and physiologically integrated with muscle cells and
are interdependent with muscle for function. Muscle resident cell
types include connective tissue fibroblasts, motor neurons and
their associated glia, and vascular cells including endothelial cells
and pericytes. Our group has recently been studying the role of
contact-­m ediated signaling via Eph/ephrin interactions on skeletal
muscle patterning, particularly between myofibers and nonmuscle
cells. While Eph/ephrin interactions are highly context-­specific, in
many cases they act to ‘sort’ populations of similar cells or to repel
migrating cells from specific regions. We have identified a specific
ephrin (ephrin-­A3) that is expressed exclusively by slow (Type I)
myofibers; conversely, its potential receptor EphA8 is expressed
exclusively by terminal Schwann cells associated with fast-­type
motor axons. We show that ephrinA3 expression on slow myofib-
ers prevents their innervation by fast motor axons during postna-
tal neuromuscular junction maturation; this repulsive interaction
preserves the fiber type pattern initially laid down during embry-
onic and fetal development which specializes each muscle for its
physiological function. Current work is aimed at identifying the
molecular mechanisms that specify fiber type-­specific Eph/ephrin
expression at the transcriptional level, and exploring other Eph/
ephrin-­m ediated interactions between muscle and nonmuscle cell
types.
D.2.2 | Bioengineering microvasculature
combining endothelial cells and muscle stem cells
to enhance volumetric muscle loss treatments
Marco Quarta1,2,3,4
1
Stanford University, School of Medicine, Dept. of Neurology and Neurological
Sciences, Stanford, USA; 2Molecular Medicine Research Institute, Sunnyvale,
USA; 3Paul F. Glenn Laboratories for the Biology of Aging, Stanford, USA;
4
Center for Tissue Regeneration, Restoration and Repair, Veterans Affairs
Hospital, Palo Alto, USA
Volumetric muscle loss (VML) is defined as an irrecoverable injury
sustained by skeletal muscle and characterized by a permanent
loss of tissue structure and function. The result is life-­l ong disa-
bility attributed to scar tissue formation and structural changes.
Unfortunately, current treatments based on scar tissue debride-
ment and placement of muscle flaps around the site of tissue
defects show limited efficacy. Novel therapeutic approaches
in regenerative medicine include the use of bioconstructs suf-
fused with muscle stem cells (MuSCs) and other muscle resident
cells (MRCs), which are effective to treat VML injuries in mice.
Among these populations of MRCs, endothelial cells (ECs) play
a critical role in sustaining the formation in vivo of de novo my-
ofibers, leading to restored function and structure within VML
injuries. In particular, freshly isolated genetically-­labeled ECs
from skeletal muscle contribute in neo-­v ascularization within
bioconstructs transplanted in VML injuries, suggesting the
role of progenitor ECs in the generation of functional micro-
circulation during the regenerative process. Incorporating ECs
together with MuSCs in these bioconstructs is necessary and
sufficient in supporting the growth of de novo muscle tissue.
Moreover, combining a physical therapy regime based on exer-
cise following these bioconstruct-­b ased treatments enhances
the neo-­m icrovasculature. Learning how to integrate functional
microcirculation in tissue engineering strategies is a critical step
to scale up to human size and eventually develop therapies for
the clinical setting.
D.2.3 | Perivascular macrophages regulate
blood flow following tissue damage
Evelina Vågesjö1; Cedric Seignez1; Gustaf Christoffersson1;
Carmen Herrera Hidalgo1; Antoine Giraud1; Olle Korsgren2;
Helene Rundqvist3; Magnus Essand2; Lena Holm1; Randall S.
Johnson3,4; Mia Phillipson1
1
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden;
2
Department of Immunology, Genetics and Pathology, Uppsala University,
Uppsala, Sweden; 3Department of Cell and Molecular Biology, Karolinska
Institute, Stockholm, Sweden; 4Department of Physiology, Development and
Neuroscience, University of Cambridge, UK
Background: Following ischemic injury, rapid reestablishment of
regulated blood perfusion is important to limit tissue damage and
support healing. Sterile inflammation is a hallmark of ischemic injury,
ABSTRACTS
as macrophages and neutrophils accumulate to support regrowth of
blood vessels and clear tissue of debris.
Methods: Blood flow dynamics in healthy and ischemic mouse mus-
cles were assessed by Laser Doppler flowmetry and in vivo confocal
microscopy during heat-­induced hyperemia, and macrophages were
characterized by flow cytometry. Targeted deletion of iNOS in mac-
rophages was attained by crossing LysM-­Cre or CX3CR1-­CreERT2
mice with iNOS-­floxed mice to attain myeloid-­cell-­specific deletion
and conditional deletion in CX3CR1 + macrophages, respectively,
where after tissue function were assessed. Tissue engineering with
DNA-­plasmids encoding specific chemokines was also investigated
as a therapeutic strategy to increase macrophage-­mediated healing.
Results: This study identified that macrophages attain perivascular
positions in ischemic muscles, where they take on blood flow regula-
tion and are crucial for limb function. While nitric oxide (NO) from
endothelial NO synthase (eNOS) no longer induced vasodilation in
ischemic muscles, loss-­of-­function experiments demonstrated that
this task instead relied on inducible NOS (iNOS) in macrophages.
Macrophages in ischemic tissues upregulated the chemokine re-
ceptor CXCR4, and gain-­of-­function experiments designed to lo-
cally overexpress the chemokine CXCL12 accelerated restoration
of regulated blood flow and concomitantly increased limb function
through increased numbers of perivascular iNOS+ macrophages.
Conclusions: We demonstrate that macrophages add blood flow
regulation to their curriculum in tissue repair, which can be thera-
peutically targeted to improve blood flow regulation and functional
recovery of injured muscles.
D.2.4 | Functional recovery of the
microcirculation during skeletal muscle
regeneration
Steven Segal1,2; DDW Cornelison3; Charmain Fernando1
1
Medical Pharmacology and Physiology, University of Missouri, Columbia, MO,
USA; 2Dalton Cardiovascular Research Center, Columbia, MO, USA; 3Biological
Sciences, University of Missouri, Columbia, MO, USA
Regeneration of myofibers following injury to skeletal muscle is
well-­documented however little is known of corresponding changes
in their microvascular supply. We hypothesized that restoration of
microvascular structure and function is integral to skeletal muscle
regeneration. In anesthetized male C57BL/6J mice (age, 4 months),
the gluteus maximus muscle was injured by local injection of bar-
ium chloride, causing myofiber degeneration and disruption of the
microcirculation. Capillary perfusion was restored at 5 days post-­
injury (dpi) as determined by imaging fluorescent dextran in the
microcirculation. Reactivity of resistance networks was evaluated
at 5, 10 and 21 dpi versus control by measuring internal diameter
of feed arteries, first-­, second-­and third-­order arterioles. All vessel
branches were dilated at 5 and 10 dpi, then recovered to control
by 21 dpi, indicating restoration of spontaneous vasomotor tone.
Across vessel branches, vasoconstriction to phenylephrine (smooth
|
      13 of 96
muscle cell recovery) and vasodilation to acetylcholine (endothe-
lial cell recovery) were minimal at 5 dpi and restored to control by
21 dpi. Functional vasodilation to rhythmic twitch contractions and
to maximal tetanic contraction were minimal at 5 dpi (degenerated
myofibers), and improved through 10 dpi (primarily new myofibers)
and 21 dpi (maturing myofibers). Isometric force production (grams)
was 60% of control at 10 dpi and not different from control at
21 dpi. We suggest that elevated resting diameter promotes tissue
perfusion as myofiber recovery begins. As new myofibers mature,
restoration of vasomotor tone along with reactivity of smooth mus-
cle cells and endothelial cells precede the recovery of functional
vasodilation to muscle contraction.
SESSION A.3 A UNIVERSE BEYOND ROS AND ATP:
NOVEL MECHANISMS OF MITOCHONDRIA AS
SECONDARY MESSENGERS
CHAIR: DR ANDREAS BEYER
A.3.1 | Mitochondrial genetics and physiology
in vascular disease
Jessica Fetterman
Boston University School of Medicine
We have demonstrated that alterations in mitochondrial genetics,
functional changes, and impairment in the turnover and replacement
of damaged mitochondria contribute to cardiovascular risk factor-­
induced vascular dysfunction.
A.3.2 | Inhibition of nitric oxide synthase (NOS)
impacts mitochondrial function differently in
brian microvascular endothelial cells and cortical
neurons
Venkata Naga Lakshmi Ramarao Sure1; Siva Sakamuri1;
Wesley Evans1,2; Jared Sperling1; Nicholas Peterson1; Walter
Murfee3; Prasad Katakam1,2
1
Department of Pharmacology, Tulane University School of Medicine, New
Orleans, Louisiana; 2Tulane Brain Institute, Tulane University, New Orleans,
Louisiana; 3J. Crayton Pruitt Family Department of Biomedical Engineering,
University of Florida, Gainesville, Florida
Objective: Experimental stroke in eNOS and nNOS knockout
mice showed diverse effects on brain injury although the underly-
ing mechanisms are unclear. Our objective is to test the hypoth-
esis that eNOS and nNOS exert diverse effects on mitochondrial
function.
Methods and Results: All experimental protocols were approved
by the IACUC of Tulane University. Primary brain microvascular
 |
14 of 96       ABSTRACTS

endothelial cells (BMECs) and cortical neurons were isolated from A.3.4 | The mitochondrial genome in
rats and exposed to either normoxic or hypoxia/reoxygenation (H/R)
endothelial injury and its propagation
conditions with or without NOS inhibitor (L-­NAME). Oxygen con-
sumptions rates measured by Seahorse bioanalyzer showed that
Mark N. Gillespie
Department of Pharmacology and Center for Lung Biology, University of South
NOS inhibition in BMECs has reduced basal and maximal respiration
Alabama College of Medicine, Mobile, AL
whereas in neurons it increased basal respiration, maximal respira-
tion, proton leak, spare respiratory capacity and non-­mitochondrial
Pharmacologic control of vascular endothelial cell (EC) dysfunction
respiration compared to control group. Interestingly, in both BMECs
in human disease has not been achieved in part because key mo-
and neurons exposed to H/R, NOS inhibition decreased all mito-
lecular targets have yet to be identified. Because mitochondrial (mt)
chondrial respiratory parameters. Superoxide measurements from
abnormalities are common in endothelial injury and since reactive
electron spin resonance spectrometry showed BMECs and neurons
oxygen species (ROS) seem to be pathophysiologically critical, we
exposed to H/R exhibit increased superoxide levels that were sensi-
tested the concept that the mt genome is a sentinel molecule in
tive to NOS inhibition indicating uncoupling of NOS. Fluorescence
which oxidative damage governs EC responses to oxidant stress.
measurements using mitochondrial membrane potential (Ѱm) sensi-
In support of this, we found that transgenic modulation of the EC
tive Rhodamine 123, showed decreased Ѱm following H/R which
mtDNA repair pathway coordinately regulates oxidative mtDNA
was recovered by NOS inhibition in both BMECs and neurons. NOS
damage, cytotoxicity, and vascular EC barrier properties. We also
inhibition has no effect on Ѱm in BMECs exposed to normoxic
discovered that oxidative mtDNA damage led to fragmentation of
conditions whereas in neurons it increased Ѱm. Conclusion. eNOS
the molecule into mtDNA Damage Associated Molecular Patterns
and nNOS have diverse effects on mitochondrial function under
(DAMPs). Mitochondrial DNA DAMPs so produced engaged a
normoxia but uncoupling of NOS promotes identical mitochondrial
feed-­forward pathway leading to unrestrained mtDNA DAMP pro-
functional impairments in anoxia. Our findings challenge the dogma
duction accompanied by their release into the extracellular envi-
attributing a protective role for eNOS and detrimental role for nNOS
ronment and injury dissemination to distant sites. Using a fusion
in ischemic injury.
protein targeting the DNA glycosylase Ogg1 to mitochondria, we
found in preclinical models that pharmacological enhancement of
mtDNA repair suppressed bacteria-­induced mtDNA DAMP release
A.3.3 | The contribution of components in and accompanying acute lung injury and multiple organ system fail-
QiShenYiQi Pills® to its potential to modulate ure. The DNA repair fusion protein also attenuated ventilator-­and
energy metabolism in protection of ischemic hyperoxia-­induced lung injury, and ischemia-­reperfusion injury in

myocardial injury heart, lung, and brain. Collectively, these finding support the con-
cept that mtDNA serves as molecular sentinel linking ROS to en-
Yuan-Chen Cui; Li Yan; Chun-Shui Pan; BaiHe Hu; Xin
dothelial dysfunction and injury propagation.
Chang; Jing-Yu Fan; Jing-Yan Han
Tasly Microcirculation Research Center, Peking University Health Science Center,
Beijing, China
S E S S I O N B . 3 LO C A L PE R FU S I O N CO NTRO L
Ischemic heart diseases remain a challenge for clinicians. Qishenyiqi I N R E N A L C I RC U L ATI O N
pills® (QSYQ) has been reported to be curative during coronary CH A I R S: PRO F N I EL S- H EN R I K H O L S TEI N -
heart diseases with modulation of energy metabolism as one of the R ATH LO U A N D CH A R LOT TE S O R EN S EN , PH D
underlying mechanisms. In this study, we detected the effect of
QSYQ and its components on rat myocardial structure, mitochon-
drial respiratory chain complexes activity and energy metabolism,
and heart function after 30 min of cardiac ischemia, with focusing B.3.1 | Autoregulation & vascular conducted
on the contribution of each component to its potential to regulate
responses—a distributed process
energy metabolism. Results showed that treatment with QSYQ and
all its five components protected myocardial structure from dam-
Will Cupples
Department of Biomedical Physiology & Kinesiology, Simon Fraser University
age by ischemia. QSYQ also attenuated release of myocardial cTnI,
and restored the production of ATP after cardiac ischemia. AS-­IV
and Rb1, but not Rg1, R1 and DLA, had similar effect as QSYQ in Renal blood flow is high to achieve high, stable filtration and delivery

regulation of energy metabolism. These results indicate that QSYQ of O2 to power reabsorption of 99% of the filtrate. Autoregulation,

may prevent ischemia-­induced cardiac injury via regulation of en- mediated by the myogenic response (MR) and by tubuloglomerular

ergy metabolism, to which each of its components contributes feedback (TGF) is the dominant controller. The MR receives no infor-

differently. mation about the adequacy of perfusion and would be unstable un-
less modulated by the slower TGF that does. Pre-­and post-­glomerular
ABSTRACTS
variable vascular anatomy suggests that autoregulation operates on a
larger scale than single nephrons. We explored perfusion of rat renal
cortical surface using laser speckle contrast imaging. We tested phase
coherence (PC [0,1]) between all possible pixel pairs in MR (0.1-­0.3 Hz)
and TGF (0.015-­0.06 Hz) frequency bands.
The MR, occasionally, and TGF, normally, showed synchro-
nized dynamics over macroscopic areas up to the Field of View
~3.5 × 5 mm. Importantly, TGF synchronization showed the car-
dinal property that it reestablishes spontaneously having been
interrupted.
To assess the role of gap junctions in synchronization, carbeno-
xolone (CBX) or glycyrrhizic acid (GZA) were infused into the renal
artery to achieve 100 μM (both). CBX, but not GZA increased spatial
and temporal heterogeneity of surface perfusion and reduced the
number of TGF edges PC > 0.6. Both dynamic (pressure-­flow trans-
fer function) and steady-­state (sequential pressure steps) autoreg-
ulation were impaired by CBX but not GZA. Consistent with these
results, CBX, but not GZA, reduces amplitude modulation of MR by
TGF in individual pixels.
The data show synchronization of TGF dynamics among hun-
dreds of nephrons implying long-­distance communication. The size
of synchronized clusters can be manipulated, making regulation
likely and a definable “unit” of autoregulation unlikely.
B.3.2 | High-­resolution laser speckle imaging of
synchronization in the renal microcirculation
Dmitry D. Postnov1,2; Donald Marsh3; Niels-Henrik
Holstein-Rathlou1; Will Cupples4; Olga Sosnovtseva1
1
Copenhagen University, Copenhagen, Denmark; 2Boston University, Boston,
USA; 3Brown University, Providence, USA; 4Simon Fraser University, Burnaby,
Canada
One of the unique aspects of renal autoregulation is nephron to
nephron interaction, which is controlled via vascular and blood flow
signaling. Simultaneous micropuncture recordings in 2-­3 nephrons have
shown that nephrons can synchronize their activity by signaling to each
other over short distances. Because of the technique being restricted to
a small number of nephrons, the question of synchronization extent and
importance has been left unanswered. In our earlier studies, we have
applied laser speckle contrast imaging (LSCI) to measure blood flow dy-
namics over the renal surface, but limitations of the commercial LSCI
system made it impossible to capture signals from efferent arterioles of
single nephrons at reasonable signal-­to-­noise ratio.
In order to resolve signals from separate arterioles, we have im-
proved methodology of renal LSCI, achieving resolution down to 0.8 μm
per pixel and imaging frequency up to 160 Hz. We apply it to record
2 dysfunction in that the vascular tone of the distal branches of the ves-
~1.7 × 1.7 mm sections of the renal surface in control and under IV
stimuli, resolving ~70 surface vessels per section. Analysis of spectral
properties, including phase and frequency locking, shows persistent
clusters of more than 20 vessels during Ang II infusion and significantly
(P < 0.05) reduced clustering in the vasodilated state. Furthermore, we
|
      15 of 96
show the dynamic evolution of clusters and the presence of the phase
waves, which are often observed in active media such as neural net-
works in the brain. To our knowledge, it is the first study to estimate the
number of vessels and nephrons in the superficial synchronized field.
B.3.3 | Kidney tissue oxygenation in acute
kidney injury
Connie P. C. Ow1,2; Jennifer P. Ngo1; Md Mahbub Ullah1;
Giannie Barsha1; Lucinda Hilliard1; Ruth C. Meex3;
Matthew J. Watt4; Vijayakumar Sukumaran ,2; Hirotsugu
Tsuchimoch2; James T. Pearson1,2; Maarten P. Koeners5,6;
Roger G. Evans1
1
Cardiovascular Disease Program and Biomedicine Discovery Institute,
Department of Physiology, Monash University, Australia; 2National Cerebral
and Cardiovascular Research Institute, Japan; 3NUTRIM School of Nutritional
and Translational Research in Metabolism, Maastricht University Medical
Centre, Department of Human Biology, Maastricht, The Netherlands;
4
Metabolism, Diabetes and Obesity Program, Biomedicine Discovery
Institute, Department of Physiology, Monash University, Australia; 5School of
Physiology, Pharmacology and Neuroscience, Biomedical Sciences, University
of Bristol, UK; 6Institute of Biomedical and Clinical Science, University of
Exeter Medical School, UK
Ischemia reperfusion injury (IRI) is the most common form of acute kid-
ney injury in the hospital setting. The inevitable ischemic insult to the
kidney is thought to render the kidney susceptible to the development
of tissue hypoxia. Indeed, tissue hypoxia has been observed in the im-
mediate hours and weeks after recovery from IRI, yet little is known
about renal tissue oxygenation during the subacute period following
IRI. We hypothesized that the kidney is hypoxic during the subacute
period (24 hours and 5 days after reperfusion) of a severe form of renal
IRI in rats and is driven by a mismatch between renal oxygen delivery
(DO2) and oxygen consumption (VO2). We employed four different
methods to assess the temporal and spatial changes in tissue oxygena-
tion during the subacute phase of renal IRI. Renal DO2 was not signifi-
cantly reduced in the subacute phase of IRI. In contrast, renal VO2 was
55% less 24 h, and 49% less 5 days after reperfusion than after sham-­
ischemia. Inner medullary tissue PO2, measured by radiotelemetry was
25 ± 12% greater 24 hours after ischemia than after sham-­ischemia.
By 5 days after reperfusion, tissue PO2 was similar to that in rats sub-
jected to sham-­ischemia. Tissue PO2 measured by Clark electrode was
consistently greater 24 h, but not 5 days, after ischemia than after
sham-­ischemia. Cellular hypoxia, assessed by pimonidazole adduct im-
munohistochemistry, was largely absent at both time-­points and tis-
sue levels of hypoxia inducible factors were downregulated following
renal ischemia. Moreover, renal microvasculature was observed to be
severely constricted and the vascular tone appears to be maintained
by EDHF. By 5 days following IRI, there was pronounced endothelial
sels could not be maintained in the presence of vasoconstrictor. Thus, in
this model of severe IRI, despite severe endothelial dysfunction, tissue
hypoxia does not appear to be an obligatory event during the subacute
phase, likely due to the markedly reduced oxygen consumption.
 |
16 of 96       ABSTRACTS
B.3.4 | Vessel wall communication in renal
descending vasa recta
Thomas Pallone
University of Maryland
Data will be presented to show that renal descending vasa recta
endothelia and pericytes are an electrical syncytium. They share
membrane potential and closely track vasoactive responses after
stimulations.
SESSION C.3 PERIVASCULAR CELLS:
CONTRIBUTIONS TO MICROVASCULAR
PATTERNING AND REGENERATION
CHAIRS: DR TARA HAAS AND MR EMMANUEL
NWADOZI
C.3.1 | Pericyte regulated basement
membranes in health and disease
Anjelica Gonzalez
Yale University
Microvascular dysfunction and disintegration is key to the genesis
and progression of many diseases of capillary rich organs. Such
organs include skin, kidney, heart and lungs, each abundant in mi-
crovasculature, and enriched in microvascular mural cells known as
pericytes. The pericyte-­elaborated basement membrane, while con-
sidered an important regulator of microvascular stability, has been
inadequately characterized in the healthy, inflamed or fibrotic state.
Further, in vivo systems have demonstrated that pericytes are capa-
ble of vascular destabilization and migration for the microvessel into
the interstitial tissue during fibrosis, though mechanisms driving this
activity have not been well elucidated.
Using engineered models of the composite microvasculature, in
conjunction with engineered human lung and skin, we have explored
the extent to which pericytes respond to proinflammatory and profi-
brotic signals, matrix protein composition, and signals generated by
mechanotransduction in mechanically altered tissue. We have used
such engineered models to determine the extent to which vascular
and mural cells can contribute to leukocyte recruitment, vascular de-
stabilization and tissue remodeling to progress and sustain a diseased
microenvironment. Lastly, we have used such models in conjunction
with human cells to determine the efficacy of currently approved and
novel therapeutics to inhibit development, slow progression and po-
tentially reverse the matrix remodeling events facilitated by pericytes,
fibroblasts and myofibroblasts in the skin and lung. Future transfor-
mation of our engineered system into high-­throughput platforms will
provide an excellent and efficient means of understanding the role of
understudied cells, like pericytes, in disease initiation and progression.
Similarly, the development of a high throughput platform will facilitate
drug screening for orphaned or incompletely characterized drugs that
would be effective in treating inflammatory and fibrotic disease.
C.3.2 | Pericyte contribution towards tumor
progression and metastasis
Hellmut Augustin1,2,3
1
Vascular Oncology and Metastasis, German Cancer Research Center, Heidelberg
(DKFZ-­ZMBH Alliance), Germany; 2Vascular Biology and Tumor Angiogenesis,
Medical Faculty Mannheim (CBTM), Heidelberg University, Germany; 3German
Cancer Consortium (DKTK), Heidelberg, Germany
Pericytes are organotypically differentiated microvascular murals
cells. They are in intimate contact with endothelial cells and thereby
control microvascular plasticity and function. The molecular analysis
of the complexity of pericyte functions is still in its infancy, which is
to this day largely due to considerable lack of pericyte research tools,
including robust cellular models, reliable and selective markers, as
well as validated genetic tools, such as Cre and CreERT2 driver mice.
At the same time, detailed comparative single cell endothelial cell
and pericyte transcriptomic analyses have yielded a wealth of novel
pericyte specific molecules whose characterization in the control of
microvascular function awaits mechanistic study. The contribution
of pericyte towards tumor progression and metastasis is contextual
and both, pro-­tumorigenic as well as anti-­tumorigenic effects have
been reported. This presentation will review the state of the art of
the role of pericytes for tumor progression and metastasis. Focusing
on the transmembrane receptors Endosialin and Tie2, novel pro-­and
antitumorigenic functions of pericytes will be presented and the po-
tential of pericytes for therapeutic intervention will be discussed.
C.3.3 | Microvascular remodelling in stroke
pathobiology and therapy
Ayman ElAli1,2
1
Department of Psychiatry and Neuroscience, Faculty of Medicine, Laval
University; 2Neuroscience Axis, CHU de Québec Research Center (CHUL), QC,
Canada
Stroke constitutes a major cause of death and disability of the adults
in the world. Unfortunately, no efficient disease-­modifying thera-
pies exist yet. Stroke triggers profound structural and functional re-
modelling of the neurovascular unit that includes endothelial cells,
pericytes, astrocytes, microglia, and neurons, working together to
enable proper brain functioning. Endothelial cells form the blood-­
brain barrier (BBB), which plays an essential role in maintaining brain
homeostasis. Pericytes play major roles in regulating the cerebral
blood flow, angiogenesis, microvasculature stability, and BBB prop-
erties. Targeting specialized mechanisms in endothelial cells to re-
store BBB integrity, and modulating pericyte function to stabilize the
vascular system, constitute important avenues in stroke therapies.
ABSTRACTS
Recombinant tissue plasminogen activator (rtPA) administration
within a narrow therapeutic window of 4.5 hours after stroke onset
constitutes the only existing approach to restore cerebral blood
perfusion. Beyond this window, rtPA worsens BBB disruption and
causes haemorrhagic transformation. Canonical Wnt pathway or-
chestrates BBB maturation during ontogeny. Using mouse models
and cell-­based assays, our recent work indicates that pathway ac-
tivity is induced specifically in endothelial cells early after stroke.
Early deactivation of the pathway aggravates BBB breakdown and
increases haemorrhagic transformation incidence. On the other
hand, pathway activation reduces the incidence of haemorrhagic
transformation associated to delayed rtPA administration via atten-
uation of BBB breakdown. Our study demonstrates that activation
of the canonical Wnt pathway constitutes a clinically relevant strat-
egy to extend the therapeutic window of rtPA by attenuating BBB
breakdown via regulation of BBB-­specific mechanisms.
Stroke triggers the formation of new microvasculature in the
lesion site via angiogenesis. Neural survival is greatest in the tissue
undergoing angiogenesis. Angiogenesis is a highly dynamic process
that involves close and finely tuned interactions between brain
endothelial cells and pericytes. Stroke triggers pericyte death and
detachment from brain endothelial cells, impairing key neurovas-
cular functions. Using mouse models and cell-­b ased assays, we
found that vascular endothelial growth factor isoform-­B (VEGF-­B),
which acts as “survival” factor, promotes the formation of stable
microvasculature within the lesion site tissue by enhancing peri-
cyte survival and interaction with brain endothelial cells. The ef-
fects of VEGF-­B are mediated via its specific receptor VEGFR-­1
that is predominately expressed in brain pericytes, unravelling
an unknown role of VEGF-­B/VEGFR-­1 signalling in regulating the
function of pericytes. Our findings suggest that stimulation of en-
dothelial cell-­p ericyte crosstalk constitute a promising approach
to promote repair after stroke.
C.3.4 | Studying the origin and function of
novel brain vascular-­associated cells
Marina Venero Galanternik1; Daniel Castranova1; Aniket
V. Gore1; Nathan H. Blewett1; Hyun Min Jung1; Amber
N. Stratman1; Martha R. Kirby 2; James Iben1; Mayumi F.
Miller1; Koichi Kawakami3,4; Richard J. Maraia1; Brant M.
Weinstein1
1
Division of Developmental Biology, Eunice Kennedy Shriver National Institute of
Child Health and Human Development, National Institutes of Health, Bethesda,
USA; 2Translational and Functional Genomics Branch, National Human Genome
Research Institute, National Institutes of Health, Bethesda, USA; 3Division of
Molecular and Developmental Biology, National Institute of Genetics, Mishima,
Japan; 4Department of Genetics, SOKENDAI (The Graduate University for
Advanced Studies), Mishima, Japan
The meninges are an external enveloping connective tissue that en-
cases the brain, producing cerebrospinal fluid, acting as a cushion
against trauma, nourishing the brain via nutrient circulation, and re-
moving waste. Despite its importance, the cell types present in the
|
      17 of 96
meninges and the function and embryonic origins of this tissue are
still not well understood. We have discovered a set of novel perivas-
cular cells closely associated with meningeal blood vessels. Based
on similarities in their morphology, location, and highly unusual
scavenger behavior, some of these cells appear to be the zebrafish
equivalent of mammalian “Fluorescent Granular Perithelial cells”
(FGPs), macrophage-­like cells about which very little is known that
likely play important roles in brain function and in a variety of CNS
pathologies. Using RNAseq of FACS-­
sorted FGPs and single-­
cell
profiling of cranial cells, we show that despite their macrophage-­like
morphology and their perivascular location these cells are molecu-
larly most similar to lymphatic endothelial cells, and lineage tracing
and time-­lapse imaging demonstrate that these unusual cells trans-
differentiate from endothelial cells lining blood vessels of the optic
choroid vascular plexus deep inside the brain, before migrating to
the brain surface. Using forward-­genetic screening of transgenic ze-
brafish for ENU-­induced recessive mutations, we recently identified
a mutant that appears to be specifically deficient in FGPs, providing
us a genetic model to explore the novel functional role of these cells.
Our findings are the first report of a non-­vessel forming, perivascu-
lar cell population in the brain that emerges by transdifferentiation
from vascular endothelium.
S E S S I O N D. 3 N OV E L AC TI O N S O F I M M U N E
C E LL S I N TH E M I C ROVA S CU L AT U R E
CH A I R S: PR O F M I CH A EL H I CK E Y A N D D R
O LG A BA R R EI R O
D.3.1 | Mechanisms of intravascular immune
surveillance by antigen-­experienced T cell
subsets during the response to acute viral
infections in the skin
Olga Barreiro1; Scott Lougghead1; Carmen Gerlach2;
Mahmoud Eljalby1; Victor Collado1; Anthonie Zwijnenburg2;
Carly Ziegler3; Nir Yosef4; Alex Shalek3; Ulrich H. von
Andrian1,5
1
Harvard Medical School, Boston, USA; 2Department of Medicine, Karolinska
Institute, Stockholm, Sweden; 3Institute for Medical Engineering and Science,
MIT, Cambridge, USA; 4Department of Electrical Engineering & Computer
Science, Center for Computational Biology, UC Berkeley, Berkeley, USA; 5The
Ragon Institute of MGH, MIT and Harvard, Cambridge, USA
Following infection, pathogen-­specific T cells proliferate and divide
in a heterogeneous manner, giving rise to effector and memory T
cells with distinct migratory patterns. This migratory division of labor
ensures that the host is efficiently scanned for ongoing or recurring
infections. Recent evidence suggests that the presumed migratory
behavior of the classical effector memory T cell subset needs re-
vision. Here, we report that terminally differentiated effector and
 |
18 of 96       ABSTRACTS
memory T cells adhere to and patrol dermal endothelium, while less
differentiated T cells do not. This behavior is preferentially observed
in arterioles and migration tends to be retrograde to blood flow.
Furthermore, patrolling T cells survey endothelium in the search for
antigen as injection of cognate antigen results in their immediate ar-
rest to help endothelium in the fight against infection. Together, this
suggests that terminally differentiated effector and memory T cells
engage in a migratory behavior that supports protective scanning of
specific vascular beds such as dermal microvasculature.
D.3.2 | The fate and functions of lung vascular
neutrophils
Bryan Yipp
University of Calgary
The lungs are an intermediary niche in the PMN lifecycle wherein
aged PMNs are regulated by B cells, which restrains their potential
to cause pulmonary pathology.
D.3.3 | Regulatory T cells differentially undergo
transmigration or prolonged intravascular
patrolling at distinct phases of the inflammatory
response
Michael J. Hickey; Latasha D. Abeynaike; Sarah L. Snelgrove
Centre for Inflammatory Diseases, Monash University Department of Medicine,
Monash Medical Centre, Clayton, VIC 3168, Australia
Regulatory T cells (Tregs) play important roles in limiting inflam-
matory responses in the skin. During these responses, Treg abun-
dance increases and interfering with their recruitment results in
exacerbation of inflammation and increased endothelial activa-
tion. However, the mechanisms whereby Tregs enter the skin re-
main poorly understood. The aim of this study was to use intravital
microscopy to investigate adhesion and transmigration of Tregs in
the dermal microvasculature in a two-­challenge model of contact
sensitivity. Using intravital confocal microscopy of Foxp3-­
G FP
mice, we visualised endogenous Tregs and assessed their inter-
actions in the dermal microvasculature at different stages of the
response. Four hours after hapten challenge, Tregs underwent
adhesion with ~25% of these cells proceeding to transmigrate, a
process dependent on CCR4. At 24 h, Tregs adhered but no longer
underwent transmigration, instead remaining in prolonged con-
tact with the endothelium, migrating over the endothelial surface.
Fours after a second challenge, Treg transmigration was restored,
although in this case transmigration was CCR4-­
independent.
Notably, at 24 hours but not 4 hours after challenge, endothelial
cells expressed MHCII. Moreover, inhibition of MHCII reduced
Treg adhesion, demonstrating an unexpected role for MHCII in
Treg attachment to the endothelium. Together these data show
that Treg adhesion and transmigration can be driven by different
molecular mechanisms at different stages of an antigen-­driven in-
flammatory response. In addition, Tregs can undergo prolonged
migration on the inflamed endothelium, potentially affording them
the opportunity to modulate endothelial adhesive function.
Animal work was approved by the Monash Medical Centre
Animal Ethics Committee.
D.3.4 | Spatiotemporal dynamics of CD8+ T
cells undergoing intrahepatic priming
Matteo Iannacone
San Raffaele Scientific Institute
No abstract or description provided.
SESSION A.4 EXTRACELLULAR VESICLES AND
MICROVASCULAR PATHOLOGY
CHAIRS: PROF GEORGES GRAU AND DR MARYAM
MOUSSAVI
A.4.1 | Modulation of brain microvascular
endothelial extracellular vesicles by plasmodium
falciparum: relevance to cerebral malaria
Georges Grau1,2,3; Elham Hosseini-Beheshti1; Nicholas H.
Hunt1; Thorey Jonsdottir1; Annette Juillard1; Pierre-Georges
Juillard1; Peter A. Lay4; Joonsup Lee 4; Stephen Rogerson5
1
Vascular Immunology Unit, Department of Pathology, School of Medical
Sciences, The University of Sydney, Australia; 2Marie Bashir Institute, The
University of Sydney, Australia; 3The Sydney Nano Institute, The University of
Sydney, Australia; 4Vibrational Spectroscopy Core Facility, School of Chemistry,
The University of Sydney, Australia; 5Doherty Institute, University of Melbourne,
Australia
Numerous pathogens infect the human brain to cause incapacitat-
ing disease, leading to death or neurological sequelae. In the case of
malaria, Plasmodium falciparum-­infected erythrocytes (IE) obstruct
cerebral microvessels and trigger massive inflammation to cause cer-
ebral malaria.
Extracellular vesicles (EV) -­essential mediators of intercellular
communication between the pathogen, the immune system and
brain endothelial cells -­include microvesicles (MV, formerly called
microparticles) and exosomes. Plasma MV numbers are dramatically
elevated at the time of cerebral malaria, and, in mice, inhibition of
MV release prevents brain pathology and its associated mortality.
We modelled cerebral malaria in vitro by co-­culturing human
brain microvascular endothelial cells with P. falciparum-­IE, and char-
acterised MV and exosomes released in the supernate. Both CD36-­
and ICAM-­1-­binding parasites dramatically enhanced MV release.
ABSTRACTS
Nanoparticle tracking analysis (NTA) confirmed that endothelial-­IE
supernates had more vesicles than other groups and were the only
group with vesicles around 500 nm. Interestingly, NTA revealed that
the majority of EV released upon contact between IE and brain en-
dothelium were in the 100-­200 nm range, i.e. the exosome range,
suggesting that smaller EV may be major players. Vibrational spec-
troscopic techniques, including FTIR and bio-­ATR spectroscopies,
identified differences in the biomolecular content of EV derived
from brain endothelial cells co-­cultured with ICAM-­1 binder versus
CD36 binder strains of P. falciparum. This in vitro model of cerebral
malaria will be used to further characterise EV and to test inhibitors
of their release in order to gain a better understanding of pathogen-
esis and to propose novel therapeutic avenues.
A.4.2 | Role of annexin A1 in resolving
thrombosis and inflammation in the brain
Felicity N. E. Gavins
Department of Molecular & Cellular Physiology, Louisiana State University Health
Sciences Center, Shreveport, LA, USA
It is essential that both thrombosis and inflammation are controlled
effectively in immune responses to maintain host homeostasis. This
is even more essential in the brain where administration of agents
that resolve the aggressive thrombo-­inflammatory states after is-
chemic insults (such as stroke) could limit irretrievable neuronal
damage. One such agent of interest is the anti-­inflammatory and
pro-­resolving endogenous mediator Annexin A1 (AnxA1). AnxA1 has
been shown to mediate its effects via the Formyl Peptide Receptor
(FPR) family, especially Fpr2 (the lipoxin A4 receptor or Fpr2/ALX)
in the brain. The objective of the study was to determine whether
Annexin A1 is effective in resolving cerebral thrombo-­inflammation
via an interaction with Fpr2/ALX.
Using Pharmacological and genetic approaches we found that
mice lacking AnxA1 (AnxA1−/−) mice displayed heightened neutro-
phil and platelet activation as well as neutrophil-­platelet aggregate
(NPA) formation within the cerebral microcirculation, as quantified
using fluorescent intravital microscopy. These effects were coupled
with increased infarct volume, blood brain barrier permeability and
changes in cytokines. Furthermore, exogenous administration of
AnxA1 was able to reduce cellular adherence to the inflamed cerebral
endothelium following stroke and aggregate formation via Fpr2/ALX.
In summary, AnxA1 is a possible therapeutic strategy for pro-
moting endogenous pro-­resolving, anti-­thrombo-­inflammatory
pathways following cerebral ischemic insults.
Poster and Oral Presentation.
(All animal experiments complied with ARRIVE (Animal
Research: Reporting In Vivo Experiments) guidelines and followed
the European Union Directive (2010/63/EU) or were approved by
LSUHSC-­S Institutional Animal Care and Use Committee and in ac-
cordance with the guidelines of the American Physiological Society.)
|
      19 of 96
A.4.3 | Invariant natural killer T cells shape
the gut microbiota and regulate neutrophil
recruitment and function during intestinal
inflammation
Sj Shen1; Kathryn Prame Kumar1; Dragana Stanley2; Robert
J. Moore3,4; Thi Thu Hao Van4; Shu Wen Wen1; Michael J.
Hickey1; Connie H. Y. Wong1
1
Centre for Inflammatory Diseases, Monash University Department of Medicine,
Monash Medical Centre, Clayton, Victoria, Australia; 2School of Health Medical
and Applied Sciences, Central Queensland University, Australia; 3Infection and
Immunity Program, Monash Biomedicine Discovery Institute and Department of
Microbiology, Monash University, Australia; 4School of Science, RMIT University,
Australia
Invariant natural killer T (iNKT) cells, the gut microbiota and neutro-
phils all play an important part in the pathogenesis of inflammatory
diseases, but their precise roles and interplay in modulating colitis re-
main unclear. This study investigates the interactions between iNKT
cells, neutrophils and gut microbiota in regulating colitis pathology.
Here, we show iNKT cell-­deficient Jα18−/− mice display reduced dex-
tran sodium sulphate (DSS)-­induced colonic inflammation compared
to wild-­t ype counterparts. We reveal that there is a distinct gut mi-
crobiota shaped by the absence of iNKT cells, which is associated
with protection from colonic inflammation. Additionally, the reduced
inflammation in Jα18−/− mice correlated with increased expression of
neutrophil chemoattractants (Cxcl1 and Cxcl2) and increased neutro-
phil recruitment. Using intravital microscopy, we showed that these
neutrophils were recruited to the colon within three days of initiation
of the model, prior to clinical signs of colitis. Further analysis showed
that these neutrophils have increased expression of anti-­inflammatory
marker Chil3 (Ym-­1). Indeed, depletion of neutrophils in DSS-­treated
Jα18−/− mice resulted in worsened pathology, demonstrating that
neutrophils confer an anti-­colitogenic effect in the absence of iNKT
cells. Thus, our data support the emerging concept that neutrophils
can mediate important regulatory roles in inflammation and highlight
the complexity of the iNKT cell-­microbiota-­neutrophil axis in regulat-
ing colonic inflammation. S. Shen and C. Wong are supported by the
Research Training Program stipend and National Heart Foundation
(Australia), respectively. All experiments were performed in accord-
ance with protocols approved by Monash Medical Centre B Animal
Ethics Committee (MMCB/2015/15).
A.4.4 | From the gut to circulation: oral
administration of bovine milk exosomes reduces
primary tumor burden but accelerates metastasis
Suresh Mathivanan
La Trobe University
The presentation includes some intriguing observations from our
group as how orally administered exosomes can survive the harsh
GI-­tract, enter circulation and accelerate cancer metastasis.
 |
20 of 96       ABSTRACTS
S E S S I O N B . 4 C Y TOS K E LE TO N DY N A M I C S
I N M I C ROVA S CU L A R TO N E G E N E R ATI O N
CH A I R S: D R A H M ED EL-YA ZB I A N D PRO F
D O N A LD W EL S H
B.4.1 | Molecular regulation of actin
polymerization and force generation
Susan J. Gunst; Youliang Huang; Yidi Wu; Wenwu Zhang
Department of Cellular and Integrative Physiology, Indiana University School of
Medicine, Indianapolis, IN, USA
Adhesion junction complexes and the actin cytoskeleton are dynamic
structures that play an integral role in regulating the development of
contractile tension and the functional properties of smooth muscle tis-
sues. Studies of many smooth muscle tissues and cells have documented
an increase in the proportion of filamentous actin in response to con-
tractile stimulation and the essential role of stimulus-­induced actin po-
lymerization and cytoskeletal dynamics in the generation of mechanical
tension. The independent inhibition of processes that regulate either
actin polymerization or myosin light chain phosphorylation prevents
tension development, indicating that their functional roles during con-
traction are distinct and separately regulated. In airway smooth muscle
tissues, contractile stimulation initiates the recruitment of proteins in-
cluding vinculin, paxillin and FAK to membrane adhesion junctions and
their assembly into an adhesome complex. Adhesome complex pro-
teins orchestrate the signaling pathways regulate the cdc42-­mediated
activation of N-­WASP and the Arp2/3 complex and the polymerization
and organization of a submembranous network of actin filaments. The
cortical actin filament network may serve to strengthen the membrane
for the transmission of force generated by the contractile apparatus to
the extracellular matrix and enable the adaptation of smooth muscle
cells to mechanical stresses. The stimulus-­induced assembly of proteins
into adhesome complexes requires non-­muscle (NM) myosin II filament
assembly and activation via a RhoA-­mediated mechanism. Tension de-
velopment in smooth muscle tissues is a complex process that requires
dynamic processes of cytoskeletal assembly and reorganization in con-
junction with smooth muscle myosin II and crossbridge cycling.
Supported by NIH grants HL029289, HL109629, HL074099, the
American Heart Association and the American Lung Association.
B.4.2 | Contribution of actin polymerization to
microvascular tone production: roles of ROCK
and PKC
Ahmed El-Yazbi
American University of Beirut, Beirut, Lebanon
Force generation during arteriolar myogenic response affords a rela-
tively constant blood flow in response to changes in perfusion pressure.
This process has been extensively studied to elucidate the pathways
triggering smooth muscle contraction. For the longest time research has
implicated Ca2+-­independent mechanisms leading to smooth muscle
contractility, particularly Rho-­associated kinase (ROCK)-­and protein ki-
nase C (PKC)-­mediated mechanisms. The size of the resistance arteries
limits the capacity to examine changes in protein phosphorylation/ex-
pression levels. A highly sensitive biochemical detection approach was
beneficial in examining these changes in the context of different force
generation mechanisms along the course of myogenic constriction. We
were able to follow up on the dynamics of myosin phosphorylation
under different combinations of contractile challenges and demonstrate
that cerebral and skeletal muscle arterioles produce active constriction
not associated with increased with myosin phosphorylation. Using the
same technique, we ruled out contribution from thin-­filament regula-
tion. Active de novo actin polymerization was detected and deemed
to be the only mechanism leading to the observed constriction. This
response was dependent on ROCK-­mediated and PKC-­evoked cofilin
and HSP27 phosphorylation, respectively. Calcium-­independent PKC
isoforms were shown to mediate this mechanism. Both pathways dem-
onstrated responsiveness to integrin-­mediated mechanosensing mech-
anisms. In rat middle cerebral arteries, these processes were estimated
to contribute about 30% of the circumferential wall stress measured
at the upper end of physiological pressure observed in this vascular
bed. These observations depict a clear and independent role of actin
cytoskeleton reorganization in arteriolar force generation that warrants
further investigation in pathological conditions.
B.4.3 | Disruption of actin cytoskeleton
contributes to impairment of cerebral
autoregulation in association with diabetes-­
related cognitive deficits
Shaoxun Wang; Feng Jiao; George Booz; Richard Roman;
Fan Fan
Department of Pharmacology and Toxicology, University of Mississippi Medical
Center, Jackson, Mississippi, USA
Diabetes mellitus is a leading risk factor for cerebrovascular disease
and dementia. However, the underlying mechanisms remain to be elu-
cidated. The present study examines whether the impaired myogenic
response and cerebral autoregulation we found in a diabetic T2DN rat
is due to disruption of the actin cytoskeleton, and if this contributes to
cognitive deficits. We found that the actin cytoskeleton was reorgan-
ized in VSMCs freshly isolated from MCAs of T2DN compared with
normal SD rats, similarly, as seen in normal primary VSMCs treated
with high glucose or H2O2. Superoxide production was elevated in
VSMCs in high glucose or freshly isolated from diabetic rats. Elderly
diabetic rats exhibit impaired pressure-­induced myogenic response
of MCA. Forced dilatation occurred at pressures above 140 mmHg in
MCAs isolated from elderly T2DN rats but not in controls. Cortical
blood flow measured by laser Doppler flowmetry rose by 137% ± 15%
vs 36% ± 5% in T2DN vs SD rats when MAP was increased from 100
ABSTRACTS
to 180 mmHg. Cerebral autoregulation curve was shifted to lower
pressures in elderly T2DN rats with breakthrough at pressures above
140 mmHg. T2DN rats exhibited higher levels of IL-­1 β and IL-­2, amy-
loid β 42 (Aβ₄₂), p-­tau (S416), and marked neurodegeneration in the
hippocampus and cortex. Elderly T2DN rats showed learning and
memory disabilities. These results indicate that actin cytoskeleton is
disrupted in cerebral VSMCs of diabetic T2DN rats, possibly due to
hyperglycemia induced superoxide production, and this contributes
to impaired cerebral vascular function and cognitive deficits. This
study was supported by grants AG050049, P20GM104357, HL36279,
DK104184 and HL138685 from the National Institutes of Health; and
16GRNT31200036 from the American Heart Association.
B.4.4 | Role of the vascular cell actin
cytoskeleton in arteriolar remodeling
Luis Martinez-Lemus
Missouri University
We will review how changes in the cytoskeletal structures of cells
within the vascular wall have determinant roles in the mechanical
and functional capacities of blood vessels.
S E S S I O N C . 4 TH E CO LL ATE R A L
C I RCU L ATI O N I N I S C H E M I C D I S E A S E
CH A I R S: PRO F JA M E S FA B ER A N D D R S H AY N
PEI R CE- COT TLER
C.4.1 | Genetic polymorphisms and variation in
abundance of the collateral circulation
James Faber
Department of Cell Biology and Physiology, University of North Carolina, Chapel
Hill, NC
Native (pre-­existing) collaterals are arteriole anastomoses, that by con-
necting a small fraction of the outer most arterioles of adjacent arterial
trees, serve as protective endogenous “bypass vessels”, i.e. their extent
(number and diameter) is an important determinant of the severity of
tissue injury in the event of acute or slowly developing arterial obstruc-
tion. Collaterals are small in diameter, averaging 25 and 90 μm in healthy
mice and humans. They thus cannot be imaged directly in humans and
are difficult to study directly in animals. Importantly, collateral blood
flow in brain, heart, and lower extremities of healthy humans varies
widely for unknown reasons. This presentation reviews studies show-
ing that: 1) healthy adult mice also have wide variation in collateral blood
flow, 2) this is primarily caused by 30 fold differences in collateral num-
ber and 3 fold differences in diameter resulting from variation in genetic
background, 3) these differences are due to differences in the vigor of
|
      21 of 96
collaterogenesis, ie, the formation of the collateral vessels that occurs
late in gestation by a unique process, 4) a major cause of this variation is
naturally occurring functional polymorphisms in a novel gene, Rabep2,
critical in the collaterogenesis pathway, 5) variants in this gene link to
differences in infarct volume in patients with acute ischemic stroke, 6)
ongoing efforts to identify other major variant “collateral genes” in mice
using the Collaborative Cross and Diversity Outbred multi-­parent ge-
netic mapping populations, 7) environmental factors, although with less
impact than genetic factors, also contribute to differences in collateral
extent by causing collateral rarefaction, ie, loss/pruning of collaterals
and smaller diameters in those that remain (aging, hypertension, other
common vascular risk factors, experimental inhibition of eNOS, and
cerebral amyloid angiopathy models of Alzheimer's disease), 8) new col-
lateral vessels can be experimentally induced to form, de novo, in adult
mice by chronic exposure to low inspired oxygen—a response that re-
quires functional Rabep2, 9) rodent species that have evolved for thou-
sands of years at high altitude have exceptionally abundant collaterals
that impart protection against experimental acute ischemic stroke, my-
ocardial infarction and peripheral arterial disease, 10) speculation that
this resulted from natural selection of high-­functioning alleles of Rabep2
and other collateral genes, 11) evidence that collaterals not only lessen
ischemia in obstructive disease but may also serve a physiological func-
tion, especially when oxygen is limiting, to optimize oxygen delivery to
match tissue oxygen demand.
C.4.2 | Targeting collaterals for stroke
treatment: influence of hypertension
Marilyn Cipolla
University of Vermont
Pial collaterals are important for stroke outcome and therapy. This
talk will present current findings on the function of collaterals and
how they are altered in models of hypertension.
C.4.3 | Beta blockers attenuate dilation of
leptomeningeal collateral arteries after middle
cerebral artery occlusion and increase infarct
volume in rats
Samantha J. McClanahan; Kelly K. Ball; David S. Henry;
Nancy J. Rusch; Sung W. Rhee
Department of Pharmacology and Toxicology, University of Arkansas for Medical
Sciences, Little Rock, AR, USA
Background: Beta-­adrenergic receptor antagonists, also called beta
blockers (BBs), are a mainstay in the treatment of hypertension,
heart failure, and angina. Although BBs are reported to increase
risk of stroke, they still remain one of the most widely-­prescribed
medications. The potential molecular mechanisms underlying ad-
verse stroke outcomes are unknown, but leptomeningeal collateral
 |
22 of 96       ABSTRACTS

arteries (LCAs) are known to provide an important alternative path-
way for blood flow after ischemic stroke. We hypothesize that vaso-
dilation of LCAs after ischemic stroke is mediated by beta-­adrenergic
receptors and that BBs attenuate this protective dilation, leading to
adverse stroke outcomes.
Methods/Results: Isolated, pressurized LCAs ex vivo and LCAs
in vivo in cranial windows in Sprague-­Dawley (SD) rats exhibited
concentration-­
dependent vasodilation in response to topical ap-
plication of isoproterenol and norepinephrine. This dilation was
attenuated by beta1-­adrenergic receptor blockers CGP20712 and
metoprolol. In cranial window in vivo, LCAs acutely dilated follow-
ing the middle cerebral artery occlusion (MCAO). This dilation post-­
MCAO was attenuated in rats suffused with topical CGP20712 or
gavaged with oral metoprolol (30 mg/kg). Similarly, cerebral blood
flow approximated by laser speckle contrast imaging was reduced
in rats given topical CGP20712 or oral metoprolol. Finally, infarct
volume measured by MRI one day post-­MCAO was three-­fold larger
in rats given oral metoprolol.
Conclusions: Our findings suggest that BBs may contribute to ad-
verse stroke outcomes by disrupting beta-­
adrenergic receptor-­
mediated dilation of LCAs.
Use of animals was approved by Institutional Animal Care and Use
Committee. Supported by NIH R01-­
HL097107, P30-­
GM110702,
American Heart Association 17GRNT33670970, and Institutional
Hornick and Fund-­to-­Cure-­Stroke awards.
accumulation and endothelial dysfunction leading to impaired CCG.
Rats underwent an RI protocol which mimics stable angina and
stimulates robust CCG in normal animals, 0-­9 days of RI. RI-­induced
cardiac CYP4F (neutrophil-­specific 20-­HETE synthase) expression
and 20-­HETE levels were increased (4-­fold) in JCR vs normal rats.
20-­HETE antagonists abolished, and neutrophil depletion (block-
ing antibodies) reduced (~60%) RI-­
induced increases in CYP4F
expression and 20-­HETE production in JCR rats. Impaired CCG in
JCR rats (collateral-­dependent blood flow using microspheres) was
completely restored by 20-­HETE antagonists ((collateral-­dependent
zone)CZ/(normal zone)NZ flow ratio was 0.76 ± 0.07 in JCR + 20-­
SOLA, 0.84 ± 0.05 in JCR + 20-­
HEDGE vs 0.11 ± 0.02 in JCR vs
0.84 ± 0.03 in normal rats). In JCR rats, elevated 20-­HETE was asso-
ciated with excessive expression of endothelial adhesion molecules
(p-­
selectin and ICAM) and neutrophil infiltration. Endothelium-­
dependent vasodilation of coronary arteries, eNOS Ser1179 phos-
dependent NO.- production and endothelial
phorylation, eNOS-­
cell survival were compromised in JCR rats. These parameters of
endothelial dysfunction were completely reversed by 20-­HETE an-
tagonism, whereas neutrophil depletion resulted in partial reversal
(~70%). We conclude that increased 20-­HETE, mainly from neutro-
phils, compromises endothelial cell survival and function leading to
impaired CCG in MetS. Furthermore, 20-­HETE plays an important
role in facilitating excessive neutrophil infiltration through upregula-
tion of p-­selectin and ICAM. Whether 20-­HETE also promotes neu-
trophil survival in the ischemic zone remains to be investigated in
future studies.

C.4.4 | Neutrophils in the regulation of

arteriogenesis
S E S S I O N D. 4 M I C RO -­T O - ­M AC RO S C A LE
G. Joseph1; A. Soler1; B. Hutcheson1; I. Hunter1; T. I M AG I N G O F TH E M I C RO C I RCU L ATI O N
Lampkin1; C. Bradford2; B. Lars1; J. Falck3; S. Proctor4; M. L.
CH A I R S: D R SA R A N U N E S VA S CO N CELOS A N D
Schwartzman1; P. Rocic1
1
D R W I LL CU PPLE S
Department of Pharmacology, New York Medical College, Valhalla, NY;
2
Department of Biology, Tuskegee University, Tuskegee, AL; 3Department of
Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX;
4
Metabolic and Cardiovascular Diseases Laboratory, Alberta Institute for Human
Nutrition, University of Alberta, Edmonton, Alberta, Canada
D.4.1 | Imaging the endothelium from inside
Coronary collateral growth (CCG) stimulated by transient, repetitive intact pressurized arteries
ischemia (RI) is impaired in metabolic syndrome (MetS). Our results John McCarron
demonstrated that while neutrophil infiltration was early and tran-
Strathclyde University
sient in normal animals, in MetS animals, neutrophil infiltration in
response to transient, RI was significantly elevated and persistent.
The endothelium controls virtually every cardiovascular function.
Dysregulated (insufficient) apoptosis was the primary mechanism
We developed a miniature wide-­field, optical probe and automated
responsible for this excessive and prolonged accumulation of neu-
image-­processing routine to visualize and quantify signaling from
trophils in MetS. Neutrophil depletion significantly improved CCG in
hundreds of cells inside pressurized arteries.
MetS indicating that this excessive accumulation had functional con-
sequences. Similarly, RI induced a moderate and transient increase in
production of 20-­hydroxyeicosatetraenoic acid (20-­HETE) in normal
animals; however, in MetS animals 20-­HETE production was mark-
edly elevated and sustained. We next determined the mechanism(s)
by which neutrophil depletion promoted CCG in MetS and whether
elevated production of 20-­HETE resulted in excessive neutrophil
ABSTRACTS |
      23 of 96

D.4.2 | Spatiotemporal dynamics of mural cell D.4.4 | MR imaging of microcirculation

Ca2+ and tissue oxygenation in awake, behaving physiology
mice Hai-Ling Cheng
1 2 2,3 1
G. Peringod ; L. Yu ; K. Murari ; G. R. Gordon University of Toronto, Toronto, Canada
1
Hotchkiss Brain Institute, Department of Physiology and Pharmacology,
Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; The microcirculation is an important part of our circulatory system,
2
Electrical and Computer Engineering, Schulich School of Engineering, University
as it is a major determinant of blood pressure and is the main site for
of Calgary, Canada; 3Hotchkiss Brain Institute, University of Calgary, Canada
the transport of materials between blood and tissue. Oxygen, nu-
trients, and other essential solutes are delivered to cells, while car-
Arteriole smooth muscle cells and contractile pericytes on pre-­
bon dioxide and other waste products are removed. This exchange
capillaries (mural cells, collectively) respond to neural signals with
is regulated in part by the permeable endothelium of capillaries.
changes in Ca2+ to regulate cerebral blood flow control. However,
However, the transport function is also determined by microvas-
little is known about the spatial dynamics of mural cell Ca2+ across
cular architecture. For example, the number, size, and arrangement
the neocortex, or how faithfully tissue oxygenation tracks mural cell
of microvessels regulate how much blood flows through tissue, and
Ca2+ in vivo in conscious animals.
the tone of vessels may be adjusted by passive and active mecha-
Here, we developed a widefield imaging system that permits
nisms in response to local changes in metabolic demands.
measurement of mural cell Ca2+ and blood volume (HbT) through in-
In this presentation, I will review the capabilities of magnetic res-
tact bone across the entire top of the cerebral cortex in awake mice
onance (MR) imaging for quantifying the function and structure of
exposed to whisker stimulation or startle. We used Cre-­Lox knock-­in
the microcirculation in relation to the parameters described above.
mice (PDGFRβ-­Cre x LSL-­GCaMP6s), to examine ‘mesoscale’ mural
The technical basis behind the clinically available MR methods is
cell Ca2+ signals.
described, and examples will be given of common pathologies for
In the resting state, we observed these signals spread from
which these methods are indicated. These clinical MR methods in-
region-­to-­region across the neocortex in wave-­like patterns, similar
clude dynamic contrast-­enhanced and dynamic susceptibility con-
to previously reported neuronal Ca2+ signals. In the barrel region,
trast imaging, both of which require the administration of a contrast
whisker stimulation produced an unexpected initial increase in mural
2+ agent, and arterial spin labeling, which is a contrast-­free approach
cell Ca followed by an expected decrease. Furthermore, HbT ex-
for neuroimaging. I will also describe the latest preclinical MR meth-
hibited a fast decrease followed by a sustained increase, which was
ods that have the capability to measure microvessel architecture and
consistent with the biphasic Ca2+ signal. Interestingly, motor cortices
vasomodulation and discuss both the clinical context in which they
showed only decreased blood flow (i.e. increase in mural cell Ca2+
are relevant and the potential for clinical translation.
and decrease in HbT) to whisker stimulation. However, startle stim-
ulation elicited a large bilateral reduction in Ca2+, reflecting wide-
spread increases in blood flow.
S E S S I O N A . 5 TH E RO LE O F LY M PH ATI C
Thus, our preliminary efforts highlight the use of PDGFRβ-­
V E S S E L S I N C A N C E R— E M E RG I N G
GCaMP6s mice to study vascular network function through the
TH E R A PEU TI C O PP O RT U N ITI E S
skull in awake and active animals. This work was supported by
CH A I R : PRO F M A RCH ACH EN
the Canadian Institutes of Health Research and approved by the
University's Animal Care Committee (#AC15-­0133).

D.4.3 | Optical coherence tomography A.5.1 | A novel role for semaphorin 7A

angiography (OCT-­A) for depth resolved imaging in normal mammary and tumor associated
of microcirculation lymphangiogenesis
Marinko Sarunic Alan Elder; Beth Tamburini; Virginia Borges; Traci Lyons
University of Colorado Anschutz Medical Campus, Aurora, USA
Simon Fraser University

Postpartum mammary gland involution is a tissue remodeling

Optical Coherence Tomography Angiography (OCT-­A) will be pre-
event that occurs in all mammals after pregnancy and lactation to
sented for visualization of the retinal microcirculation. Results will
return the gland to the pre-­p regnant state. We and others have
include clinical imaging with a concentration on the retinal capillaries
shown that the tissue microenvironment created by involution is
in diabetic retinopathy.
tumor promotional. The environmental cues that promote meta-
static phenotypes in tumor cells during postpartum involution
includes activation of pro-­inflammatory programs, extracellular
 |
24 of 96       ABSTRACTS
deposition of collagen, macrophage infiltration and secretion of
immunosuppressive cytokines, and induction of lymphangiogen-
esis—all of which occur as a part of the normal involution process.
We have shown that semaphorin 7a (SEMA7A) is expressed on
mammary epithelial cells during involution. Our results presented
herein suggest that SEMA7A signaling results in macrophage ex-
pression of podoplanin (PDPN), integrin-­d ependent macrophage
motility, and macrophage adherence to lymphatic endothelial
cells. In addition, we show a significant decrease in the number
of macrophages, CD45 + F480 + MerTK+PDPN+ or “PDPN+ mac-
rophages” and lymphatic vessel density (LVD) in SEMA7A knock-
out mouse mammary glands. Additionally, we show that tumors
that develop during involution, and those that have been engi-
neered to overexpress SEMA7A, promote the presence of PDPN+
macrophages in the tumor microenvironment and increased LVD.
Finally, co-­expression of SEMA7A, PDPN, and macrophage marker
CD68 predicts for decreased distant metastasis free survival in a
cohort of over 600 cases of breast cancer. Together, our results
indicate that SEMA7A expression in the mammary gland during
postpartum involution is an important part of the tumor promo-
tional landscape and that SEMA7A may orchestrate a macrophage
mediated lymphatic remodeling, which in turn drives metastasis.   Hogan BM, Fox SB, Mueller SN, Simpson KJ, Achen MG, Stacker SA
A.5.2 | The molecular control of vascular
remodeling
Steven A. Stacker; Steve Williams; Rae Farnsworth; Michael
Halford; Michael He; Sara Lamont; Tara Kanezis; Ramin
Shayan; Sophie Pacquet-Fifeild; Natalia Davydova; Sidney
Levy; Nicole Harris; Carol Caesar; Sally Roufail; You-Fang
Zhang; Marc G. Achen
Tumour Angiogenesis and Microenvironment Program, Peter MacCallum Cancer
Centre, Melbourne, Victoria, Australia
The remodelling of blood and lymphatic vessels is a critical aspect of
human pathology and is associated with inflammation, infection and
malignancy. The discovery and characterisation of the lymphangio-
genic growth factors vascular endothelial growth factor-­C (VEGF-­C)
and VEGF-­D and of their receptor on lymphatic endothelial cells,
VEGFR-­3, has provided an understanding of the molecular mecha-
nisms controlling the growth of lymphatic vessels. Our growing un-
derstanding of the molecular mechanisms controlling the blood and
lymphatic systems has led to the isolation and characterisation of nu-
merous families of growth factors and receptors that in part control
the growth and remodelling of the vasculature (Stacker et al, 2014).
To gain a further understanding of how complex signalling net-
works in endothelial cells work together to generate the growth,
differentiation and remodelling of lymphatic vessels, we have estab-
lished approaches to identify novel, functionally important genes in
both blood and lymphatic endothelial cells and markers to distinguish
these cells. Using microarray expression analysis of purified lymphatic
endothelial cell populations from vessels within tumour-­
involved
lymph nodes and collecting lymphatic vessels carrying metastatic
tumour cells we have identified two novel pathways for modulating
tumour metastasis (Farnsworth et al, 2011; Karnezis et al, 2012).
Furthermore, we have used genome-­wide siRNA screening ap-
proaches to identify molecules which are important for the movement
and differentiation of lymphatic endothelial cells (Williams et al, 2017).
These screens are allowing us to map key signalling networks for the
control of vessel remodelling in the lymphatics. Ongoing screens will
identify functionally important molecules in the development of ther-
apeutic resistance to anti-­angiogenic drugs in cancer and eye diseases.
References: Farnsworth RH, Karnezis T, Shayan R, Matsumoto M,
Nowell CJ, Achen MG, Stacker SA (2011). Cancer Res 71: 6547-­6557
Karnezis T, Shayan R, Caesar C, Roufail S, Harris NC, Ardipradja
K, Zhang YF, Williams SP, Farnsworth RH, Chai MG, Rupasinghe
TW, Tull DL, Baldwin ME, Sloan EK, Fox SB, Achen MG, Stacker SA
(2012). Cancer Cell 21: 181-­195
Stacker SA, Williams SP, Karnezis T, Shayan R, Fox SB, Achen MG
(2014). Nat Rev Cancer 14: 159-­172
Williams SP, Odell AF, Karnezis T, Farnsworth RH, Gould CM, Li
J, Paquet-­Fifield S, Harris NC, Walter A, Gregory JL, Lamont SF, Liu
R, Takano EA, Nowell CJ, Bower NI, Resnick D, Smyth GK, Coultas L,
(2017) Science Signaling 10
A.5.3 | Assessing angiogenic response of 3D
blood vessel mimics to tumour spheroids in a
microfluidic co-­culture model
Noosheen Walji1,2; Edmond Young1,2
1
Mechanical and Industrial Engineering, University of Toronto, Canada; 2Institute
of Biomaterials and Biomedical Engineering, University of Toronto, Canada
Angiogenesis occurs when a tumour reaches a critical size, result-
ing in its vascularization and subsequent growth. In vitro tumour-­
vasculature platforms have been explored in literature but are
often limited in physiological relevance and spatiotemporal control.
Microfluidic models have demonstrated high physiological relevance
by recapitulating 3D structures while allowing for characterization
and manipulation of the surrounding microenvironment. This work
aims to investigate the interactions between tumour spheroids and
endothelial lumens using a microfluidic model optimized for 3D
tumour-­vasculature co-­culture to observe chemical and physical re-
sponses across multiple time points.
In this work, lung cancer (A549) and breast cancer (MCF-­7 ) spher-
oids of varying angiogenic potential are co-­cultured with human um-
bilical vein endothelial cell (HUVEC) lumens. Angiogenic potential
is characterized by areas of hypoxia, necrosis, and proliferation, as
well as production of key signalling molecules. Angiogenic response
is measured in terms of endothelial migration distance as well as
sprout quantity, location, and morphology. Preliminary results show
that spheroids with higher angiogenic potential had a detrimental
ABSTRACTS
effect on nearby endothelial cells. In contrast, spheroids with low
angiogenic potential induced sprouting in the adjacent lumen.
The observations made in this research will shed light on inter-
actions between tumour growth and vasculature, enabling observa-
tions of the biological programming of tumour behaviour at a cellular
level that have been previously inaccessible.
This work is supported by the Ontario Ministry of Research,
Innovation and Science Early Researcher Award and an NSERC
Discovery Grant to EY, and the Queen Elizabeth II Graduate
Scholarship in Science and Technology to NW.
A.5.4 | Role of lymphatic vessels in
immunosuppression in cancer
Melody Swartz
University of Chicago
No abstract or description provided.
S E S S I O N B . 5 E X E RC I S E A N D S H E A R
S TR E S S : LI N K I N G TH E B E N E FIT S TO TH E
M AC RO -­ A N D M I C ROVA S CU L AT U R E
CH A I R : PRO F K A R EN B I RCH
B.5.1 | Exercise training: vascular adaptations
in function-­structure and the role of shear
Dick Thijssen
Radboud University, Nijmegen, The Netherlands
This lecture discusses the distinct hemodynamic signals during exer-
cise, the inter-­relationships between these signals, the nature of the
adaptive responses under different physiological conditions and the
implications for human health.
B.5.2 | Hemodynamic regulation of blood-­brain
barrier integrity
Peter Galie
Rowan University
Previous studies have demonstrated that exercise increases blood
flow in the brain but interrogating the effect of cerebral blood flow
on vascular integrity is difficult in the complex in vivo microenvi-
ronment. Here, using a three-­dimensional model of the blood-­brain
barrier, we demonstrate that the augmented shear stress exerted
by elevated cerebral blood flow affects the integrity of the barrier.
The model mimics several aspects of the in vivo microenvironment,
|
      25 of 96
including the presence of tight junctions between endothelial cells,
a robust basement membrane, and interaction between the en-
dothelial cells and surrounding astrocytic endfeet. Microparticle
image velocimetry provides insight into the flow regimes present
within the three-­dimensional vessels and demonstrates that both
fluid shear stress and vascular strain can be controlled using this
system. Permeability testing suggests that shear stress strength-
ens the barrier function of the endothelial monolayer compared to
static controls. Immunofluorescence indicates localization of ZO-­1,
a tight junction scaffolding protein, to the cell-­cell junctions in the
presence of shear stress. Additionally, the use of CRISPR/Cas9 to
knockout CD44, a cell surface receptor for hyaluronan, implicates
its role in the mechanotransduction of shear stress in cerebral en-
dothelial cells. Taken together, these results provide insight into the
beneficial effects of exercise on blood-­brain barrier integrity.
B.5.3 | Endothelial Hba1 is a regulator of
exercise fitness in mice
Alexander S. Keller1,2; Steven Brooks3; Aditi Islam1; T. C.
Stevenson Keller IV1,4; Angela K. Best1; Sara Murphy1;
Henry Askew Page1; Miriam M. Cortese-Krott5; Zhen Yan1,4;
Hans Ackerman3; Brant E. Isakson1,4
1
Robert M. Berne Cardiovascular Research Center, University of Virginia School
of Medicine; 2Department of Pharmacology, University of Virginia School of
Medicine; 3Laboratory of Malaria and Vector Research, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland, USA; 4Department of Molecular Physiology and Biophysics, University
of Virginia School of Medicine; 5Cardiovascular Research Laboratory, Division of
Cardiology, Pneumology, and Vascular Medicine, Medical Faculty, Heinrich Heine
University of Düsseldorf, Düsseldorf, Germany
Hemoglobin alpha (Hbα), encoded by the redundant genes Hba1 and
Hba2, is uniquely expressed in the endothelial cells (ECs) of resistance
arteries. We have previously shown that endothelial Hbα is an impor-
tant regulator of nitric oxide (NO) signaling in the vascular wall via its
functional coupling with endothelial nitric oxide synthase (eNOS) and
hypothesized that endothelial Hbα may function as a nitrite reductase
in hypoxic conditions. To study this question, we flanked exons 2 and
3 of the Hba1 gene with loxP sites (Hba1 fl/fl) and bred this Hba1 fl/fl
mice with Cdh5-­Cre/ERT2 + mice to produce a tamoxifen-­inducible,
endothelial cell-­specific genetic model of Hba1 deletion (EC Hba1 fl/
fl). In another novel mouse model, we used CRISPR-­Cas9 technology
to delete the nucleotides encoding four amino acids of the putative
eNOS binding region from the Hba1 gene (Hba1Δ36-­39/WT). We
compared both models against Hba1−/− (global Hba1 deletion) mice
and found that both Hba1−/− and EC Hba1 fl/fl, but not Hba1Δ36-­39/
WT mice, showed reduced Hbα expression in the endothelium of re-
sistance arteries. However, whereas Hba1−/− mice displayed reduc-
tions in hemoglobin concentration, hematocrit, red cell volume, and
other blood parameters, EC Hba1 fl/fl and Hba1Δ36-­39/WT blood
parameters were normal. We then used a treadmill running test to
measure the effect of each genetic model on acute endurance ca-
pacity. Hba1−/− and EC Hba1 fl/fl, but not Hba1Δ36-­39/WT mice,
 |
26 of 96       ABSTRACTS
displayed significant reductions in exercise capacity versus littermate
controls. We conclude that endothelial Hba1 is an important regulator
of vascular function and skeletal muscle performance, independent of
the interaction between Hbα and eNOS.
B.5.4 | Exercise, shear, and endothelial
progenitor cells
Mark Ross1; Eva Malone1; Richard Simpson2; Geraint
Florida-James1
1
School of Applied Sciences, Edinburgh Napier University, Edinburgh, UK;
2
Department of Nutritional Sciences, Department of Pediatrics, Department
Immunobiology, University of Arizona, Tucson, USA
Endothelial progenitor cells (EPCs) were discovered in 1997 (Asahara
et al., 1997), and have been a focus of research scientists interested in
the regeneration and growth of vascular beds, in health and disease.
Individuals with low circulating number and function of EPCs are at
considerably higher cardiovascular disease risk than those with higher
levels. However, exercise may promote cardiovascular health via the
potential to modulate EPC biology. We have conducted a number of
studies showing that acute bouts of aerobic (Ross et al., 2017) exercise
and resistance exercise (Ross et al., 2014) have the ability to acutely
increase circulating number and chemokine receptor expression of
these cells. However, we have also shown that the deleterious effect
of advancing age on these cells is not counteracted by maintaining
high levels of cardiorespiratory fitness (Ross et al., 2017). Despite this,
a plethora of studies have shown significant effect of acute bouts (Van
Craenenbroeck et al., 2008; Harris et al., 2017), and training interven-
tions on these cells (Xia et al., 2012a), which may be due to regular
exercise-­induced elevations in shear (Xia et al., 2012b). Therefore, ex-
ercise, acutely and chronically, may promote EPC number and function
which may, in turn, promote vascular growth and repair.
S E S S I O N C . 5 M I C ROVA S CU L A R
R E M O D E LI N G — PE R I C Y TE S H AV E G OT IT
W R A PPE D U P!
CH A I R : PRO F S T UA RT EG G I NTO N
C.5.1 | Pericytes are a key player in skeletal
muscle remodelling
Birgitte Høier
University of Copenhagen
Skeletal muscle tissue derived pericytes contain, absorb and subse-
quently release VEGF when conditioned with skeletal muscle me-
dium. Thus, pericytes may be central in relaying cell-­cell cross talk
in angiogenesis.
C.5.2 | Discovering pericyte dynamics during
angiogenesis
Walter Murfee
Tulane University
This presentation will highlight discoveries that link pericyte dy-
namics during angiogenesis to stem cell therapies, novel modes of
growth, and lymphangiogenesis. The results motivate questions re-
garding pericyte identity and function.
C.5.3 | Nutrient overload stimulates leptin
production by skeletal muscle pericytes by
provoking commitment to an adipogenic lineage
Emmanuel Nwadozi1; Andrew Ng1; Anna Strömberg2; Karl
Olsson2; Thomas Gustafsson2; Tara Haas1
1
School of Kinesiology and Health Science, York University, Toronto, Canada;
2
Department of Clinical Physiology, Karolinska Institutet, Stockholm, Sweden
Previously, we showed that leptin signaling regulates physiologi-
cal skeletal muscle angiogenesis. Ectopic production of the adi-
pokine leptin in nutrient-­overloaded skeletal muscle was reported
~20 years ago. However, the cellular source and regulation of
leptin production in skeletal muscle remains unknown. A sub-­
population of pericytes demonstrates multipotent capacity to dif-
ferentiate into various mesodermal lineages including adipocytes.
Thus, we hypothesized that pericytes are a source of leptin in skel-
etal muscle, and leptin production occurs in proportion to adipo-
genic potential. Immunohistological analyses of murine and human
skeletal muscle was utilized to define the cellular localization of
leptin. Leptin immunoreactivity predominantly localized around
capillaries but was neither present on myocytes nor associated
with BODIPY-­
p ositive staining (demarcating lipid-­
laden cells).
Consistently, leptin mRNA was expressed in isolated capillaries but
not cultured myocytes or endothelial cells from skeletal muscle.
Pericytes were identified by platelet derived growth factor recep-
tor beta (PDGFRβ) immunoreactivity, and significantly overlapped
with leptin staining. Leptin transcripts were detected in isolated
PDGFRβ+ cells. Notably, PDGFRβ+ cells also expressed transcripts
of the adipogenic precursor marker Pdgfra. Nutrient-­overload in-
duced by 2 weeks of high fat (HF) diet significantly increased the
Leptin mRNA in PDGFRβ+ cells, coinciding with increased levels
of the adipogenic commitment factor Zfp423. However, mRNA
of adipogenic regulators Cebpa and Pparg were unaltered by HF
feeding. These data suggest that HF feeding stimulates leptin
production in pericytes by provoking adipogenic commitment,
providing mechanistic insight into the regulation of the skeletal
muscle capillary network in response to nutrient-­overload. Ethics
approved for all studies. Funded by NSERC.
ABSTRACTS
C.5.4 | Pericyte therapy of ischaemia: the
mechanistic pathway toward clinical translation
Paolo Madeddu
University of Bristol
Pericytes are attracting much interest for the scope of cardio-
vascular regenerative medicine. In the last 5 years we have been
engaged in a translational research to generate a clinical grade
product made by pericytes. These mural cells are isolated and
expanded from a small piece of vascular tissue obtained as the
leftover of coronary artery bypass surgery. We set up a standard
operating procedure that reliably allows us to obtain high purity
pericytes based on immunosorting for the surface marker CD34.
The pericytes are then expanded in adhesion on fibronectin-­
coated dishes to reach a yield of million cells. Similarly, we expand
pericytes from single cell sorted preparations, which confirms
clonogeneity. The cell population showed consistent purity and
was functionally tested in vitro before performing a series of pre-
clinical studies of efficacy in mouse models of limb ischemia and
myocardial infarction. In addition, we compared efficacy of peri-
cytes and mesenchymal stromal cells, with results showing better
outcomes and a safer profile of pericytes. The combination of cKit
stromal cells was additive in some cardiac outcomes but the cells
showed contrasting paracrine activities too. We next performed a
blind, controlled trial in pigs with myocardial ischemia-­reperfusion.
Results confirmed improvement in angiogenesis and fibrosis but
did not show any benefit on contractility. We are now considering
to upgrade the dosage of pericytes in a comparative study with
other regenerative cell preparations in immunosuppressed pigs
with myocardial infarction.
S E S S I O N D. 5 OX YG E N O N D E M A N D :
I N EQ UA LIT Y A N D CO N S EQ U E N C E S
CH A I R S: PRO F S COT T E A R LE Y A N D D R
FA B R I CE DA B ERTR A N D
D.5.1 | Microscopic foundation of multimodal
human imaging
Anna Devor
University of California San Diego
Today, most major programs in systems Neuroscience and Psychology
have their own functional imaging systems and laboratories. We can
assess hemodynamic changes with functional Magnetic Resonance
Imaging (fMRI) and functional Near-­Infrared Spectroscopy (fNIRS),
broad regional electrical activity with magneto/electroencephalog-
raphy (MEG/EEG), and metabolism/neurochemistry with Positron
Emission Tomography (PET). And yet, despite this widespread
|
      27 of 96
adoption, the power of available human neuroimaging methods re-
mains limited, leaving a gap between the macroscopic activity pat-
terns available in humans and the rich, detailed view achievable in
model organisms (1). Thus, a central challenge facing neuroscience
today is leveraging these mechanistic insights from animal studies
to accurately draw physiological inferences from non-­invasive sig-
nals in humans, essentially asking the fundamental question: what
information about local neuronal circuit activity and the state of
neuromodulation can we reliably determine from non-­invasive func-
tional imaging in humans? On the essential path towards this goal
is the development of a detailed “bottom-­up” forward model bridg-
ing neuronal activity at the level of cell-­t ype-­specific populations to
non-­invasive imaging signals (2, 3). The general idea is that specific
neuronal cell types have identifiable signatures in the way they drive
changes in cerebral blood flow, cerebral metabolic rate of O2 (meas-
urable with quantitative fMRI), and electrical currents/potentials
(measurable with magneto/electroencephalography, MEG/EEG) (4).
This forward model would then provide the “ground truth” for the
development of new tools for tackling the inverse problem—estima-
tion of neuronal activity from multimodal non-­invasive imaging data.
References: 1. Devor A, et al. (2013). Neuron 80(2):270-­274.
2. Uhlirova H, et al. (2016) Philos Trans R Soc Lond B Biol Sci
371(1705).
3. Gagnon L, et al. (2015) J Neurosci 35(8):3663-­3675.
4. Uhlirova H, et al. (2016) Elife 5.
D.5.2 | Two-­photon microscopy imaging
of capillary red blood cell flux in mouse
brain reveals vulnerability of white matter to
hypoperfusion
Baoqiang Li1; Ryo Ohtomo2; Martin Thunemann3; Jing
Yang1; Buyin Fu1; Chongzhao Ran1; Jonathan R. Polimeni1;
David A. Boas1,4; Anna Devor1,3,5; Eng H. Lo2; Ken Arai2;
Sava Sakadzic1
1
Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General
Hospital, Harvard Medical School, Charlestown, MA, USA; 2Departments of
Radiology and Neurology, Massachusetts General Hospital, Harvard Medical
School, Charlestown, MA, USA; 3Department of Radiology, University of
California San Diego, La Jolla, CA, USA; 4Department of Biomedical Engineering,
Boston University, Boston, MA, USA; 5Department of Neurosciences, University
of California San Diego, La Jolla, CA, USA
We employed two-­photon microscopy (TPM) to measure capillary
red blood cell (RBC) flux in both gray and white matter, in isoflurane-­
anesthetized C57BL/6 mice, through a sealed cranial window. The
deep capillary RBC flux measurements were enabled by a red-­
shifted fluorophore Alexa 680 and photon-­counting detection. All
experiments were approved by the MGH IACUC.
Our results showed that in control animals, the mean capillary
RBC flux at baseline in the cerebral white matter (67 ± 6 RBC/s)
was significantly higher than that in the gray matter (48 ± 4 RBC/s).
However, during cerebral hypoperfusion induced by bilateral
 |
28 of 96       ABSTRACTS
common carotid artery stenosis, the white matter capillary RBC
flux was significantly reduced (43 ± 8 RBC/s) relative to baseline,
while the gray matter capillary RBC flux (45 ± 8 RBC/s) remained
comparable with the control values. The opposite trends of RBC
flux changes in gray and white matter were observed in response
to mild hypercapnia.
We demonstrated that the white matter capillary RBC flux could
be measured using standard TPM available to many investigators in
this research field. Our results show significant differences between
the white matter and gray matter RBC flux, both at baseline, and in
response to hypoperfusion and mild hypercapnia. We hypothesize
that blood flow regulation in the upstream-­located gray matter in re-
sponse to global blood flow perturbation may cause disproportional
downstream pressure drops in arterioles supplying the white matter.
This could make the white matter significantly more susceptible to
hypoperfusion than the gray matter, facilitating the white matter
deterioration in conditions involving global cerebral hypoperfusion.
D.5.3 | In vivo optogenetic control of brain
mural cells
Andy Shih
Center for Developmental Biology and Regenerative Medicine, Seattle Children's
Research Institute, Seattle, Washington
Cerebral blood flow regulation is critical for the maintenance of brain
energetics. More than 90% of the brain's microvasculature is covered
by capillary pericytes, a mural cell type with uncertain contribution
to blood flow regulation. Using in vivo two-­photon imaging in mouse
cerebral cortex, we show that selective optogenetic stimulation of
pericytes throughout the capillary bed leads to reduction in capillary
diameter. Pericyte contractility is slower and smaller in magnitude
than upstream smooth muscle cells, yet sufficient to impede blood
cell flow. This constriction can be mitigated by the clinically-­used
vasodilant, Fasudil. Conversely, selective optical ablation of capillary
pericytes results in prolonged local dilation and increased blood flow
in a subset of capillaries with smaller basal diameter. Capillary peri-
cytes are thus sufficient and necessary to alter capillary diameter in
vivo, and their slow dynamics suggest a role in setting constant re-
sistance and enduring blood flow patterns in the brain's capillary bed.
D.5.4 | Contractile pericytes determine the
direction of blood flow at capillary junctions
Albert Gonzales
University of Vermont
The current work tests the hypothesis that contractile pericytes,
localized at capillary junctions proximal to the feeding arteriole, dy-
namically regulate the distribution of RBCs within in the capillary
network.
S E S S I O N A . 6 M O LEC U L A R M EC H A N I S M S
R EG U L ATI N G LY M PH ATI C FU N C TI O N
CH A I R : PRO F PI ER R E-Y V E S VO N D ER W EI D
A.6.1 | Molecular and ionic mechanisms
involved in the propulsion of lymph
Jorge Castorena-Gonzalez; Michael Davis; Min Li; Scott
Zawieja
Department of Medical Pharmacology and Physiology, University of Missouri,
Columbia, MO, USA
The spontaneous contractions of collecting lymphatics are impor-
tant for the propulsion of lymph. These contractions are driven by
action potentials (APs) in lymphatic smooth muscle cells (LMCs) and
their frequency is regulated by intraluminal pressure. The prevail-
ing dogma, based on studies of unstretched vessel strips, main-
tains that APs are initiated from the summation of small-­amplitude
(1-­2 mV) transient depolarizations (STDs) that summate to bring the
LMC membrane potential from a stable baseline to the AP thresh-
old. We studied the ionic basis of lymphatic pacemaking in pressur-
ized collectors from rat, human and mouse, focusing on the latter
species for mechanistic insights into the role of putative mecha-
nosensitive mechanisms. In each species we recorded nearly-­
linear, diastolic depolarizations leading to AP firing in the absence
of STDs. Smooth muscle (SM)-­specific deletion of the mechano-
sensitive cation channel Piezo1, or global deletion of the second-­
messenger regulated cation channel TRPC6, failed to significantly
impair pressure-­induced changes in frequency. Pressure-­regulated
pacemaking was also normal in TRPC3−/− and TRPV4−/− vessels.
Selective TRPM4 inhibitors had no significant effect on pacemak-
ing. Genetic deletion of T-­t ype VGCCs (Cav3.1, Cav3.2, or both),
previously suggested to regulate pacemaking, did not significantly
alter pressure-­induced changes in frequency. However, SM-­specific
deletion of the calcium-­activated Cl-­channel Ano1 (TMEM16a)
reduced basal contraction frequency by 2-­3 fold and abrogated
pressure-­induced chronotropy. SM-­specific deletion of the L-­t ype
VGCC, Cav1.2, abolished all spontaneous contractions. The collec-
tive evidence suggests that Ano1 contributes a depolarizing cur-
rent to whatever combination of unidentified ionic currents drives
diastolic depolarization to trigger the Cav1.2-­mediated AP.
A.6.2 | Novel regulatory mechanisms of
lymphatic muscle contraction
Mariappan Muthuchamy
Texas A&M University
Lymphatic muscle is uniquely composed of both smooth and stri-
ated muscle components and studies elucidating their contractile
ABSTRACTS
behavior have unequivocally established that this is a new class of
muscle that is distinct from smooth and cardiac/skeletal muscle
types. Lymphatic muscle exhibits rapid, phasic contractile activ-
ity that drives the intrinsic lymphatic pumping in addition to the
slower, tonic form of contractions. We have previously reported
that inhibition of myosin light chain kinase did not alter phasic
contraction amplitude of mesenteric lymphatics, but decreased
tone. Additionally, activation of protein kinase C signaling induced
strong contractions and increased the calcium sensitivity of the
contractile apparatus in lymphatic muscle. Cardiac troponin C
(cTnC), the Ca2+-­binding subunit of Tn complex, which is present
in cardiac muscle thin filament, is found in the lymphatic muscle.
A Ca2+ sensitizing drug, bepridil that acts through cTnC increases
force development in lymphatic myofilaments. Furthermore,
caldesmon targeted peptide, IK29C increases force generation
of lymphatic myofilaments. Our data suggest that cTnC that is
present in lymphatic muscle may be responsible for the regula-
tion of the phasic component of the lymphatic contractions. We
propose that caldesmon may play an essential role along with the
cardiac thin filament regulatory protein (cTnC) in modulating the
Ca2+ sensitivity and cooperativity of lymphatic myofilament and
allow the lymphatic vessels to undergo the fast/phasic contractile
cycle. Additionally, our data show that the inflammatory signaling
including immune cells and specific miRNAs modulate lymphatic
muscle contractile activity under different disease conditions,
such as metabolic syndrome.
A.6.3 | Role of microvascular lymphatic
clearance in Alzheimer's disease
Liudmila Romanova; Christopher Janson
Department of Neurology and Rehabilitation, College of Medicine, University of
Illinois at Chicago
The meningeal lymphatic system is a recently discovered intrac-
ranial circulatory system which participates in clearance of fluid
and solutes from the brain. While it is already established that
the cerebral microcirculation at the blood-­b rain barrier is closely
involved with cerebral amyloidosis in aging and Alzheimer's, very
little is known about the contributions of this parallel lymphatic
system to clearance or accumulation of brain amyloid. In normal
aging, one of the key amyloid peptides which is cleared from the
brain is Aβ. In Alzheimer's disease, however, the physiological
clearance system is impaired, which leads to buildup of toxic Aβ
peptide in microvessels and throughout the brain. Thus far, no
studies have been done to quantify Aβ accumulation in meningeal
lymphatic vessels in the setting of Alzheimer's disease, or to com-
pare pathology observed in these vessels alongside the progres-
sion of Alzheimer's brain pathology. We have used an Alzheimer's
transgenic rat to explore the contributions of the meningeal lym-
phatic vasculature to clearance of Aβ, considered the earliest bio-
marker for Alzheimer's disease. This work demonstrates that the
|
      29 of 96
meningeal lymphatic system is an early warning system for ini-
tiation and progression of Alzheimer's, and may be a therapeutic
target for preventing or reversing the disease pathology. In other
words, a healthy meningeal lymphatic microvasculature may be a
prerequisite for healthy aging.
A.6.5 | Calcium release-­activated calcium
(CRAC) channels are responsible for the shear
stress-­induced intracellular calcium mobilization
and downstream signalings in HDLECs
Hongjiang Si; Kate Kearney; Meng-Hua Zhou; Xueyang
Zhang; Xu Peng; David Zawieja; Shenyuan Zhang
Department of Medical Physiology Texas A&M, Temple, USA
The lymphatics transport body fluid and immune cells in most or-
gans and the lymphatic endothelial cells (LECs) are responsible for
the dynamic modulation of lymphatic functions in response to the
lymph flow in physiological and pathological conditions. We previ-
ously reported the intracellular calcium dynamics under different
fluid shear stress (FSS) levels in LECs and found the involvement of
both intracellular calcium store and extracellular calcium sources
in this process. However, the channel molecules mediating these
calcium responses remain elusive. In this present study, we de-
termined that calcium release-­activated calcium (CRAC) channels,
formed by Orai1 and STIM1 proteins, were responsible for mediat-
ing FSS-­evoked calcium entries in human dermal LECs (HDLECs).
Pharmacological blockade of CRAC channels and various molecu-
lar suppressions targeting STIM1 or Orai1 all abolished the FSS-­
induced calcium entry as well as the calcium-­dependent nuclear
translocation of nuclear factors of activated T-­cells (NFAT) for the
regulation of the downstream gene expression. Our pilot data also
suggested that Piezo1 or Piezo2 was not the major mechanosensory
receptor in our model to relay the flow stimulation to calcium dy-
namics. Our results demonstrated that CRAC channels served as a
dominant signal transducer in the LECs to convert physical stimula-
tions (shear stress) to intracellular electrophysiological signals and
paved the road for the future investigations of FSS sensing mecha-
nisms in the lymphatics. In conclusion, our research shed light into
the better understanding of FSS-­induced regulations of lymphatic
functions (e.g., modified fluid homeostasis and immune surveil-
lance) under healthy or disease conditions.
 |
30 of 96       ABSTRACTS
SESSION B.6 ANGIOGENESIS AND
R E M O D E LI N G : E M E RG I N G TO PI C S
CH A I R S: D R PH O EB E S TA PLE TO N A N D D R
J OS H UA B U TCH ER
B.6.1 | Directing angiogenesis and vessel
function with mechanical cues
Jonathan W. Song
Department of Mechanical and Aerospace Engineering, The Ohio State
University, Columbus, OH; Comprehensive Cancer Center, Columbus, Ohio
Recent advancements in microfabrication and biological integra-
tion have helped propel the design and implementation of micro-
fluidic systems as highly modular in vitro cell culture model systems.
Moreover, increased sophistication and characterization of microflu-
idic systems that are intelligently applied to the most pressing ques-
tions in pathophysiology will continue to support their adoption to
help bridge basic science with translational research for advancing
drug discovery and personalized medicine. Here, I will discuss the
application of microfluidic systems for understanding the physical
dynamics of vascular and tumor biology. Specifically, the talk will
focus on understanding how: i) local flow dynamics controlling ves-
sel permeability and angiogenesis and ii) genetic lesions to stromal
fibroblasts reprogramming the physical characteristics of the tumor
microenvironment. Further advancement of microfluidic systems in
recapitulating tissue‐level phenomena in vitro, controlling important
physicochemical and biological parameters, and integrating cellular
and molecular analysis will help further enhance their application
within the microcirculation research community.
B.6.2 | Vascular NOX1 as a target for
preventing obesity-­derived hypertension and
renal injury
1 1 1,2
Joshua T. Butcher ; James Mintz ; David J. Fulton ; David
W. Stepp1,3
1
Vascular Biology Center, Medical College of Georgia/Augusta University,
Augusta, GA, USA; 2Department of Pharmacology, Medical College of Georgia/
Augusta University, Augusta, GA, USA; 3Department of Physiology, Medical
College of Georgia/Augusta University, Augusta, GA, USA
Obesity compromises cardiovascular function; being associated
with vascular dysfunction, hypertension and chronic kidney disease.
In humans, exercise or pharmaceutical inhibition of the NOX family
blunts increases in the NOX enzymes and restores obesity-­induced
vascular dysfunction. Our lab identified NOX1 as a specific regu-
lator of endothelial function in obesity. It is upregulated in resist-
ance arterioles of obese mice and blunts NO-­mediated vasodilation.
We have shown that myostatin deletion (used to mimic exercise),
pharmaceutical inhibition, or germline deletion of NOX1 improves
obesity-­induced endothelial dysfunction. However, research has not
translated into functional cardiovascular outcomes. The objective of
this study was to determine the effect of NOX1 on blood pressure
regulation and renal injury in the db/db mouse, in combination with
myostatin or NOX1 deletion.
This study found that inhibition of NOX1 prevents hypertension
in the db/db mouse. Blood pressure was significantly elevated with
obesity compared to lean control (117.5 ± 1.2 vs 107.8 ± 1.5 mmHg)
and significantly improved with myostatin deletion (108.4 ± 1.8) or
NOX1 deletion (106.8 ± 2.0). Further, the significant polydipsia and
polyuria that accompanies obesity is improved with deletion of myo-
statin or NOX1. Renal injury and urinary oxidant load was increased
with obesity and significantly decreased with myostatin deletion.
Important confounding variables were found to be unchanged
(weight, fat profiles, activity, heart rate), thus the improved cardio-
vascular outcomes occur despite full presentation of the classic db/
db phenotype. To conclude, these results suggest that NOX1 inhi-
bition could serve as an effective target for prevention/reversal of
obesity-­derived changes to renal and cardiovascular function.
Support: Stepp; NHLBI:RO1HL124773. Butcher;
AHA:17POST33410322.
B.6.3 | Angiogenesis in ischemic muscle and
tumors: novel insights revealed by intravital
microscopy
John-Michael Arpino; J. Geoffrey Pickering
Robarts Research Institute, Departments of Medicine, Biochemistry, and Medical
Biophysics, Schulich School of Medicine and Dentistry, Western University and
London Health Sciences Centre, London, ON, Canada
Rationale: Angiogenesis is fundamental to tissues rendered ischemic
from occlusive vascular disease. However, strategies to stimulate an-
giogenesis in adult tissues, including skeletal muscle, have failed as a
therapeutic approach. Importantly, to appropriately deliver oxygen,
a precisely organized and responsive microcirculation must form.
However, current understanding of the structure and function of
new microvascular networks in adult tissues, and methodologies for
delineating network function, are limited.
Approach and Results: To elucidate the performance attributes of a
regenerated microvascular network, we established a model of com-
plete microcirculatory regeneration after ischemia-­induced oblitera-
tion in the mouse hindlimb extensor digitorum longus (EDL) muscle.
The structure and function of the regenerated microvasculature was
interrogated using real-­time imaging of RBC transit. This revealed
that, despite extensive regeneration of flowing neo-­microvessels,
there were profound flaws in network geometry, microcircula-
tory hierarchy, RBC transit, flow control by hypoxia, and smooth
muscle cell wrapping around arterioles. Furthermore, mapping all
skeletal muscles in the ischemic hindlimb revealed unanticipated
heterogeneity in skeletal muscle and microvascular regeneration,
ABSTRACTS
with neo-­vessels forming exclusively in zones of muscle infarction
and regeneration.
Conclusions: Despite extensive microcirculatory regeneration in in-
farcted and regenerated skeletal muscle, the neo-­microvasculature
fails to recapitulate the anatomy and physiology of a normal micro-
vasculature. These findings have relevance to strategies for restor-
ing oxygen content in pathological tissues.
Sources of Funding: CIHR, Heart and Stroke Foundation of Canada.
B.6.4 | Pericyte migration and investment
during developmental blood vessel remodeling
John Chappell
Virginia Tech Carilion Research Institute, Roanoke, Virginia, USA
Pericytes synchronize their activity with endothelial cells to build
and remodel their local microenvironment during angiogenic
sprouting. Endothelial cells are sensitive to disruptions in vascular
endothelial growth factor-­A (VEGF-­A ) signaling, while pericytes
depend on platelet-­d erived growth factor-­B (PDGF-­B) secreted
from the endothelium. Recently, we found that loss of VEGF-­A
regulation via genetic deletion of the decoy VEGF-­
A receptor
Flt-­1/VEGFR1 disrupts pericyte coverage along developing ves-
sels. Pericytes did not appear to express any VEGF-­A receptors
including Flt-­1 or Flk-­1. Furthermore, decreased pericyte coverage
occurred despite an increase in PDGF-­B production from endothe-
lial cells. Dynamic imaging of pericytes migrating along endothelial
cells during a VEGF-­A gain-­of-­function scenario revealed a sig-
nificant decrease in pericyte migration persistence. Under normal
conditions, pericyte migration was entrained with endothelial cell
migration, while pericyte proliferation correlated with endothelial
sprout elongation. Interestingly, as endothelial sprouts extended
and retracted, pericyte movement closely matched endothelial
cell migration dynamics. These observations suggested the po-
tential for dynamic secretion and remodeling of a provisional ex-
tracellular matrix (ECM), produced by endothelial cells to support
pericyte migration during angiogenic sprouting. Taken together,
these data support a model in which sprouting endothelial cells
shape their local microenvironment via ECM synthesis and growth
factor deposition to facilitate pericyte migration. Pericytes syn-
chronize their migration with endothelial cells, while simultane-
ously secreting ECM components that contribute to the vascular
basement membrane and promote vessel stability.
|
      31 of 96
SESSION C.6 EBB & FLOW OF BRAIN CAPILLARIES
CHAIRS: PROF ANDY SHIH AND MR IAIN LAMB
C.6.1 | Perfusion characteristics of the capillary
bed—homogeneities, heterogeneities and
erythrocyte trajectories
Matthew Barrett1,2; Patrick Jenny3; Dominik Obrist4; Franca
Schmid1,3; Bruno Weber1,2
1
Institute of Pharmacology and Toxicology, University of Zurich, Zurich,
Switzerland; 2Neuroscience Center Zurich, University and ETH Zurich, Zurich,
Switzerland; 3Institute of Fluid Dynamics, ETH Zurich, Zurich, Switzerland;
4
ARTORG Center for Biomedical Engineering Research, University of Bern, Bern,
Switzerland
Microvascular perfusion is crucial for nutrient and oxygen sup-
ply in all tissues. Nonetheless, topological details and perfusion
characteristics for many tissues are largely unknown. We use nu-
merical simulations1,2 in realistic microvascular networks 3 (MVN)
in combination with in vivo measurements to study flow, topology
and the impact of red blood cells (RBCs) in the vasculature of the
mouse brain.
The RBC velocity and hematocrit distributions reveal that the
perfusion of the capillary bed is highly heterogeneous. However, at
individual divergent bifurcations RBC velocities differ by only 27%.
We conclude that velocities are balanced locally while on the net-
work scale the velocity heterogeneity remains. Both characteris-
tics, the hematocrit heterogeneity and the local velocity balancing,
prove to be a direct consequence of the bi-­phasic nature of blood
(plasma and RBCs). Further analysis on the velocity balancing sug-
gests that well-­
balanced bifurcations (velocity difference < 20%)
promote robustness of perfusion and might be key for local regula-
tion mechanisms.
Furthermore, we are interested in depth-­
dependent differ-
ences1. The trajectories of individual RBCs show that RBCs tend to
enter and exit the capillary bed at the same cortical depth. This is
surprising because commonly the capillary bed is described as a ho-
mogeneous 3D network3,4 where a preferential flow direction would
not be expected. Further differences with respect to depth are: a
decrease in RBC velocity with depth and a shift of the location of
the largest pressure drop from capillaries to descending arterioles.
These aspects are relevant for the vascular supply capability and for
the up-­regulation of flow.
References: 1. Schmid et al., PLOS Computational Biology, 2017.
2. Schmid et al., AJP -­Heart and Circ, 2015. 3. Blinder et al., Nature
Neuroscience, 2013. 4. Schmid et al., Neuroimage, 2017. 5. Schmid
et al., submitted, 2018.
 |
32 of 96       ABSTRACTS
C.6.2 | Capillary-­to-­arteriole electrical signaling
in small vessel disease of the brain
Fabrice Dabertrand1,2,3; Osama F. Harraz3; Masayo Koide3;
Thomas A. Longden3; Anne Joutel4,5; Mark T. Nelson3,6
1
Department of Anesthesiology, University of Colorado, Anschutz Medical
Campus, CO, USA; 2Department of Pharmacology, University of Colorado,
Anschutz Medical Campus, CO, USA; 3Department of Pharmacology, University
of Vermont, College of Medicine, VT, USA; 4Institute of Psychiatry and
Neuroscience of Paris, INSERM U894, Université Paris Descartes, France; 5DHU
NeuroVasc, Sorbonne Paris Cité, Paris, France; 6Institute of Cardiovascular
Sciences, University of Manchester, Manchester, UK
Small vessel diseases (SVD) of the brain are a leading cause of vas-
cular cognitive decline and stroke, but without treatment. Cerebral
autosomal dominant arteriopathy with subcortical infarcts and leu-
koencephalopathy (CADASIL) is a hereditary form of SVD caused by
dominant mutations in the NOTCH3 gene. Recent studies have re-
vealed significant alterations in neurovascular coupling (NVC), which
underlies brain functional hyperemia, at an early stage of the dis-
+
ease. During normal NVC, potassium (K ) released by neural activity
+
stimulates inward-­rectifier K (Kir2.1) channel in capillary endothelial
cells (cECs) which initiates a membrane hyperpolarization propagat-
ing to the upstream arteriole where it causes vasorelaxation, hence
increase in local blood flow. However, in CADASIL mice, stimula-
+
tion of capillaries with 10 mM K failed to increase red blood cell
flux measured in vivo using two-­photon laser-­scanning microscopy.
Capillaries stimulation also failed to dilate upstream arteriole in an
innovative ex vivo capillary-­parenchymal arteriole preparation from
CADASIL mice. Finally, using conventional whole cell patch clamp,
we measured a 50% reduction in Kir2.1 current in cECs from the
disease model compared to control animals. We previously identi-
fied phosphatidylinositol 4,5-­bisphosphate (PIP2) as a crucial Kir2.1
regulator and found that addition of diC16-­PIP2, a soluble form of
the phospholipid, restored Kir2.1 current density to control levels.
Confocal microscopy with a fluorophore-­labelled PIP2 confirmed
that exogenous PIP2 was taken up by cECs ex vivo, and fluorescence
recovery after photobleaching (FRAP) confirmed its mobility in the
plasma membrane. Finally, addition of exogenous PIP2 restored K -­
induced upstream arteriolar dilation in ex vivo preparations from
CADASIL mice. In conclusion, our study supports the concept that
exogenous PIP2 restores Kir2.1-­mediated currents and capillary-­to-­
arteriole electrical signaling in CADASIL mouse model.
C.6.3 | Imaging single cell blood flow in the
living retina
Jesse Schallek1,2,3
1
The Flaum Eye Institute, University of Rochester, Rochester, NY, USA; 2Center
for Visual Science, University of Rochester, Rochester, NY, USA; 3Department of
Neuroscience, University of Rochester, Rochester, NY, USA
Objective: Using an adaptive optics camera to correct for the blur of
the eye, we image single blood cells in the living eye. Combined with
15 kHz camera capture, we image and measure the velocity of single
blood cells in the living retina of mice and humans.
Methods: Using a custom adaptive optics scanning light ophthalmo-
scope (AOSLO) we measure and correct for the aberrations of the
anterior eye of humans and mice. Split-­detector phase contrast im-
aging enabled imaging of single blood cells without the need for ex-
ogenous contrast agents. Automated measures of single cell speed
were performed by a custom Radon algorithm optimized to measure
single cell velocity. In humans, we imaged single cell blood flow in the
healthy eye to show the dynamics of pulsatile flow in arterioles and
venules of the retinal circulation. In mice, we imaged single blood
cell flux, velocity and measured total flow in vessels of all size in the
anesthetized and awake-­behaving mouse retinae.
Results: The smallest through largest vessels were visualized in the
mouse ranging from ~3-­45 micrometers in diameter. In vessels of every
caliber, we were able to visualize and measure the speed of single blood
cells as they flowed from complex inter-­digitation of the largest vessels,
to the parachute shape visualized in single cell flow capillaries. Flow in
mouse vessels ranged four orders of magnitude in difference from the
smallest to largest vessels of the retina (0.0002-­1.55 μL/min). Flow and
velocity rates along with heart rate were dramatically reduced in the
mouse retina in response to isoflurane anesthesia. Single cell blood flow
showed pulsatile flow in both arterioles and venules of the human retina
and demonstrated blunted laminar profiles across the vessel lumen.
Conclusion: AOSLO imaging is an emerging functional technology
that enables blood flow at the single cell level. Visualization of the
complete vascular network of the eye without contrast agents opens
possibilities to study the dynamics of the retinal circulation in a num-
ber of animal species. The automated imaging and analysis pipeline
enables measures of millions of blood cell speeds in the full spectrum
of vessels of the living eye in a non-­invasive way enabling the study
of single cell hemodynamics in conditions of health, disease and re-
sponse to therapy.
Research Support: Research was supported by the National Eye Institute
of the National Institutes of Health under Award No. EY028293 and
P30 EY001319. The content is solely the responsibility of the authors
+ and does not necessarily represent the official views of the National
Inst. of Health. This study was also supported by an Unrestricted Grant
to the University of Rochester Department of Ophthalmology, a Stein
Innovation Award and a Career Development Award (J. Schallek) from
Research to Prevent Blindness, New York, NY. Work was also sup-
ported by the Dana Foundation-­David Mahoney Neuroimaging Grant
and a research grant from Hoffmann-­La Roche Inc.
C.6.4 | Mapping and manipulating the fate of
clogged cerebral capillaries
Craig Brown
Division of Medical Sciences, University of Victoria
Cortical capillaries are prone to obstruction, which over time, could
have a major impact on brain angioarchitecture and function.
ABSTRACTS
Unfortunately, what long-­term fate awaits these capillaries remains a
mystery. Here we estimate that ~0.12% of cortical capillaries are ob-
structed each day (lasting > 20 min), preferentially in superficial layers
and lower order branches. Tracking natural or microsphere induced ob-
structions revealed that most capillaries recanalize by pushing obstruc-
tions back into the circulation rather than through the capillary wall.
Remarkably, 30% of all obstructed capillaries were pruned by 21 days
through endothelial regression, which was not compensated for by
sprouting. Using this information, we were able predict capillary loss
with aging that closely matched experimental estimates. From a mech-
anistic perspective, VEGF-­R2 signaling was a critical factor in dictat-
ing capillary recanalization and subsequent pruning. Thus, our studies
reveal the incidence, mechanism and long-­term outcome of capillary
obstructions which can also explain age related capillary rarefaction.
SESSION D.6 THE CONFLUENCE OF BASIC AND
CLINICAL SCIENCE IN THE DISCOVERY OF INOCA
(ISCHEMIA WITH NO CORONARY ARTERY
DISEASE)
CHAIRS: DR WILLIAM CHILIAN AND ASST PROF
VAHAGN OHANYAN
D.6.1 | Role of atherosclerosis and endothelial
dysfunction in INOCA
Janet Wei
Cedars Sinai Medical Center
This talk will describe the association between atherosclerosis and
endothelial dysfunction in patients with INOCA, review the prog-
nostic significance of these disorders, and recommend appropriate
therapies.
D.6.2 | What a mouse model of INOCA reveals
in mechanisms of cardiac dysfunction
William Chilian
Northeast Ohio Medical University
The goal of this presentation is to highlight the important role of the
coronary microcirculation and impaired myocardial perfusion in the
epidemic of heart failure.
|
      33 of 96
D.6.3 | miRNA92a plays a key role in regulating
LPP3 and eNOS expression in adipose arterioles
from CAD patients
Dawid Chabowski1,3; Andreas Beyer1,2; David Gutterman1,3
1
Department of Medicine, Medical College of Wisconsin, Wausau, WI, USA;
2
Department of Physiology, Medical College of Wisconsin, Wausau, WI, USA;
3
Department of Pharmacology, Medical College of Wisconsin, Wausau, WI, USA
We have previously shown that knockdown of lipid phosphate
phosphatase 3 (LPP3) changes the mediator of flow-­induced di-
lation (FID) from nitric oxide to hydrogen peroxide as observed
in arterioles from patients with coronary artery disease (CAD).
Blocking miR92a, a modulator of LPP3, restores NO-­
mediated
FID in subjects with CAD. These functional studies have not been
confirmed on a molecular basis. Our objective was to compare ex-
pression of LPP3 in arterioles from CAD and non-­C AD subjects to
provide support for the hypothesis that miR92a operates through
LPP3 to restore NO-­mediated FID in arterioles from CAD patients.
Protocols for tissue acquisition/processing were approved by the
MCW IRB. Human left ventricle from CAD and non-­C AD patients
and adipose arterioles (treated for 48 hours with miR92a inhibitor)
from CAD patients were prepared for immunoblotting and qPCR to
evaluate expression of LPP3 and eNOS.
LPP3 protein (arbitrary units: non-­
C AD 1.11 ± 0.08, n = 4 vs
CAD 0.39 ± 0.03 n = 8, P < 0.05) but not transcript levels were sig-
nificantly decreased in CAD hearts. LPP3 transcript levels were sig-
nificantly decreased in arterioles from CAD subjects (fold change:
Non-­C AD 1 ± 0.16 n = 13 vs CAD 0.61 ± 0.14 n = 11, P < 0.05) CAD
arterioles treated with miR92a inhibitor showed increase in LPP3
(untreated: 1 ± 0.28, n = 5 vs treated: 2.51 ± 0.87, n = 5) and eNOS
(untreated: 1 ± 0.25, n = 6 vs treated: 3.75 ± 1.35, n = 7, P < 0.05)
transcript levels, respectively.
We conclude that LPP3 is downregulated in heart and arteri-
oles from CAD subjects, and that miR92a plays a key role in reg-
ulating LPP3 and eNOS in CAD arterioles. These findings identify
novel targets for improving endothelial function in subjects with
CAD.
D.6.4 | Cardiac autonomic dysfunction in
women with coronary microvascular dysfunction
Puja Mehta
Emory University Medical School
The talk will focus on findings of peripheral vasoreactivity and auto-
nomic function in women with no obstructive coronary artery dis-
ease who have coronary microvascular dysfunction.
 |
34 of 96       ABSTRACTS
S E S S I O N A .7 I NTEG R ATI V E M O D E LI N G
O F B LO O D FLOW CO NTRO L A N D TI S S U E
OX YG E N ATI O N
CH A I R : D R N I KO L AOS T S O U K I A S
A.7.1 | A dynamic model of blood flow, oxygen
transport and flow regulation in skeletal muscle
Daniel Goldman; Dwayne Jackson; Zahra Farid; Sameh
Khan; Baran Serajelahi
Medical Biophysics, University of Western Ontario, London, Canada
The microcirculation is key in maintaining normal tissue function,
due to its role in both blood-­tissue transport and the regulation of
blood flow distribution. The function of the microcirculation, in turn,
depends on both its structure and its integrated response to various
local and global stimuli. While microvascular structure changes rela-
tively slowly, regulation is a dynamic process that involves a number
of interacting mechanisms and is also highly dependent on network
structure and flow properties. Here we present a flow regulation
model that incorporates a number of known regulatory mechanisms
and can utilize arteriolar and venular geometry derived from in vivo
data in rat skeletal muscle. The model includes time-­dependent
descriptions of two-­phase (plasma and red cell) blood flow, oxygen
transport, and regulation of arteriolar diameters. Results will be pre-
sented for temporal variability produced by the model, and these
will be discussed in the context of the challenges that remain in de-
veloping a model that can produce complex behavior similar to that
observed in vivo. Supported by Natural Sciences and Engineering
Research Council of Canada (NSERC) Grants R4081A03 (D.G.) and
R4218A03 (D.N.J.).
A.7.2 | Neurovascular coupling and the
arteriole-­to-­endfoot communication in awake
behaving mice
Cam Ha Tran1; Grant Gordon2
1
Reno School of Medicine, University of Nevada, Reno, USA; 2Cumming School of
Medicine, University of Calgary, Calgary, Canada
Continuous and constant blood supply is critical for the survival
and normal neuronal computation in the brain. Insufficient supply
of nutrients will lead to cell death and consequent sensory, motor
and cognitive deficits. To accomplish optimal perfusion for the
brain moment-­to-­m oment functions, it is equipped with a number
of control mechanisms, among which is neurovascular coupling
typically referred to as functional hyperemia. Albeit tremendous
efforts have been put forward, the relationship among the three
essential elements of the neurovascular coupling unit remains
poorly understood. In particular, how astrocytes integrate signals
from synapses, neuromodulatory networks and the vasculature to
regulate blood flow under physiological conditions is elusive. We
used two-­p hoton imaging in the barrel cortex of fully awake mice
and examined vascular responses and astrocyte Ca2+ dynamics
caused by sensory stimulation or volitional behaviors. We found
that sensory stimulation induced fast local arteriole dilation fol-
lowed by astrocytic Ca2+. These astrocyte Ca2+ transients were
reliant on excitatory synaptic transmission and dependent on the
behavioral state of the animal. Interestingly, we found that local
blood flow but not astrocytic endfoot Ca2+ relied on COX2 and
neuron-­d erived nitric oxide. Using Cre-­L ox technologies, optoge-
netic and chemogenetic approach, we manipulated the vasculature
and found that changes in blood flow generated small elevations
in astrocyte endfoot Ca2+ that was facilitated by vascular derived
nitric oxide. These data show that there is a tight relationship be-
tween synapses, neural networks and microvasculature in neuro-
vascular coupling and reveal a dynamic interaction between the
vasculature and the astrocytes in active animals.
A.7.3 | Requirements for regenerative
conduction of hyperpolarization in cerebral
capillaries: a model analysis
Arash Moshkforoush1; Asad Mirza1; Thomas Longden2;
Fabrice Dabertrand2; Osama Harraz2; Mark Nelson2,3;
Nikolaos Tsoukias1
1
Department of Biomedical Engineering, Florida International University, Miami,
Florida; 2Department of Pharmacology, University of Vermont, Burlington,
Vermont; 3Institute of Cardiovascular Sciences, University of Manchester,
Manchester, UK
Limited energy reserves of neurons in the brain necessitates the ex-
istence of tightly-­regulated mechanisms aiming at in-­time delivery of
oxygen and nutrients in response to elevated neuronal activity. This
phenomenon is referred to as neurovascular coupling (NVC) or func-
tional hyperemia and is disrupted in a variety of neurological conditions.
Recent findings point to an active role of capillary endothelial cells
(cECs) in NVC through inwardly rectifying potassium (KIR) channels. Ex-­
vivo and in-­vivo data suggest that a long-­range, capillary-­mediated vas-
odilatory signal is conducted to upstream parenchymal arterioles (PAs)
following a modest increase in local extracellular potassium concentra-
tion ([K+]o), a bi-­product of neuronal activity. The underlying mecha-
nisms of such conducted responses are yet to be fully understood and
await more thorough evaluation. In this study, we utilize an integrative,
bottom-­up computational modeling approach to investigate the bio-
physical determinants of cEC-­mediated NVC in brain vasculature. To
that end, detailed mathematical models of cEC, PA-­EC, and PA smooth
muscle cells (PA-­SMCs) are created. Cells are coupled via gap junctions
to form a network of cECs connected to an upstream PA. Both local and
distal hyperpolarization are analyzed in response to a local K+ challenge.
Results indicate that the ratio of outward to inward currents (primarily
through KIR and TRPV4, respectively), determines the sensitivity of cEC
ABSTRACTS
to [K+]o and the propagation of electrical signals. Simulations suggest
a novel role of cECs as amplifiers of incoming hyperpolarizing signals,
enabling them to promote regenerative and directional conduction of
vasoactivity in a network.
A.7.4 | Local vs long range signaling in the
ongoing network adaptation necessary for
adequate perfusion
Jens Brings Jacobsen
University of Copenhagen, Copenhagen, Denmark
Aim: To investigate how vessel remodeling enables vascular net-
works to meet long-­term spatial variation in tissue demand, with
focus on local versus long-­range endothelial signals.
Background: Tissue regions situated closely together can have dif-
ferent long-­term requirements for flow. The vascular network must
adjust continuously to meet such differences. Structural reduction
in vessel radius (inward remodeling), can effectively limit flow to
down-­stream regions following permanent reduction in demand. In
contrast, local outward remodeling is less efficient regarding per-
manently increasing local flow. In the latter case, a vasodilatory sig-
nal must be conveyed into and influence the surrounding resistance
network. Effective network restructuring therefore requires a com-
bination of mechanisms operating locally and globally. The vascular
endothelium is in a key position in that regard. It can sense and react
to local stimuli (e.g. flow) and convey information on local meta-
bolic status into the surrounding network, as vascular conducted
responses (VCR's).
Simulations were performed on networks where perfusion/metab-
olism mismatch induces VCR's that influence network structure at
distant sites.
Results & Conclusion: Starting from capillary-­sized vessels, remode-
ling and growth caused a realistic network to emanate. Subsequently,
metabolic activity was permanently changed in small regions.
Matching flow to reduced demand was achievable by local inward
remodeling. Similar matching to an increase in demand was difficult
to achieve solely by local vasodilatation. In both cases, flow mis-
match was induced in neighboring regions. Introducing a metabolism-­
dependent VCR allows for network restructuring that satisfy
high-­demand regions without compromising the ability to maintain
normal or low flow in nearby regions.
|
      35 of 96
S E S S I O N B .7 TA RG E TI N G TH E
PATH O PH YS I O LO G I C A L R E S P O N S E S
O F I S C H E M I A- ­R E PE R FU S I O N I N J U RY I N
D I FFE R E NT O RG A N S
CH A I R : D R FELI CIT Y G AV I N S
B.7.1 | Selective endothelin-­a receptor
antagonism prevents the progression of acute
kidney injury to chronic kidney disease
Neeraj Dhaun
University of Edinburgh
Acute kidney injury (AKI) is common and often progresses to chronic
kidney disease (CKD). We examined the role of endothelin-­1 (ET-­1) in
the transition of AKI to CKD.
B.7.2 | Cerebral microvessel alterations in the
acute period following focal cerebral ischemia
Gregory Del Zoppo
Department of Medicine and Department of Neurology, University of
Washington, Seattle, WA, USA
Our understanding of fundamental events in the cerebral microvas-
culature during the acute period of ischemic stroke, including cell-­
cell, cell-­matrix, and microvessel-­neuron (the “neurovascular unit”)
interactions, is still limited. The neurovascular unit is a structural and
conceptual framework that recognizes functional interactions among
microvessels, intervening astrocytes, and the neurons they serve.
This presentation examines several of these events and interactions
that are acutely altered by ischemia. While neuron stimulation can
alter flow through dependent cerebral microvascular beds, evidence
that the microvessel endothelium–matrix–astrocyte complex com-
municates with the neuron is so far sparse. Compartment sensitiv-
ity to ischemia and innate inflammation are central to this discussion.
Processes affected acutely in response to focal ischemia include mi-
crovessel patency, the microvessel permeability barrier, new vessel
formation (angiogenesis), and new neuron generation (neurogenesis).
This presentation will consider new information about the temporal
responses and interconnections of i) inter-­endothelial cell interactions
within cerebral microvessels, the permeability barrier, and signaling
processes established by microvessel endothelial cell β1-­integrin–ma-
trix interactions that affect permeability, ii) microvessel-­associated
signals (e.g. VEGF, αv-­integrins) generated within 1-­2 hours of focal
ischemia onset and their potential consequences, and iii) activation of
hemostasis (and inflammation) that cause acute changes in the vessel
wall in addition to intramicrovascular occlusion.
 |
36 of 96       ABSTRACTS
B.7.3 | Aging induces colonic dysfunction in
stroke to promote development of post-­stroke
infection
Shu Wen Wen; Raymond Shim; Connie Wong
Centre for Inflammatory Diseases, Department of Medicine, School of Clinical
Sciences, Monash University, Clayton, Victoria, Australia
Development of bacterial lung infection following stroke increases
dramatically with age and is a leading cause of death in elderly stroke
patients. The reason for this age-­dependent effect remains unclear.
Here, we provide evidence that aging exacerbates gut dysfunction in
stroke and can potentiate the development of lung infections. Using
an experimental mouse model of transient ischaemic stroke, we
firstly noted that aged mice (12 months) have a 100-­fold increase of
culturable lung bacteria when compared to young mice (8-­10 weeks)
after stroke. Despite similar brain infarct sizes between the two co-
horts, aged stroke mice showed worsening of clinical symptoms and
systemic inflammation (IL-­6). To examine the contribution of gut per-
meability in worsening disease outcome, we assessed the translo-
cation of orally gavaged fluorescein-­isothiocyanate labelled dextran
into the bloodstream in young and aged post-­stroke mice. Colonic
permeability was increased exclusively in aged post-­stroke mice but
remain unchanged in young stroke cohorts when compared to sham-­
operated animals. Additionally, the colon of aged stroke mice showed
significant breakdown of barriers that help maintain gut integrity,
including reduced expression of mucin and tight junction proteins.
Furthermore, when we inoculate a strain of streptomycin-­resistant
derivative of Escherichia coli into young and aged post-­stroke mice,
this strain was found only in the lungs of aged stroke mice. Taken
together, our results suggest that aging promotes the breakdown of
colonic barriers after stroke, allowing for the translocation of com-
mensal bacteria for systemic dissemination. (Monash Medical Centre
Animal Ethics Committee approved (MMCB/2016/10)).
B.7.4 | Innate immunity in sterile injury and
repair
Paul Kubes
University of Calgary
Understanding the role of the immune system in repairing injured
tissue with emphasis on the microcirculation will be discussed. The
role of neutrophils, monocytes and macrophage will be examined.
S E S S I O N C .7 U N D E R S TA N D I N G VA S C U L A R
B E D E LEC TR I C A L R E M O D E LI N G : N OV E L
M EC H A N I S M S A N D TA RG E T S
CH A I R : D R M . TER E SA PÉR E Z G A RCÍ A
C.7.1 | Orai channels in smooth muscle
Martin Johnson
Penn State College of Medicine
Vascular smooth muscle (VSM) remodeling contributes to the patho-
genesis of hypertension, atherosclerosis, restenosis, and asthma. I
will discuss the role of the proteins STIM and Orai in VSM remodeling.
C.7.2 | Membrane lipid KIR2.x channel
interactions enable hemodynamic sensing in
cerebral arteries
Maria Sancho1; Sergio Fabris1; Bjorn Hald2; Donald Welsh1
1
Robarts Research Institute, Department of Physiology & Pharmacology, Schulich
School of Medicine and Dentistry, University of Western Ontario, London, ON,
Canada; 2Department of Neuroscience, University of Copenhagen, København
N, Denmark
This study sought to characterize the unique regulation of cere-
bral arterial KIR by membrane lipids such as phosphatidylinositol-­
bis-­phosphate (PIP2) and cholesterol, and whether these signaling
molecules confer distinct mechanosensitivity to each cellular pool.
Endothelial and smooth muscle cells were freshly isolated from rat
cerebral arteries; patch-­clamp electrophysiology, Q-­PCR and im-
munohistochemistry defined KIR channel activity and expression.
Electrophysiology revealed a Ba2+-­sensitive KIR current in smooth
muscle and endothelial cells, while Q-­PCR and immunohistochem-
istry confirmed KIR2.x mRNA and protein expression respectively.
Each cellular pool of KIR channels was sensitive to particular mem-
brane lipids and hemodynamic forces. Endothelial KIR current was
dependent on PIP2 levels and interestingly, laminar flow activated
this channel pool in a PIP2 -­
dependent manner. In comparison,
smooth muscle KIR responded to cholesterol/ caveolae perturba-
tions, and pressure stimuli (e.g. hyposmotic challenge or stretch)
modulated its activity in a cholesterol-­dependent manner. The flow
and pressure sensitivity of KIR channels was confirmed in intact cer-
ebral arteries with the aid of vessel myography. Finally, a computa-
tional modeling was employed to predict at a conceptual level how
smooth muscle and endothelial KIR interact to set arterial VM/ tone.
In summary, while both vascular cell types express KIR2.x channels,
each pool is uniquely regulated by membrane lipids and hemody-
namic stimuli, driving together an integrated KIR, which helps con-
trol cerebral arterial tone. Funding support from CIHR and Rorabeck
Chair in Molecular Neuroscience and Vascular Biology.
ABSTRACTS
C.7.3 | Mechanisms involved in Kv1.3 signaling
to proliferation
Dra Pilar Cidad; Miguel Ángel De la Fuente; Esperanza
Alonso; José Ramón López-López; Dra Maria Teresa
Pérez-García
Instituto de Biología y Genética Molecular. Universidad De Valladolid, Valladolid,
Spain
Vascular smooth muscle cells (VSMCs) have the ability to switch
from a contractile to a proliferative phenotype, in a process known
as phenotypic modulation (PM). We have previously demonstrated
that changes in the expression of voltage-­dependent potassium (Kv)
channels associated with this proliferative phenotype. These results
were validated in transfected HEK293 cells: Kv1.5 decreased prolif-
eration and Kv1.3 increased proliferation independently of ion flux.
We also identified the molecular determinants of Kv1.3-­induced
proliferation at the cytosolic-­C-­terminal domain and two individual
point mutations of phosphorylation sites (Y447A and S459A) that
abolished proliferation.
Our data suggested that voltage-­dependent transitions of Kv1.3
could be coupled to signaling pathways leading to proliferation. To
test this hypothesis we explored: 1) the effects of VM variations on
Kv1.3-­induced proliferation and 2) the interactions of Kv1.3 with
proteins that activate signaling pathways.
We explored the effects of VM variations on Kv1.3-­
induced
HEK cells proliferation. Kv1.3 were co-­transfected with WT-­K ATP
channels (composed of SUR1 + Kir6.2) or gain of function (GOF-­
KATP), ATP–insensitive channels (SUR1 + Kir6.2G334D). Resting
VM was significantly hyperpolarized and Kv1.3-­induced prolifera-
tion was abolished in cells expressing GOF-­K ATP. Increasing [K ]e
was able to restore Kv1.3-­induced proliferation in cells expressing
Kv1.3 + GOF-­K ATP. Moreover, we found that Kv1.3-­induced prolif-
eration and Kv1.3 phosphorylation was impaired by MEK/ERK signal
inhibition. And both, Kv1.3 phosphorylation and Kv1.3 interaction
with a scaffold protein involved in proliferation (IQGAP3) were facil-
itated by membrane depolarizations. Altogether, these data showed
the signaling pathway linking Kv1.3 to proliferation requires voltage-­
induced conformational changes.
Supported by grant BFU2016-­75360-­R (MINECO) and VA114P17
(Junta Castilla y León).
C.7.4 | KATP channels and vascular pathology:
novel insights from cantu syndrome
Colin Nichols
Washington University, Saint Louis, Missouri, USA
Much of the vasculature is surrounded lined by layers of smooth
muscle (VSM), essential to the generation of vascular tone and
hence blood pressure. VSM is electrically active, generating spon-
taneous action potentials that cause Ca2+ entry via activation of
|
      37 of 96
L-­t ype voltage-­sensitive Ca2+-­channels (Nelson, 1995 #2584). As
in all excitable tissues, voltage-­gated and non-­voltage-­gated potas-
sium channels regulate the duration and frequency of these action
potentials. Activation of K-­
channels promotes membrane hyper-
polarization, hence decrease in voltage-­dependent Ca2+-­entry and
vasodilation (Nelson, 1995 #2584). Alterations in potassium channel
activity have been associated with various vascular diseases, both
as a result of genetic mutations and as a consequence of upstream
defects.
What are the defects in these channels and what is the relation-
ship between these defects and cardiovascular consequences? It
is likely that the primary relationship between altered ion channel
activity and vascular contractility is relatively linear, but perhaps un-
likely that the net effect in the animal is nearly as simple. There are
strong hormonally and neuronally driven feedback mechanisms that
will modulate cardiac and vascular tissues to combat aberrant blood
pressure.
One way to dissect and understand the primary and secondary
consequences of altered ion channel and electrical activity on car-
diovascular function is through the study of monogenic diseases in
which the primary defect is the ion channel itself. One such example
is Cantu Syndrome, a very complex syndrome involving multiple car-
diovascular pathologies, all arising from single mutations in the ATP-­
sensitive potassium channels that are prominent in vascular smooth
muscle. By comparison of human disease phenotypes to those iden-
tified in engineered mice that genocopy the human mutations, we
have provided a mechanistic links between the primary vascular
effects and secondary cardiac consequences. This presentation will
consider how this syndrome can be dissected and understood from
+ the molecular basis to cellular consequences to whole organism out-
comes, and then consider mechanism-­based therapies in treatment.
S E S S I O N D.7 A DVA N C E D I M AG I N G
TEC H N O LO G Y FO R D I S S EC TI N G T U M O R
M I C RO C I RC U L ATI O N A N D M E TA B O LI S M
CH A I R S: PRO F M A KOTO S U EM AT S U A N D D R
DA I FU K U M U R A
D.7.1 | Dissecting tumor microenvironment
using advanced optical imaging techniques
Dai Fukumura
Deputy Director of Edwin L. Steele Laboratories, Massachusetts general Hospital;
Associate Professor, Harvard Medical School, Boston, MA, USA
Intravital microscopy such as multiphoton microscopy, Doppler op-
tical coherence tomography and short-­wave infrared imaging and
sophisticated animal models have been providing unprecedented
molecular, cellular, anatomical and functional insights in tumor
biology.
 |
38 of 96       ABSTRACTS
Tumor microvasculature is structurally and functionally abnor-
mal, hindering nutrients and drug delivery, and causing ineffective-
ness of anti-­tumor treatments. These pathophysiological features
are results of pro-­and anti-­angiogenic factors’ Imbalance in tumors.
We demonstrated that restoring the balance of these factors in tu-
mors “normalizes” tumor vasculature, improves its function and mi-
croenvironment, and enhances the efficacy of cytotoxic therapies.
In addition to the abnormal angiogenesis, altered physical prop-
erties of solid tumors can also cause the derangement of tumor
microcirculation. We have shown that the abnormal deposition of
extracellular matrix (ECM) together with the growing stress of tu-
mors elevates stiffness and compressive force in tumors. Enzymatic
ECM depletion rescued the compromised perfusion in tumors and
prolonged survival in combination with cytotoxic therapy.
TME also suppresses anti-­
t umor immunity and hinders the
effect of immunotherapies. We uncovered novel immunosup-
pressive roles of nonclassical monocytes in tumors. Intravital
microscopy revealed that a chemokine signaling mediates the
transmigration of nonclassical monocytes across the endothelium.
Moreover, these monocytes further recruit additional immuno-
suppressive innate immune cells and inhibit adaptive immunity.
Prevention of these innate immune cells enhanced the efficacy of
anti-­t umor treatment.
Collectively, abnormal tumor vasculature and microenvironment
form formidable barriers to anti-­tumor therapies. Normalization of
TME such as reprogramming vasculature, ECM and immune cell pro-
file would enhance delivery and efficacy of existing and novel anti-­
tumor treatment strategies.
References: 1. Fukumura D, et al. (2018). Nature Reviews Clinical
Oncology 15(5):325-­340. PMCID: PMC5921900
2. Rahbari NN, et al. (2016) Science Translational Medicine
8(360):360ra135.
3. Jung K et al. (2017) Journal of Clinical Investigation 127:3039-­
51. PMCID: PMC5531423
4. Jung K et al. (2017) PNAS pnas.org/cgi/doi/10.1073/
pnas.1710754114.
D.7.2 | Next generation intravital imaging in the
short-­wave infrared (SWIR)
Oliver Bruns
Helmholtz Zentrum München
The short-­wavelength infrared region (SWIR; 1000-­2000 nm) pro-
vides several advantages for in-­vivo imaging. To demonstrate the ca-
pabilities, we generate detailed three-­dimensional quantitative flow
maps of brain vasculature by intravital microscopy.
D.7.3 | Gold-­nanofève substrate-­enhanced
raman spectroscopy visualizes cancer boundaries
for automated on-­tissue pathological diagnosis
Takako Hishiki1; Akiko Kubo1; Masayuki Naya2; Megumi
Shiota2; Makoto Suematsu1; Takeharu Tani2; Shogo
Yamazoe2
1
Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan;
2
Frontier Core-­Technology Laboratories, Research & Development Management
Headquarters, FUJIFILM Corporation, Kanagawa, Japan
Large-­
a rea surface-­
e nhanced Raman spectroscopy (SERS) is a
promising technology that enables to visualize small-­m olecular
metabolites without any chemical treatment (e.g. staining, labeling
or ionization) on frozen tissues with square-­centimeter regions (1).
Although refinement of the technology might benefit automated
on-­
t issue pathological diagnosis, difficulty exists in fabricating
robust electromagnetic fields serving as SERS excitation sources
that enable to detect many fingerprint peaks as biomarker sig-
nature to recognize pathological states such as cancer. We have
herein applied an oblique gold deposition technique to develop-
ment of highly porous and anisotropic island growth of gold nano-­
particles (2). The device is named gold-­nanofève (GNF) substrate
after the anisotropic shape of the gold nanostructure and benefits
highly sensitive large-­area SERS imaging that is suitable for visual-
izing distinct features of metabolic systems in cancer models in
mice. Two experimental cancer models were tested: xenografting
human colon cancer in super-­immunodeficient mice and syngeneic
mouse glioblastoma model. SERS peaks predominantly occurring
in tumor regions and those in the surrounding regions were sepa-
rately collected to establish database as “teacher's data” accord-
ing to manually annotated histological diagnosis by professional
pathologists. Such a database combined with a newly-­d eveloped
digital image-­p rocessing method to calculate tumor boundaries
allowed us to predict localization of tumors on frozen tissues
without any subjective evaluation with an excellent accuracy.
Refinement of our GNF-­S ERS technology combined with enforced
learning software will thus provide automated on-­t issue diagnosis
of cancer in clinical tissue samples.
References: 1. Yamazoe S, et al. ACS Nano (2014).
2. Shiota M, et al. Nature Communications (2018).
D.7.4 | Deciphering metabolism of reactive
sulfur species in cancer tissues by SERS imaging
Makoto Suematsu
Keio University School of Medicine, Shinjuku-­ku, Japan
Reactive sulfur species (RSS) is a class of highly reductive sulfur-­
containing metabolites that include reduced glutathione (GSH),
hypotaurine (HT), H2S and polysulfides (PS). Methionine uptake
drives re-­methylation cycle and partly supports trans-­sulfuration
ABSTRACTS
(TS) pathway under the support of serine/glycine cleavage sys-
tems to provide cysteine and RSS. Cystathionine β-­synthase (CBS)
and cystathionine γ-­lyase (CSE) have been thought to account for
major H2S generators. In some cancers, CD44/xCT complex func-
tions to incorporate extracellular cysteine to be catalyzed by CBS
or CSE and to generate GSH for survival (1). Atmospheric-­pressure
(AP)-­MALDI-­MS (Shimadzu Inc., Kyoto) allowed us to suggest that
CBS-­derived H2S modulates development of colon cancer HCT116
xenografts; observation suggested that the glutathione persulfide
(GSSO3-­)/glutathione sulfide (GSO3-­) ratio becomes smaller in CBS-­
knockdown cancer xenografts concurrently with their increasing
sizes versus wild-­t ype zenografts (2). However, labile properties of
RSS against artificial auto-­oxidation hamper detection of HT, H2S,
and PS by AP-­MALDI-­MS. By contrast, surface-­enhanced Raman
spectroscopy equipped with a unique gold nano-­
fève substrate
(GNF-­SERS) enables us to directly visualize GSH, HT, and PS simul-
taneously in frozen tissue sections without any labeling/staining
procedures (3). SERS imaging combined with 13C6-­labeled glucose-­
based fluxome analyses in different cancer cells suggest that, under
stress conditions such as CD44 blockade, a considerable C1 flux
driven by activated glycolysis through Warburg effect towards TS is
consumed to generate cysteine-­derived RSS to protect cancer cells
under stress conditions.
References: 1. Ishimoto T, et al. Cancer Cell (2011).
2. Yamamoto T, et al. Nature Communications (2014).
3. Shiota M, et al. Nature Communications (2018).
S E S S I O N A . 8 N E W A N D N OTA B LE I N S I G HT S
I N FO U N DATI O N A L M I C RO C I RC U L ATO RY
R E S E A RC H
CH A I R : D R CO R A L M U R R A NT
A.8.1 | Caveolae link CaV3.2 channels to BKCa-­
mediated feedback in vascular smooth muscle
Ahmed Hashad1; Osama Harraz2; Suzanne Brett1; Monica
Romero3; Mario Kassmann4,5; Jose Puglisi3; Sean Wilson3;
Maik Gollasch4,5; Donald Welsh1
1
Robarts Research Institute, and Department of Physiology & Pharmacology,
Schulich School of Medicine and Dentistry, University of Western Ontario,
London, ON Canada; 2Department of Pharmacology, University of Vermont,
Burlington, VT, USA; 3Division of Pharmacology, Loma Linda University, Loma
Linda, CA, USA; 4Charité—Universitätsmedizin Berlin, Experimental and Clinical
Research Center (ECRC), a joint cooperation between the Charité Medical Faculty
and the Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany;
5
DZHK (German Centre for Cardiovascular Research), partner site Berlin,
Germany
This study examined whether caveolae position CaV3.2 (a T-­t ype
Ca2+ channel), sufficiently close to ryanodine receptors (RyR) for
extracellular Ca2+ influx to trigger Ca2+ sparks and large conduct-
ance Ca2+-­a ctivated K+ (BKCa)-­m ediated feedback. Using smooth
|
      39 of 96
muscle cells from mouse mesenteric arteries, the proximity liga-
tion assay confirmed that CaV3.2 reside within 40 nm of caveo-
lin 1, a key caveolae protein. Methyl-­β -­c yclodextrin, a cholesterol
depleting agent that disrupts caveolae, suppressed CaV3.2 activ-
ity along with BKCa-­
m ediated spontaneous transient outward
currents (STOCs) in cells from C57BL/6 but not CaV3.2−/− mice.
Genetic deletion of caveolin 1, a perturbation that prevents cave-
olae formation, also impaired STOC production, but did so without
impairing Ca2+ channel activity including CaV3.2. These observa-
tions indicate a mistargeting of CaV3.2 in caveolin 1−/− mice, a
view supported by a loss of Ni2+-­s ensitive Ca2+ spark generation
and co-­l ocalization signal (CaV3.2-­R yR) from the proximity ligation
assay. Vasomotor and membrane potential measurements con-
firmed that cellular disruption of the CaV3.2-­R yR axis functionally
impaired the ability of BKCa to set tone in pressurized caveolin
1−/− arteries. In conclusion, Caveolae play a critical role in protein
targeting and preserving the close structural relationship between
CaV3.2 and RyR needed to drive negative feedback control in re-
sistance arteries.
Funding: This work is supported by the Canadian Institute of
Health Research, Alberta Innovates Health Solutions (AIHS), the
American Heart Association. The Deutsche Forschungsgemeinschaft
(DFG) and the Deutsche Akademische Austauschdienst (DAAD).
A.8.2 | The cystic fibrosis transmembrane
conductance regulator (CFTR) as a potential
link between the myogenic response and the
circadian clock
Chloe Ng; Jeff Kroetsch; Darcy Lidington; Steffen-Sebastian
Bolz
University of Toronto, Department of Physiology, Toronto, ON, Canada
Cerebral autoregulation maintains constant cerebral perfusion de-
spite variations in mean arterial pressure (MAP). The primary mecha-
nism underlying cerebral autoregulation is the myogenic response,
the intrinsic ability of cerebral resistance arteries to adapt their tone
to prevalent pressures. Circadian oscillations in MAP challenge cer-
ebral autoregulation in a rhythmic fashion; we therefore hypothesize
that the molecular clock within cerebral arteries adjusts cerebrovas-
cular myogenic reactivity in anticipation of these circadian MAP
oscillations.
Myogenic and phenylephrine-­
stimulated vasoconstrictions were
determined in mouse olfactory arteries using pressure myography
at Zeitgeber times (ZT) 3, 7, 11, 15, 19, and 23. Expression of micro-
vascular clock gene mRNA (Bmal1, Clock, and Per2) was analyzed
by qPCR. Bmal1, Clock, and Per2 show typical oscillations, indica-
tive of normal peripheral clock function. We examined the mRNA
expression of 3 key myogenic signalling targets (sphingosine kinase
1, S1P receptor 2 and cystic fibrosis transmembrane regulator,
CFTR). We found that only CFTR mRNA oscillates and confirmed
 |
40 of 96       ABSTRACTS
the differential expression at ZT11 and ZT23 at the protein level.
Myogenic vasoconstriction exhibited circadian rhythmicity, with a
peak and trough that matched CFTR's function as a negative regula-
tor of myogenic reactivity. In contrast to myogenic vasoconstriction,
agonist-­induced vasoconstriction by phenylephrine did not exhibit
circadian rhythmicity.
In summary, cerebral microvessels exhibit circadian rhythmicity
in myogenic responsiveness. This provides a functional basis to cope
with circadian oscillations in systemic input pressures (i.e., MAP). At
the molecular level, our data suggest that rhythmic expression of the
key myogenic signalling element CFTR is responsible for the func-
tional phenotype.
A.8.3 | The regulatory role of chloride ions
to the contractile responses changes during
maturation in rat saphenous artery
1,2 1,2
Dina K. Gaynullina ; Daria S. Kostyunina ; Anastasia A.
Shvetsova1,2; Olga S. Tarasova1,2
1 2
Lomonosov Moscow State University, Moscow, Russia; Institute for Biomedical
Problems, Russian Academy of Sciences, Moscow, Russia
Opening of Cl-­channels in arterial smooth muscle (ASM) leads to
Cl-­efflux from the cell thereby causing depolarization and con-
traction. The functioning of ASM changes significantly during
maturation. However, whether the regulatory role of Cl-­ to the
contractile responses changes during early postnatal develop-
ment is not clear, despite maturational changes in Cl-­homeosta-
sis were shown previously in neurons. Thus, we hypothesized the
role of Cl-­in the regulation of arterial contractile responses dif-
fers in young and adult rats. We used the muscular-­t ype saphe-
nous artery from male Wistar rats aged 10-­12 days (young) and
3-­4 months (adult). All procedures were approved by Russian insti-
tutional committee on animal welfare. Endothelium-­d enuded arte-
rial segments were mounted in isometric myograph (DMT A/S).
Contractile responses were evoked by α1-­a drenoreceptor agonist
methoxamine (10 −8-­10 −4 mol/L). The effects of Cl-­removal from
the solution (equimolar substitution of NaCl and KCl by respec-
tive aspartate salts), NKCC1 blockade (bumetanide, 30 μmol/L),
Cl-­channels blockade (MONNA, 3 μmol/L) were studied. Removal
of Cl-­decreased contractile responses to methoxamine, and the
effects were more prominent in arteries of young rats compared
to adults. Bumetanide in normal solution as well as bicarbonate-­
free solution did not change the contractile responses in either
young or adults. MONNA reduced contractile responses stronger
in arteries of young animals in comparison to adults. The obtained
results demonstrate that Cl-­is more important for the arterial con-
tractile responses in 10-­12-­day old rats than in adult rats. Study
was supported by RFBR (grant N 18-­015-­0 0216-­a).
A.8.4 | Control of vascular network structure
and function: role of axial communication
Ed Van Bavel
Amsterdam University Medical Centers, Amsterdam, The Netherlands
Objective: Proper regulation of local perfusion requires control of
both tone and structure of the myriad of segments that make up a
resistance vessel network. We aimed to evaluate the role of con-
ducted vasoactive responses and their structural equivalent, “con-
ducted remodeling”, in network control.
Methods: Experiments (ethical approval obtained) were aimed at
demonstrating that axial conduction of vasoactive responses has
a sustained component, as a possible drive for conducted remod-
eling. A novel pressure myograph setup was developed that al-
lowed localized stimulation of a cannulated rat small mesenteric
artery by dividing the superfusion bath into two compartments.
Obtained data were used as input for network modeling, ranging
from simple conceptual models to actual networks based on imag-
ing cryomicrotome data on the human coronary microcirculation
with incorporation of mechanics and local functional and struc-
tural adaptation.
Results: In concentration-­
response tests, local phenylephrine-­
induced constriction and acetylcholine-­induced dilation were con-
ducted into the remote compartment over distances of several mm
and these remote responses had a clear sustained (5 minutes) compo-
nent. Absence of remote stimulation blunted the local PE response.
Constriction to high potassium was not conducted. Incorporation of
such responses into network simulation models reveals their pos-
sible importance for maintenance of microvascular arcades and col-
laterals, and protection against AV-­shunting.
Conclusion: Vascular tone control is integrated along the resistance
vessel wall, and possibly between mother and daughter segments
in a network. This provides a possible base for spatial integration of
vascular remodeling and maintenance of resistance artery network
architecture.
Funded: EU under the GA 606998 SmArteR.
A.8.5 | Purinergic/Pannexin signaling in
capillaries: implications for skeletal muscle blood
flow regulation
Iain R. Lamb; Nicole M. Novielli; Coral L. Murrant
Department of Human Health and Nutritional Sciences, University of Guelph,
Guelph, Ontario, Canada
Skeletal muscle's dynamic requirement for O2 requires precise co-
ordination of tissue blood flow. Evidence suggests capillaries (CAPS)
are critical in flow coordination given their ability to increase their
perfusion by conducting a vasodilatory signal to their associated
upstream arteriole. Conducted vasodilation has been shown to be
mediated via gap junctions (GJs) in the microvasculature; however,
ABSTRACTS |
      41 of 96

vasodilation initiated by muscle contraction fails to display key
characteristics of GJ-­mediated responses, suggestive of a second
signaling pathway. Pannexin (PanX) channels can facilitate cell-­cell
signaling by releasing ATP which activates purinoceptors on neigh-
boring cells. We sought to characterize the involvement of PanX-­
and GJ-­dependent signaling during CAP stimulation. Using intravital
microscopy of the hamster cremaster we stimulated CAPS via mi-
cropipette application of vasodilators relevant to muscle contrac-
tion (potassium chloride (KCl), adenosine (ADO), nitric oxide (NO)
and acetylcholine (ACh)) while observing diameter changes in the
associated upstream arteriole. Application of PanX blocker, meflo-
quine, between the CAP and upstream arteriole, blunted the con-
ducted vasodilation of KCl, ADO and ACh by 111%, 15% and 33%,
respectively. Application of purinoceptors antagonist, suramin at-
tenuated the vasodilation elicited by KCl, ADO and ACh by 114%,
63% and 70%, respectively. Separately, application of GJ uncou-
pler halothane inhibited the conducted vasodilation elicited by NO
and ACh by 107% and 87%, respectively. These data demonstrate
the existence of a purinergic/PanX-­dependent signaling pathway
through which CAPS can communicate with their upstream arteri-
ole. These data imply that during muscle contraction both pannexin
and gap-­junction mediated signaling pathways play a role in blood
flow coordination.
to 3.17 (5 days), 3.35 (10 days), 3.57 (21 days), and 3.15 (35 days)
segments/mm, respectively. Neither the number of arterioles with
diameter > 15 μm nor the total length of networks differed at re-
spective time points. We suggest that proliferation of small (termi-
nal) arterioles promotes capillary perfusion during regeneration of
muscle fibers. (Support: MU Fellowship, AHA 17PRE33410260 &
NIH R37HL041026).
S E S S I O N B . 8 M O LEC U L A R M O D U L ATI O N
O F M I C ROVA S CU L A R BA R R I E R FU N C TI O N
CH A I R S: PRO F J I N G -YA N H A N A N D D R
Q I AO B I N G H UA N G
B.8.1 | Role of Nrf2 signaling in diabetes-­
associated microvessel dysfunction
Pingnian He; Xinghai Xia; Kyle LaPenna; Yunpei Zhang
College of Medicine, Pennsylvania State University, Hershey, USA
Increased reactive oxygen species (ROS) have been implicated
to play important roles in disease-­a ssociated microvascular dys-
function. Nrf2 (NF-­E 2-­r elated factor 2) is a master transcription

A.8.6 | Microvascular network remodeling factor regulating antioxidant defenses. The objective of this
study is to investigate the role of Nrf2 in microvessel perme-
during skeletal muscle regeneration
ability under normal and diabetic conditions. Experiments were
Nicole Jacobsen1; Rebecca Shaw1; Charmain Fernando1; conducted in individually perfused mesenteric venules in wild
Robert Mazzeo2; Steven Segal1
type (WT) and Nrf2 knock out (Nrf2−/−) rats. Microvascular per-
1
Medical Pharmacology and Physiology, University of Missouri, Columbia, MO,
meability was determined by measuring hydraulic conductivity
USA; 2Integrative Physiology, University of Colorado, Boulder, CO, USA
(Lp). Plasma H2O2 was measured from fresh blood using perox-
ide assay. Results showed significantly elevated plasma H2O2 in
Following injury to skeletal muscle, damaged myofibers regenerate
both WT and Nrf2−/− diabetic rats compared to normal controls.
via activation of resident stem (satellite) cells, which proliferate, dif-
Nrf2−/− rats showed higher plasma H2O2 with higher basal Lp and
ferentiate into myotubes, and mature into muscle fibers. However,
Lp responses to PAF than those in WT rats under both normal and
little is known regarding concomitant adaptations of the microvas-
diabetic conditions. WT female rats had lower responses to PAF
cular supply. We hypothesized that the smallest branches of arteri-
than WT male rats, but such gender differences were abolished
olar networks would proliferate during the early stages of skeletal
in diabetic WT rats and Nrf2−/− rats under both normal and dia-
muscle regeneration in order to supply a robust increase in muscle
betic conditions. Importantly, the Lp values are positively corre-
capillarity. With IACUC approval, the gluteus maximus muscle (GM)
lated with the plasma H2O2 levels in all animal groups (R2 > 0.92).
of male C57Bl/6 mice (age, 4 months; n = 5-­7/group) was injured by
These results suggest that Nrf2 and its downstream target genes
local injection of barium chloride (1.2%, 75 μL) and evaluated at 5,
play important roles in counterbalancing the ROS levels and pro-
10, 21, & 35d post-­injury vs Control (0d). During anesthesia, fluores-
tect microvessel barrier function. The Nrf2−/− with increased
cent wheat germ agglutinin was injected retro-­orbitally to label the
oxidative stress resembles diabetic conditions and increases the
endothelium. A GM was excised, pinned at in situ dimensions, then
microvessel susceptibility to inflammation. Our study reveals an
fixed and cleared to visualize and map entire microvascular resist-
important role of plasma ROS levels in the regulation of microves-
ance networks, from feed arteries through terminal arterioles, on a
sel permeability, indicating plasma H2O2 as a potential predictor
Vesselucida-­based imaging system (MBF Bioscience, Williston, VT).
of microvessel barrier function. Supported by HL130363 and
Throughout networks, the number of small arterioles (internal diam-
DK097391.
eter ≤ 15 μm) increased at 5 day, remained elevated from 10-­21 days
(P < 0.05) and began to subside at 35 days. Normalized to total
network length, the smallest arterioles increased from 2.2 (0 day)
 |
42 of 96       ABSTRACTS
B.8.2 | Sphingosine-­1-­phosphate protects
endothelial barrier function following
hemorrhagic shock and hypoxic injury
Natascha G. Alves; Jerome W. Breslin; Sarah Y. Yuan
Department of Molecular Pharmacology and Physiology, Morsani College of
Medicine, University of South Florida, Tampa, FL, USA
Our lab has previously demonstrated that S1P reduces hemorrhagic
shock and resuscitation (HSR)-­induced microvascular hyperperme-
ability. HSR can cause poor perfusion and hypoxia in the gut, leading
to endothelial apoptosis and injury to the endothelial surface layer
(glycocalyx). In the current study our objective was to investigate
the extent to which S1P can prevent apoptotic signaling, shedding of
the endothelial surface layer (glycocalyx), and elevated microvascu-
lar permeability in response to HSR in vivo or hypoxia in a cultured
endothelial cell model. For our in vivo studies, we used a rat model
of HSR, in which S1P was administered in the intravenous resuscita-
tion fluid. The mesenteric microvasculature was dissected, and pro-
tein was extracted for the assessment of pro-­apoptotic proteins by
western blot.
We also assessed mitochondrial membrane potential using JC-­
1. We performed immunofluorescence of the mesenteric micro-
vasculature to evaluate paracellular junction protein interaction.
As an indicator of glycocalyx health, we used an intravenous in-
jection of FITC-­labeled lectin and measured the amount of lectin
bound to the microvascular endothelium. For the in vitro studies,
we placed cultured primary endothelial cells in a low-­oxygen envi-
ronment and applied S1P during the hypoxic challenge. We tested
whether S1P has the potential to protect barrier function of en-
dothelial cells grown on Transwell membranes (0.4 μm pores) from
hypoxia-­induced hyperpermeability by determining permeability to
FITC-­albumin. We have found that HSR did not cause an increase
in pro-­apoptotic protein expression levels in vivo. However, HSR
caused a significant disruption in mitochondrial membrane po-
tential. The effects of S1P on mitochondrial function still need to
be investigated. Besides that, we found that the amount of lectin
bound to the mesenteric microvasculature is higher in HSR animals
treated with S1P compared to HSR only. In our in vitro studies, we
found that S1P prevents hypoxia-­induced permeability in different
types of endothelial cells, characterized by a decrease in endothe-
lial permeability. Taken together, our results suggest that S1P has
the potential to protect microvascular barrier dysfunction caused
by HSR by preventing glycocalyx shedding. However, how S1P re-
duces the activation of apoptotic cascade following HSR remains
elusive. Supported by NIH grants R01HL098215, R01GM120774,
R01HL070752 and R01GM097270.
B.8.3 | QiShenYiQi, a compound chinese
medicine, improved albumin leakage from cardiac
venules induced by ischemia-­reperfusion in rats
Jing-Yan Han
Peking University Health Science Center
QSYQ, a traditional Chinese medicine, prevent I/R induced micro-
vascular hyperpermeability and endothelial barrier damage through
the regulating coronary microvascular endothelial caveolae and
junctions.
B.8.4 | Interaction of phospho-­moesin and
CD44 in pericytes attenuated the maturation of
neovessels in AGE-­induced angiogenesis
Qiaobing Huang; Shuangshuang Zhang; Lixian Chen; Yun
Cui; Xiaohui Liu; Xiaohua Guo
Guangdong Provincial Key Laboratory of Shock and Microcirculation, School of
Basic Medical Sciences, Southern Medical University, Guangzhou, China
Background: This study was to investigate AGE-­induced angiogen-
esis and neovessel maturation.
Methods: AGE-­treated model was developed in WT & RAGE(−/−)
mice and in WT postnatal mice by intraperitoneal injection of AGE-­
BSA. Aorta rings were also used to detect AGE-­induced angiogen-
esis and pericyte coverage. Primary pericytes were used to detect
the proliferation and migration under AGE treatment. Colocalization
and interaction between CD44 and phospho-­moesin was detected
by confocal laser scanning microscopy and Co-­IP.
Results: The number of neovessels in retina and the phosphorylation
of moesin were increased in WT mice, as well as in WT postnatal
mice after AGE treatment. RAGE(−/−) mice did not show obvious
angiogenesis after AGE treatment. Both in vivo and in vitro AGE
treatment could also enhanced the phosphorylation of moesin and
promoted neovessel sprouting in WT mouse aortic ring, while there
were not significant changes in RAGE(−/−) aortic ring. The pericyte
coverage was reduced in neovessels in mouse aortic ring and retina
after AGE treatment. AGE-­
BSA increased moesin phosphoryla-
tion and migration in pericytes and promoted CD44 and phospho-­
moesin interaction. ROCK inhibitor decreased AGE-­induced moesin
phosphorylation and subsequently attenuated pericyte migration,
suppressed the formation and colocalization of CD44-­
phospho-­
moesin complex. The inhibiting mutant of phosphorylation of Thr
558 in moesin suppressed AGE-­induced pericyte migration, while
the activating mutant of moesin phosphorylation of Thr 558 pro-
moted cell migration.
Conclusions: AGEs promote angiogenesis in mice and this is ac-
companying with moesin phosphorylation. The interaction of
phospho-­moesin and CD44 participates in AGE-­induced pericyte
migration through ROCK activation and might hamper the neovessel
maturation.
ABSTRACTS |
      43 of 96

S E S S I O N C . 8 N EU ROVA S C U L A R C.8.2 | Age-­related impairment of

DYS FU N C TI O N I N AG I N G A N D neurovascular coupling: new targets for
A L ZH E I M E R ' S D I S E A S E
prevention of cognitive decline
CH A I R S: PRO F G R A NT G O R D O N A N D PRO F
ZO LTA N U N G VA R I A. Csiszar1,2; G. A. Fulop1,2; W. E. Sonntag1,2; S.
Tarantini1,2; Z. Ungvari1,2; N. M. Varcarcel-Ares1,2; Andriy
Yabluchanskiy1,2
1
Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences
Center, Oklahoma City, OK, USA; 2Translational Geroscience Laboratory, Donald

C.8.1 | The cellular deconstruction of W. Reynolds Department of Geriatric Medicine, University of Oklahoma Health
Sciences Center, Oklahoma City, OK, USA
neurovascular coupling
Adam Institoris1; Cam Ha Tran1,2; David Rosenegger1; Moment-­to-­m oment adjustment of cerebral blood flow (CBF) via
Govind Peringod1; Grant R. Gordon1 neurovascular coupling (NVC) is essential to maintain healthy
1
Hotchkiss Brain Institute, Department of Physiology and Pharmacology, cognitive function. Aging, increased oxidative stress and cer-
Cumming School of Medicine, University of Calgary, Calgary, Canada;
2
ebromicrovascular endothelial dysfunction impairing NVC, con-
University of Nevada, Reno School of Medicine, Reno, USA
tributing to age-­related decline of higher cortical functions. This
concept is supported by studies showing that pharmacologically-­
Neurovascular coupling is a process by which neurons and astrocytes
induced NVC impairment associate with cognitive dysfunction.
release vasoactive messengers to control local blood flow. However,
We will present new data that in aged laboratory rodents NVC
the exact contribution of neurons and astrocytes during functional
and endothelium-­
d ependent cerebromicrovascular dilation can
hyperemia is uncertain. We aimed to elucidate the role of neurons
be rescued, which represents a potential therapeutic target for
and astrocytes during various lengths of functional hyperemia. To
the promotion of healthy brain aging. In particular, the potential
observe the temporal dynamics of neuronal and astrocyte activity
role of mitochondrial oxidative stress in NVC dysfunction will be
in response to sensory stimulation, we imaged synthetic and geneti-
2+ discussed. Increasing evidence indicates mitochondrial oxidative
cally encoded cell-­t ype specific Ca indicators with in vivo 2-­photon
stress plays a critical role in many age-­related cellular impairments.
fluorescence microscopy through a closed cranial window over the
To test the hypothesis that attenuation of mitochondrial oxidative
barrel cortex of awake mice. 5 seconds air puff to the contralateral
stress may exert beneficial effects on NVC responses in aging,
whiskers elicited neuronal Ca2+ responses prior to arteriole dilation,
2+ 24-­m onth-­old C57BL/6 mice were treated with a cell permeable,
while astrocyte Ca levels increased 2-­3 seconds later. 30 seconds
mitochondria-­t argeted antioxidant peptide (SS-­31; 10 mg/kg/day,
air puff elicited a larger arteriole dilation with prolonged astrocyte
i.p.) or vehicle for 2 weeks. We found that NVC responses were
endfoot and process Ca2+ signals. In acute neocortical slices, low
significantly impaired in aged mice. Treatment with SS-­31 signifi-
intensity, 5 seconds electrical stimulation evoked neuronal Ca2+ el-
2+ cantly improved NVC responses by increasing NO-­m ediated cere-
evation and vasodilation without astrocyte endfoot Ca increase.
bromicrovascular dilation, which was associated with significantly
Clamping intracellular Ca2+ concentration in the astrocyte network
improved spatial working memory, motor skill learning and gait co-
by patch-­infusing BAPTA had no effect on short stimulation-­induced
ordination. These findings are paralleled by the protective effects
vasodilation even at higher stimulation frequency and intensity. This
of SS-­31 on mitochondrial production of reactive oxygen species
purely neuronal response was mediated through AMPA receptors
and mitochondrial respiration in cultured cerebromicrovascular
and partially via cyclooxygenase-­
2-­
derived prostaglandin E2, ac-
endothelial cells derived from aged animals. Thus, mitochondrial
tivating EP4 receptors. Importantly, inhibition of most vasoactive
oxidative stress contributes to age-­related cerebromicrovascular
pathways attributed to astrocytes failed to reduce vasodilation to
dysfunction, exacerbating cognitive decline. We propose that the
5 seconds stimulation. Surprisingly, intense stimulation for 30 sec-
mitochondria-­t argeted antioxidants could be considered for pre-
onds activated astrocytes, and clamping astrocyte Ca2+ reduced
vention/treatment of age-­related vascular cognitive impairment
arteriole dilation. Astrocyte-­related vasodilation to enduring stimu-
(VCI).
lation was reduced by inhibiting epoxyeicosatrienoic acid produc-
tion. We propose that neurons initiate while astrocytes sustain
vasodilation during functional hyperemia.
 |
44 of 96       ABSTRACTS
C.8.3 | Transient receptor potential vanilloid
4 channels are important regulators of cognitive
function and parenchymal arteriole dilation
Janice M. Diaz-Otero1; Ting-Chieh Yen1; Amna Ahmad1;
Erinn Laimon-Thomson1; Bana Abolibdeh1; Kara Kelly1;
Matthew T. Lewis2; Robert W. Wiseman2,3; William F.
Jackson1; Anne M. Dorrance1
1 with genetical, pharmacological, biochemical, and in vivo physiol-
Department of Pharmacology and Toxicology, Michigan State University, East
Lansing, MI USA; 2Department of Physiology, Michigan State University, East
Lansing, MI USA; 3Department of Radiology, Michigan State University, East
Lansing, MI USA
Hypertension-­
associated parenchymal arteriole (PA) dysfunc-
tion reduces cerebral blood flow and impairs cognition. We have
shown that Transient Receptor Potential Vanilloid 4 (TRPV4)
channels mediate PA endothelium-­d ependent dilation and that
TRPV4-­m ediated dilation is impaired in hypertension. We tested
the hypothesis that TRPV4 channels are important regulators of
cognition and PA structure and dilation. 10-­12-­m onth-­old male
TRPV4−/− rats were compared to Wistar rats. Assessment of cer-
ebral blood flow using MRI with arterial spin labeling showed that
TRPV4−/− rats have reduced cerebral perfusion. TRPV4 deletion
did not change blood pressure. Cognitive function was assessed
using the Barnes maze. TRPV4−/− rats have an increased latency
to escape the barnes maze suggesting impaired cognitive func-
tion; however, the TRPV4−/− rats moved significantly less and
were slower than control rats. The TRPV4−/− rats also had gait
abnormalities and showed anxiety-­
like behaviors. PA structure
and function were assessed by pressure myography. TRPV4 dele-
tion did not cause PA remodeling as shown by the lack of changes
in the outer diameter, lumen diameter and wall thickness. TRPV4
deletion did not change PA myogenic tone development, but the
PAs from TRPV4−/− had severely blunted responses to carbachol
(CCh) and the TRPV4 agonist GSK1016790A. The TRPV4 inhibi-
tor, GSK2193874 (10 −7 mol/L) was used to confirm the importance
of TRPV4 activation in CCh-­m ediated dilation in Wistar rats. Our
data indicate that TRPV4 channels play a critical role in cerebral
arteriole dilation and cognitive function. Impaired TRPV4 function
in hypertension may increase the risk for the development of vas-
cular dementia. Supported by the NIH R01-­HL-­137694-­01.
C.8.4 | Brain microvascular mechanisms linking
aging to Alzheimer's disease
Veronica Galvan
School of Medicine, Department of Cellular and Integrative Physiology,
Barshop Institute for Longevity and Aging Studies and the Nathan Shock
Center of Excellence in the Biology of Aging, Glenn Biggs Institute for
Alzheimer's and Neurodegenerative Diseases, University of Texas Health San
Antonio
We and others showed that the mammalian/mechanistic target-­of-­
rapamycin (mTOR) inhibitor rapamycin, a drug that delays aging in
mice, halts and even reverses memory deficits and restores cere-
bral blood flow (CBF) in several different mouse Alzheimer's mod-
els, in models of atherosclerosis, and in a model of advanced aging
in rats. Decreasing mTOR restored cortical network activation and
neurovascular coupling. To define the mechanisms by which mTOR
drives cerebrovascular and neuronal dysfunction in models of ad-
vanced aging and AD we used in vitro and in vivo models combined
ogy and imaging approaches. Moderate, but not drastic reduction
of TORC1 assembly in neurons (to levels similar to those achieved
by rapamycin treatment), promoted synaptic bouton remodeling.
This was linked to enhanced memory and increased brain glucose
metabolism. The restoration of CBF by mTOR attenuation in AD
mice was dependent on nitric oxide synthase (NOS) activation,
suggesting that NO-­d ependent vasodilation may be required for
the maintenance of CBF during AD-­like progression. In agreement
with these observations, our in vitro studies showed that mTOR
potently inhibits NOS activation. Taken together, our data indi-
cate that the mechanisms by which TOR attenuation restores CBF,
neuronal network activity, and memory may be common to dif-
ferent models of age-­associated neurological disease and to brain
aging, and single out (a) cerebrovascular NO release, and (b) syn-
aptic bouton remodeling as key mechanisms by which TOR drives
brain aging and mechanistically contributes to the pathogenesis of
dementias modeled in mice.
SESSION D.8 DYNAMIC CALCIUM CONTROL IN
THE VESSEL WELL
CHAIRS DR POONEH BAGHER AND PROF AVRIL
SOMLYO
D.8.1 | Endothelial cell calcium: location,
location, location
Pooneh Bagher1; Lyudmyla Borysova2; Kim A. Dora2;
Christopher J. Garland2; Hamish A. L. Lemmey2; Chloe
Powell2; Xi Ye2
1
Department of Medical Physiology, Texas A&M University Health Science
Center, Temple, TX, USA; 2Department of Pharmacology, University of Oxford,
Oxford, UK
Endothelial cell-­mediated feedback dilation has a significant influ-
ence on vascular smooth muscle tone. Calcium signals underlie this
feedback-­dilation and can be observed as focal or global increases
in intracellular calcium following activation of either smooth mus-
cle cells or endothelial cells. Focal calcium signals can occur within
myoendothelial projections, where smooth muscle and endothelial
cells make direct physical contact. By imaging intracellular calcium
fluxes via laser scanning confocal microscopy in isolated cannulated
rat cremasteric arterioles or freshly isolated endothelial cell tubes, we
ABSTRACTS
provide evidence that activation of L-­t ype voltage-­dependent calcium
channels (VDCCs) specifically in smooth muscle cells is sufficient to
trigger endothelial cell calcium activity. These VDCC-­
dependent
Endothelial Cell calcium transients, or VECTors, were focal in the pres-
ence of heparin, an IP3 receptor blocker, but amplified to form propa-
gating waves in the absence of IP3 receptor block. This endothelial
cell calcium activity stimulates voltage-­
insensitive, intermediate-­
conductance, calcium-­
activated potassium channels, which spread
hyperpolarization to the smooth muscle suppressing vasoconstriction
as well as promoting vasomotion. Thus, smooth muscle cell calcium
increase can lead to activation of the underlying endothelium result-
ing in feedback dilation, preserving tissue blood flow.
D.8.2 | PIP2 Toggles Electrical and Ca2+
Signaling Modalities in Brain Capillaries
1 1
Osama F. Harraz ; Thomas A. Longden ; Fabrice
Dabertrand1; Mark T. Nelson1,2
1
Department of Pharmacology, University of Vermont, Burlington, VT, USA;
2
Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK
Brain capillaries play a critical role in sensing neural activity and
translating it into dynamic changes in cerebral blood flow to serve
the metabolic needs of the brain. To assume their role, capillary en-
dothelial cells are equipped with the molecular machinery—inward-­
rectifier K+ (KIR 2.1) channel and Gq-­
protein-­
coupled receptors
(GqPCRs)—necessary to respond to factors—K+ and GqPCR ago-
nists—that are implicated in neurovascular coupling (NVC). Here, we
demonstrate that phosphatidylinositol 4,5-­
bisphosphate (PIP2) is
necessary for capillary KIR 2.1-­mediated electrical signaling. GqPCR
activation depletes PIP2 levels and should therefore affect KIR 2.1
channels. In capillaries, application of GqPCR agonists that are impli-
cated in NVC deactivated KIR 2.1 channel currents and crippled the
associated electrical signaling. In addition, these agonists triggered
capillary Ca2+ signals that are in part due to Ca2+ influx through tran-
sient receptor potential vanilloid (TRPV4) channels. TRPV4 channel
activation was caused by relief of PIP2 inhibition. In conclusion, this
study supports the concept that PIP2 acts as a bi-­directional modu-
latory hub to balance depolarizing (TRPV4) and hyperpolarizing
(KIR 2.1) influences in the brain capillary endothelium. GqPCR activa-
tion therefore acts as a molecular switch that alters the balance be-
tween electrical and Ca2+ signaling, an observation with potentially
profound significance for the control of blood flow into the brain.
Supported by Postdoctoral Fellowship (17POST33650030-­O.F.H.)
and Scientist Development Grant (17SDG33670237-­
T.A.L.) from
the American Heart Association; the United Leukodystrophy
Foundation, the Totman Medical Research Trust, Fondation Leducq,
the EU Horizon 2020 Programme (666881, SVDs@target, M.T.N.);
and National Institutes of Health Grants R01-­
HL-­
136636 (F.D.),
4P20 GM103644/4-­5, P30-­GM-­103498, P01-­HL-­095488, R01-­
HL-­121706, R37-­DK-­053832, 7UM-­HL-­1207704, R01-­HL-­131181
(M.T.N.)
|
      45 of 96
D.8.3 | Calcium dynamics in the lymphatic wall:
uncovering endothelium-­dependent mechanisms
of lymphatic contractile function modulation
Jorge Castorena; Scott Zawieja; Min Li; Michael Davis
Department of Medical Pharmacology and Physiology, University of Missouri,
Columbia, MO, USA
Lymphatic system deficiencies can result in abnormal accumulation
of fluid and protein causing edema. Efficient lymph propulsion re-
lies critically on the intrinsic spontaneous contractions of lymphatic
muscle cells (LMCs), which are initiated and entrained by action po-
tentials that propagate rapidly along the LMC-­layer.
We recently identified Cx45 as the critical connexin isoform
mediating the propagation of pacemaking signals between LMCs.
In contrast to the arteriolar wall, our results show that the major
endothelial connexins are dispensable for the initiation and entrain-
ment of spontaneous contractions and reveal an apparent lack of
electrical coupling between lymphatic endothelial cells (LECs) and
LMCs. Therefore, our objective was to investigate whether signals
can be conducted along the lymphatic endothelium and to assess if
those signals modulate LMC contractility.
The calcium indicator GCaMP6f was selectively expressed in the
LMC or LEC layer of murine lymphatic vessels and imaged ex vivo
using pressure myography. Acetylcholine (ACh) was applied focally
via glass micropipette.
While the LMC layer consistently showed spontaneous, rhyth-
mic global calcium events, the endothelium remained quiescent.
Focal ACh application induced an endothelial calcium wave that
propagated between LECs at 60-­100 μm/s for ~0.2-­0.5 mm. When
imaging calcium in the LMC layer, focal LEC stimulation resulted in a
silencing wave that transiently stopped all intracellular events and, in
contracting vessels, inhibited spontaneous contractions.
In conclusion, our results show that focal LEC stimuli can be
conducted as calcium, and perhaps electrical, waves along the en-
dothelium to regulate the contractile function of LMCs, presumably
through production of nitric oxide.
D.8.4 | Local increase in peroxynitrite impairs
TRPV4‐dependent endothelial calcium signaling
in obesity
Eric L. Cope1; Leon J. DeLalio1; Kwangseok Hong1; Brant E.
Isakson1; Wolfgang B. Liedtke2; Corina Marziano1; Matteo
Ottolini1; Swapnil K. Sonkusare1
1
University of Virginia, Charlottesville, USA; 2Duke University, Durham, USA
In resistance‐sized systemic arteries, localized Ca2+ influx signals
through TRPV4 channels (TRPV4 sparklets) at myoendothelial projec-
tions (MEPs) drive endothelium‐dependent vasodilation. Moreover,
a kinase anchoring protein 150 (AKAP150) enhances the activity
of TRPV4 sparklets at the MEPs through protein kinase C (PKC)
 |
46 of 96       ABSTRACTS
anchoring. We hypothesized that impaired AKAP150‐PKC‐TRPV4
vasodilatory signaling at MEPs contributes to obesity‐induced loss of
endothelium‐dependent vasodilation. Mean arterial pressure (MAP)
was elevated in endothelium‐specific AKAP150‐/‐ or TRPV4‐/‐ mice,
suggesting a key role of endothelial AKAP150 (AKAP150EC) and
TRPV4 (TRPV4EC) channels in blood pressure regulation. In a high‐
fat diet fed mouse model of obesity, endothelium‐dependent vaso-
dilation was impaired and MAP elevated when compared to normal
fat diet‐fed mice. Additionally, AKAP150EC‐PKCTRPV4EC vasodila-
tory signaling was impaired specifically at the MEPs in obese mice.
Reactive nitrogen species peroxynitrite (PN) has previously been
implicated in obesity‐induced endothelial dysfunction, although
the underlying mechanisms remain unknown. Lowering PN levels
restored TRPV4EC sparklet activity at the MEPs, rescued endothe-
lium‐dependent vasodilation in a TRPV4EC‐dependent manner, and
lowered MAP in obese mice. The staining for PN marker nitrotyros-
ine was increased exclusively at MEPs in obese mice. Moreover, ex-
ogenous peroxynitrite inhibited the activity of TRPV4EC sparklets at
MEPs in wild‐type mice but not in AKAP150EC−/− mice suggesting
that PN targeted AKAP150EC to inhibit TRPV4EC channel activ-
ity. Taken together, these results indicate that local elevation of PN
levels inhibits AKAP150EC‐PKC‐TRPV4EC sparklet activity at MEPs
and impairs endothelium‐dependent vasodilation, thus contributing
to obesity‐induced hypertension.
S E S S I O N A .9 N E W A N D N OTA B LE I N S I G HT S
I N TR A N S L ATI O N A L M I C RO C I RC U L ATO RY
R E S E A RC H
CH A I R : D R M A R I A N N E TA R E
A.9.1 | Increased stroke severity worsens
outcome via activation of the sympathetic
nervous system
R. Shim1; S. W. Wen1; S. E. Phillips2,3; G. W. Lambert2,3; C. G.
Sobey4; C. H. Y. Wong1
1
Centre for Inflammatory Diseases, Department of Medicine, School of Clinical
Sciences, Monash University, Australia; 2Inversion Health Innovation Research
Institute, Faculty of Health, Arts and Design, Swinburne University of Technology,
Australia; 3Human Neurotransmitters Laboratory, Baker Heart and Diabetes
Institute, Australia; 4Department of Physiology, Anatomy and Microbiology,
School of Life Sciences, La Trobe University, Australia
Infection is a leading cause of mortality in stroke patients, account-
ing for around one-­t hird of post-­s troke deaths. Emerging evidence
demonstrates that infection is a result of stroke-­induced immuno-
suppression, which may be mediated by effector molecules of the
sympathetic nervous system (SNS), such as norepinephrine (NE).
However, the relationship between infarct size, post-­s troke im-
munosuppression and the SNS remains unclear. To study this, we
used a model of ischemic stroke in 8-­10-­week-­old male mice and
varied stroke severity by adjusting the period of middle-­cerebral
arterial occlusion (MCAO): mild (30 minutes MCAO), moderate
(60 minutes MCAO) and severe (permanent; pMCAO). At 24 hours
post-­s troke, a dramatic increase in bacteria was detected in the
lungs, with a greater frequency of infection in mice that under-
went pMCAO. This corresponded with decreased spleen weights
in all severities of MCAO. Additionally, infarct size inversely cor-
related with the number of circulating leukocytes, suggestive of
stroke-­induced immunosuppression. Increased levels of plasma
NE was detected 3 hours following pMCAO, consistent with a dys-
function in SNS regulation after severe stroke. Indeed, blocking
the action of the SNS with propranolol following pMCAO reduced
the incidence of infection at 24 hours. These findings indicate that
stroke severity dictates the extent of immunosuppression and in-
fection after stroke, which may be mediated by effector molecules
of the SNS. All animal procedures were approved by the Monash
Medical Centre Animal Ethics Committee (MMCB/2016/10). This
work is funded by the National Health and Medical Research
Council (Australia).
A.9.2 | An ex vivo approach to mimic the
vascular dysfunction of preeclampsia: assessing
potential drug treatments
Sarah Marshall1,2; Chen-Huei Leo1; Marianne Tare3,4; Natalie
Hannan5; Laura Parry1
1
School of BioSciences, University of Melbourne, Parkville, Australia;
2
Department of Obstetrics and Gynaecology, The Ritchie Centre, Monash
University, Clayton, Australia; 3Department of Physiology, Monash University,
Clayton, Australia; 4Monash Rural Health, Monash University, Clayton,
Australia; 5The Translational Obstetrics Group, Mercy Hospital for Women,
Department of Obstetrics and Gynaecology, The University of Melbourne,
Heidelberg, Australia
Preeclampsia (PE) affects 1 in 20 pregnancies and remains a lead-
ing cause of maternal and fetal morbidity and mortality worldwide.
PE is characterised by hypertension after 20 weeks gestation
coupled with proteinuria, uteroplacental dysfunction and/or
maternal organ dysfunction. A characteristic endpoint of PE is
widespread maternal vascular dysfunction caused by placental-­
derived factors such as soluble fms-­like tyrosine kinase-­1 (sFlt-­1).
This anti-­angiogenic factor is often significantly upregulated in
the circulation of women with PE and is positively correlated with
the severity of maternal vascular dysfunction. The main aims of
this study were to: i) establish an ex vivo model to deliver del-
eterious placental-­d erived factors to arteries to cause vascular
dysfunction, and ii) investigate if co-­
incubation with potential
drugs to treat PE protects the vasculature. Experiments were
approved by the University of Melbourne Animal Experimental
Ethics Committee 1212387 and the Mercy Health Human
Research Ethics Committee R11/34. Small mesenteric arteries
from C57BL/6J mice were incubated for 24 hours at 37°C in nor-
mal or conditioned media (exposed to placental explants: PEM,
ABSTRACTS
or isolated primary trophoblast: TCM). Both media types contain
sFlt-­1 levels > 20 ng/mL in combination with other placental re-
leased factors that can contribute to endothelial dysfunction in
PE. PEM and TCM induced vascular dysfunction, characterised
by reduced agonist-­induced endothelium-­d ependent relaxation,
without affecting responses to the endothelium-­independent dila-
tor sodium nitroprusside (n = 7-­8/media). Co-­incubation with TCM
and the pregnancy hormone relaxin (10-­
15 nmol/L) prevented
the development of vascular dysfunction. In conclusion, exposing
mesenteric arteries to media containing high levels of placental-­
derived sFlt-­1 induces dysfunction commonly associated with PE.
A.9.3 | Antenatal corticosteroid exposure alters
placental glucocorticoid receptor profile and
modulates fetal vascular cystathionine gamma-­
lyase activity
1,2,3 4 2,5
Rebecca Dyson ; Vicki Clifton ; Hannah Palliser ; Max
Berry1; Xin Ni6; Ian Wright2,3,7
1
Department of Paediatrics & Child Health and Centre for Translational
Physiology, University of Otago, Wellington, New Zealand; 2Mothers &
Babies Research Centre, Hunter Medical Research Institute, Newcastle,
Australia; 3Discipline of Paediatrics & Child Health, School of Medicine and
Public Health, University of Newcastle, Newcastle, Australia; 4Mater Medical
Research Institute, University of Queensland, Brisbane, Australia; 5School of
Biomedical Sciences & Pharmacy, University of Newcastle, Newcastle, Australia;
6 trials. Transplantation of PPs in the kidney capsule of immuno-
Department of Physiology, Second Military Medical University, Shanghai, China;
7
Discipline of Paediatrics, Graduate Medicine, Faculty of Science Medicine and
Health, University of Wollongong, Australia
Background: Hydrogen sulphide (H2S) is a potent vasodilator pro-
duced endogenously via cystathionine gamma-­
lyse (CSE). H2S
contributes to the dysregulation of microvascular tone associated
with poor outcome following preterm birth, particularly in males.
Previous reports suggest glucocorticoids such as those routinely
given to mothers at risk of preterm labour may suppress CSE activ-
ity. We hypothesise that these antenatal corticosteroids (ACS) may
contribute to the relative “protection” of preterm females by reduc-
ing production of pro-­vasodilatory molecules such as H2S.
Method: All animal procedures were approved by the University of
Newcastle Animal Ethics Committee. Preterm (GA62) and full-­term
(GA69) guinea pig fetuses were studied. Guinea pig dams received
vehicle or betamethasone (ACS, 1 mg/kg) at 24 and 12 hours prior
to tissue collection. Real-­time H2S production capacity of fetal vas-
culature (skin) was assessed using a microrespiration system. CSE
contribution was investigated by inhibition via propargylglycine.
Results: CSE contribution to vascular H2S production increases with
advancing gestational age. Exposure to clinically-­relevant doses of
ACS reduces CSE activity, and total H2S production, by approxi-
mately 50% in female preterm and term fetuses, and may be related
to differential expression profiles of the placental glucocorticoid re-
ceptor in relation to preterm delivery, sex and ACS exposure.
Conclusions: ACS may play a role in preventing potent vasodilation
via CSE-­mediated pathways in females, potentially contributing to
|
      47 of 96
their greater cardiovascular stability and improved outcomes follow-
ing preterm birth compared to males of the same gestational age.
Funding: John Hunter Hospital Charitable Trust; Hunter Medical
Research Institute Children's Research Exchange Visit Award.
A.9.4 | Using pre-­vascularization strategies for
stem cell therapy for the treatment of type 1
diabetes
Yasaman Aghazadeh1,2; Maria Cristina Nostro1,2,3; Sara
Nunes Vasconcelos1,4
1
Toronto General Hospital Research Institute, University Health Network,
Toronto, Ontario; 2McEwen Center for Regenerative Medicine, Toronto,
Ontario; 3Department of Physiology, University of Toronto, Toronto, Ontario;
4
Institute of Biomaterials & Biomedical Engineering, University of Toronto,
Toronto, Ontario
Type 1 Diabetes (T1D) is an autoimmune disease characterized
by destruction of the beta cells and impaired insulin production.
Using the Edmonton protocol, donor-­
d erived islets perfused
into the portal vein successfully restore glycemia in 58% of T1D
patients, however donor scarcity and poor engraftment (60%-­
70% loss immediately post transplantation) limit the therapeutic
applications. To overcome these challenges, human stem cell-­
derived pancreatic progenitors (PP) are being tested in clinical
deficient mice leads to formation of insulin secreting islet-­like
structures. While insulin-­
p roducing cells appear 6 weeks after
transplantation, restoration of normoglycemia occurs ~5 months
post-­t ransplantation, suggesting poor connection to the host vas-
culature. Moreover, pancreas developmental studies indicate that
interactions with islet capillaries trigger dorsal pancreas budding,
beta cell maturation, glucose sensing and insulin secretion. Our
goal is to accelerate PP cell functionality and glucose normalization
by increasing vascularization. We tested endothelial cells (ECs) or
ready-­made microvessels (MVs) from adipose tissue; which were
mixed with PPs and transplanted subcutaneously in a T1D mouse
model. The PP + MV grafts inosculated with host vasculature in
the first week post-­t ransplantation resulting in higher rate of PP
proliferation, while PP + EC grafts had significantly lower perfu-
sion and proliferation rates. Furthermore, recipients of PP + MVs,
reached normoglycemia 8 weeks post-­t ransplantation, responded
to glucose stimulated insulin secretion and displayed significantly
higher human insulin levels (c-­p eptide) than PP + ECs. Our future
studies will focus on characterizing the mechanisms through which
vascularization improves PP performance in vivo.
 |
48 of 96       ABSTRACTS

A.9.5 | Microcirculatory perfusion disturbances A.9.6 | Remote microvascular dysfunction

and endothelial hyperpermeability following precedes tissue injury in feces-­induced peritonitis
cardiopulmonary bypass persist in the early model of sepsis
postoperative period Paulina Kowalewska1,2; Stephanie Milkovich1,2; Lynn Wang3;
Nicole A. M. Dekker1,2; Anoek L. I. van Leeuwen1,2; Peter L. Shawn Whitehead3; Christopher Ellis1,2
Hordijk 2; Alexander B. A. Vonk3; Christa Boer1; Charissa E.
1
Department of Medical Biophysics, University of Western Ontario, London,
van den Brom1,2 Canada; 2Robarts Research Institute, University of Western Ontario, London,
1
Canada; 3Department of Anatomy & Cell Biology, University of Western Ontario,
Department of Anesthesiology, Amsterdam Cardiovascular Research,
London, Canada
Amsterdam University Medical Centers – location VUmc, Amsterdam,
The Netherlands; 2Department of Physiology, Amsterdam Cardiovascular
Research, Amsterdam University Medical Centers—location VUmc, Amsterdam, Sepsis is an overwhelming systemic inflammatory response to infec-
The Netherlands; 3Department of Cardiothoracic Surgery, Amsterdam tion that may lead to multiple organ failure. The objective of this
Cardiovascular Research, Amsterdam University Medical Centers—location
VUmc, Amsterdam, The Netherlands
study was to determine whether peripheral microvascular dysfunc-
tion precedes histopathological alterations in septic organs in a rat
model of feces-­induced peritonitis (FIP). After intraperitoneal in-
Objective: Endothelial hyperpermeability following cardiopulmo-
jection of a fecal slurry, male Sprague-­Dawley rats were prepared
nary bypass (CPB) could contribute to microcirculatory perfusion
for intravital video microscopy (IVVM) of the right extensor digi-
disturbances and postoperative organ dysfunction. We investigated
torum longus (EDL) muscle (protocols were approved by Western
whether microcirculatory perfusion disturbances continue after
University). The microvasculature was examined for changes in
CPB and are paralleled by endothelial barrier dysfunction.
hemodynamics and erythrocyte oxygen saturation using dual wave-
Methods: Microcirculatory perfusion was measured in seventeen
length IVVM. Red blood cell dynamics were altered in septic animals
patients undergoing cardiac surgery with CPB using sublingual side-
compared to saline-­injected controls as early as 2.5-­4 hours after
stream dark-­field imaging to calculate the percentage of perfused
infection, which was marked by significantly reduced red blood cell
vessels. Plasma was obtained before (pre-­CPB) and several time
velocity (125 ± 24 vs 277 ± 79 μm/s, respectively), supply rate (8 ± 2
points after CPB (post-­CPB) to study the effect of plasma exposure
vs 21 ± 9 cells/s, respectively) and oxygen saturation (52 ± 6 vs
on in vitro renal and pulmonary microvascular endothelial barrier
67% ± 8%, respectively), and a decreased number of capillaries with
using electric cell-­substrate impedance sensing. Plasma angiopoie-
continuous flow. Immunohistochemical labelling of brain sections for
tin-­2 and soluble Tie2 levels were measured with ELISA.
rat MHC class II did not show signs of inflammatory microglial activa-
Results: CPB decreased the proportion of perfused vessels from
tion and intravenous injection of Evans Blue dye indicated that the
92 ± 6 to 69% ± 9% (P < 0.001), which did not restore in the first
blood-­brain barrier was not broken in septic animals. Septic rats had
72 postoperative hours (71 ± 5 vs 92% ± 6%, P < 0.001). In parallel,
evidence of increased dye leakage in liver and kidneys which was not
1 hour post-­CPB plasma decreased renal and pulmonary endothe-
observed in the EDL muscles. Histopathological alterations were not
lial resistance (0.84 ± 0.1 to 0.78 ± 0.1 and 0.77 ± 0.1 to 0.36 ± 0.1;
observed in septic livers and kidneys. The findings of this study show
P < 0.001) compared to pre-­
CPB plasma. The reduction in renal
that, in our rat FIP model of sepsis, microvascular dysfunction occurs
and pulmonary endothelial resistance was even more severe when
exposed to plasma obtained 72 hours post-­
CPB (0.84 ± 0.1 vs before detectable organ injury. Funding: CHRP & NSERC.
0.64 ± 0.1 and 0.77 ± 0.1 vs 0.34 ± 0.1, P < 0.001; respectively).
Moreover, circulating angiopoietin-­2 levels were negatively associ-
ated with the proportion of perfused vessels (r = −0.28; P = 0.04) S E S S I O N C .9 U N I Q U E VA S CU L AT U R E S I N
and renal endothelial resistance (r = −0.28; P = 0.047). H E A LTH A N D D I S E A S E
Conclusion: Microcirculatory perfusion disturbances in patients CH A I R : A S S I S T PR O F ER I K A B O ER M A N
undergoing CPB persist in the first postoperative days and are par-
alleled by prolonged renal and pulmonary endothelial hyperperme-
ability and increased levels of angiopoietin-­2. Future research should
reveal whether targeting endothelial barrier could preserve micro- C.9.1 | Flow with the go: the bladder
circulatory perfusion and organ function in patients undergoing car-
vasculature as a regulator of bladder function
diac surgery with CPB.
Nathan Tykocki; Adrian Bonev; Thomas Longden; Thomas
Heppner; Mark Nelson
University of Vermont, Burlington, VT, USA

Prolonged decreases in urinary bladder blood flow are linked to lower

urinary tract symptoms (LUTS), including both bladder overactivity
ABSTRACTS
and underactivity. Although ample evidence links impaired bladder
blood flow to LUTS, a fundamental gap exists in our understanding
of bladder blood flow regulation and its effects on bladder function.
We recently found that urinary bladder arterioles, unlike vessels of
similar size from other vascular beds, do not develop myogenic tone
in response to increases in intraluminal pressure but did respond to
multiple contractile agonists. This lack of myogenic tone was caused
by strong inward-­rectifier K+ (KIR) channels in arteriolar smooth
muscle cells: both blockade or knockout of smooth muscle KIR
channels “restored” pressure-­induced constriction and membrane
depolarization. However, knockout of smooth muscle KIR channels
alone caused symptoms of bladder overactivity, suggesting that this
absence of myogenic tone is paramount to proper bladder function.
Together, these data suggest that: (1) the regulation of vascular tone
in the bladder is independent of pressure, (2) control of vascular
tone in the urinary bladder is likely neurohumoral in nature, and (3)
changes in blood flow alone can cause the development of LUTS.
These results potentially re-­frame LUTS treatment to include both
vascular and neurological therapeutic interventions.
C.9.2 | How the eye views inflammation and
diabetes: microvessel adaptations in the retina
and cornea
Shayn M. Cottler
University of Virginia, Charlottesville, VA, USA
The microvessels of the eye are highly responsive to chronic in-
flammatory signals, such as those present in diabetic retinopathy,
which is the leading cause of blindness in adults in the United
States. No matter how effective a diabetic patient is at control-
ling blood sugar, over time, the retinal microvasculature becomes
destabilized, leading to edema and fibrosis. We and others have
found evidence that pericytes, cells that wrap around and stabilize
microvessels, malfunction very early in diabetes and may cause
many of the vascular complications that arise later on. We have
recently identified a subset of pericytes in the retina that exhibit
atypical morphology, such as off-­vessel cell bodies and cell pro-
cesses extending to neighboring capillaries. We have observed
that these atypical pericytes are: (1) significantly more abundant in
diabetic conditions, (2) produce collagen IV basement membrane
bridges that connect across capillaries, and (3) able to be modu-
lated by Ang-­2 , PDGF-­B B, and blood sugar. Our in vivo dynamic
imaging of injured murine limbal vessels in the cornea reveals the
progression of this atypical morphology, implicating this behavior
as a key event in eye inflammation. We hypothesize that this atypi-
cal morphology exhibited by pericytes during sustained inflamma-
tion represents an intermediate pericyte phenotype that can be
therapeutically targeted to prevent subsequent diabetes-­induced
vascular dropout.
|
      49 of 96
C.9.3 | Coronary microvascular smooth muscle
cells isolated from type 2 diabetic db/db mice
display higher contractile forces and larger
spread areas
Aaron J. Trask1,2; Youjin Cho3; Samir N. Ghadiali3; Patricia E.
McCallinhart1
1
Center for Cardiovascular Research, The Research Institute at Nationwide
Children's Hospital, Columbus, Ohio, USA; 2Department of Pediatrics, The Ohio
State University, Columbus, Ohio, USA; 3Department of Biomedical Engineering,
The Ohio State University, Columbus, Ohio, USA
Previous data from both our lab and the literature show that type
2 diabetic coronary microvascular smooth muscle cells (MVSMCs)
are less stiff, but diabetic coronary microvessels display enhanced
myogenic tone. The goal of the present study was to evaluate the
hypothesis that primary diabetic coronary MVSMCs that display
reduced stiffness are able to generate enhanced contractile forces
compared to normal. All animal experiments were approved by
the institutional animal and care and use committee at Nationwide
Children's Hospital. Primary normal or diabetic coronary MVSMCs
were isolated from normal or type 2 diabetic db/db mice and were
evaluated by traction force microscopy (TFM). Cells were cultured
on polyacrylamide gels containing red fluorescent microspheres/
beads and grown overnight. Beads were imaged before and after
trypsinization, bead displacements were determined using a parti-
cle tracking algorithm, and the data were analyzed using COMSOL
finite element software. The median maximum traction stress in
diabetic coronary MVSMCs, 1075 Pa [625-­
2483 Pa], was signifi-
cantly larger than in controls, 689 Pa [377-­998 Pa] (P < 0.01, n = 32)
and the median spread area in diabetic MVSMCs, 8.5 × 10−9 m2
[4.3 × 10−9 − 12 × 10−9 m2], was also significantly larger than in
controls, 5 × 10−9 m2 [3.7 × 10−9 − 0.8 × 10−9 m2] (P = 0.03, n = 32).
There were no statistical differences in the average force or contrac-
tile moments. These data demonstrate that primary diabetic coro-
nary MVSMCs display enhanced contraction and larger spread areas
and further suggest that less stiff coronary MVSMCs can retain a
more contractile phenotype in the setting of diabetes. Funding: (NIH
R00116769 and S10 OD023438 and Nationwide Children's Hospital
to AJT).
C.9.4 | Unique mechanisms regulating the
pulmonary circulation: acid sensing ion channel 1
in pulmonary hypertension
Nikki Jernigan
Vascular Physiology Group, Department of Cell Biology and Physiology,
University of New Mexico Health Sciences Center, Albuquerque, NM, USA
Pulmonary hypertension (pHTN) is a complex, progressive con-
dition leading to increased pulmonary vascular resistance, right
heart failure and ultimately death. Although pHTN arises from a
 |
50 of 96       ABSTRACTS
variety of genetic and pathogenic causes, it is widely recognized
that structural alterations in the vascular wall contribute to all forms
of pHTN. A chronic shift in cellular metabolism from mitochondrial
oxidative phosphorylation to aerobic glycolysis underlies the hyper-
proliferative and anti-­apoptotic phenotype within the pulmonary
vasculature. These metabolic derangements are accompanied by
H+ extrusion creating an alkalotic intracellular pH while acidifying
the extracellular microenvironment that could activate the H+-­gated
acid sensing ion channel 1 (ASIC1). ASIC1 conducts both Na+ and
Ca2+ and activation leads to membrane depolarization and variety of
intracellular Ca2+ signaling events. Our previous studies show ASIC1
contributes to the development of pHTN; however the role of ASIC1
to metabolic-­mitochondrial dysfunction is unknown. We hypothe-
size ASIC1 contributes to metabolic dysfunction in pHTN as a result
of altered subcellular localization and regulation of plasma mem-
brane and mitochondrial membrane potential. In pulmonary arterial
smooth muscle cells from hypertensive animals, we found greater
localization of ASIC1 at the plasma membrane and loss of ASIC1 in
the mitochondria. Furthermore, ASIC1 contributes to the persistent
plasma membrane depolarization following pHTN, and loss of ASIC1
leads to mitochondrial hyperpolarization. Future studies will define
a role for ASIC1 in regulating mitochondrial dynamics and metabolic
dysfunction and will enable a fundamental understanding of ASIC1
in various proliferative and degenerative diseases and permit future
studies to evaluate the therapeutic potential of ASIC1.
S E S S I O N D.9 M EC H A N OTR A N S D U C TI O N I N
A N G I O G E N E S I S A N D R E M O D E LI N G
CH A I R S: D R CH A R LE S TH O D E TI A N D D R LI YA
YIN
D.9.1 | Microengineered physiological
biomimicry: human organs-­on-­chips
Dongeun Huh
Wilf Family Term Assistant Professor, Department of Bioengineering, University
of Pennsylvania, Philadelphia, PA, USA
Remarkable progress in life science and technology in the past cen-
tury has advanced our fundamental understanding of the human
body beyond our imagination. The ever-­increasing knowledge of
human anatomy and biology, however, has done surprisingly little
to improve the way we emulate and probe the complex inner work-
ings of the human body. Even today, our ability to model human
physiological systems relies on the century-­old practice of cell cul-
ture or animal experimentation that has raised significant scien-
tific, economic, and ethical concerns. The paucity of predictive and
human-­relevant model systems is emerging as a critical impediment
to our scientific endeavors for a wide variety of biomedical applica-
tions. This talk will present interdisciplinary research efforts in my
laboratory to develop advanced in vitro models engineered to recon-
stitute the structural and functional complexity of human organs.
Specifically, I will talk about (1) bioinspired microsystems that mimic
the alveoli and airways of the human lung during health and disease,
(2) a blinking eye-­on-­a-­chip microdevice that emulates the ocular
surface of the human eye, and (3) microengineered physiological
models of human reproductive organs.
D.9.2 | Mechanosensitive mechanism of
angiogenesis in lung regeneration and pathology
Akiko Mammoto
Medical College of Wisconsin, Milwaukee, WI, USA
Angiogenesis, the formation of new blood capillaries, plays a key role
in organ development and regeneration. Inhibition of angiogenesis
impairs compensatory lung growth after unilateral pneumonectomy
(PNX). Mechanosensitive transcription co-­activator, Yes-­associated
protein (YAP1) controls organ size and regeneration. However, the
role of endothelial YAP1 in lung vascular and alveolar morphogen-
esis remains unclear. We have found that knockdown of YAP1 in
endothelial cells (ECs) decreases angiogenic factor expression, and
inhibits EC sprouting and epithelial cell budding in vitro and vascu-
lar and alveolar morphogenesis in the gel implanted on the mouse
lung. The expression levels of YAP1 increase in ECs isolated from
the remaining mouse lungs after unilateral PNX and vascular for-
mation is stimulated in the post-­PNX mouse lungs. Knockdown of
endothelial YAP1 inhibits compensatory lung growth and vascular
and alveolar morphogenesis after unilateral PNX. Mechanical en-
vironment altered by unilateral PNX controls YAP1 expression and
contributes to post-­PNX lung growth. These findings suggest that
mechanosensitive endothelial YAP1 is required for lung vascular and
alveolar regeneration and modulation of YAP1 in ECs may be novel
interventions for the improvement of lung regeneration.
D.9.3 | How the secreted matrix
metalloproteinase ADAMTS5 inhibits
angiogenesis?
Bhuvanasundar Ranganathan; Dogan Can Kirman; Ruowen
Ge
Department of Biological Sciences, National University of Singapore, Republic of
Singapore
ADAMTS5 (A Disintegrin And Metalloproteinase with
ThromboSpondin motifs 5) is an endogenous angiogenesis inhibitor
previously identified by us. It is a member of the ADAMTS family
of secreted matrix metalloproteinase which digests multiple pro-
teoglycans in the extracellular matrix such as aggrecan, versican,
and brevican. Although its antiangiogenic function has been dem-
onstrated to be independent of its metalloproteinase activity, how
ABSTRACTS
ADAMTS5 inhibits angiogenesis remain unresolved. In this presen-
tation, we identify the cell-­surface nucleolin as a functional receptor
for ADAMTS5 in human umbilical vein endothelial cells (HUVECs).
ADAMTS5 triggers HUVEC apoptosis via interaction with the cell-­
surface nucleolin as demonstrated by antibody neutralization and
RNAi knockdown experiments. Fluorescent microscopy imaging,
proximity ligation assay (PLA) as well as biochemical fractionation
investigations show that both the mammalian cell produced 45 kDa
truncated ADAMTS5 (p45) and E. coli produced rTSR1 are quickly
endocytosed into HUVECs and then dominantly localized to the
nucleus. Using various chemical inhibitors to different endocytosis
pathways, we revealed that ADAMTS5 is internalized into HUVECs
mainly through a macropinocytosis pathway rather than classical en-
docytosis pathway through clathrin. How the internalized ADAMTS5
reach the nucleus and how the nucleus-­ADAMTS5 trigger HUVEC
apoptosis are currently under investigation. This work is funded
by the Singapore Ministry of Education grant MOE2014-­T2-­2-­150
awarded to Ruowen Ge.
D.9.4 | Mechanical control of vascular growth
and integrity
Charles Thodeti
Northeast Ohio Medical University, Rootstown, OH, USA
This talk will summarize our work on mechanosensitive ion chan-
nel TRPV4 mediated mechanotransduction in the regulation of en-
dothelial cell mechanosensitivity, vascular growth and integrity in
physiological and pathological angiogenesis
P OS TE R PR E S E NTATI O N S , M O N DAY
S E P TE M B E R 10 , 2 018
2+
MOPE003 | CaV3.1, T-­type Ca channels,
facilitate myogenic tone development in
mesenteric arteries
Naman Arora; Sergio Fabris; Timothy Hsu; Maria Sancho;
Donald G. Welsh
Robarts Research Institute, Department of Physiology and Pharmacology,
Schulich School of Medicine and Dentistry, University of Western Ontario,
London, ON, Canada
CaV3.1 is a T-­t ype Ca 2+
channel expressed in mesenteric vascu-
lature whose role in tone development remains uncertain. Using
wildtype and global CaV3.1−/− mice, this study ascertained the
linkage between CaV3.1 and myogenic tone development and
whether its contribution is dependent on the sex of the animal.
Functional phenotyping initially revealed that blood pressure was
|
      51 of 96
substantively lower in male/female CaV3.1−/− mice compared to
wild type animals. Whole body metabolism (assessed by the res-
piratory exchange ratio), was similar among the two mouse strains;
there was, however, a difference in VO2 and VCO2 was observed
in the CaV3.1−/− mice (females > males). Pressure myography re-
vealed that all mesenteric vessels (male & female) generated myo-
genic tone. Intriguingly, that portion of this response insensitive to
L-­t ype Ca2+ channel blockade was absent in CaV3.1−/− mice. Such
findings suggest that Ca2+ influx through Cav3.1 facilitates tone
development by elevating global [Ca2+] or initiating Ca2+ waves
from the sarcoplasmic reticulum (SR). While work remains prelimi-
nary, immuno-­labeling approaches (proximity ligation assay) note
that CaV3.1 co-­localizes with the inositol 1,4,5-­t risphosphate re-
ceptor type 1 (IP 3R1). The latter protein is the principal SR Ca2+
release channel responsible for repetitive Ca2+ wave generation.
In closing, our observations highlight a key role for CaV3.1 setting
cytosolic Ca2+ dynamics, myosin light chain phosphorylation and
basal tone generation in the resistance vasculature of male and
female mice. Supported by NSERC.
MOPE004 | TRPML1 regulates Ca2+ spark
activity and contractility of cerebral vascular
smooth muscle cells
Pratish Thakore1; Harry Pritchard1; Paulo Pires1; Evan
Yamasaki1; Yumei Feng1,2; Scott Earley1
1
Department of Pharmacology, Center for Molecular and Cellular Signaling in the
Cardiovascular System, University of Nevada, Reno School of Medicine, Reno, NV,
USA; 2Department of Physiology and Cell Biology, University of Nevada, Reno
School of Medicine, Reno, NV, USA
Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+ perme-
able, non-­selective cation channel localized to the membranes of
late endosomes and lysosomes. We investigated a potential role for
TRPML1 in the regulation of Ca2+ signaling activity in smooth muscle
cells (SMCs) of the cerebral vasculature. Super-­resolution microscopy
revealed significant nanoscale colocalization of TRPML1 with type-­2
ryanodine receptors (RyR2s) on the sarcoplasmic reticulum. Ca2+ re-
lease through RyR2 clusters generates high-­amplitude Ca2+ sparks
that are functionally coupled with large conductance K+ (BK) channel
clusters on the plasma membrane which conduct outward K+ currents
that hyperpolarize SMCs and counteract vasoconstriction. We hypoth-
esized that Ca2+ released from TRPML1 could stimulate RyR2 through
Ca2+-­induced Ca2+ release and regulate contractility. We found that
spontaneous Ca2+ sparks were absent in SMCs from global TRPML1
knockout (TRPML1−/−) mice. Perforated patch-­clamp electrophysi-
ology revealed a corresponding reduction in BK channel activity as
observed through decreased frequency of spontaneous transient
outward currents (STOCs). Furthermore, the TRPML activator com-
pound MK6-­83 increased STOC frequency in SMCs from wild-­type
but not TRPML1−/− mice. Ex vivo pressure myography experiments
demonstrated hypercontractility of cerebral resistance arteries from
 |
52 of 96       ABSTRACTS

TRPML1−/− mice in response to increasing intraluminal pressure and
the thromboxane A2 receptor agonist U46619. Comparable results
were obtained in human mesenteric arteries following knockdown of
TRPML1. Radio-­telemetry blood pressure recordings revealed sys-
tolic hypertension in TRPML1−/− mice, suggesting elevated systemic
vascular resistance. We conclude that TRPML1 is critically important
for the regulation of SMC contractility and vascular resistance, and
ultimately blood pressure. Funded by R01HL091905; R01HL137852;
R01HL139585; R0HL113785; K99HL140106.
MOPE007 | Diastolic dysfunction and AST as
markers of coronary artery disease
Ana-Maria Vintila1; Mihaela Horumba2; George Gabriel
Cristea2; Cristina Bulei2; Stefan Dragos Tudorica3; Vlad
Damian Vintila4; Cezar Cornel Tudorica2
1
Carol Davila University of Medicine and Pharmacy, Coltea Clinical Hospital,
Department of Internal Medicine, Bucharest, Romania; 2Coltea Clinical Hospital,
Internal Medicine and Cardiology Department, Bucharest, Romania; 3CMI
Tudorica Steluta, Bucharest, Romania; 4Carol Davila University of Medicine and
Pharmacy, University Emergency Hospital, Department of Cardiology, Bucharest,
Romania

MOPE006 | Adipose tissue-­produced Echocardiography and coronary computed tomography angiogra-

inflammation contributes to the impaired flow-­ phy (CCTA) are resources that have become readily available in di-
agnosing coronary artery disease even in the absence of coronary
induced dilations in visceral and subcutaneous
catheterization.
adipose arterioles in morbidly obese adults
We performed a retrospective study encompassing a total of
Abeer Mahmoud1; Mohamed Ali1; Dina Naquiallah1; Richard 57 patients out of whom 14 women (24.56%) and 43 men (75.43%)
Severin1; Francesco Bianco2; Antonio Gangemi2; Mario with a mean age of 64.5 ± 9.34 years. None of the patients suffered
Masrur2; Chandra Hassan2; Shane Phillips1
1
an acute coronary syndrome at the time of data collection; most
Department of Physical Therapy, University of Illinois at Chicago, Chicago, IL,
exhibited at least one cardiovascular risk factor: either high blood
USA; 2Department of Surgery, University of Illinois at Chicago, Chicago, IL, USA
pressure, dyslipidemia or type 2 diabetes mellitus. Hypertension
was the most prevalent, with 37 out of 57 patients (65%) undergoing
Introduction: Obesity is a major risk factor for cardiovascular dis-
some sort of antihypertensive treatment be it either beta-­blockers,
ease. We previously demonstrated reductions in microvascular
diuretics, CCB, ACEi/ARBs or a combination of the above, followed
flow-­induced dilation (FID) in adipose tissues (AT) from obese adults
closely by dyslipidemia (58% of patients). Laboratory assays and data
(OB). We now hypothesize a role of AT-­produced adipokines in the
from echocardiographic and CCTA studies were made available for
impaired FID.
all patients. Further analysis showed a bivariate correlation between
Methods: We obtained subcutaneous (SAT) and visceral AT (VAT) bi-
total calcium score and diastolic dysfunction (E/A and E/E′ ratio),
opsies from bariatric-­surgery patients (n = 22; age: 36 ± 7 years; BMI:
P < 0.001. Moreover, AST but not ALT levels correlated with total
50.7 ± 8.7 kg/m2) and gluteal SAT from non-­obese (NOB) sedentary
coronary calcium mass for left main, circumflex and right coronary
adults (n = 8; age: 35 ± 4 years; BMI: 26.4 ± 3 kg/m2). This NIH-­
arteries, P < 0.001.
funded research was approved by UIC's Institutional Review Board,
Diastolic dysfunction, as a consequence of longstanding hyper-
and written consents were obtained. AT-­isolated arterioles were
tension, correlates with higher coronary calcium scores and should
tested for FID (pressure gradients of ∆10-­∆100 cm H2O) ± NFκB/
guide the clinician towards treating risk factors more aggressively so
TNFα-­inhibitory peptides, superoxide dismutase mimetic (Tempol) or
as to slow down the progression of coronary artery disease. Patients
nitric oxide (NO)-­synthase inhibitor (LNAME). We measured NO and
with known coronary artery disease and high levels of AST in the
reactive oxygen species (ROS) in the arterioles and gene expression
absence of acute coronary syndrome may require closer monitoring
of leptin, TNFα, IFNγ, IL1β, IL6, and IL8 in the AT.
as this rise may indicate lesions of the main coronary arteries.
Results: FID was 35-­50% higher in NOB than OB across all pres-
sure gradients (P < 0.05). LNAME reduced FID in NOB arterioles
(−55%, P < 0.001) and to a lower extent in OB (−16%, P = 0.01).
NFκB/TNFα inhibition and Tempol improved FID in OB arterioles; MOPE008 | Does coronary microvascular
to a greater extent in VAT (48-­60%) compared to SAT (35-­45%). dysfunction cause heart failure?
They also increased NO production and arteriolar sensitivity to
Vahagn Ohanyan; Tatevik Hakobyan; Lindsey Rinker; Molly
LNAME and decreased ROS generation. Adipokines were 2-­4-­fold Enrick; Tigran Gyulkhasyan; Punita Peketi; Christopher L.
higher in OB compared to NOB and correlated positively with FID Kolz; Liya Yin; William M. Chilian
and NO production (r = 0.82, 0.91; P < 0.001) and negatively with Northeast Ohio Medical University, Rootstown, OH, USA
ROS (r = −0.71, P < 0.01). Conclusion: Our results demonstrate an
impaired microvascular reactivity in obese adults that may be attrib- Heart failure (HF) is a leading cause of death in the USA and
uted to higher levels of AT-­produced adipokines. Canada. Unfortunately, optimal medical therapies only slow the
Conclusion: Our results demonstrate an impaired microvascular re- progression of the disease. No current treatment stops or reverses
activity in obese adults that may be attributed to higher levels of the progression. Our thesis is that if the causal mechanism was
AT-­produced adipokines. targeted the progression of the disease could be stopped and
ABSTRACTS
potentially reversed. Although the current treatments for HF
decrease cardiac work, we hypothesize that a better treatment
should increase blood flow to the heart. This hypothesis is based
on the view that heart failure is a disease of insufficient myocardial
perfusion in micro-­areas of the heart. Over time these microscopic
areas of injury accumulate, leading to failure. Accordingly, we de-
termined the relationship between MBF and cardiac work (wall
stress-­r ate product [WSRP]) in three groups: control mice (CTRL),
mice with HF (induced by transaortic constriction), and HF mice
treated with the coronary vasodilator, Chromonar (2 weeks of
treatment). MBF and WSRP were measured during norepinephrine
infusion (to increase cardiac work) in anesthetized mice. The rela-
tionship between WSRP and MBF is uncoupled in HF, i.e., when
work increases MBF does not. Chromonar treatment recoupled
work with MBF and also produced coronary dilation (increased
MBF per unit of work). These changes in flow paralleled cardiac
function in the HF and HF+Chromonar groups. Based on these
findings, we propose that a cause of HF is inadequate MBF to meet
the metabolic demands of the working heart. Pharmacological
coronary vasodilation with Chromonar increased MBF in HF and
reversed the functional decline cardiac function.
MOPE009 | Isolated microvessels promote
human induced pluripotent stem cell-­derived
cardiomyocyte (hiPSC-­CM) survival and cardiac
functional recovery in infarcted rat hearts
1 2 1,2 1,3,4,5
Xuetao Sun ; Jun Wu ; Ren-ke Li ; Sara S. Nunes
1
Toronto General Hospital Research Institute, University Health Network,
Toronto, ON, Canada; 2Division of Cardiovascular Surgery, Department of
Surgery, University Health Network and University of Toronto, Toronto, ON,
Canada; 3Laboratory of Medicine and Pathobiology, University of Toronto,
Toronto, ON, Canada; 4Heart & Stroke/Richard Lewar Centre of Excellence,
University of Toronto, Toronto, ON, Canada; 5Institute of Biomaterials and
Biomedical Engineering, University of Toronto, Toronto, ON, Canada
Background: While human stem cell-­derived cardiomyocytes (hSC-­
CMs) are a promising source for CMs for tissue regeneration post-­
myocardial infarction (MI), its poor survival post-­
transplantation
hinders its application. Death of transplanted CMs occurs in the first
2-­3 days post-­transplantation due to ischemia. Attempts to promote
blood perfusion via addition of endothelial cells and/or angiogenic
factors require weeks for new vessels to form and to carry blood.
Our objective is to develop a strategy to accelerate vascularization
and promote the survival of transplanted CMs.
Methods and Results: We used ready-­
made microvessels from
adipose tissue to form a vasculature and to carry blood within
the first day post implantation. We co-­implanted hiPSC-­CMs and
microvessels into the hearts of male immuno-­compromised rats
that underwent left anterior descending artery (LAD) ligation to
mimic MI and assessed cardiac function and histology. Compared
to hiPSC-­CM transplantation alone, microvessels promoted a sig-
nificant increase in hiPSC-­
CM survival. Echocardiography and
|
      53 of 96
pressure–volume (PV) loop analysis performed 4 weeks post-­MI
revealed that hiPSC-­CM transplantation attenuated post-­infarct
ventricular dilation and enhanced left ventricular contractility. Co-­
transplantation of hiPSC-­CMs with microvessels showed signifi-
cantly superior functional recovery compared to hiPSC-­CMs alone.
Conclusions: Microvessels improve the survival of transplanted
hiPSC-­CMs and ameliorates cardiac function. Given the ability to
use microvessels harvested from adipose tissue from patients, this
study presents a new, personalized approach to cell-­based therapy
for heart failure post-­MI whereby improving hiPSC-­CM survival it is
possible to promote remuscularization.
Acknowledgements: This work was funded by CIHR (137352,
143066 and 153160) and a J.P. Bickell foundation (1013821).
MOPE012 | Down regulation of gamma-­
adducin in the cerebral vasculature promotes
blood-­brain barrier leakage and contributes to
hypertension-­related cognitive deficits
Fan Fan; Shaoxun Wang; George Booz; Richard Roman
Department of Pharmacology and Toxicology, University of Mississippi Medical
Center, Jackson, MS, USA
We recently identified an inactivating mutation in Add3 in FHH
rats that impairs the myogenic response and cerebral autoregula-
tion. The present study examined whether it promotes BBB leak-
age and contributes to hypertension-­related dementia. The inner
diameter of MCA decreased in control but not in FHH rats when
perfusion pressure was increased from 60 to 140 mm Hg. The
pressure in terminal pial arteries was increased in FHH rats when
MAP was increased from 100 to 160 mm Hg, and the impaired cer-
ebral autoregulation in FHH rats was rescued by introduction of
wildtype Add3. The penetrating arterioles were larger and capil-
lary density was reduced in FHH rats. BBB leakage was greater in
FHH rats after induction of hypertension. Hypertensive FHH rats
exhibited marked neurodegeneration in the neocortex and hip-
pocampus. Gap junctions were damaged in hypertensive FHH rats
in association with neuronal and mitochondrial degeneration in the
vacuolated area near leaky capillaries. The hypertensive FHH rats
took 2.5 times longer to escape from an eight-­arm water maze than
hypertensive control rats suggesting spatial learning and memory
dysfunction. The expression of p-­PKC was reduced in the brain of
FHH than control rats. These findings suggest that the K572Q mu-
tation in FHH rats decreases the expression and activity of Add3
which is involved in phosphorylation of PKC. This contributes to the
impaired myogenic response and cerebral autoregulation, therefore
elevating the transmission of pressure in penetrating arterioles
that promotes BBB leakage and neurodegeneration, all of which
contribute to dementia with hypertension and aging. This study
was supported by grants AG050049, P20GM104357, HL36279,
DK104184 and HL138685 from the National Institutes of Health;
and 16GRNT31200036 from the American Heart Association.
 |
54 of 96       ABSTRACTS
MOPE014 | Rapid clinical assessment of the
sublingual microcirculation – visual scoring using
microVAS in comparison to standard semi-­
automated analysis
1 2 1
Joel Sardinha ; Sean MacKinnon ; Christian Lehmann
1
Department of Anesthesia, Pain Management and Perioperative Management,
Dalhousie University, Halifax, NS, Canada; 2Department of Psychology and
Neuroscience, Dalhousie University, Halifax, NS, Canada
Rationale: Standard microcirculation analysis using
automated analysis is expensive, time consuming, and expertise
dependent making it clinically unfeasible. We proposed a novel vis-
ual scoring system (microVAS) for the analysis of microcirculation
videos that can be performed at the patient bedside in real time.
Objective: Validate our microVAS score by training health profes-
sionals unfamiliar with the microcirculation field to use our micro-
VAS score and compare their scores to the standard method of
semi-­automated analysis using AVA3 software.
Methods: Using a prospective double-­blind study design, we re-
cruited and trained 20 participants to use our microVAS score.
Participants scored 40 videos (from 22 healthy and 18 septic pa-
tients) for MFI and PPV. The same 40 videos were analyzed by an
expert using the gold standard semi-­automated method of analysis.
Results: Overall correlation of MFI was r = 0.3283 (95% CI 0.27-­
0.39), P < 0.05; overall correlation of PPV was r = −0.1123 (95% CI
−0.18 to −0.04), P < 0.05. The Krippendorff's alpha for MFI was
0.56 (healthy videos: α = 0.34, sepsis videos: α = 0.31). For PPV
Krippendorff's alpha was 0.43 (healthy videos: α = 0.56, sepsis vid-
eos: α = 0.17).
Conclusions: Overall for both MFI and PPV, there was a small cor-
relation between our microVAS score and AVA 3 scores. Regarding
inter-­rater reliability both MFI and PPV showed fair agreement be-
tween raters. Going forward multiple improvements to the micro-
VAS scoring system as well as the training program are suggested to
improve reliability and consistency.
Funding: Dalhousie Medicine.
MOPE015 | The presence of synchronized
perfusion dips in the microcirculation of the
resting nail bed
Robin Mirdell1,2,3; Aukje Nienke Lemstra-Idsardid3; Simon
Farnebo1,2; Erik Tesselaar1,4
1
Department of Clinical and Experimental Medicine, Faculty of Health Sciences,
Linköping University, Linköping, Sweden; 2Department of Plastic Surgery, Hand
Surgery, and Burns, Linköping University, Linköping, Sweden; 3University of
Twente, Enschede, The Netherlands; 4Department of Medical Radiation Physics,
Department of Medical and Health Sciences, Linköping University, Linköping,
Sweden
Objectives: Laser speckle contrast imaging (LSCI) has seen limited
use in the study of perfusion dynamics such as vasomotion. The aim
of this study was to investigate the effects of a prolonged seated
position on perfusion dynamics in the nail bed using LSCI.
Methods: Perfusion was recorded in digits II to IV bilaterally for
20 minutes during two separate sessions in ten healthy volunteers.
The acclimatization period was 5 minutes for the 1st session and
20 minutes for the 2nd. Perfusion variability and the presence of re-
curring perfusion dips were analyzed. A digital nerve block was done
to verify suspected nervous origin of phenomenon.
Results: Synchronized phases of vasoconstriction were observed in
all subjects with perfusion dips in all digits bilaterally and simultane-
semi-­
ously. Application of a digital nerve block abolished perfusion dips.
The frequency of this phenomenon increased by 25.0% (95% CI:
1.6-­49.2%) in the left-­hand digits after a prolonged seated position.
Perfusion variability increased by 11.6% (95% CI: 2.6-­20.3%) in the
digits of the left hand. Perfusion changes in right-­hand digits did not
significantly increase. During the 1st session, temperature increased
by 2.7°C (1.1-­4.2) while it decreased by 1.3°C (0.2-­2.4) during the
2nd session.
Conclusion: The observed perfusion dips are of a centrally mediated
nervous origin but are also affected by local factors. They are af-
fected by seating duration and differ between left and right hands,
likely because of local micro perfusion dips. This phenomenon seems
related to digital thermoregulation.
MOPE016 | Interobserver reliability of laser
speckle contrast imaging in the assessment of
burns
Robin Mirdell1,2; Simon Farnebo1,2; Folke Sjöberg1,2; Erik
Tesselaar1,3
1
Department of Clinical and Experimental Medicine, Faculty of Health Sciences,
Linköping University, Linköping, Sweden; 2Department of Plastic Surgery, Hand
Surgery, and Burns, Linköping University, Linköping, Sweden; 3Department
of Medical Radiation Physics, Department of Medical and Health Sciences,
Linköping University, Linköping, Sweden
Objectives: Laser speckle contrast imaging (LSCI) is an emerging
technique for the assessment of burns and interobserver differences
have not been studied. The aim of this study was to compare assess-
ments of perfusion images by different professional groups.
Methods: Twelve observers, without previous training in LSCI, were
evenly recruited from three professional groups: plastic surgeons
with experience in assessing burns, nurses with experience in treat-
ing burns, and junior doctors with limited experience of burns. Ten
cases were included, and each case consisted of one digital photo
pre-­marked with a region of interest (ROI) and two unmarked perfu-
sion images. The perfusion values acquired were used to generate
a LSCI recommendation. Each observer estimated the burn depth
using all available information.
Results: Perfusion values and perfusion trends between the dif-
ferent observers had an intraclass correlation (ICC) of 0.96 (95%
CI 0.91-­
0.99). The assessment of burn depth by the observers
yielded an ICC of 0.53 (95% CI: 0.31-­0.80) and an accuracy of 0.53
ABSTRACTS |
      55 of 96

(weighted kappa). LSCI recommendations had an ICC of 0.95 (95%
CI: 0.90-­0.99).
Conclusion: Observers can reliably identify the same ROI, which
results in observer-­independent perfusion measurements, irrespec-
tive of their experience with burns. To make accurate assessments
of the burn depth was more difficult and was not improved by previ-
ous experience. The LSCI recommendation was more accurate in all
groups of observers. Introducing LSCI measurements would be likely
to facilitate further understanding among clinicians and improve
early assessment of burns.
MOPE019 | Inflammatory responses to
chlorinated lipids mimic those invoked by cecal
ligation and puncture in rat mesentery
Hong Yu1; Meifang Wang1; Theodore J. Kalogeris1; Ricardo
J. Restrepo1; David A. Ford2; Ronald J. Korthuis1,3
1
Department of Medical Pharmacology and Physiology, University of Missouri
School of Medicine, Columbia, MO, USA; 2Edward A. Doisy Department of
Biochemistry and Molecular Biology, Center for Cardiovascular Research,
Saint Louis University School of Medicine, St. Louis, MO, USA; 3The Dalton
Cardiovascular Research Center, and University of Missouri School of Medicine,
Columbia, MO, USA

Sepsis is a life-­
threatening syndrome characterized by dysregu-
MOPE017 | Passive movement training as a lated systemic inflammatory responses that leads to organ dys-
treatment for non-­healing diabetic foot ulcers: a function. We have recently suggested that the chlorinated lipid,
microcirculatory perspective 2-­chloropalmitic acid (ClPA) and 2-­chlorohexadecanal (ClHDA), may
1,2 1 1 serve as mediators of the inflammatory responses to sepsis. The ob-
Tue Smith Jørgensen ; Hans Gottlieb ; Hans Gottlieb ; Stig
Brorson3; Ylva Hellsten2; Birgitte Høier2 jective of this study was to compare the effects of cecal ligation
1
Department of Orthopedic Surgery, Herlev University Hospital; 2Department and puncture (CLP), a polymicrobial peritonitis model that mimics
of Nutrition Exercise and Sports, Faculty of Science, University of Copenhagen; sepsis in humans, to those invoked by mesenteric superfusion with
3
Department of Orthopedic Surgery, Zealand University Hospital, Department of ClPA or ClHDA on leukocyte-­endothelial cell interactions, platelet-­
Clinical Medicine, University of Copenhagen
endothelial cell interactions, mast cell activation, and microvascular
barrier function in rat mesenteries. All animal protocols were ap-
Background: Diabetic foot ulcers are serious complications in diabe-
proved by the Institutional Animal Care and Use Committee at the
tes mellitus. Only two-­thirds eventually heal, and 15-­20% ultimately
University of Missouri. Six hours after CLP or sham surgery (control)
requires amputation. High oxygen tension and perfusion of the limb
in anesthetized rats, adhesive responses, venular protein leakage,
is essential for wound healing, and interventions that increase blood
and mast cell activation were quantified using intravital microscopic
flow and promote capillary growth may be useful in wound treat-
methods. In other groups of rats, the exposed mesenteries were su-
ment and prevention of amputation.
perfused with ClPA or ClHDA at 10 μmol/L (a dose within the range
Objective: To investigate the correlation between passive training of
of plasma concentrations reported for septic patients or rats) for
the lower limbs, and wound healing in diabetic patients.
60 minutes, during which time these same inflammatory responses
Materials and Methods: A randomized clinical trial of wound heal-
were monitored. CLP or mesenteric superfusion with ClPA or ClHDA
ing with passive movement training (1 hour/3 times per week for
induced marked increases in the numbers of rolling and firmly ad-
8 weeks) as an intervention. Nine and 7 participants were included
herent leukocytes and platelets relative, mast cell activation, and
in the training and control group, respectively. Wound size was as-
significant albumin leakage that were similar in magnitude. Thus, our
sessed using Image J. Femoral bloodflow was measured by Doppler,
results indicate that the putative septic mediators, ClPA and ClHDA,
skin perfusion pressure by photocell technique, and distal blood
provoke proinflammatory and prothrombogenic effects that mimic
pressure by strain gauge method. Muscle samples will be analyzed
the responses to CLP. Supported by NIH grant GM-­115553.
for proteins and capillaries.
Results: Wound area was reduced 77% in the training group com-
pared to 36% in the control group (P = 0.099). Resting perfusion of
the limb was lower in the training group compared to the control
group; femoral bloodflow (138 vs 191 mL/min), skin perfusion pres-
sure (77 vs 98 mm Hg) and distal pressure (98 vs 107 mm Hg).
Conclusion: We found a statistically non-­significant, but clinically
relevant effect on wound healing after passive movement training.
The passive movement training did not result in an increased resting
bloodflow.
Ethics: Approved by the Committee of Ethics project H-­15008102
and the Danish Data Protection Agency journal nr: 03953. og ID-­nr.:
HGH-­2015-­023.
 |
56 of 96       ABSTRACTS

MOPE020 | Determining the significance of MOPE022 | Alterations in ghrelin and GHSR

perivascular cell infection by oncolytic virus with cardiovascular inflammation in duchenne
Madison Turk1,2,3; Victor Naumenko1,4; Franz Zemp1,2,3; muscular dystrophy
Jahanara Rajwani2,3,5; Jeff Biernaskie2,6,7; Doug Maedeh Naghibosadat1; Tyler Lalonde2; Leonard G.
Mahoney1,2,3,5; Craig Jenne1,8 Luyt2,3,4; Lisa M. Hoffman5,6,7; Savita Dhanvantari1,6,7,8
1
Department of Microbiology, Immunology and Infectious Diseases, Faculty 1
Dept of Pathology and Laboratory Medicine, Western University, London, ON,
of Medicine, University of Calgary, Calgary, AB, Canada; 2Alberta Children's
Canada; 2Dept of Chemistry, Western University, London, ON, Canada; 3London
Hospital Research Institute, Calgary, AB, Canada; 3Arnie Charbonneau Cancer
Regional Cancer Program, London, ON, Canada; 4Depts of Oncology and
Institute, Calgary, AB, Canada; 4National University of Science and Technology
Medical Imaging, Western University, London, ON, Canada; 5Dept of Anatomy
“MISIS”, Moscow, Russia; 5Department of Biochemistry and Molecular Biology,
and Cell Biology, Western University, London, ON, Canada; 6Dept of Medical
Faculty of Medicine, University of Calgary, Calgary, AB, Canada; 6Hotchkiss
Biophysics, Western University, London, ON, Canada; 7Imaging Program, Lawson
Brain Institute, University of Calgary, Calgary, AB, Canada; 7Department of
Health Research Institute, London, ON, Canada; 8Metabolism and Diabetes
Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine,
Program, Lawson Health Research Institute, London, ON, Canada
University of Calgary, Calgary, AB, Canada; 8Department of Critical Care
Medicine, Faculty of Medicine, University of Calgary, Calgary, AB, Canada
Introduction: Duchenne muscular dystrophy (DMD) is a severe

Oncolytic Viral Therapy (OVT) is an emerging cancer treatment that neuromuscular disease of skeletal and myocardial degeneration.

has captured the interest of both medical and non-­medical personal Eventually, dilated cardiomyopathy develops from ischemia, inflam-

alike. The concept of using viruses to treat cancer is a very intrigu- mation and fibrosis. Both cardiomyocytes and vascular endothelial

ing alternative to current cancer therapies; which are often associ- cells produce the hormone ghrelin and its receptor, the growth hor-

ated with unpleasant side effects. The current dogma of oncolytic mone secretagogue receptor (GHSR) which may be indicators of car-

viruses (OV) describes them as having the ability to infect and lyse diovascular inflammation in DMD.

tumour cells while essentially ignoring healthy host cells. Tumor lysis Methods: We used a murine model of DMD (mdx:utrn−/−) in which

causes direct destruction of cancer cells and subsequently exposes cardiomyopathy had been detected at 15-­17 weeks of age (n = 3)

tumor antigens. The host immune system will recognize these anti- (ethics approval #2008-­
067). Myocardial tissues were assessed

gens and prime an anti-­tumor attack against them. Although it seems for GHSR using the fluorescent peptide analog Cy5-­ghrelin(1-­19).

straightforward, the specific mechanisms of OVT and how exactly Des-­acyl ghrelin binding was detected with Cy5-­des-­acyl ghrelin(1-

it enhances tumor clearance are yet to be explained. Intravital ­19). To determine whether GHSR and ghrelin levels correlate with

Microscopy (IVM) can help reveal physiological information about inflammation, ghrelin, interleukin-­6 (IL-­6) and the fatty acid trans-

OVT in ways that in-­vitro assays cannot. With the use of IVM, our porter CD36 were quantified by immunofluorescence. Fibrosis was

lab has determined that tumor cell infection and lysis may not be the detected using Masson's trichrome staining.

center of the OVT story. We do not often see OV infecting tumor Results: GHSR (P < 0.002), IL-­6 (P < 0.001) and CD36 (P < 0.005) lev-

cells by IVM; however, we consistently see tumor-­associated peri- els were increased in the myocardium of mdx:utrn−/− mice. There

cytes getting infected. These findings have been reproduced in sev- was a strong positive correlation between GHSR and IL-­6 (P < 0.04,

eral tumor cell lines and using different viruses. This suggests a shift r = 0.86). CD36 correlated positively with fibrosis and necrosis

of importance to mechanisms other than tumor lysis in the efficacy (P < 0.02, r = 0.925). Interestingly, large cardiac vessels, but not mi-

of OVT. Establishing the significance of infected pericytes could il- crovessels, were strongly positive for Cy5-­desacyl ghrelin (1-­19) in

luminate some of the unknown mechanisms of OVT and thus help to mdx:utrn−/− mice (P < 0.02).

make the therapy more effective in a clinical setting. Conclusion & Significance: We report that GHSR associates with

Research funded by a grant from Women in Insurance Cancer cardiac inflammation and that CD36 associates with vascular inflam-

Crusade (WICC). mation in mdx:utrn−/− mice. Des-­acyl-­ghrelin could be a marker of

inflammation in large blood vessels in the heart. We propose that
GHSR and des-­acyl ghrelin are markers of cardiovascular inflam-
mation in DMD. Supported by the Canadian Institutes for Health
Research (CIHR).
ABSTRACTS
MOPE023 | High fat diet promotes perfusion
recovery and a pro-­regenerative phenotype of
the ischemic skeletal muscle
Matthew De Ciantis1; Emmanuel Nwadozi1; Vrati Mehra1;
Stephanie Milkovich2,3; Christopher Ellis2,3; Tara Haas1
1
School of Kinesiology & Health Science, York University, Toronto, ON, Canada;
2
Department of Medical Biophysics, University of Western Ontario, London, ON,
Canada; 3Robarts Research Institute, University of Western Ontario, London,
ON, Canada
Peripheral artery disease generates an ischemic muscular environ-
ment that is characterized by tissue hypoxia, cellular death and
compromised vascular remodeling. Long term viability of ischemic
muscle relies on effective re-­establishment of blood flow and local
tissue repair. High fat (HF) diet and obesity can cause low-­grade sys-
temic inflammation and microvascular adaptations that may impact
ischemia outcomes. We hypothesized that HF diet would impair
microvascular hemodynamics, exacerbate inflammation and dimin-
ish blood flow recovery and the regenerative capacity of ischemic
muscle. C57BL/6 male mice (ethically approved) were fed HF or
normal chow (NC) diets (9 weeks). Intravital microscopy of skeletal
muscle was used to measure resting capillary hemodynamics in one
group of mice. Surprisingly, capillaries of HF mice exhibited higher
red blood cell velocities, supply rates and O₂ saturations compared
to NC. A second group underwent unilateral common femoral ar-
tery ligation and muscle was collected at 4-­or 14-­days post-­ligation.
Laser-­Doppler imaging showed blood-­flow recovery was greater in
HF mice at days 7, 11 and 14 compared to NC mice. Interestingly,
capillary-­to-­fiber ratio was elevated by HF diet alone. At day 14, cap-
illary density increased in the ischemic muscle of both NC and HF
mice. 14 days post-­ligation, pro-­inflammatory macrophage marker
(Emr1) remained elevated only in the ischemic muscle of NC mice,
corresponding with higher mRNA levels of pro-­fibrotic gene Col1a1
compared to HF mice. Our data suggest that moderate duration HF
diet promotes a more favorable microenvironment that potentially
improves regeneration of ischemic muscle. Funded by Heart and
Stroke Foundation.
MOPE024 | Oxygen dependence of oxygen
consumption in rat muscle for diabetic and
hypertensive animals
Roland Pittman; Sami Dodhy; Alexander Liles; Habiba Shah;
Aleksander Golub
Department of Physiology and Biophysics, Virginia Commonwealth University,
Richmond, VA, USA
Tissue oxygenation, in terms of PO2, depends upon the balance be-
tween oxygen demand and supply. Oxygen demand is determined
by the activity of the mitochondria. This study sought to determine
differences in the PO2 dependence of oxygen consumption (VO2)
between diabetic or hypertensive animals and healthy controls. The
|
      57 of 96
PO2 dependence of VO2 in vitro is described by Michaelis-­Menten
kinetics, where Km is about 0.1 mm Hg. A recent in vivo finding is
that Km is 10 mm Hg or about 100 times higher than the in vitro
results. The question arises whether the PO2 dependence of VO2
is different for healthy animals vs their diseased counterparts. Two
studies were performed, with a model of type-­2 diabetes (Goto-­
Kakizaki or G-­K rats vs Wistar) and with a model of hypertension
(Spontaneously Hypertensive Rats, SHR vs Wistar Kyoto, WKY).
VO2 was measured in the spinotrapezius muscle, using phospho-
rescence quenching microscopy. VO2 vs PO2 data were plotted and
fit to Hill's equation to obtain values for Vmax and Km. Results for
Vmax (nl O2 cm3/s) were 216 ± 23 (G-­K ), 132 ± 9 (Wistar), 170 ± 18
(SHR), and 149 ± 14 (WKY). Results for Km (mm Hg) were 8.5 ± 0.5
(G-­
K ), 8.0 ± 0.6 (Wistar), 22.3 ± 3.6 (SHR), and 8.9 ± 0.8 (WKY).
What stands out among these results is that Vmax is significantly
higher for G-­K rats than their controls and Km is significantly higher
for SHRs than their controls. Thus, in two animal models of disease,
differences in the oxygen demand component of oxygen transport
which might have important implications for tissue oxygenation.
(Funding VCU).
MOPE025 | Reduced mtDNA copy number
and expression in A7r5 smooth muscle cells
contributes to Angiotensin II-­mediated changes
in cell cycle regulation
Andrew D. Pauls1; Pola Kalinowski1; Farzad Sharif1; Bob E.
Mulamba1; Damon Poburko1,2
1
Department of Biomedical Physiology and Kinesiology, Simon Fraser University,
Burnaby, BC, Canada; 2Centre for Cell Biology, Development and Disease, Simon
Fraser University, Burnaby, BC, Canada
Elevated angiotensin II (AngII) stimulates vascular hypertrophy via
Akt-­
dependent circumvention cell-­
cycle checkpoints, which in-
creases pulse pressure in primary hypertension. AngII also causes
mitochondrial fragmentation and oxidative damage of mitochondrial
DNA (mtDNA), and mtDNA depletion in other cell types causes ab-
errant cell cycle regulation. We hypothesized that reduced mtDNA
content and gene expression contribute to the effect of AngII on
the smooth muscle cell cycle and ploidy. We treated rat A7r5 cells
for 1-­4 weeks with AngII (10 nmol/L), H2O2 (3 μmol/L), rotenone
(10 nmol/L) or 2,3-­
dideoxycytidine (ddC, 3 μmol/L). A7r5 prolif-
eration, cell cycle, ploidy (Hoechst-­33342), DNA synthesis (Edu),
mtDNA density (anti-­double-­stranded DNA), mitochondrial polari-
zation (MitoTracker Orange) and α-­smooth muscle actin were im-
aged on 96-­well plates and correlated with mtDNA copy number and
mRNA expression (ND1, ND3 and ND4) measured by droplet digital
PCR. mtDNA copy number and mRNA were reduced by all stress-
ors at 7 and 14 days, and upregulated at 28 days. DNA synthesis did
not differ between treatments (~9% of cells), but AngII, H2O2 and
rotenone increased the fraction of S-­phase and G2/M/4N cells vs
control (17%) at 7 days. Cells with high levels of mtDNA encountered
 |
58 of 96       ABSTRACTS
an intra-­S-­phase check point, whereas cells with mtDNA depletion
exhibited M-­phase arrest or tetraploidy. AngII initially (7 days) in-
creased mtDNA immunoreactivity paired with loss of mitochondrial
gene expression and eventually (28 days) decreased mtDNA and
more M-­phase/tetraploid cells. In conclusion, altered mtDNA con-
tent and expression may contribute to smooth muscle polyploidy in
response to AngII. Funding: NSERC Discovery grant.
MOPE26 | Acid sensing ion channel
1 contributes to angiotensin II-­induced
hypertension
Selina M. Garcia; Nancy L. Kanagy; Nikki L. Jernigan
Vascular Physiology Group, Department of Cell Biology and Physiology,
University of New Mexico Health Sciences Center, Albuquerque, NM, USA
The acid sensing ion channel 1 (ASIC1) is part of the amiloride-­
sensitive degenerin/epithelial sodium channel superfamily and
conducts both Na+ and Ca2+. ASIC1 is expressed in the vascu-
lar smooth muscle cells and endothelial cells of various vascular
beds. However, little is known about the role of these ASIC1 in
the regulation of vascular resistance and mean arterial blood pres-
sure (MABP). Based on preliminary experiments showing ASIC1
contributes to acetylcholine (Ach)-­induced endothelial cell Ca2+
entry and vasodilation in small mesenteric arteries, we hypoth-
esize that ASIC1 contributes to blood pressure homeostasis and
that a loss of ASIC1 leads to increased MABP and exaggerated
angiotensin II (Ang II)-­
induced hypertension. To test this hy-
pothesis, aged (60 weeks) male wild type (+/+) and ASIC1 global
knockout (−/−) mice were implanted with radiotelemeters to as-
sess baseline MABP and responses to angiotensin II (600 ng/
kg/d for 28 days). Average 24-­h our baseline MABP were slightly
higher in aged ASIC1−/− mice (118 ± 1 mm Hg; n = 6) compared to
ASIC1+/+ (109 ± 6 mm Hg; n = 7). In contrast to our hypothesis,
Ang II (28 days following infusion) significantly increased MABP
in ASIC1+/+ (Δ of 36 ± 9 mm Hg) but did not significantly increase
MABP in ASIC1−/− mice (Δ of 6 ± 3 mm Hg), suggesting loss of
ASIC1 is protective against Ang II-­induced hypertension. Future
studies will examine the mechanism by which ASIC1 contributes
to Ang II-­induced hypertension.
Supported by National Heart, Lung, and Blood Institute Grants
R01 HL-­111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta).
MOPE029 | Conducted vasodilation initiated
by capillary TRPA1 channels in the cerebral
microcirculation
Paulo Pires1; Harry Pritchard1; Pratish Thakore1; Tom
Longden2; Mark Nelson2,3,4; Scott Earley1
1
School of Medicine, University of Nevada at Reno, Reno, NV, USA;
2
Department of Pharmacology, University of Vermont, Burlington, VT, USA;
3
Department of Surgery, University of Vermont, Burlington, VT, USA; 4Institute
of Cardiovascular Sciences, University of Manchester, Manchester, UK
A recent study showed that cerebral capillary endothelial cells (ECs)
can initiate functional hyperemia in the brain through a mechanism
that requires the activity of inwardly-­rectifying K+ channels. Here we
tested the hypothesis that additional molecular sensors are present in
brain capillary ECs that can also orchestrate dilation of upstream pa-
renchymal arterioles (PAs). We found that Ca2+-­permeable transient
receptor potential ankyrin 1 (TRPA1) cation channels are present and
functional in native cerebral capillary ECs. Using a recently developed
ex vivo capillary-­parenchymal arteriole microvascular preparation, we
observed that picospritzing capillaries with the TRPA1 activator allyl
isothiocyanate (AITC) induced dilation of upstream PAs. This effect
was inhibited by the selective TRPA1 antagonist HC-­030031 and was
absent in microvascular preparations obtained from EC-­specific TRPA1
knockout (eTRPA1−/−) mice. The vasodilatory response was insensitive
to block of the nitric oxide synthase and cyclooxygenase pathways but
was abolished by inhibition of small-­and intermediate-­conductance
Ca2+-­activated K+ channels with TRAM34 and apamin. Using EC-­
specific Tek:GCaMP6f Ca2+ biosensor mice, we also found that pi-
cospritzing capillaries with AITC initiated intercellular Ca2+ waves that
propagated from capillaries to upstream PAs. Ca2+ wave propagation
was inhibited by HC-­030031 and the P2X receptor blocker PPADS.
Using laser Doppler flowmetry, we found that functional hyperemic
responses induced by somatosensory stimulation were smaller in
eTRPA1−/− mice compared with TRPA1 fl/fl littermates. We conclude
that stimulation of TRPA1 channels on capillary ECs initiates retro-
grade propagating Ca2+ waves that induce dilation of upstream PAs
and contribute to functional hyperemia in the brain. R01HL091905;
R01HL137852; R01HL139585; K99HL140106; 15POST2472002.
MOPE030 | High-­fidelity computational
modeling of cellular-­scale blood flow in
microvascular networks
Prosenjit Bagchi; Peter Balogh
Mechanical and Aerospace Engineering, Rutgers, The State University of New
Jersey
Most computational studies of red blood cells flow in microcirculation
have considered vessels of simple geometry, such as straight tubes
of circular or rectangular cross-­section. In contrast, microvascular
networks in vivo are characterized by vessels that are constantly bi-
furcating, merging and curving. There has been very little work on
ABSTRACTS
high-­fidelity modeling of flow of blood cells in such geometrically
complex vasculature. We recently have developed a high-­
fidelity
direct numerical simulation technique that is capable of modeling
flow of many deformable red blood cells, leukocytes, and platelets
through physiologically realistic microvascular networks comprised
of multiple winding, bifurcating, and merging vessels. The vascu-
lature data are obtained from in vivo images in the literature. Our
model is based on the immersed-­boundary methods. The flow and
deformation of every blood cells flowing in the networks are modeled
with high accuracy. Good agreement is observed between the time
time-­averaged hemodynamic quantities and published in vivo data.
However, at the cellular-­scale, we predict several novel phenomena.
The cells are observed to frequently jam at vascular bifurcation, re-
sulting in large temporal spikes in vascular resistance. This, in turn,
results in negative pressure-­flow correlations, implying a deviation
from Poiseuille's law. Using the simulation data, we present analysis
of red blood cell partitioning at bifurcations and show that the plasma
skimming mechanism alone cannot accurately predict the distribution
of the cells through the bifurcations. We further show that cell-­free
layer is essentially three-­dimensional and highly non-­symmetric along
the vessel length, unlike blood flow in long, straight tubes.
MOPE031 | 3D imaging, mapping and analysis
of microvascular networks in skeletal muscle
Rebecca Shaw; Charmain Fernando; Steven Segal
Medical Pharmacology and Physiology, University of Missouri, Columbia, MO,
USA
Microvascular resistance networks control the distribution and mag-
nitude of tissue blood flow, however there is a lack of readily available,
standardized methods for quantifying network architecture. Further,
little is known of how or where microvascular networks remodel
following injury due to technical challenges associated with such
measurements. To address this challenge, we developed methods
for evaluating microvascular architecture in healthy skeletal muscle
and applied them to muscle injury. With IACUC approval, the glu-
teus maximus muscle (GM) of male C57BL/6J mice (age, 16 weeks;
n = 6-­7) was injured by local injection of barium chloride solution
(1.2%, 75 μL). Under anesthesia, control and 5d post-­injury mice re-
ceived retro-­orbital injection of fluorescent wheat germ agglutinin
(1 mg/mL; 200 μL) to stain the luminal surface of endothelial cells
throughout the vasculature. Individual GM were excised, fixed in 4%
paraformaldehyde at in situ dimensions, cleared in glycerol, mounted
on a prepared slide and secured on the joystick-­controlled stage of a
microscope (Nikon E600) instrumented for creating 3D maps using
Vesselucida software (MBF Bioscience; Williston, VT). Via interface
with a personal computer, Vesselucida acquires X,Y,Z information ac-
counting for vessel branch points (nodes) set by the user. Applying
user-­defined criteria, data are compiled for vessel segment counts, di-
ameters, lengths, branch angles, tortuosity, surface area, volume and
anastomoses. At 5d, GM networks exhibit proliferation of arterioles
|
      59 of 96
having diameters ≤15 μm), consistent with supporting capillary pro-
liferation during the initial stages of myofiber regeneration. (Support:
Mulligan Professorship, AHA 17PRE33410260 & NIH R37HL041026).
MOPE033 | Optimization of vessel diameters
to mimic flow changes in microvascular networks
during activation
Robert Epp1; Franca Schmid1,2; Bruno Weber2; Patrick
Jenny1
1
Institute of Fluid Dynamics, ETH Zurich, Zurich, Switzerland; 2Institute of
Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland
The brain is capable of up-­regulating cerebral blood flow in response
to local changes in neural activity. Recently, it was reported that also
the homogenization of capillary flow might be very relevant. However,
the precise pattern of the underlying vasodynamics is still poorly un-
derstood. Our goal is to improve our understanding of the impact of
diameter changes on blood flow regulation in the vasculature.
We present a numerical framework, which is based on a previ-
ously developed blood flow model and on well-­established optimi-
zation algorithms. The strength of our model is that we can compute
diameter changes necessary to achieve desired flow distributions.
It allows us to investigate different scenarios related to regulation,
e.g. by finding the most likely diameters which lead to agreement
between computed and specified local flow rates.
We performed simulations in artificial and realistic microvascu-
lar networks to compute the vessel diameters required to either in-
crease the mean flow or decrease the standard deviation of the flow
at different locations in the network. First results reveal that vessel
diameters are adjusted only locally to reduce the standard deviation
of the capillary flow, whereas many more vessels are involved in up-­
regulating the mean flow rate.
These findings suggest that not only arteriole, but also pericyte
mediated capillary dilation may play an important role in the local
regulation of cerebral blood flow.
Funding for this research is provided by the Swiss National
Science Foundation Grant No. 166707
MOPE034 | Does hind limb temperature affect
capillary perfusion of murine sural nerve?
Anete Dudele1,2; Nina Kerting Iversen1; Eugenio Gutiérrez
Jiménez1; Troels Staehelin Jensen2,3; Leif Østergaard1,4
1
Center for Functionally Integrative Neuroscience and MINDLab, Aarhus
University Hospital, Aarhus, Denmark; 2International Diabetic Neuropathy
Consortium, Aarhus University, Aarhus, Denmark; 3The Danish Pain research
Centre, Aarhus University Hospital, Aarhus, Denmark; 4Department of
Neuroradiology, Aarhus University Hospital, Aarhus, Denmark
It is well known that temperature affects nerve conduction and
function. This becomes especially important when performing
 |
60 of 96       ABSTRACTS
neurophysiological measurements in rodents, as they rapidly lose
body heat when anesthetized due to the high ratio of body surface
area to volume. While it is reasonable to assume that temperature
changes not only nerve conduction but also perfusion, data to sup-
port this in mice models is currently absent. Therefore, we aim to
assess how hind limb temperature affects nerve perfusion in murine
sural nerves.
Mature (12-­18 weeks old, n = 8) C57 male mice were anesthe-
tized using isoflurane and the right hind limb was fixed in a custom-­
made holder with an adjustable heating element. A temperature
probe was placed in the muscle tissue within 5 mm of the sural
nerve. The sural nerve was carefully exposed and capillary perfu-
sion assessed by two-­photon in vivo microscopy at three hind limb
temperatures – 28, 32 and 36°C – in a random order. Study was ap-
proved by The Danish Animal Experiments Inspectorate.
Preliminary data suggests that sural nerve capillary diameter, red
blood cell velocity and flux change with altering temperatures, but
data analysis is still ongoing. We also estimate how temperature af-
fects mean transit time and capillary transit time heterogeneity in
these nerves.
Our results enable us to understand the importance of regulating
and reporting temperature during studies that investigate peripheral
nerve capillary function in mice.
Research was funded by the International Diabetic Neuropathy
Consortium, which is supported by a Novo Nordisk Foundation
Challenge Programme grant (NNF14OC0011633).
MOPE037 | Multiscale modeling of
neurovascular coupling: from ion channel activity
to BOLD fMRI responses
Asad Mirza; Arash Moshkforoush; Baarbod Ashenagar;
Manuel Russo; Nikolaos Tsoukias
Department of Biomedical Engineering, Florida International University, Miami,
FL, USA
Neuronal activity signals local changes in blood flow such that is
matches metabolic demands for O2 and nutrients, referred to as
Neurovascular Coupling (NVC). We propose an integrative mod-
eling approach to model microcirculatory responses to NVC me-
diators and their effect on the regulation of blood flow, tissue
perfusion and oxygenation. Membrane electrophysiology, signaling
pathways, and ionic dynamics, are captured in single cell models
of endothelial (EC), smooth muscle (SMC), and pericyte cells (PC).
Multicellular models of microcirculatory vessels simulate the effect
of ions, flow, pressure, and agonist stimuli. Multiple capillary and
arteriolar segments are coupled together to form a microvascular
network that examines the effect of localized stimuli. Simulations
are extended at a macroscale level, incorporating dynamic regula-
tion of vessel diameter by propagating electrical signals, wall shear
stress (WSS), and pressure. The model predicts changes in tissue
perfusion and O2 distribution in response to neuronal activity.
Simulations suggest that the inwardly rectifying potassium chan-
nel (KIR ) as an important mediator in NVC. A localized K+ challenge
(10 mmol/L) can hyperpolarize nearby capillary ECs. The hyperpo-
larizing current can be transmitted upstream to feeding arterioles
leading to dilation and increase blood flow. The resulting changes
in WSS and pressure feedback to alter microcirculatory tone and
diameter throughout the vascular network. The theoretical frame-
work presented will allow for testing of proposed NVC mechanisms
and assist in the interpretation of macroscale functional responses
in health and in disease.
MOPE038 | Brain hemodynamics in awake
mice
Eugenio Gutiérrez Jiménez1; Nina Kerting Iversen1; Signe
Kirk Fruekilde1; Peter Mondrup Rasmussen1; Irene Klærke
Mikkelsen1; Luca Bordoni1,2; Susanne Smith Christensen1;
Leif Østergaard1,3
1
Center of Functionally Integrative Neuroscience, Clinical Institute, Aarhus
University, Aarhus, Denmark; 2Department of Biomedicine, Aarhus University,
Aarhus, Demark; 3Dept. of Neuroradiology, Aarhus University Hospital. Aarhus,
Denmark
Changes in brain hemodynamics accompany neuronal activation,
and the majority of these effects have been examined in anesthe-
tized rodents. However, anesthesia affects the cerebral autoregula-
tion. In this project, we evaluated steady-­state hemodynamics and
oxygenation in the brain barrel cortex in awake mice. The experi-
ments were approved by the Danish Animal Inspectorate. Imaging
was performed in head-­restrained C57BL/6 mice (n = 6), through a
chronic sealed cranial window centered over the C2 whisker bar-
rel. We measured intravascular oxygen partial pressure (ptO₂) and
mean transit-­time (MTT) using two-­photon microscopy. Additionally,
we quantified the relative changes in cerebral blood flow (CBF) and
ptO₂ during functional activation by whiskers stimulation (10 s).
During steady state, MTT was 0.46 ± 0.14 seconds, arterial ptO₂
93.8 ± 17.9 mm Hg, venous ptO₂ 41.1 ± 3.6 mm Hg, and the esti-
mated oxygen extraction fraction (OEF) was 0.55 ± 0.07. During
functional activation, CBF significantly increased by 6.8 ± 4.0%, and
ptO₂ increased in both the arterial and venous network by 2.7 ± 3.0%
and 8.6 ± 2.6%, respectively. The estimated OEF showed a decrease
of 12.4% ± 2.9% during functional activation. Our study describes
brain hemodynamics in awake mice by the use of a combination of
several optical imaging techniques. The suggested combination of
optical imaging techniques can be applied to various murine disease
models to evaluate the effect of the pathology on brain oxygenation
and hemodynamics.
ABSTRACTS
MOPE039 | Capillary dilation and constriction
during brain activation in the somatosensory
cortex of awake mouse
Hiroki Suzuki1; Hiroshi Takeda1; Takuma Sugashi1; Hiroyuki
Takuwa2; Ji Bin2; Naruhiko Sahara2; Tetsuya Suhara2;
Makoto Higuchi2; Iwao Kanno2; Kazuto Masamoto2,3
1 Adrian Sackheim1; Nuria Villalba Isabel2; Adrian Bonev2;
Graduate School of Informatics and Engineering, National Institute of
Radiological Sciences, Inage, Chiba, Japan; 2Department of Functional Brain
Imaging Research, National Institute of Radiological Sciences, Inage, Chiba,
Japan; 3Brain Science Inspired Life Support Research Center, University of
Electro-­Communications, Chofu, Tokyo, Japan
Cerebral capillary responses to cortical neural activity play a key
role in adjusting regional blood flow to meet local demand in the
brains. Here, capillary morphometry was conducted with a cus-
tom written Matlab software for two-­photon microscope images
(258 × 258 × 200 μm) of microvessels captured in the somatosen-
sory cortex of transgenic mice (Cre-­C aMKII/GCaMP3, N = 7). The
experimental protocol was approved by the institutional Animal
Ethics Committee of National Institute of Radiological Sciences and
University of Electro-­Communications. The animals expressed fluo-
rescence calcium indicator GCaMP3 in the cortical neurons, while
sulforhodamine 101 was intraperitoneally infused to label blood
plasma. Air-­puff stimulation (30 seconds) was applied to the con-
tralateral side of whiskers with variable frequency (1, 4, 8 Hz), while
activated neurons and microvessels were simultaneously imaged
over depths of 50-­320 μm from the cortical surface. The images
were three-­
dimensionally reconstructed and capillary diameters
and number of activated neurons were quantified. For each volume
images, 3,000-­7,000 measurement points were extracted along a
center line of the capillary. Our preliminary results showed that
84 ± 8% of capillary locations showed no detectable responses to
stimuli, whereas other locations showed dilation (9 ± 7%) or con-
striction (7 ± 6%). These capillary locations had different diameters
at rest (without stimulation); a mean of 5.0 ± 1.2 μm (unchanged),
4.6 ± 1.0 μm (dilated), and 5.4 ± 1.4 μm (constricted). Furthermore,
detailed spatial analysis of the responses revealed that a portion
of the single capillaries reacts to the stimulation irrespective of
distances from the connecting arterioles or venules over depths
measured.
|
      61 of 96
MOPE041 | Traumatic brain injury disrupts
phosphatidylinositol 4,5-­bisphosphate
homeostasis resulting in inward rectifier
potassium channelopathy and microvascular
dysfunction
Mark Nelson2,3; Kalev Freeman1,2
1
Department of Surgery, University of Vermont, Burlington, VT, USA;
2
Department of Pharmacology, University of Vermont, Burlington, VT, USA;
3
Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK
Severe trauma, including traumatic brain injury (TBI), can result in
coagulopathy, multi-­organ failure and death. The inward-­rectifier
potassium channel (KIR) is an essential component of flow-­induced
vasodilation and signal transduction in mesenteric arteries (MAs);
however, its function following trauma has not been assessed.
Phosphatidylinositol 4,5-­bisphosphate (PIP₂) is a membrane phos-
pholipid which is required for KIR function in MAs. We hypothesized
that oxidative stress was driving down PIP₂ levels, causing KIR chan-
nelopathies. We studied male rats 24 hours after fluid percussion
TBI. Plasma oxidative-­
reduction potential (ORP), an indicator of
oxidative stress, was significantly elevated in TBI rats. We measured
KIR currents in isolated endothelial cells (ECs) from MAs, using the
whole-­cell patch-­clamp technique. KIR currents were significantly
diminished in ECs from TBI rats and were rescued with the addi-
tion of PIP₂ when compared to untreated TBI cells and controls.
Next, diameter responses were measured by eliciting dilations using
10 mmol/L K+, which were significantly reduced following TBI. Basal
KIR function was assessed using the antagonists BaCl₂ and ML133.
Following TBI, arteries constricted significantly less to both BaCl₂
and ML133. Next, the metabolomic phenotype was measured by
liquid chromatography-­mass spectrometry (LC-­MS) in plasma. Rats
exhibited a profound metabolopathy and an increase in PIP₂ degra-
dation products following TBI. Phospholipase A₂ (PLA₂) activity was
significantly elevated in plasma from TBI rats. These data suggest
oxidative stress increases PLA₂ activity, which subsequently de-
grades PIP₂, and disrupts KIR function through second messenger and
lipid signaling pathways following TBI. Funding Acknowledgements:
Totman Trust and NIH (UM1-­HL-­120877, R01-­GM-­123010).
MOPE042 | In vivo two-­photon imaging of
early post-­natal cerebrovascular development
Vanessa Coelho-Santos1,2; Andy Shih1,2
1
Seattle Children's Research Institute, Center for Developmental Biology
and Regenerative Medicine, Seattle, WA, USA; 2University of Washington,
Department of Pediatrics, Seattle, WA, USA
The blood-­brain barrier (BBB) is a highly selective vascular interface
that regulates brain access and homeostasis. The stages involved
in postnatal maturation of the BBB remain poorly understood. Yet
 |
62 of 96       ABSTRACTS
this process is essential to study given the unknown permeability
of many drugs used to treat ailments in mothers and their infants,
including depression and anxiety. Most studies of BBB development
have used histological methods or acute preparations that do not
adequately capture the dynamics of cellular interactions involved in
construction of the BBB. Here, we report a neonatal reinforced thin‐
skull preparation for time-­lapse in vivo imaging microvasculature in
mice using two‐photon microscopy. In contrast to other protocols,
we demonstrate the use of iterative shaving with scalpel blades to
thin the delicate calvarium of early postnatal pups (P0 to P9), per-
mitting imaging to 100-­250 μm below the cortical surface without
breaching the intracranial cavity. The restraining head caps for head-­
fixation are light-­weight and do not hinder the movement of pups
between imaging sessions, providing a system that is conducive to
repeated imaging over multiple days. As a proof of principle for the
ability to visualize cells in the developing BBB, we tracked tdTomato-­
positive capillary pericytes in the vasculature of transgenic mice. We
observed a marked shift in the pericyte morphology and endothe-
lial coverage between P2 to P8, suggesting active remodeling at
the pericyte-­endothelial interface, and potentially the BBB, over a
relatively short period of time. Our ongoing studies will characterize
in vivo BBB permeability during this critical period in brain vascular
development.
MOPE043 | Dynamic remodeling of pericytes
in vivo maintains capillary coverage in the adult
mouse brain
Andree-Anne Berthiaume1,2; Andy Shih2,3
1
Medical University of South Carolina, Department of Neuroscience, Charleston,
SC, USA; 2Seattle Children's Research Institute, Center for Developmental
Biology and Regenerative Medicine, Seattle, WA, USA; 3University of
Washington, Department of Pediatrics, Seattle, WA, USA
Direct contact and communication between pericytes and en-
dothelial cells is critical for the maintenance of cerebrovascular
health. Capillary pericytes have thin processes that span hundreds
of micrometers along the capillary bed. The processes of adja-
cent pericytes are in close proximity to each other with minimal
overlap, yielding a cellular chain with discrete territories occu-
pied by individual cells. Little is known about whether this peri-
cyte chain is structurally dynamic in the adult brain. Using in vivo
two-­p hoton imaging in adult mouse cortex, we show that while
pericyte somata were immobile, the tips of their processes under-
went extensions and/or retractions over days, while still maintain-
ing a non-­overlapping arrangement. Because pericyte abundancy
decreases in age-­related diseases such as stroke and Alzheimer's
disease, we wanted to investigate the cellular response to acute
pericyte loss. We found that the selective ablation of single peri-
cytes provoked exuberant extension of processes from neighbor-
ing pericytes to contact uncovered regions of the endothelium.
Uncovered capillary segments had normal barrier function but
were consistently dilated until pericyte contact was regained. This
type of pericyte structural plasticity may be critical for cerebro-
vascular health and homeostasis, warranting detailed investiga-
tion in the context of aging and pathology.
MOPE045 | CFTR therapeutics normalize
cerebral perfusion deficits in mouse models of
heart failure and subarachnoid hemorrhage
Darcy Lidington1; Anja Meissner1; Jessica Fares1; Danny
Dinh1; Jeff Kroetsch1; Meghan Sauve1; Firhan Malik1;
Arman Adel1; Abdul Momen2; Hangjun Zhang1; Roozbeh
Aschar-Sobbi3; Warren Foltz1; Manabu Sumiyoshi1; R. Loch
Macdonald4; Christine Bear1; Peter Backx5; Scott Heximer1;
Steffen-Sebastian Bolz1
1
University of Toronto, Department of Physiology, Toronto, ON, Canada;
2
Toronto General Hospital Research Institute, Toronto, ON, Canada;
3
University of Toronto, Departments of Physiology and Medicine, Toronto,
ON, Canada; 4Division of Neurosurgery, St. Michael's Hospital, Toronto, ON,
Canada; 5Division of Cardiology, University Health Network, Toronto, ON,
Canada
Heart failure (HF) and subarachnoid hemorrhage (SAH) induce a
chronic reduction in cerebral perfusion that negatively impacts pa-
tient prognosis and clinical outcome. Our recent work demonstrates
a strong relationship between cerebral artery cystic fibrosis trans-
membrane conductance regulator (CFTR) expression and altered
cerebrovascular reactivity in HF and SAH. We sought to determine
whether CFTR corrector therapeutics improve cerebral perfusion in
experimental models of HF and SAH.
HF was induced by left anterior descending coronary artery liga-
tion; SAH by injecting arterial blood into the basal cistern. Vascular
reactivity was assessed by pressure myography. We utilized stan-
dard procedures to measure CFTR protein expression, systemic pa-
rameters, cerebral perfusion and neuronal injury.
We found that disrupting CFTR function augments cerebral ar-
tery myogenic tone and decreases cerebral perfusion, without af-
fecting skeletal muscle resistance artery myogenic tone and total
peripheral resistance. In HF and SAH, CFTR corrector compounds
(C18 or lumacaftor) normalize the pathologically reduced cerebral
artery CFTR expression, myogenic responsiveness and cerebral per-
fusion. In SAH, the normalization of cerebral perfusion following
C18 or lumacaftor treatment correlates with a significant reduction
in neuronal injury. Mechanistically, both C18 and lumacaftor exert a
proteostatic effect on wild-­t ype CFTR expression, as evidenced by
a transcription-­independent increase in CFTR protein expression in
cultured cells and cerebral arteries.
In summary, we provide the first direct evidence that CFTR
regulates cerebrovascular reactivity and hence, cerebral perfusion.
Therapeutics that increase CFTR expression, therefore, are valuable
tools to specifically manage cerebrovascular dysfunction, impaired
cerebral perfusion and neuronal injury, with minimal impact on sys-
temic hemodynamics.
ABSTRACTS
MOPE046 | Reprogramming resident NG2+
pericytes in adult mouse cochlea: new vessel
growth and NG2+ pericytes integral for vascular
integrity and hearing
Xiaohan Wang; Han Jiang; Guangshuai Li; Na Sai; Allan
Kachelmeier; Xiaorui Shi
Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck
Surgery, Oregon Health & Science University, Portland, OR, USA
Rationale: Angiogenesis is critical to tissue regeneration and repair
under wound healing, hypoxic, and chronic ischemia conditions. Can
vessels be regenerated in the damaged adult ear? The question is
important because loss in vessel density is seen in a wide variety
of hearing disorders, including in loud sound-­induced hearing loss,
ageing-­related hearing loss, and genetic hearing loss. Progression
in blood vessel pathology can parallel progression in hair cell and
hearing loss. However, new vessel growth in the ear has not been
studied, nor has the role of angiogenesis in hearing.
Objective: To determine whether new vessels can be regenerated
from adult mouse cochlea.
Methods and Results: In this study, using integrated approaches
which include primary cochlear cell lines, we demonstrate, for the
first time, that new vessels can be regenerated in adult mouse coch-
lea by activation of vascular endothelial growth factor-­A (VEGF-­A)
signaling. Most important, we discovered the progenitors of tip
cells for new vessel growth are not pre-­existing endothelial cells but
converted NG2-­derived pericytes. Depletion of NG2+ pericytes by
pharmacological and genetic approaches impair sprouting angiogen-
esis in vitro and cause vascular regression and high frequency hear-
ing loss in vivo.
Conclusions: Our data highlight the vital role pericytes play in adult
vascular regeneration. Resident pericytes self-­convert to tip cells
and lead sprouting angiogenesis. Resident pericytes are also essen-
tial for the maintenance of normal microvascular structure critical
for auditory function.
This research was supported by NIH/NIDCD R21 DC016157, NIH/
NIDCD R01 DC015781 and Action on Hearing Loss Flexi Grant 2017
MOPE048 | Assessment of the developmental
stage of embryonic vascular network based on a
deep learning model
1 1 2
Qing Pan ; Shenjia Zhao ; Bianca Nitzsche ; Fei Lu ; Luping 1 by a reduced fetal number, pup and placental weight, and increased
Fang1; Wolfgang Kuebler2; Axel Pries2,3; Gangmin Ning4
1
College of Information Engineering, Zhejiang University of Technology,
Hangzhou, China; 2Institute of Physiology, Charité Universitätsmedizin
Berlin, Berlin, Germany; 3Deutsches Herzzentrum Berlin, Berlin, Germany;
4 model to quantify fluid flow across the intact placenta after ENM
Department of Biomedical Engineering, Key Laboratory of Biomedical
Engineering of MOE, Zhejiang University, Hangzhou, China
Background: The developmental stage of embryonic vascular sys-
tems can be characterized by distinct morphological and topological
|
      63 of 96
properties. Here, we developed a deep learning model to differenti-
ate between the developmental stages of chicken embryos based on
microvascular characteristics and assess the primary morphological
changes during the developmental procedure.
Methods: After incubation of fertilized eggs for 3 and 4 days, re-
spectively, chicken embryonic vascular networks were scanned
using intravital microscopy. Convolutional neural network (CNN), an
established deep learning model, was applied to classify the devel-
opmental stage of the vascular networks based on the local mor-
phology of the vasculature. A gradient-­weighted Class Activation
Mapping (Grad-­C AM) algorithm was developed to identify the re-
gions that contribute primarily to the classification.
Results: Two-­
dimensional (2D) square images of the embryonic
vasculature were used for model training and testing. Images with
different physical side lengths (1000-­1500 μm, step: 100 μm) were
used for examining the role of size on the performance. The ac-
curacies of classification were 83.10%, 83.53%, 86.83%, 89.37%,
90.63%, 90.57% depending on the size of input images, respectively.
The Grad-­C AM algorithm identified the bifurcating pattern as the
most relevant morphological properties to the correct classification
of the vasculature.
Conclusion: Deep learning models can differentiate between de-
velopmental stages of embryos based on 2D images and allow the
quantitative study of primary morphological changes during the de-
velopmental procedure. Thus, it can serve as a novel tool for the
investigation of angiogenesis.
Fundings: National Natural Science Foundation of China (Grant Nos.
81401491, 81271662).
MOPE049 | Development of intrauterine
growth restriction after maternal environmental
exposure
J. N. D'Errico1; S. B. Fournier2; C. L. Doherty2; N. Renkel2; B.
Buckley2; P. A. Stapleton1,2
1
Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy,
Rutgers University, Piscataway, NJ, USA; 2Environmental and Occupational
Health Sciences Institute, Piscataway, NJ, USA
One of the most unique and acutely demanding circulations is the
uterine vasculature to support fetal development. In our model, ma-
ternal engineered nanomaterial (ENM) exposure has led to the de-
velopment of intrauterine growth restriction (IUGR) characterized
number of reabsorption sites (analogous to miscarriage). This condi-
tion is often associated with altered placental hemodynamics.
We recently developed a novel rat ex-­vivo placental perfusion
exposure. Sprague-­Dawley rats (gestational day 20) were anesthe-
tized, the uterus was dissected and a placental unit isolated. In a
modified isolated vessel chamber, the proximal and distal ends of
the maternal uterine artery and the vessels of the umbilical cord
 |
64 of 96       ABSTRACTS

were cannulated, secured and perfused with PSS. The proximal uter- Funding: NIH-­R00-­ES024783 (PAS); T32-­ES007148 (JTS, LMA); P3
ine artery and umbilical artery were pressurized at 80 mm Hg and 0-­ES005022.
50 mm Hg, respectively, for countercurrent flow. After equilibration,
a 900 μL bolus dose of gold or polystyrene ENM was introduced into
the proximal maternal artery. Distal uterine and umbilical vein efflu-
MOPE051 | Effect of gestational diabetes on
ents were collected every 10 m for 180 m to quantify fluid dynamics
and ENM transfer. In preliminary studies, we were able to quantify
endothelium-­dependent vasodilation of human
a significant reduction in fluid transfer to the fetal compartment myometrial and omental arteries
70 minutes after gold (−44 ± 13%) or 140 minutes after polystyrene Timothy V. Murphy1; Leo Leader2; Shaun L. Sandow1,3
(−52 ± 16%) infusion. 1
Department of Physiology, School of Medical Sciences, University of New
This novel methodology may be widely incorporated into studies South Wales (UNSW), Sydney, Australia; 2School of Women's and Children's
Health and Royal Hospital for Women, UNSW, Sydney, Australia; 3Faculty of
of placental physiology. These outcomes provide evidence of de-
Science, Health, Education and Engineering, University of the Sunshine Coast,
creased blood flow and ENM transfer to the fetal compartment after Queensland, Australia
maternal ENM exposure.
Funding:NIH-­K99-­ES024783(PAS);P30-­ES005022;T32-­ES007148. Microvascular dysfunction contributes to the deleterious effects of
diabetes. Relatively few studies have examined the effects of diabe-
tes on human microvasculature. This study investigated endothelial
function of myometrial and omental arteries isolated from women
MOPE051 | Altered placental angiogenic with gestational diabetes (GD).
signaling in response to engineered nanoparticle Arterioles were obtained from caesarean-­section non-­diabetic
and GD women at term (internal diameter approx. 200 μm). Protein
exposure
expression was assessed using immunohistochemistry and func-
J. T. Szilagyi1; L. M. Aleksunes1,2; P. A. Stapleton1,2 tional effects with pressure (60 mm Hg) myography; arteries were
1
Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, pre-­constricted with vasopressin (3-­10 nmol/L). Protocols were ap-
Rutgers University, Piscataway, NJ, USA; 2Environmental and Occupational
Health Sciences Institute, Piscataway, NJ, USA
proved by UNSW and Health District Human Ethics Committees.
In myometrial arteries, GD decreased the maximum endothelium-­
dependent vasodilation induced by bradykinin (Control 86.8 ± 2.6%,
Intrauterine growth restriction (IUGR) is a condition where the
n = 8; GD 69.7 ± 5.6%, n = 12, P < 0.05). GD reduced the potency of
fetal growth rate is reduced leading to low birth weight and an
bradykinin in omental arteries, but not the maximum response (Control
increased risk for future cardiometabolic disease. Placental insuf-
pEC50 8.61 ± 0.10, n = 9; GD 8.04 ± 0.12, n = 5, P < 0.05). GD did not
ficiency involving impaired vascular function and dysregulation
alter endothelium-­independent vasodilation (sodium nitroprusside) in
of angiogenesis has been proposed as a critical factor in IUGR.
either vessel. In myometrial arteries from non-­diabetic women, nitric
Elevated placental cell expression of HIF-­1α, eNOS, and VEGF
oxide (NO) pathway inhibition with L-­NAME (100 μmol/L) and ODQ
along with reduced hemodynamic control has been reported with
(10 μmol/L) inhibited bradykinin-­induced vasodilation, as did inhibi-
the development IUGR. We have previously demonstrated that
tors of the intermediate-­and small-­conductance Ca2+-­activated K+-­
maternal inhalation of titanium dioxide nanoparticles (TiO₂) during
channels (I-­and SKCa), TRAM-­34 (1 μmol/L) and apamin (0.1 μmol/L)
pregnancy led to the development of IUGR in rats, impairing fetal
respectively. In contrast NO-­and IKCa-­inhibition had no effect on
and progeny health through impaired uterine artery function. In
bradykinin-­induced responses in GD, while vasodilation induced by
the current study, we sought to determine whether TiO₂ exerts di-
the IKCa activator, SKA-­31, was also decreased.
rect effects on placental cell viability and angiogenic gene expres-
These studies suggest that GD inhibits endothelium-­dependent
sion. For this purpose, BeWo b30 trophoblast carcinoma cells and
vasodilation through inhibition of nitric oxide production and IKCa
human term placental explants were treated with increasing doses
activity.
of TiO₂ (1, 10, 100 μg/mL) for 24 hours and analyzed for changes
This project was supported by Diabetes Australia Grant
in angiogenic gene expression by qPCR. Treatment with TiO₂ did
#Y16G-­MURT.
not alter trophoblast viability or syncytialization/oxidative stress
mRNA markers. Interestingly, treatment with TiO₂ up-­r egulated
HIF-­1α, eNOS, and VEGF mRNAs (up to 2-­fold) in BeWo b30 cells
and placental explants. Results from these studies indicate an im-
balance of pro-­a ngiogenic factors in placental cells after TiO₂ ex-
posure. Unique to this study, these outcomes exist in the absence
of impaired hemodynamic control. Overall, direct TiO₂ exposure
alters angiogenic placental signaling, possibly contributing to the
development of IUGR.
ABSTRACTS
MOPE054 | Neonatal hyperthyroidism
increases anticontractile influence of NO in rat
arteries
Dina K. Gaynullina1,2; Anastasia A. Shvetsova1,2; Ekaterina
K. Selivanova1,2; Andrey A. Martyanov1,2; Olga S. Tarasova1,2
1
Institute for Biomedical Problems, Russian Academy of Sciences, Moscow,
Russia; 2Lomonosov Moscow State University, Moscow, Russia
Endothelium synthesizes NO that can reduce arterial contractile
responses thereby exhibiting anticontractile influence. This influ-
ence is highly pronounced in 2-­week-­old rats compared to adults.
Recently we have shown that thyroid deficiency during early stages
of ontogenesis decreases the anticontractile effect of NO in arter-
ies of 2-­week-­old rats. However, the impact of thyroid hormones
excess on the anticontractile effect of NO in young arteries has
not been studied yet. Thus, we hypothesized that neonatal hyper-
thyroidism would increase the anticontractile influence of NO in
arteries of 2-­week-­old rats. We studied isometric contractions of
endothelium-­intact saphenous artery from hyperthyroid (injections
of 15 μg T3/100 g body weight every day starting from the 2nd day
of life, s.c.) and control (injections of saline in equivalent volume)
until 2-­week age. All procedures were approved by Russian insti-
tutional committee on animal welfare. Contractile responses to α1-­
adrenoreceptor agonist methoxamine were similar in control and
hyperthyroid groups. NO-­synthase inhibitor L-­NNA (100 μmol/L)
significantly enhanced the contractile responses in both groups.
However, in the presence of L-­NNA the contractile responses to
methoxamine were stronger in arteries of hyperthyroid animals,
despite significantly decreased responses to exogenous NO-­donor
DEA/NO in comparison to control. Our results demonstrate that
neonatal hyperthyroidism increases anticontractile influence of NO
in arteries of 2-­week-­old rats that is followed by compensatory re-
duction of arterial NO-­responsiveness. The study was supported by
the Russian Science Foundation (grant N14-­15-­0 0704).
MOPE055 | QTc dispersion in T2DM patients
with high glycemic variability
Cornel Cezar Tudorica1; Ariel Florentiu2; Ana Maria Vintila3;
Stefan Tudorica4; Vlad Vintila5
1
Coltea Clinical Hospital, Department of Internal Medicine, Bucharest, Romania;
2
“N. C. Paulescu” National Institute of Diabetes, Nutrition and Metabolic
Diseases, Bucharest, Romania; 3Carol Davila University of Medicine and
Pharmacy, Coltea Clinical Hospital, Department of Internal Medicine, Bucharest,
Romania; 4CMI Tudorica Steluta, Bucharest, Romania; 5Carol Davila University
of Medicine and Pharmacy, University Emergency Hospital, Department of
Cardiology, Bucharest, Romania
Objective: Glycemic variability (GV) is one more tool to explain re-
lation between hyperglycemia and increased cardiovascular risk in
diabetic patients. Increased QT interval dispersion (QTd) on the sur-
face ECG has been linked to increased heterogeneity of ventricular
repolarization, implicated in the genesis of ventricular arrhythmias.
|
      65 of 96
The study aim was to assess the correlation between QTc dispersion
and glycemic variability in T2DM.
Method: Thirty noninsulin treated T2DM patients, 18 men, aged
between 48 and 64, T2DM duration 7.8 years were included in the
last year. They didn't have documented coronary disease or systemic
hypertensive. In all the patients were evaluated HbA1c (in the last
3 months), continuous glucose monitoring (CGM) in the last month
and 12 lead standard ECG concomitant with CGM. GV was meas-
ured by overall standard deviation (SD). Were included only patients
with HbA1c between 7-­7.5%.
Results: In 16 pts overall SD was in normal range (10-­26 mg/dL), in
14 out of range. Prolonged QT-­dispersion defined as QT-­dispersion
>50 ms, was present in 42% of the patients almost equally distrib-
uted between the two groups. Prolonged QTc interval defined as
QTc-­max >440 ms, was present in 36% of the patients.
Conclusion: In this T2DM selected patients the percentage of in-
creased QTc and QTc dispersion in type 2 diabetic patients is rela-
tively high but not correlated with GV. The limitation of the study is
low number of patients so further studies are needed for establish if
there could be some relationship between these parameters.
MOPE056 | The assessment of
microalbuminuria in patients with uncontrolled
type 2 diabetes
Cornel Cezar Tudorica1; Ana Maria Vintila2; Stefan
Tudorica3; Ariel Florentiu4; Mihaela Horumba5
1
Coltea Clinical Hospital, Department of Internal Medicine, Bucharest, Romania;
2
Carol Davila University of Medicine and Pharmacy, Coltea Clinical Hospital,
Department of Internal Medicine, Bucharest, Romania; 3CMI Tudorica Steluta,
Bucharest, Romania; 4“N. C. Paulescu” National Institute of Diabetes, Nutrition
and Metabolic Diseases, Bucharest, Romania; 5Coltea Clinical Hospital, Internal
Medicine and Cardiology Department, Bucharest, Romania
Objective: Microalbuminuria is considered to be an early stage of
diabetic nephropathy and also a predictor for cardiovascular disease.
Our aim was to assess the percentage of microalbuminuria in uncon-
trolled T2DM in a cross-­sectional study.
Method: T2DM uncontrolled patients (with T2DM duration >2 years)
were assessed between March 2016 and December 2017 in a cross-­
sectional study. All the patients were evaluated by clinical examina-
tion, paraclinical exams (HbA1c in the last 3 months, fasting plasma
glucose – FPG, microalbuminuria defined as at least one positive
urine albumin-­creatinine ratio test in the last 6 months).
Results: 332 patients, 49% male, mean age 61.3 years, mean duration
of DM 7(3.7) years, HbA1c 8.2% (1.7), FPG: 204.9(69.3) mg/dL were
included. 28% of the patients had an HbA1c<7.5% and were consid-
ered the control group. The other 72% had a mean HbA1c of 8.9%.
Patients were treated for DM with metformin (88.5%), sulphonylu-
reas (81%), dipeptidyl peptidase 4 inhibitors (7.4%), glinides (2.9%) in
monotherapy or combination. Patients with insulin treatment were
excluded. 28.5% of patients had microalbuminuria in total group, 37%
in those uncontrolled, 13% in pts with HbA1c<7.5%.
 |
66 of 96       ABSTRACTS

Conclusion: It is well known that most of the complications in diabe- these processes is suggested as an adjunct to traditional antimi-
tes arise from damages to small blood vessels due to chronic hyper- crobial therapy.
glycemia and hypoglycemic episodes. Our study confirms that the Funding: EPSRC Network for Anti-­
Microbial Resistance and
long-­term exposure to hyperglycemia is correlated with an increased Infection Prevention.
percentage with patients with microalbuminuria. It is absolutely nec-
essary to screen more often these patients and to maintain their gly-
caemic control on long term. TUPE004 | The use of continuous wave
spectroscopy for the evaluation of microvascular
P OS TE R PR E S E NTATI O N S – T U E S DAY, dysfunction in sepsis
S E P TE M B E R 11 , 2 018 Justin Piazza1; Paulina Kowalewska1,2; Stephanie
Milkovich1,2; Richard Sové1; Asher Mendelson1,2;
Christopher Ellis1,2
1
Department of Medical Biophysics, University of Western Ontario, London, ON,
Canada; 2Robarts Research Institute, University of Western Ontario, London,
TUPE002 | Endothelial cells capture bacteria ON, Canada

are acutely activated by quorum molecules and

Sepsis is a dysregulated host inflammatory response to infection
establish biofilms under shear stress in infective
potentially leading to life-­
t hreatening organ-­
d ysfunction. The
vascular disease study objective was to determine whether early microvascular
Victoria Humbert1; Jemma JA Paterson1; Dario Carugo2; dysfunction (MVD) in septic skeletal muscle can be detected using
Myron Christodoulides1; Timothy M. Millar1 continuous wave spectroscopy (CWS) and with similar efficacy as
1
Clinical and Experimental Sciences, Faculty of Medicine, University of dual wavelength intravital video microscopy (IVVM). Under proto-
Southampton, UK; 2School of Engineering, Faculty of Engineering and Physical
cols approved by Western University, male Sprague-­D awley rats
Sciences, University of Southampton, UK
were prepared for IVVM of the right extensor digitorum longus
muscle and injected intraperitoneally with either a fecal slurry
Systemic vascular infections can cause organ failure and progress
(feces-­induced peritonitis) or normal saline (control group). IVVM
rapidly to septic shock and death. Bacteria subvert normal en-
and CWS recordings were taken over a period of 6 hours after
dothelial functions to setup a protected surface niche or become
induction of sepsis. While macrohemodynamic parameters were
internalised and hidden from the immune system. Bacteria that
only found to significantly differ between septic and control ani-
colonise the endothelial surface can also activate the clotting cas-
mals 4.5-­6  hours after the induction of sepsis, microhemodynamic
cade. These biofilms are difficult to treat and growing antimicrobial
measurements (RBC velocity, supply rate and oxygen saturation)
resistance is an additional burden. However, the initiating events
derived from IVVM recordings were significantly decreased in
of biofilm formation are less well characterised. We hypothesised
septic animals by 2.5-­4 hours post infection. Fourier analysis of
that host pathogen interactions acutely activate the endothelium
the mean ΔODiso of 6 isosbestic wavelengths in CWS exhibited
to provide an environment on which biofilms form. Our objective
a significant shift in the power spectrum towards low frequency
was to simulate the environment of vascular compartments and
oscillations in hemoglobin by the 2.5-­4 hours time point in septic
investigate the host pathogen interaction required to establish
animals that resembled oscillations in microhemodynamic meas-
biofilms. Using microfluidic assays of bacteria-­
e ndothelial and
urements and mean frame intensity collected with IVVM. These
bacteria-­m atrix interaction, we show that S. pyogenes binds to the
oscillations were not readily apparent in control animals during
endothelium under static and increasing shear stresses. Bacteria
the 6 hours experiment. This study showed that dynamic changes
also attached to endothelial cells under more complex pulsatile
in hemoglobin levels detected with CWS are comparable to data
flows. Membrane changes in the endothelium were observed on
collected with IVVM and that changes in the power spectrum
adherence of bacteria and resembled active endosomes. Bacteria
may be a useful diagnostic tool for non-­invasively detecting early
also adhered to extracellular matrix proteins under laminar and
MVD in septic patients. Funding: Collaborative Health Research
pulsatile flows. Quorum molecules used by bacteria in forming bi-
Projects, CIHR & NSERC.
ofilms were shown to acutely activate the endothelium via release
of the clotting protein von Willebrand factor (vWF) while S. pyo-
genes enhanced thrombin mediated platelet aggregations. Host
pathogen interactions that include bacteria-­e ndothelial adhesion
and quorum signals therefore are a potential mechanism for cap-
ture and establishment of biofilms in the vasculature. Targeting
ABSTRACTS
TUPE006 | PR3 and HLE contribute to
microcapillary damage in sepsis
Eric K. Patterson1; Gediminas Cepinskas1,2; Carolina Gillio-
Meina3; Douglas D. Fraser1,3
1
Centre for Critical Illness Research, Lawson Health Research Institute, London
(ON), Canada; 2Department of Medical Biophysics, Western University, London
(ON), Canada; 3Department of Pediatrics, Western University, London (ON),
Canada
Sepsis is a detrimental systemic response to infection which com-
monly leads to vascular barrier dysfunction and associated microcapil-
lary endothelial leak. Polymorphic neutrophils (PMNs) are thought to
mediate some aspects of vascular instability, but evidence in sepsis is
lacking. The serine proteases Proteinase 3 (PR3) and Human Leukocyte
Elastase (HLE) are abundant in PMN granules released upon degranu-
lation and have overlapping substrates. We hypothesized that PR3 and
HLE contribute to vascular barrier dysfunction in sepsis.
Plasma was obtained from severe sepsis patients admitted to the
intensive care unit at Victoria Hospital under a protocol approved by
the Western University Ethics Committee. We used ELISA and real-­
time PCR to quantify HLE and PR3 concentrations and their source
in leukocytes, respectively. Exogenous HLE or PR3 was applied to
HUVEC monolayers to determine endothelial leak in response to
each enzyme using fluorescently-­
labelled dextran (3000 MW).
Furthermore, electrical resistance was used to provide real-­time data
on endothelial monolayer permeability in response to each enzyme.
Both PR3 and HLE were significantly increased in plasma from pa-
tients with severe sepsis, and their genes in leukocytes. Additionally,
both enzymes significantly increased HUVEC monolayer permeabil-
ity to dextrans, as well as decreased HUVEC monolayer integrity in a
time and dose-­dependent manner.
Taken together, these results suggest that PR3 and HLE at
concentrations relevant to conditions at and near the site of PMN
degranulation can contribute to the vascular barrier dysfunction as-
sociated with sepsis. This further suggests that inhibiting these en-
zymes may be beneficial to treating patients suffering capillary leak.
TUPE007 | CORM-­3 prevents sepsis-­induced
skeletal muscle microvascular dysfunction
Gediminas Cepinskas1,2; Christopher Ellis2; D. D. Fraser1,3;
G. H. (Barry) Janssen1,2
1
Centre for Critical Illness Research, Lawson Health Research Institute, London
(ON), Canada; 2Department of Medical Biophysics, Western University, London
(ON), Canada; 3Department of Pediatrics, Western University, London ON,
Canada
Impaired microvascular perfusion contributes to sepsis-­
induced
organ injury/dysfunction. Studies have indicated that carbon mon-
oxide releasing molecules (CORM) offer protection in various mod-
els of systemic inflammation.
In an experimental model of an acute minimally resuscitated
poly-­microbial sepsis (Fecal-­Induced Peritonitis; FIP) in the rat, the
|
      67 of 96
effects of water-­soluble CORM (CORM-­3) on microvascular perfu-
sion in skeletal muscle were investigated.
Experiments were approved by the local Animal Care and Use
Committee of Western University. Male Sprague-­Dawley rats were
treated with 3 mg/kg (iv), CORM-­3 (FIP n = 7; 34 Fields of View
(FOV), SHAM n = 7; 45 FOV) or inactive CORM-­3 (iCORM-­3; FIP
n = 8; 37 FOV, SHAM n = 6; 32 FOV) 30 minutes before induction
of sepsis. Microvascular perfusion in the peroneus muscle was as-
sessed by intravital video microscopy. Furthermore, the effects of
CORM-­3 on systemic hemodynamic parameters and animal survival
were also assessed.
CORM-­3 treatment significantly improved the 5-­hour survival in
FIP-­challenged animals and significantly delayed the onset of micro-
vascular dysfunction. Four hours after induction of sepsis, 55% of
the micro-­vessels in CORM-­3 group maintained continuous blood
flow, while in the iCORM-­3 pretreated group only 43% of the ves-
sels were continuously perfused (P: 0.03: Mann–Whitney U test).
CORM-­3 improved myocardial contractility, augmenting mean arte-
rial pressure, as well as systolic and pulse pressure, without affecting
heart rate and diastolic pressure.
Our findings indicate that CORM-­
3 pre-­
treatment minimizes
micro-­vessel dropout in skeletal muscle, improves systemic hemo-
dynamic parameters, and animal survival in a model of acute and
minimally resuscitated sepsis.
Funding: Lawson Health Research Fund and HSFO (G-­
17-­
0018622) (GC).
TUPE009 | Chloropalmitic acid, a putative
septic mediator, produces leukocyte rolling/
adhesion responses similar to cecal ligation and
puncture or LPS exposure
Meifang Wang1; Derek Wang1; David A. Ford2; Ronald J.
Korthuis1,3
1
Department of Medical Pharmacology and Physiology, Saint Louis University
School of Medicine, St. Louis, MO, USA; 2Edward A. Doisy Department of
Biochemistry and Molecular Biology, Center for Cardiovascular Research,
Saint Louis University School of Medicine, St. Louis, MO, USA; 3The Dalton
Cardiovascular Research Center, and University of Missouri School of Medicine,
Columbia, MO, USA
We recently showed that chlorinated lipids, which increase in plasma
of septic patients and animals, produced marked proinflammatory
effects in the intestinal microcirculation. In this study, we com-
pared leukocyte rolling and stationary adhesion responses invoked
by Chloropalmitic Acid (ClPA), LPS, and Cecal Ligation Puncture
(CLP). LR and LA were quantified in postcapillary venules in both the
small intestinal wall (SIW) and in Peyer's Patch (PP) using intravital
fluorescence microscopy 6, 24 and 48 hours after CLP, 50 minutes
after ClPA, and 24 hours after LPS. Our data showed that LR and
LA were increased in SIW venules and PP after ClPA, LPS, or CLP (at
6 hours) by a similar magnitude. The increases in LR and LA noted
6 hours after CLP were restored to control levels when assessed 24
 |
68 of 96       ABSTRACTS
and 48 hours later. Interfering with toll-­like receptor signaling inhib-
ited the pro-­adhesive responses to CLP or LPS but failed to block
increased LR and LA induced by ClPA. Our data indicates that the
putative septic mediator ClPA mimics the adhesive responses in-
voked by CLP or LPS but provokes these responses by a mechanism
independent of toll-­like receptor signaling. This work was supported
by a grant from the National Institutes of Health (GM-­115553).
TUPE010 | The combination of 3, 4-­dihydroxyl-­
phenyl lactic acid and notoginsenoside R1
protects heart from ischemia/reperfusion injury
via antioxidation and energy regulation
Li Yan; Ke He; Chun-Shui Pan; Yuan-Chen Cui; Yu-Ying Liu;
Bai-He Hu; Xin Chang; Xiao-Hong Wei; Jing-Yan Han
Tasly Microcirculation Research Center, Peking University Health Science Center,
Beijing, China
This study was designed to investigate the effect of 3,
4-­dihydroxyl-­phenyl lactic acid (DLA) and notoginsenoside R1 (NR1),
as the two major ingredients of Cardiotonic pill (CP), against I/R-­
induced microcirculatory disturbance and myocardial damage, with
emphasis on the underlying mechanism.
In this study, we detected the effect of DLA, NR1 and its combi-
nation (DR) on myocardial blood flow, vascular diameter, velocity of
red blood cells, and albumin leakage after reperfusion in rat heart.
Myocardial infarction and myocardial histology were evaluated.
ATP and AMP content were assessed by ELISA. P-­AMPK, ATP5D,
phosphorylated-­Myosin Light Chain (P-­MLC), apoptosis and oxida-
tive stress-­related molecules were determined by Western blotting.
Results showed that pre-­treatment with DR significantly atten-
uated I/R-­induced infarct size and myocardial microcirculatory dis-
turbance, including decreased coronary blood flow and red blood
cells velocity in venules, and albumin leakage from venules. In ad-
dition, DR significantly ameliorated I/R-­induced myocardial damage
and apoptosis. Moreover, I/R caused the decrease in the ratio of
ATP/ADP and ATP/AMP, accompanying with reduction of ATP 5D
expression and increase of P-­MLC and P-­AMPK, all of these were
ameliorated by DR. Furthermore, DLA or NR1 alone exhibited simi-
lar effects as their combination (DR) but to a lesser extent with NR1
being more effective for enhancing energy metabolism, while DLA
was more effective for counteracting oxidative stress.
In conclusion, the combination of DLA and NR1 exerts a more
potent effect against myocardial I/R injury than their alone, due to
the interaction of its active components through a multi-­component
and multi-­t arget mode
TUPE011 | Sphingosine-­1-­phosphate
protects endothelial barrier function following
hemorrhagic shock and hypoxic injury
Natascha Alves; Sarah Yuan; Jerome Breslin
Department of Molecular Pharmacology and Physiology, Morsani College of
Medicine, University of South Florida, Tampa, FL, USA
Our lab has previously demonstrated that S1P reduces hemorrhagic
shock and resuscitation (HSR)-­induced microvascular hyperperme-
ability. HSR can cause poor perfusion and hypoxia in the gut, leading
to endothelial apoptosis and injury to the endothelial surface layer
(glycocalyx). In the current study, our objective was to investigate
the extent to which S1P can prevent apoptotic signaling, shedding of
the endothelial surface layer (glycocalyx), and elevated microvascu-
lar permeability in response to HSR in vivo or hypoxia in a cultured
endothelial cell model. For our in vivo studies, we used a rat model
of HSR, in which S1P was administered in the intravenous resuscita-
tion fluid. The mesenteric microvasculature was dissected, and pro-
tein was extracted for the assessment of pro-­apoptotic proteins by
western blot.
We also assessed mitochondrial membrane potential using JC-­
1. We performed immunofluorescence of the mesenteric micro-
vasculature to evaluate paracellular junction protein interaction.
As an indicator of glycocalyx health, we used an intravenous in-
jection of FITC-­labeled lectin and measured the amount of lectin
bound to the microvascular endothelium. For the in vitro studies,
we placed cultured primary endothelial cells in a low-­oxygen envi-
ronment and applied S1P during the hypoxic challenge. We tested
whether S1P has the potential to protect barrier function of endo-
thelial cells grown on Transwell membranes (0.4 μm pores) from
hypoxia-­induced hyperpermeability by determining permeability
to FITC-­albumin. We have found that HSR did not cause an in-
crease in pro-­apoptotic protein expression levels in vivo. However,
HSR caused a significant disruption in mitochondrial membrane
potential. The effects of S1P on mitochondrial function still need
to be investigated. Besides that, we found that the amount of lectin
bound to the mesenteric microvasculature is higher in HSR animals
treated with S1P compared to HSR only. In our in vitro studies, we
found that S1P prevents hypoxia-­induced permeability in different
types of endothelial cells, characterized by a decrease in endothe-
lial permeability. Taken together, our results suggest that S1P has
the potential to protect microvascular barrier dysfunction caused
by HSR by preventing glycocalyx shedding. However, how S1P re-
duces the activation of apoptotic cascade following HSR remains
elusive.
Supported by NIH grants R01HL098215, R01GM120774,
R01HL070752 and R01GM097270.
ABSTRACTS
TUPE013 | Microalbuminuria, a marker of
microvascular inflammation, could improve risk
prediction of stroke in patients with TIA and
minor stroke
Angela Shore; Nicola Wedge; Luke Mounce; Salim Elyas;
Martin James; David Strain
University of Exeter Medical School, Royal Devon and Exeter Hospital
Background: Stroke is the biggest cause of disability world-­wide.
23% of stroke suffers have a Transient Ischaemic Attack (TIA) in
the preceding 90 days, giving a short window to identify high risk
people and intervene. Urinary albumin excretion rate, or its surro-
gate albumin:creatinine ratio (ACR), is a marker of systemic micro-
vascular inflammation. ACR, measured on a spot-­test of urine, gives
a clinically accepted proxy for systemic vascular inflammation. We
hypothesised that ACR after TIA would predict progression to full
stroke.
Methods: Basic demographics were recorded in 2,408 patients
attending a clinic within a month of TIA or minor stroke. Urinary
ACR was measured on a bench-­top analyser (Affion AS300, Alere,
Dundee). Patients were followed up at day 7, 30 and 90 to determine
recurrent stroke, cardiovascular events or death.
Results: 2,262 were included in the final analysis. 287 (12.7%) pa-
tients had an outcome. Participants with events were older (72.9
vs. 70.8 years; P = 0.003) but otherwise similar. ACR was higher in
those with events compared to those without (2.4(95% CI 2.1-­2.7)
vs 1.9(1.8-­1.9) mg/mmol respectively, P = 0.001) independent of age
and sex. An elevated ACR (≥2.5 mg/mmol for men; ≥3.5 mg/mmol
for women) predicted recurrent events (OR = 3.35; 95% CI 1.64-­
6.83; P = 0.001) and was associated with a 5.25-­fold (1.74-­15.82;
P = 0.003) higher 90-­day mortality.
Conclusion: After TIA or minor stroke, an elevated ACR predicts
evolution to full stroke or premature mortality. This assists with risk
stratification after TIA and may present a novel therapeutic target
for stroke prevention.
TUPE014 | Rapid treatment of grade 2-­3
hypertension in previously untreated patients
is accompanied by increased total and perfused
skin capillary density
Angela Shore; Andrew Sharp; Christine Anning; Nicola
Pamphilon; Claire Ball; Kim Gooding; Dave Mawson;
Christopher Clark; Andrew Jordan
University of Exeter Medical School, Royal Devon and Exeter Hospital
Introduction: Reduced skin capillary density (rarefaction) occurs in
patients with, or at risk of, essential hypertension and can be reversed
by 6 months antihypertensive medication in mild hypertension 1.
|
      69 of 96
Aim: To assess whether capillary numbers increased in treatment
naïve individuals with grade 2-­3 hypertension undergoing a short
intensive 18-­week treatment regime.
Methods: 54 treatment naive individuals (18-­
8 0 years) (office
systolic BP ≥ 170 mm Hg and ambulatory daytime average sys-
tolic BP ≥ 150 mm Hg) completed 18 weeks of stepwise treat-
ment intensification every 2-­
4 weeks if office BP remained
≥ 140/90 mm Hg. At weeks 0 and 18 skin capillary density was
measured by intravital capillaroscopy on the dorsal finger skin
at rest and following venous occlusion to measure total capillary
numbers.
Results: Treatment increased resting capillary number from 83.8
mean (24.1 SD) to 92.2 (23.6) caps/mm2 P = 0.004), total capil-
lary number from 104.4(23.8) to 111.5 (23.3) caps/mm2 P = 0.003.
Capillary reserve was not altered by treatment, %change of capillary
number from resting levels to post occlusion, 27.3 (21.4) % pre vs
23.6 (18.0)%, P = 0.291, post treatment. Rarefaction was associated
with higher plasma glucose and HbA1c (r = −0.374 and r = −0.383;
P < 0.006), higher LVM (r = −0.512, P < 0.001). Capillary density pre-­
treatment was not associated with the change in systolic or diastolic
BP with treatment but correlated with the change in LVM r = 0.41;
P = 0.005.
Conclusions: Skin capillary density is increased in patients with
grade 2-­3 hypertension by a short, 18-­week, period of intensive
adjustment of blood pressure sufficient to bring 69% of patients to
target office blood pressure.
TUPE015 | Laser speckle contrast imaging for
intraoperative assessment of tissue perfusion in
autologous breast reconstruction with DIEP flaps
Johan Zötterman1; Dries Opsomer2; Simon Farnebo1; Phillip
Blondeel2; Stan Monstrey2; Erik Tesselaar3
1
Department of Plastic Surgery, Hand Surgery, and Burns, Linköping University,
Linköping, Sweden; 2Department of Plastic and Reconstructive Surgery,
University Hospital Ghent, Belgium; 3Department of Medical Radiation Physics,
Department of Medical and Health Sciences, Linköping University, Linköping,
Sweden
Introduction: Partial flap necrosis is common after free flap surgery.
We used laser speckle contrast imaging (LSCI) to map the perfusion
in deep inferior epigastric artery perforator (DIEP) flaps for breast
reconstruction in relation to the main perforator and to study the
value of LSCI in predicting postoperative complications.
Method: Sixteen patients who underwent a unilateral DIEP proce-
dure were included in the study. Perfusion was measured in four
zones according to Hartrampf; at baseline, after raising the flap, after
anastomosis and after shaping the flap.
Results: At baseline, there were no differences in mean perfusion
between zones except between zone II and III. After raising the flap,
zone I showed the highest perfusion (65 ± 10 PU), followed by zone
II (58 ± 12 PU), and zone III (53 ± 10 PU) and IV (45 ± 10 PU). The
perfusion in zones III and IV were significantly lower than in zone I.
 |
70 of 96       ABSTRACTS
After anastomosis, perfusion was similar in zone I (64 ± 16 PU), while
it was increased in the other zones, compared to before anastomosis.
Zone IV had significantly lower perfusion than zone I (P < 0.0001),
zone II (P = 0.01) and zone III (P = 0.02). All flaps with perfusion re-
gions <30 PU had partial or fat necrosis postoperatively.
Conclusion: As expected, perfusion was highest in zone I. Contrary
to some previous studies, we found no difference in perfusion be-
tween zone II and III. Perfusion <30 PU intraoperatively was associ-
ated with partial flap necrosis. LSCI is a promising tool for assessing
flap perfusion and risk for postoperative ischemia.
TUPE016 | Breast cancer metastasis to bone:
the role of the perivascular niche in regulating
tumour cell dormancy
1,2 1,2
Nicola Jane Brown ; Russell Hughes ; Gloria Allocca ; 2 lyze microvascular flow. Each video clip was assessed for quality
Penny Ottewell2; Jamie Hobbs3; Ingunn Holen2
1
Microcirculation Research Group, University of Sheffield, Sheffield, UK;
2
Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK;
3
Department of Physics and Astronomy, University of Sheffield, Sheffield, UK
Tumour cell dissemination to bone is an early event in breast can-
cer with approximately 30% of patients having disseminated tumour
cells in their bone marrow at the time they receive treatment for
their primary tumour. However, only a proportion of patients de-
velop metastasis, often following an extended period of dormancy.
The perivascular niche within bone plays an important role in regu-
lating the dormancy of disseminated tumour cells (DTC), but the cel-
lular and molecular components of this perivascular niche remain to
be clearly defined.
We have established two in vivo mouse models of breast cancer
bone metastasis using MB-­MDA-­231 cells (Home Office Authority
PPL70/8964) where breast cancer cells arriving in bone either un-
dergo outgrowth (6-­week old animals with high bone turnover) or
enter dormancy (12-­week old animals with mature skeleton). We use
confocal microscopy, immunohistochemistry and qPCR to quantify
differences in both cellular composition and gene expression signa-
tures, with particular emphasis on the perivascular niche.
The bone microvasculature and perivascular niche are mark-
edly different in these two models. We show that significantly in-
creased numbers of Thrombospondin-­1 + cells, vascular remodeling
and reduced numbers of CD31+ blood vessels, Osterix+ and αSMA+
osteoprogenitors, and CD169+ macrophages are associated with
the dormancy of DTCs in bone. We also show the differential ex-
pression of such dormancy-­regulating genes as Thrombospondin-­1,
Collagen-­IV, Periostin, Tenascin-­C and Osteopontin between the
outgrowth and dormancy-­promoting niches.
Our data indicate that tumour cell dormancy in bone is sup-
ported by the mature microvasculature, a reduced bone turnover
and alterations in the immune system.
TUPE017 | Sublingual microcirculation
characteristics in patients with a left ventricular
assist device
Andrew Deitchman1; Michael McCurdy1; Michael Mazzeffi2
1
Department of Medicine, University of Maryland School of Medicine;
2
Department of Anesthesiology, University of Maryland
Background: Microvascular flow abnormalities exist in many disease
states; however, microcirculatory characteristics in continuous-­flow
left ventricular assist devices (LVADs) are unknown. We report our
observational experience imaging the sublingual microcirculation of
10 hemodynamically stable patients with LVADs using point-­of-­care
(POC) microvascular analyses.
Methods: A single operator at the patient's bedside used the
Cytocam (Bradeus, Netherlands) to acquire, measure, and ana-
using the Massey quality score. Acceptable measurements were
evaluated for a microvascular flow index (MFI) and a Point Of carE
Microcirculation (POEM) score, a recently developed POC scoring
algorithm to determine flow and heterogeneity. Total vessel density
(TVD) was calculated using the Bradeus imaging analysis software in
addition to a POC visual estimate of TVD.
Results: All patients had normal MFI (>2.6). Nine of 10 patients ex-
amined had normal or near-­normal POEM scores (4 or 5). One pa-
tient who was postoperative day 3 from LVAD insertion had a POEM
score of 3 and a borderline normal MFI of 2.625. All other patients in
the cohort were >14 days postoperative.
Conclusions: Patients with LVADs represent a novel patient popu-
lation with alterations in normal physiologic blood flow. Patients
>2 weeks since device implantation had normal microvascular flow.
An acute postoperative period may be associated with microvascular
flow abnormalities, although the onset, resolution, and clinical impli-
cations of this finding require further investigation.
TUPE018 | Neovascular homing peptides
dimeric GX1 showed antiangiogenesis to diabetic
retinopathy and gastric cancer
Xiaoli Hui1; Yingying Luo1; Zhijie Lei2; Wei Cui1; Kaichun
Wu2
1
Department of Geriatrics, First Affiliated Hospital of Xi'an Jiaotong University,
Xi'an, Shaanxi Province, China; 2Xijing Hospital of Digestive Diseases & State
Key Laboratory of Cancer Biology, Air Force Medical University, Xi'an, Shaanxi
Province, China
Background: Antiangiogenesis has become an important approach
for the therapy of tumor and diabetic retinopathy. Some common
receptors and drug targets are observed in both tumor and dia-
betic retinopathy. GX1 peptide (nation patent peptide) screened by
in vivo phage display technology was fully confirmed with binding
ability to vasculature endothelial cells of human gastric cancer and
antiangiogenesis, and its binding affinity was further improved by
ABSTRACTS
dimerization. In this study, dimeric GX1 was synthesized to evaluate
its antiangiogenesis ability to retina and gastric cancer.
Methods: Antiangiogenesis of dimeric GX1 on retinal micro-
vasculature endothelial cells (RMEC) and HUVEC with gastric
cancer endothelium characteristics generated by culturing in tumor-­
conditioned medium (Co-­HUVEC) was analyzed by CCK-­8 assay, mi-
gration assay, and tube formation assay.
Results: Dimeric GX1 showed more significant inhibition effect on
cell proliferation, micro-­tube formation and migration of RMEC and
Co-­HUVEC in a dose-­dependent manner than GX1 monomer and di-
meric control peptides. In addition, more significant inhibition effect
of dimeric GX1 was observed on RMEC than Co-­HUVEC, especially
at the dose of 100 μm(P < 0.05).
Conclusions: Dimeric GX1 owned more significant antiangiogenesis
on RMEC and Co-­HUVEC than GX1 monomer, and more significant
antiangiogenesis on RMEC than Co-­HUVEC. Dimeric GX1 was more
promising to be explored for effective antiangiogenesis targeting
drug to diabetic retinopathy and gastric cancer than GX1 monomer.
Sources of funding: National Foundation of Natural Science, China
(No. 30900704).
General project of key research and development program in Shaanxi
Province (2013K12-­09-­03, 2017SF-­104)
Scientific research fund project of Shaanxi Health and Family
Planning Commission (2016D066).
This study was supported by General project of key research and de-
velopment program in Shaanxi Province (2013K12-­09-­03, 2017SF-­
104), Scientific research fund project of Shaanxi Health and Family
Planning Commission (2016D066), and Scientific research fund pro-
ject of Xi'an City (201805095YX3SF29(6)).
TUPE019 | A preliminary study examining
albuminuria, macular thickness and glycocalyx
shedding in early stages of diabetic retinopathy in
type 2 diabetes
Kim Gooding; Daniel Chapman; Francesco Casanova; Claire
Ball; Jacqueline Whatmore; Roland Ling; Timothy McDonald;
Angela Shore
University of Exeter Medical School, Royal Devon and Exeter Hospital
Proliferative diabetic retinopathy (DR) is associated with macu-
lar thickening and greater risk of nephropathy in type 2 diabetes
(T2DM). A potential explanation underlying these associations is
widespread glycocalyx perturbations. However, little is known about
early DR.
This preliminary study aims to examine whether macular thick-
ness, albuminuria (albumin:creatinine ratio, ACR) and glycocalyx
shedding (plasma hyaluronic acid levels) are altered in early DR in
T2DM.
Individuals with T2DM with either no DR (n = 214) or mild/back-
ground DR (n = 23) were selected from the Exeter SUMMIT cohort
(Ethics reference: 10/H0206/67). ACR was assessed from a random
|
      71 of 96
urine sample; macular thickness by optical coherence tomography;
fasting plasma hyaluronic acid levels by ELISA. Between group anal-
ysis was performed using Mann-­Whitney U test.
ACR was higher in the DR group compared to non-­DR group (DR
group median (25th, 75th quartiles): 1.5 8 (1.23, 8.12) vs non-­DR
group: 0.97 (0.49, 1.66) mg/mol, P = 0.005). Outer quadrants of the
macular were thicker in the DR group compared to non-­DR group (P
values ≤0.042); there was no difference in fovea thickness. Plasma
hyaluronic acid was higher in the DR group (DR group: 88.5 (51.6,
109.7) ng/mL vs non-­DR group: 51.8(36.5, 86.1) ng/mL, P = 0.017).
Age was similar, but the DR group had a longer duration of diabetes
and higher HbA1c.
This preliminary study suggests that even in the early stages of
DR there are subtle increases in permeability in both the retinal and
systemic vascular beds, as reflected by an increase in ACR and outer
macula thickness, possibly resulting from early perturbations in the
glycocalyx.
Funders: Innovative Medicines Initiative (SUMMIT consortium,
IMI-­2008/115006) and Northcott Devon Medical Foundation.
TUPE020 | Examining the relationship between
skin microvascular reactive hyperaemia and
urinary albumin excretion, extending into the
low-­level range
Daniel Chapman; Kim Gooding; Francesco Casanova;
Damilola Adingupu; Timothy McDonald; Kunihiko Aizawa;
David Mawson; Rebecca Lear; Abigail Chui; Christine
Anning; W. David Strain; Angela Shore
University of Exeter Medical School, Royal Devon and Exeter Hospital
Increasing urinary albumin excretion, commencing within the nor-
mal range, is a recognised risk factor for cardiovascular conditions
in the general population. However, research into the relationship
between early stages of vascular disease and low levels of albumin
excretion is limited due to routine assays being unable to quantify
low levels of albumin.
Aim: To investigate the relationship between skin microvascular
reactive hyperaemia and urinary albumin excretion, encompassing
the low-­level range. 186 participants were recruited (70% male; 65%
with type 2 diabetes; 40% with cardiovascular disease, age range:
46-­86 years) (Ethics reference: 10/H0206/67). Reactive hyperaemia
(peak height and time to peak) was assessed on the dorsal surface of
the foot following arterial occlusion (4 minutes) using laser Doppler
fluximetry. Mean albumin excretion rate (AER) was reported from
two overnight urine collections. AER data transformed by 1/square
root, thus correlations are presented inversely.
Peak height and time to peak were associated with AER (peak
height: r = −0.14; P = 0.051; time to peak: r = 0.15; P = 0.046).
Further examination demonstrated that the association between
peak height, but not time to peak, and AER remained after adjust-
ment for age, gender, smoking status, HDL-­
cholesterol, systolic
 |
72 of 96       ABSTRACTS
blood pressure, history of CVD, HbA1c and waist circumference
(standardised beta = −0.21, P = 0.008). This was replicated when re-
peated within the normal AER range (n = 157).
Increasing urinary albumin excretion, commencing in the previ-
ously undetectable range, is associated with an increase in the peak
hyperaemic response to arterial occlusion; suggesting that increas-
ing levels of albumin excretion is associated with a generalised im-
pairment in microvascular autoregulation.
Supported by Innovative Medicines Initiative (SUMMIT consor-
tium, IMI-­2008/115006).
TUPE021 | mDia1 is crucial for advanced
glycation end product-­induced endothelial
hyperpermeability
Xiaohua Guo; Xiaoyan Zhou; Jie Weng; Qiaobing Huang
Department of Pathophysiology, Key Laboratory for Shock and Microcirculation
Research of Guangdong Province, Southern Medical University, Guangzhou,
510515, China
Aims: Disruption of endothelial barrier integrity in response to
advanced glycation end-­
product (AGE) stimulation contributes
to vasculopathy associated with diabetes mellitus. Mammalian
diaphanous-­related formin (mDia1) has been reported to bind to
the cytoplasmic domain of the receptor for advanced glycation end
products (RAGE), which induces a series of cellular processes. This
study directly evaluated the participation of mDia1 in AGE-­induced
hyperpermeability and revealed the precise intracellular signal trans-
ductions of this pathological process.
Methods: Human umbilical vein endothelial cells (HUVECs) were
used in the in vitro studies. Trans-­e ndothelial electric resistance
and permeability coefficient for dextran (Pd) were measured to
analyze cell permeability. Western blotting, immunofluorescence
staining and flow cytometry assay were performed to investi-
gate the underlying mechanism. Dextran flux across the mes-
entery in mice was monitored to investigate in vivo microvessel
permeability.
Results: We found that AGEs evoked Nox4 membrane translo-
cation, reactive oxygen species production, phosphorylation of
Src and VE-­c adherin, dissociation of adherent junction and even-
tual endothelial hyperpermeability though RAGE-­m Dia1 binding.
Cells overexpressing mDia1 by recombinant adenovirus infection
showed stronger cellular responses induced by AGEs. Down-­
regulation of mDia1 by infection with an adenovirus encoding
siRNA or blockade of RAGE-­m Dia1 binding by transfection with
RAGE mutant plasmids into HUVECs abolished these AGE-­induced
effects. Furthermore, knockdown of mDia1 using an adenovirus or
genetic knockout of RAGE in C57 mice rescued AGE-­evoked mi-
crovascular hyperpermeability.
Conclusion: Our study revealed that mDia1 plays a critical role in
AGE-­induced microvascular hyperpermeability through binding to
RAGE.
TUPE024 | Dissecting two-­way control
mechanisms of cerebral microcirculation:
optogenetic studies for neuron or astrocyte-­
specific mechanisms of functional hyperemia
Nao Hatakeyama1; Miyuki Unekawa2; Hiroyuki Takuwa3;
Iwao Kanno3; Ko Matsui4; Kenji F Tanaka5; Yutaka Tomita2,6;
Norihiro Suzuki2; Jin Nakahara2; Kazuto Masamoto3,7
1
Graduate School of Informatics and Engineering, University of Electro-­
Communications, Chofu, Tokyo, Japan; 2Department of Neurology, Keio
University School of Medicine, Shinjuku, Tokyo, Japan; 3Department of
Functional Brain Imaging Research, National Institute of Radiological Sciences,
Inage, Chiba, Japan; 4Division of Interdisciplinary Medical Science, Tohoku
University Graduate School of Medicine, Sendai, Miyagi, Japan; 5Department
of Neuropsychiatry, Keio University School of Medicine, Shinjuku, Tokyo,
Japan; 6Tomita Hospital, Okazaki city, Aichi, Japan; 7Brain Science Inspired
Life Support Research Center, University of Electro-­C ommunications, Chofu,
Tokyo, Japan
Functional brain activation leads to localized increases in cerebral
blood flow (CBF), allowing for non-­invasive measurements of acti-
vated regions of the brains with modern neuroimaging techniques.
Accumulating evidence suggests that multiple-­cellular mechanisms,
including neurons, astrocytes, and vascular cells, are involved in
spatiotemporally-­tuned vascular responses. In the present study,
we aimed to compare the spatiotemporal evolution of functional
hyperemia induced by cell-­type specific mechanisms of vasodila-
tion. Transgenic mice that expressed photosensitive cation channels,
channelrhodopsin-­2, in muscarinic acetylcholine receptor (mAChR)
M4-­
expressing neurons (N = 32) or Mlc1-­
expressing astrocytes
(N = 27) were used with experimental protocols approved by the
institutional Animal Ethics Committee of Keio University, School of
Medicine, and University of Electro-­Communications. Transcranial
photo-­stimulation was focally applied to the somatosensory cor-
tex, while spatiotemporal changes in CBF were monitored with
laser speckle flow-­graphy. Significantly larger increases in CBF were
observed for neural stimulation, compared to those for astrocytic
stimulation. In contrast, wider areas exhibited the changes in CBF
for stimulation to astrocytes than neurons. Direct imaging of arte-
rial diameters with two-­photon microscopy further revealed higher
involvement of pial arterial responses to astrocytic stimulation but
less for neural stimulation. Finally, pharmacological manipulations
showed that different signaling mechanisms participate in the vas-
cular responses to neural or astrocytic stimulation. Namely, astro-
cytic stimulation leads to localized and conducted dilation of arterial
networks via barium ion and carbenoxolone-­sensitive mechanisms,
whereas vasodilatory responses to mAChR neural stimulation were
not inhibited. In conclusion, cell-­type specific mechanisms deter-
mine either localized or conducted arterial dilation in functional
hyperemia.
ABSTRACTS
TUPE026 | A well-­known agent from a new
perspective: the impact of nimodipine on
neurovascular coupling in the ischaemic rat brain
Orsolya M. Tóth1; Dóra Hantosi1; Viktória É. Varga1;
Ákos Menyhárt1; Máté Gyöngyösi1; László Janovák 2; Imre
Dékány2; Eszter Farkas1; Ferenc Bari1
1
University of Szeged, Faculty of Medicine, Department of Medical Physics
and Informatics, Szeged, Hungary; 2University of Szeged, Faculty of Medicine,
Department of Medical Chemistry, Szeged, Hungary
Under cerebral ischaemia, disruption of neurovascular coupling
t ype Ca2+-­channel
promotes vasoconstriction. Nimodipine, an L-­
blocker was shown to be neuroprotective and a vasodilator through
vascular smooth muscle cell hyperpolarization. Our aim was to as-
sess the potentially beneficial action of nimodipine on the efficacy
of neurovascular coupling under ischaemia, and to design a novel
strategy of drug administration. Nanoparticles are emerging carriers
of drug delivery, which release drugs in response to specific signals.
We propose to utilize ischemic tissue acidosis as a signal to initiate
drug release.
Anesthetized Sprague-­
Dawley rats (n = 35) were used (eth-
ics certification: XXXII/3128/2017). After washing nimodipine (in
solution or associated to nanoparticles; 100 μmol/L) or its vehicle
(alone, or containing nanoparticles) on the exposed brain surface,
both common carotid arteries were permanently occluded (2VO) to
create global forebrain ischaemia. Spreading depolarizations (SDs)
were elicited by 1 M KCl. Local field potential, cerebral blood flow
(CBF) and tissue pH-­variations were recorded from the cerebral
cortex. Nimodipine significantly increased baseline CBF before
(131 ± 43 vs. 104 ± 12% nimodipine vs. vehicle) and after (113 ± 31
vs. 80 ± 9%) 2VO. In contrast, nimodipine carried by nanoparticles
caused significant CBF elevation (60 ± 24 vs. 35 ± 6%) only after tis-
sue pH reduction associated with 2VO. Nimodipine shortened the
duration of both SD itself (54.9 ± 37.1 vs. 106.9 ± 65.5 seconds), and
the associated tissue acidosis (62.6 ± 30.8 vs. 139.5 ± 64.7 seconds),
moreover it enhanced the SD-­related hyperaemia (4543.8 ± 2339.6
vs. 3003.6 ± 1793.7%*s).
Our results show that nimodipine-­treatment maintains neuro-
vascular coupling in the ischaemic brain, which could be achieved
by use of a novel, pH-­dependent, nanoparticle-­based drug delivery
system.
Funding: NKFIH (K120358, K111923); Ministry of Human Capacities
of Hungary Nr. 34232-­
3/2016/INTFIN, to DH; GINOP-­
2.3.2-­
15-­
2016-­0 0006; EFOP-­3.6.1-­16-­ 2016-­0 0008.
|
      73 of 96
TUPE028 | Cell-­cell communication optimizes
arterial network function and conserves blood
flow homeostasis during cerebrovascular
challenges
A. Zechariah1; C. H. Tran2; B. O. Hald3; S. L. Sandow4,5; U. I.
Tuor6; G. R. Gordon6; D. G. Welsh1
1
Robarts Research Institute, Department of Physiology and Pharmacology,
Schulich School of Medicine and Dentistry, University of Western Ontario,
London, ON, Canada; 2Reno School of Medicine, University of Nevada, Reno,
USA; 3Department of Neuroscience, University of Copenhagen, København N,
Denmark; 4Department of Physiology, School of Medical Sciences, University
of New South Wales (UNSW), Sydney, Australia; 5Faculty of Science, Health,
Education and Engineering, University of the Sunshine Coast, Queensland,
Australia; 6Hotchkiss Brain Institute, Cumming School of Medicine, University of
Calgary, Calgary, AB, Canada
Rationale: Cerebral arterial networks match blood flow delivery with
neural activity. Neurovascular coupling begins with a stimulus and
a focal vessel response, which by itself is inconsequential to blood
flow magnitude until it influences the contractile status of multiple
arterial segments. Mechanisms that coordinate these integrated vas-
cular responses are incompletely understood.
Objective: To test the hypothesis that intercellular communication
enables vasomotor coordination and is a crucial regulator of blood
flow homeostasis during cerebrovascular challenges.
Methods and Results: Electron microscopy and immunohisto-
chemistry revealed that cerebrovascular cells are structurally
coupled, and gap junctional subunits are expressed at cell inter-
faces, enabling intercellular signaling. Indeed, robust conduction
was detected in human and mice cerebral arteries after focal ves-
sel stimulation. This response was intimately tied to gap junctions
as genetic deletion of connexin40 attenuated this behavior. An
in-­silico model of a cerebral arterial network demonstrated that
conducted responses can ascend from penetrating arterioles,
influencing the contractile status of surface vessels. Ascending
responses observed in vivo after whisker stimulation, were signifi-
cantly attenuated in connexin40 knockout mice, confirming that
gap junctions drive integrated vessel responses. Diminished com-
munication impaired the ability of cerebral vasculature to maintain
blood flow homeostasis and hence tissue viability after stroke, as
evidenced by MRI.
Conclusions: Using multi-­variant approaches, we demonstrate that
conducted responses are crucial in transcribing focal stimuli into
coordinated changes in cerebrovascular contractile activity. Our
findings establish the integral role for intercellular signaling in neu-
rovascular coupling and expose, a hitherto unknown, mechanism for
flow regulation after stroke. Funding: CIHR and Rorabeck Chair in
Molecular Neuroscience and Vascular Biology.
 |
74 of 96       ABSTRACTS
TUPE030 | ADGRL4, an adhesion G protein-­
coupled receptor involved in angiogenesis
Koon Hwee Ang1; Helen Sheldon1; Esther Bridges1;
David Mordechai Favara1; Massimo Masiero2; Demin Li2;
Francesco Pezzella2; Alison H Banham2; Adrian L Harris1
1 The increase of Slc1a3 + cells lead to impaired penile blood flow
Department of Oncology, University of Oxford, Oxford, UK; 2Nuffield Division
of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of
Oxford, Oxford, UK
ADGRL4 is an orphan adhesion G protein-­coupled receptor whose
expression is upregulated in the tumour endothelium. Its silencing in
endothelial cells impairs angiogenesis and tumour growth in vivo, iden-
tifying ADGRL4 as a novel therapeutic target. This project aims to fur-
ther elucidate ADGRL4's role in angiogenesis. Using Cre-­Loxp genetic
manipulation system, both constitutive (Tie2-­
Cre) and tamoxifen-­
inducible (Pdgfb-­Cre/ERT2) endothelial cell-­specific Adgrl4 knockout
mouse models have been generated. These mice are viable and no
differences in architectural features and vessel morphology of major
organs in the Adgrl4 knockout mice were observed. Syngeneic tu-
mour models of breast cancer (E0771) and colorectal cancer (MC38)
have been conducted in constitutive endothelial cell-­specific Adgrl4
knockout model. Despite no striking difference in tumour growth in
the two tumour models, interestingly, there was an increase in hy-
poxia and necrosis as well as lower microvessel density in the knock-
out group compared to control. This is consistent with Adgrl4's role in
tumour angiogenesis. Aortic rings from the constitutive endothelial
cell-­specific Adgrl4 knockout mouse model showed no difference
in the area of vessel outgrowths in culture; however, those from the
tamoxifen-­inducible endothelial cell-­specific showed drastic lack of
outgrowths compared to control. This suggests that there might be
a functional compensatory mechanism, enabling adaptation to the
loss of Adgrl4, in the constitutive knockout model. Currently, we are
conducting syngeneic tumour models in the tamoxifen-­inducible en-
dothelial cell-­specific Adgrl4 knockout mice. All animal work has been
conducted under the relevant ethics guidelines for animal care and
experimentation. This project is funded by Cancer Research UK.
TUPE031 | Perivascular cells mediate penile
erection
Eduardo Guimaraes1; David Oliveira Dias1; Evelina Vågesjö2;
Daniel Holl1; Mia Phillipson2; Christian Göritz1
1
Department of Cell and Molecular Biology, Karolinska Institutet, SE-­171 77
Stockholm, Sweden; 2Department of Medical Cell Biology, Division of Integrative
Physiology, Uppsala University, 751 23 Uppsala, Sweden
Penile erection is dependent on increased blood inflow and re-
duced outflow due to a veno-­occlusive system, resulting in expan-
sion of the corpora cavernosa. We discovered that the corpora
cavernosa contained a specific, proliferative, subpopulation of
perivascular cells that express the sodium-­dependent glutamate/as-
partate transporter Slc1a3. In old or diabetic mice, both conditions
associated with dysfunctional penile erection, a reduced number
of Slc1a3 + perivascular cells and disturbed penile blood perfu-
sion were observed. In contrast, when the number of Slc1a3 + cells
were increased by cell-­
specific genetic stimulated proliferation,
spontaneous long-­lasting erections called priapism were observed.
response to vasodilators and reduced the response to vasocon-
strictors. Furthermore, cell specific light-­
induced stimulation of
Slc1a3  +  perivascular cells expressing channelrhodopsin 2 increased
penile blood flow and penile swelling, demonstrating a direct effect
of these cells in erection. In conclusion, Slc1a3 + perivascular cells
mediate erection through modulation of vasodilation.
All experimental procedures were carried out in accordance to
the Swedish and European Union guidelines and approved by the
institutional ethical committee (Stockholms Norra Djurförsöksetiska
Nämnd). Christian Göritz is a Hållsten Academy Fellow, Knut and
Alice Wallenberg Fellow and Ming Wai Lau Centre Fellow.
TUPE032 | Super-­resolution imaging visualized
organized spatial patterns of activated β2
integrins in arresting neutrophils
Zhichao Fan1; William Bill Kiosses2; Dirk M. Zajonc3,4; M.
Amin Arnaout5,6,7,8; Edgar Gutierrez9; Alex Groisman9; Klaus
Ley1,10
1
Division of Inflammation Biology, La Jolla Institute for Allergy and
Immunology, La Jolla, CA, USA; 2Microscopy Core Facility, La Jolla Institute for
Allergy and Immunology, La Jolla, CA, USA; 3Division of Immune Regulation,
La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA; 4Department
of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent
University, Ghent, Belgium; 5Harvard Medical School, Boston, Massachusetts,
USA; 6Leukocyte Biology and Inflammation Program, Massachusetts General
Hospital, Boston, Massachusetts, USA; 7Division of Nephrology, Department
of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA;
8
Center For Regenerative Medicine, Medical Services, Massachusetts General
Hospital, Boston, Massachusetts, USA; 9Department of Physics, University of
California San Diego, San Diego, CA, USA; 10Department of Bioengineering,
University of California San Diego, La Jolla, California, USA
The transition from leukocyte rolling to firm adhesion is called ar-
rest. β2 integrins are required for neutrophil arrest. Chemokines can
trigger neutrophil arrest in vivo and in vitro. Resting integrins exist in
a “bent-­closed” conformation, i.e., not extended (E-­) and not high af-
finity (H-­), unable to bind ligand. Electron microscopic images of iso-
lated β2 integrins in “open” and “closed” conformations inspired the
switchblade model of integrin activation from E-­H-­to E+H-­to E+H+.
Recently, we discovered an alternative pathway of integrin activation
from E-­H-­to E-­H+ to E+H+. Spatial patterning of activated integrins is
thought to be required for effective arrest, but so far only diffraction-­
limited localization maps of activated integrins exist. Here, we com-
bine super-­resolution microscopy with molecular modeling to identify
the molecular patterns of H+E-­, H-­E+, and H+E+ activated integrins
on primary human neutrophils. At the time of neutrophil arrest, E+H+
integrins form oriented (non-­random) nanoclusters that contain a
total of 4,625 ± 369 E+H+ β2 integrin molecules.
ABSTRACTS
TUPE033 | A role for Ca2+-­induced Ca2+-­
release in smooth muscle differentiation
BaRun Kim1; Renato Molina1; Gaby Jensen1; Damon
Poburko1,2
1
Department of Biomedical Physiology and Kinesiology, Simon Fraser University,
Burnaby, British Columbia, Canada; 2Centre for Cell Biology, Development and
Disease, Simon Fraser University, Burnaby, British Columbia, Canada
Vascular smooth muscle cells (VSMC) shift between a differenti-
ated, contractile phenotype and a de-­differentiated, proliferative
phenotype. Phenotypic switching contributes to wound repair but
also pathological remodelling and involves differential expression
of contractile and Ca 2+
handling proteins. Myocardin is a key tran-
scriptional regulator of VSMC phenotype. Studying A7r5 cells in an
in vitro model of differentiation, we hypothesized that altered Ca2+
signaling contributes to the up-­regulation of myocardin during dif-
ferentiation. Culturing A7r5 cells in differentiation media (DM: 1%,
5 mmol/L glucose) vs growth media (GM: 10% serum, 25 mmol/L
glucose) caused growth arrest, increase expression of myofilament
proteins and electrical coupling. Imaging fluo2-­loaded cells in 96-­
well plates, we found that proliferating A7r5 cells predominantly
exhibit asynchronous, IP3 receptor-­mediate Ca 2+
signalling that is
inhibited by cyclopiazonic acid (5 μmol/L), while differentiating cells
2+
electrically couple and exhibit synchronized Ca oscillations driven
by voltage-­gated Ca2+ channels and ryanodine receptors that are
sensitive to nifedipine (1 μmol/L) and dantrolene (1.25-­5 μmol/L).
Correlative immunofluorescence revealed that upregulation of myo-
cardin was detectable after 2 days of differentiation, while altered
Ca2+ signaling and actin expression and organization required an
additional two to four days. Chronic treatment with nifedipine, but
not dantrolene, further increased myocardin and actin expression
in DM-­treated but not GM-­treated cells. Thus, onset of a differen-
tiated Ca2+ signaling phenotype and up-­regulation of myofilament
proteins lags myocardin upregulation upon initiation of differentia-
tion. Further, the spontaneous Ca2+ signaling in differentiated cells
mediated negative feedback on myocardin expression in this in vitro
model of differentiation. Funding: NSERC Grant.
TUPE035 | Shear stress modulates notch
signaling-­mediated vascular repair
Kyung In Baek1; Chih-Chiang Chang1; Shyr-Shea Chang2;
Rongsong Li3; Marcus Roper2; Tzung Hsiai1,3
1
Department of Bioengineering, University of California Los Angeles;
2
Department of Mathematics, University of California, Los Angeles; 3Division of
Cardiology, Department of Medicine, University of California, Los Angeles
Zebrafish (Danio rerio) shares a conserved cardiovascular develop-
mental signaling pathway with mammals and demonstrates robust
structural recovery in response to anatomical amputation, thereby
providing a genetically tractable model to investigate mechanisms
underlying vascular injury and repair. The Notch signaling is a
|
      75 of 96
conserved flow-­responsive mechanism playing a pivotal role in en-
dothelial proliferation and migration during embryogenesis. Using our
transgenic zebrafish model of tail amputation, we seek to elucidate
flow-­sensitive mechanisms whereby Notch signaling is implicated in
vascular repair after injury. Embryonic zebrafish at 1-­2 cell stage were
micro-­injected with or without Gata1a morpholino oligonucleotide
(MO) or erythropoietin (EPO) mRNA to genetically manipulate the
level of hematopoiesis and subsequent wall shear stress. The control
group developed complete vascular repair between the dorsal aorta
and the dorsal longitudinal anastomotic vessel at 3 days post tail am-
putation (dpa), while reduction of viscosity-­mediated shear stress via
Gata1a MO injection attenuated Notch activity on the injured site
and resulted in impaired vascular repair. In contrast, erythropoietin
(EPO) mRNA as a means to augment wall shear stress up-­regulated
endothelial Notch activity and restored vascular repair. To further
corroborate flow-­responsive Notch signaling and its role in vascu-
lar repair, the level of Notch activation was manipulated by micro-­
injecting Notch Intracellular Cytoplasmic Domain (NICD) mRNA or
dominant negative (DN)-­Notch1b mRNA. Micro-­injection of NICD
mRNA to overexpress Notch signaling promoted vascular repair and
restored impaired vascular repair in the presence of (DN)-­Notch1b
mRNA. As a corollary, co-­injection of Gata1a MO with NICD mRNA
rescue restored endothelial Notch activity and vascular repair.
TUPE036 | Identification of a subpopulation
of F4/80 + ve, NG2 + ve pericytes on healthy
murine vasa recta
Rebecca Lilley; Scott Wildman; Claire Peppiatt-Wildman
Urinary Systems Physiology Unit, University of Kent, Chatham Maritime, UK
Blood flow to the renal medulla (MBF) is provided by the network
of vasa recta capillaries, perfusion of which is controlled by contrac-
tile perivascular pericytes. Pericyte dysregulation and migration is
implicated in renal pathophysiology¹ and in recent years, emerging
evidence has shown that these pericytes have a role in the innate
immune response. This includes communicating with innate immune
cells (IIC), and co-­expressing pericyte and IIC markers². The aim here
is to identify if healthy renal medullary NG2 +ve pericytes express
macrophage (MΦ) marker F4/80, in order to elucidate if medullary
pericytes participate in the innate immune response.
Methods: Animal experiments were conducted in accordance with
the United Kingdom Home Office Scientific Procedures Act (1986).
Kidney slices were taken from adult male C57BL/6J mice and sub-
jected to immunohistochemistry, where slices were co-­stained with
anti-­neural glial 2 (NG2; pericytes), anti-­F4/80 (MΦ), and Hoechst
33342, and then imaged using confocal microscopy.
Results: A small population of vasa recta pericytes in healthy tissue
were co-­stained with the F4/80 MΦ marker implying they act as MΦ.
Pericytes and MΦs not co-­stained have close associations with each
other demonstrating paracrine signalling is feasible.
 |
76 of 96       ABSTRACTS
Conclusion: MΦ and pericytes are closely associated with each
other, with a very small subpopulation of pericytes positive for both
NG2 and F4/80 in healthy tissue. This data implies a subset of med-
ullary pericytes could also act innate immune cells. Activation of
these cells in disease could lead to dysregulation of MBF and com-
pounding of the deleterious effect of the innate immune response.
TUPE038 | Balancing arteriolargenesis and
angiogenesis to achieve functional control of
blood flow
1,2 1,2
David O. Bates ; Andrew V. Benest ; Rachel Boardman ;
Costanza Emmanueli3; Maria J. C. Machado1,4; Federica Riu1
1 bioactive lipid implicated in disease including cancer and atheroscle-
Tumour Vascular Biology Laboratories, Cancer Biology, Division of Cancer
and Stem Cells, School of Medicine, Queen's Medical Centre, University of
Nottingham, UK; 2COMPARE University of Birmingham and University of
Nottingham, UK; 3National Heart & Lung Institute, Imperial College London, UK;
4 potentially regulates endothelial function in inflammatory diseases
Department of Medical Biophysics, Schulich School of Medicine & Dentistry,
Western University, London, Ontario, Canada
Arteriolargenesis, the muscularization of pre-­
existing capillaries,
can be stimulated by concomitant NO-­Tie signaling in a model of
physiological angiogenesis (Stone et al, 2017). We hypothesized that
stimulation of NO-­Tie signaling would have a positive impact on
the recovery of blood flow in a murine model of unilateral hindlimb
ischemia.
Mice injected with Ad.eNOS+Ad.Ang1 (NO-­Tie) had a faster rate
of blood flow recovery compared to control mice, as assessed by
laser speckle imaging. After 21 days of ischemia, blood flow in the
control virus-­injected mice had recovered to that of NO-­Tie mice.
There was no change in capillary density amongst the ischemic mus-
cles, but arteriole density was higher in NO-­Tie mice than control
(assessed by αSMA staining). Given the previously documented ben-
eficial effect of VEGF signaling, we tested whether mice injected
with Ad.eNOS+Ad.Ang1 + Ad.VEGF would show further improve-
ment. Surprisingly, these mice recovered no differently than control.
There was no difference in arteriole density and capillary density
was lower than control.
Because Dll4 is a driver of arterial specification, we hypothesized
that Notch1 expression would be involved in this process. There was
a significant upregulation of Notch1 transcripts in NO-­Tie-­VEGF
treated compared with NO-­Tie treated mice. Using the Notch acti-
vator, soluble Dll4 (sDll4), we stimulated Notch signaling in the isch-
emic muscles of mice. NO-­Tie-­sDll4 mice had significantly increased
capillary and arteriole density, while presenting a worst outcome in
blood flow recovery.
We conclude that a local balance between angiogenesis and ar-
teriolargenesis needs to occur for efficient recovery of blood flow
after ischemia.
TUPE038 | oxLDL blocks angiogenesis and
endothelial migration, potentially via S1P and
ERK mediated signalling
Jemma Paterson; Michael Olding; Alan Hunt; Timothy M
Millar
Clinical and Experimental Sciences, Faculty of Medicine, University of
Southampton, UK
Introduction: Psoriasis is a skin disease affecting 3% of the world
population and is associated with inflammation, hyperproliferation,
hyperlipidaemia and obesity. Angiogenesis is a hallmark of the char-
1 acteristic skin lesions and these plaques bare some resemblance to
those seen in atherosclerosis. Sphingosine-­1-­phosphate (S1P) is a
rosis and is elevated in psoriasis. We have shown previously that oxi-
dised LDL (oxLDL) regulates endothelial function. Hyperlipidaemia
such as psoriasis and we hypothesise that the mechanism involves
S1P.
Objective: To define the role of S1P in angiogenesis and endothelial
function to identify novel lipid/receptor targets for the treatment
of psoriasis.
Methods: In vitro assays of angiogenesis and cell migration plus
Western blotting.
Results: At high concentrations (10 μmol/L), exogenous S1P pre-
vented endothelial migration and angiogenesis. These results mim-
icked those seen with oxLDL. S1P caused a rapid, concentration
dependent phosphorylation of Extracellular signal-­regulated kinase
(ERK 1/2) and inhibition of ERK1/2 phosphorylation with the inhibi-
tor of the upstream kinase MEK, corroborated a role for ERK1/2 in
migration. Fingolimod, an analogue of the S1P precursor sphingosine,
changed the nature of endothelial migration but not angiogenesis.
Inhibition of three S1P receptors had varying effects on regulating
ERK phosphorylation and migration, suggesting different receptors
link to distinct downstream pathways to control angiogenesis. These
results suggest perturbation of the S1P signalling cascade can regu-
late pathologic endothelial function.
Funding/Ethics: Funding from The Gerald Kerkut Charitable Trust
and Psoriasis Association. Ethical approval REC REF: 07/H0502/83.
TUPE039 | Connexin-­formed channels provide
critical pathways for initiation and coordination
of the Ca2+ signaling involved in endothelial cell
migration
Hilda Espinoza; Alexandra Córdova; Xavier F. Figueroa
Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia
Universidad Católica de Chile, Santiago 8330025, Chile
Endothelial cell migration is a key process in endothelium repair,
tube formation and angiogenesis. The initiation and development of
ABSTRACTS
endothelial cell migration depends on Ca2+ entry, but the molecular
pathways involved in the activation and coordination of this intracel-
lular signaling have not been determined. We analyzed the participa-
tion of connexin-­formed hemichannels and gap junction channels in
the Ca2+ signaling associated to endothelial cell migration. Primary
cultures of mesenteric endothelial cells were used to evaluate cell
migration by the wound healing assay, hemichannels activity by
measuring ethidium uptake and gap junction communication by as-
sessing dye coupling. In addition, endothelial cells were loaded with
2+ 2+
Fluo-­4 to record changes in intracellular Ca concentration [Ca ]i.
This study was approved by the Institutional Bioethics Committee.
The scratch of the endothelial cell monolayer with a micropipette tip
induced a strong increase in [Ca2+]i and ethidium uptake by cells of
the migration front, but not those of the monolayer. Although this
stimulation evoked a reduction of the gap junction-­mediated com-
munication in the monolayer, the endothelial cells of the migration
front remained highly coupled. Treatment with 37,43GAP27, a con-
nexin blocking peptide, or 18-­β-­Glycyrrhetinic acid (βGA), a general
connexin-­formed channel blocker, blunted the increase in [Ca2+]i and
ethidium uptake. βGA also reduced the endothelial cell migration
rate. These results suggest that Cx37 or Cx43 hemichannels con-
tribute to the Ca2+ entry that triggers the endothelial cell migration
and gap junction are likely involved in the coordination of the Ca2+
signaling along the endothelial cell migration front.
Grants: FONDECYT-­1150530 and CONICYT-­21170977.
TUPE040 | Cigarette smoke-­induced BACH1
regulates he cross-­talk between NRF2 and AHR
through inhibiting microRNA-­125b
1,2 1 of 158 (values in mlO₂/min/mm Hg) far exceeds observed values for
Mohamad Musbah Almedawar ; Sindy Giebe ; Melanie
Brux1; Coy Brunssen1; Henning Morawietz1
1
Division of Vascular Endothelium and Microcirculation; 2Dresden International
Graduate School for Biomedicine and Bioengineering, Technische Universität
Dresden, Germany
Background: MicroRNA (miR)-­
125b has been shown to regulate
the cross-­talk between NRF2 and AHR by inhibiting AHRR in the
anti-­oxidative response. MiR-­125b has been also shown to be down-­
regulated by cigarette smoke but the mechanism and the conse-
quences on endothelial apoptosis have not been studied. We aim to
understand whether the transcriptional regulation of miR-­125b by
cigarette smoke plays a role in anti-­oxidative defense and apoptosis.
Methods: Primary human umbilical vein endothelial cells (HUVEC)
were exposed to a cigarette smoke extract (CSE) under static and
unidirectional shear stress (USS) conditions. Levels of reduced
glutathione (GSH) were measured using the fluorescent probe
o-­
phthalaldehyde. MiR-­
125b overexpression was achieved by
transfecting mimics. Inhibition of BACH1 was done using siRNA
transfection. Apoptosis was assessed using Caspase 3/7 assay.
Results: Exposure of HUVEC to CSE led to a 2-­fold reduction in
GSH and an increased expression of anti-­oxidative genes HMOX1
|
      77 of 96
(18.18 ± 1.8) and NQO1 (16.12 ± 1.99) (P < 0.001). The expression
of miR-­125b was down-­regulated after CSE exposure in HUVEC
(0.39 ± 0.09; P < 0.05) despite a higher nuclear:cytosolic ratio of
the upstream transcription factor NRF2 (1.33 ± 0.05; P < 0.01). We
also show that overexpression of miR-­125b or pre-­treatment with
1 mmol/L N-­acetyl cysteine (GSH donor) can reverse CSE-­induced
apoptosis. CSE-­induced AHRR expression was also reduced by miR-­
125b overexpression. Inhibition of CSE-­induced BACH1 increased
miR-­125b and HMOX1 while reducing AHRR expression.
Conclusion: These results show that cigarette smoke-­
induced
BACH1 regulates the anti-­oxidative response through inhibiting miR-­
125b. Cigarette smoke-­induced apoptosis can be reduced by restor-
ing miR-­125b expression.
Funding: Dresden International Graduate School for Biomedicine
and Bioengineering, Technische Universität Dresden, Germany
TUPE041 | Determinants of lung oxygen
diffusing capacity: roles of morphology,
intracapillary transport, and flow heterogeneity
Tuhin Roy1; Timothy Secomb2
1
Dept. of Anesthesiology, Mayo Clinic, Rochester MN; 2Dept. of Physiology,
University of Arizona, Tucson AZ
The lung oxygen diffusing capacity (DLO₂) governs the rate at which
oxygen can be taken up into the bloodstream and is a critical de-
terminant of arterial oxygen content in exercise and disease. Prior
methods for estimating DLO₂ include the morphometric method,
which accounts for alveolar membrane area and erythrocyte unload-
ing rate, but not the particulate nature of blood. The resulting DLO₂
humans at rest and during exercise. To better characterize diffusing
capacity, we developed a theoretical model based on simplified cap-
illary and erythrocyte geometry, accounting for effects of capillary
diameter and hematocrit. When uniform perfusion is assumed, the
model predicts DLO₂ = 102 for typical hematocrit and capillary di-
ameter, which is lower than the morphometric value, but higher than
observed values in healthy individuals at rest and exercising under
normoxic and hypoxic conditions. Including effects of perfusion het-
erogeneity with a coefficient of variation of approximately 0.61 in
the model gives results in agreement with literature data on exercis-
ing humans under normoxic and hypoxic conditions, corresponding
to an effective DLO₂ = 74. These results show that a high degree of
pulmonary flow heterogeneity may be tolerable under resting nor-
moxic conditions, but that in exercise or hypoxic conditions, tighter
control of flow heterogeneity and ventilation/perfusion matching in
the lung is necessary. This may also suggest that impaired flow regu-
lation leading to higher levels of heterogeneity could limit oxygen
uptake and impair oxygen delivery to tissue in disease. Supported by
NIH U01 HL133362.
 |
78 of 96       ABSTRACTS
TUPE042 | Novel functional imaging algorithm
to determine capillary hematocrit in vivo
Jaryd Christie1; Isaac Kong1; Laura Mawdsley1,2; Jason
Baek1; Ande Doornekamp1; Christopher Ellis1,2; Richard
Sové1
1 protocols were approved by the Institutional Bioethics Committee.
Department of Medical Biophysics, University of Western Ontario, London,
Canada; 2Robarts Research Institute, University of Western Ontario, London,
Canada
The ability to study and quantify the hemodynamic parameters of
the microcirculation can help us to better understand the regula-
tion of oxygen and nutrient delivery to the surrounding tissue. In
addition, diseases such as sepsis and diabetes have been associated
with the impairment of microvascular function. Japee et al. (2005)
have previously demonstrated a method to quantify the hemato-
crit of capillaries; however, it is manual and time-­consuming. Our
main objective is to develop an automated algorithm to calculate
capillary hematocrit from intravital video microscopy of rat skel-
etal muscle. We implemented a user-­f riendly MATLAB algorithm
that uses a ratio of optical densities to calculate the hematocrit
on a pixel by pixel basis and generate a corresponding functional
image to visualize hematocrit. Each vessel segment was then
validated through linear regression to the method described by
Japee et al. We show that the hematocrit calculated by the algo-
rithm was positively correlated to Japee et al. (y = 1.185x − 2.967;
r² = 0.8393). The algorithm was then used to demonstrate the re-
duction in functional capillary density and the redistribution of
blood flow during the progression of sepsis in rat models, which
is consistent with previous studies. Due to the close correlation
to the previous method, the algorithm developed in this study ac-
curately calculates hematocrit. In addition, our methodology pro-
vides the spatial distribution of hematocrit in capillaries, making
this tool useful for studying both physiological and pathological
states of the microcirculation in vivo.
Funding: NSERC Discovery Grant.
TUPE045 | Intercellular propagation of
astrocyte Ca2+ waves by coordinated activation
of glutamate receptors
Manuel Muñoz; Xavier Figueroa
Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia
Universidad Católica de Chile, Santiago 8330025, Chile
Brain functions depend on a fine regulation of cerebral blood flow.
The intercellular propagation of Ca 2+
waves among astrocytes lo-
cated between neurons and parenchymal arterioles mediates the
neurovascular coupling. Astrocytic Ca 2+
signals spread via gap junc-
tions and ATP release-­mediated purinergic receptor activation, but
in addition to ATP, astrocytes may also release D-­serine and glu-
tamate. We evaluated if activation of metabotropic glutamate re-
ceptors (mGluR) and NMDA receptors (NMDAR) contribute to the
propagation of astrocytic Ca2+ signals in primary cultures of rat
cortex astrocytes loaded with the Ca2+ indicator Fluo-­4. Changes in
[Ca2+]i induced by glutamate and t-­ACPD, a mGluR agonist, were an-
alyzed and Ca2+ waves were initiated in a single astrocyte by apply-
ing a controlled mechanical stimulation (MS) using a micropipette. All
Glutamate and t-­ACPD induced a rapid increase in astrocyte [Ca2+]i,
which was abolished by MPEP, a mGluR5 antagonist, and attenuated
by DL-­AP5, a NMDAR antagonist; phenazine methosulfate (Phen-­
Meth), a serine racemase inhibitor; or D-­amino acid oxidase (DAOX),
an enzyme that deaminates D-­
serine. Interestingly, Blockade of
connexin-­
based hemichannels with the peptide 37,43Gap27 or
pannexin-­1-­formed channels with the peptide 10Panx also attenu-
ated the glutamate-­elicited increase in [Ca2+]i. In addition, the in-
tercellular propagation of the Ca2+ signaling initiated by MS was
inhibited by MPEP, DL-­AP5 or Phen-­Meth. These results suggest
that glutamate and D-­serine release through Cx43 hemichannel and
Panx-­1 channels contributes to the propagation of astrocytic Ca2+
waves by activation of mGluR5 and NMDAR.
Grants: FONDECYT 1150530 and CONICYT 21160646.
TUPE046 | Pharmacological activation of
endothelial small conductance Ca2+-­activated K+
channels enhances nitric oxide availability and
limits superoxide production
Stephen Gust1; Ran Wei1; Stephanie Lunn1; Paul Kerr2;
Frances Plane1
1
Department of Pharmacology, Faculty of Medicine and Dentistry University of
Alberta, Edmonton, AB; 2Department of Nursing Science, Faculty of Nursing,
MacEwan University, Edmonton, AB
Background: Endothelial cells play a protective role in the cardiovas-
cular system by releasing nitric oxide (NO) which mediates vasodila-
tion, regulates cell growth and inhibits inflammatory processes. NO
bioavailability is limited by its interaction with superoxide (O₂-­). NO
release occurs concurrently with hyperpolarization of the endothe-
lial cell membrane potential, an event mediated by opening of Ca2+-­
activated K+ (KCa) channels. We have tested the hypothesis that
pharmacological activation of endothelial small conductance (SKCa)
channels will enhance NO-­mediated responses in isolated mesen-
teric resistance arteries and reduce O₂-­ levels.
Method: Functional, electrophysiological and biochemical tech-
niques were used to investigate the effects of cyclohexyl-­N-­[2-­(3,5-­
dimethyl-­pyrazol-­1-­yl)-­6-­methyl-­4-­pyrimidinamine (CyPPA), a small
molecule modulator of endothelial SKCa channels, on contractile
responses and O₂-­ production in rat isolated mesenteric resistance
arteries. Rats were euthanized under a protocol approved by the
Animal Care and Use Committee (HS1) at the University of Alberta.
Results: Our data show that CyPPA caused: (i) endothelium-­
dependent smooth muscle hyperpolarization (n = 5), (ii) dilated myo-
genically active mesenteric arteries via a NO-­dependent mechanism
ABSTRACTS
(n = 6), (iii) enhanced shear stress-­evoked, NO-­mediated inhibition of
sympathetic vasoconstriction in the intact mesenteric vascular bed
(n = 4), and (iv) reduced production of O₂-­ (n = 6). All effects of CyPPA
were blocked by the SKCa channel inhibitor apamin (50 nmol/L).
Conclusion: These findings support the proposal that endothelial
cell membrane potential may play a key role in vascular health and
that targeting SKCa channels could provide a novel approach to re-
ducing O₂-­.
This work was supported by the Heart and Stroke Foundation of
Canada.
TUPE047 | Microvascular response to methyl
nicotinate in the skin
Sherif Elawa
Department of Hand Surgery, Plastic Surgery and Burns, Linköping University,
Linköping, Sweden
Background: Methyl nicotinate (MN) induces a local cutaneous er-
ythema in the skin and may be used as a local provocation in the
assessment of skin viability. The aims were to characterize MN's
dose-­response effect, to find the most reproducible dose, day-­to-­
day and site-­to-­site, to study its vasodilatory mechanisms and varia-
tions between skin sites.
Methods: Microvascular responses to topically applied MN at dif-
ferent concentrations were measured in 12 healthy subjects on
two separate days, using laser speckle contrast imaging (LSCI).
MN effects were measured on different body sites. By blocking
NO-­mediated (L-­NMMA), nerve-­mediated (lidocaine/prilocaine),
and cyclooxygenase-­mediated (NSAID) vasodilation through topi-
cal administration or iontophoresis, we explored MN vasodilatory
mechanisms.
Results: The concentration of 20 mmol/L MN proved the most re-
liable with respect to reproducibility day-­
to-­
day site-­
to-­
site and
resulted in a plateau response between 5 and 20 minutes after ap-
plication. There was no significant difference in responses between
epigastric region and arm (P = 0.892) and between arm and hand
(P = 0.142), but a significant difference in response between other
sites. Topically applied NSAID reduced MN-­induced vasodilatation
with 64% (P < 0.01), whereas lidocaine/prilocaine reduced it with
25% (P < 0.01). L-­NMMA did not affect the microvascular response
to MN.
Conclusion: When MN is to be used to give a reproducible mi-
crovascular response in the skin, 20 mmol/L is the optimal dose.
Responses vary between different body sites and the prostaglandin
pathway seems to be the dominant mechanism by which MN acts
|
      79 of 96
TUPE049 | Long-­term administration of the
endothelial KCa channel activator SKA-­31
improves cardiovascular function in aged rats
Cini Mathew John1; Rayan Khaddaj Mallat1; Ramesh C.
Misha1; Channakeshava S. Umeshappa2; Heike Wulff3;
Andrew P. Braun1
1
Depts. of Physiology and Pharmacology, Cumming School of Medicine,
University of Calgary; 2Microbiology, Immunology and Infectious Diseases,
Cumming School of Medicine, University of Calgary; 3Dept. of Pharmacology, UC
Davis, Davis, California
Aging promotes the development of cardiovascular disease (i.e.
hypertension, coronary artery disease and atherosclerosis), and is
typically associated with vascular endothelial dysfunction. We have
previously reported that SKA-­31, an endothelial KCa channel acti-
vator, inhibits myogenic tone in small resistance arteries, enhances
endothelium-­dependent vasodilation and restores endothelial func-
tion in tissues from Type 2 Diabetic rats. In the current study, SKA-­
31 (10 mg/kg, I.P. injection) was administered daily for 8 weeks to
aged male Sprague Dawley rats (~18 months). Following treatment,
cardiovascular structure and function were examined, along with
the potential effects of SKA-­31 on the immune system. All experi-
mental work received ethics approval by the University of Calgary
animal care committee.
SKA-­31 treatment improved endothelium-­dependent vasodila-
tion in mesenteric resistance arteries compared with vehicle treated
rats. Echocardiographic analyses revealed that declining cardiac
function (i.e. ejection fraction, stroke volume and fractional short-
ening) could be restored to the levels of young rats (5 months of age).
Vascular expression of endothelial KCa channels and associated sig-
naling molecules at the protein and mRNA levels was also improved
following SKA-­31 treatment. Immune system characterization re-
vealed no pro-­inflammatory changes in T cell subpopulations, NFAT
signaling, T cell proliferation or plasma cytokines in SKA-­31 treated
rats. SKA-­31 administration also did not promote tissue toxicity in
kidney, liver, spleen and brain.
Collectively, these data demonstrate that long-­term SKA-­31 ad-
ministration is well tolerated and can improve cardiovascular func-
tion (i.e. endothelium-­dependent vasodilation, cardiac performance)
in aged male rats, without adversely affecting the immune system.
Study supported by CIHR funding to APB.
TUPE050 | Role of TRPC3 channels in
endothelium-­dependent vasodilation of rat
mesenteric resistance arteries
Sarah Wright
School of Medical Sciences, UNSW Sydney
Objective: Activation of transient receptor potential canonical-­
3
channels (TRPC3) may mediate endothelium-­dependent vasodila-
tion (EDD) of arteries. We examined the role of TRPC3 channels in
 |
80 of 96       ABSTRACTS
EDD evoked by acetylcholine (ACh) and intra-­luminal flow (ILF) in rat
3rd order mesenteric resistance arteries, utilizing a selective TRPC3
blocker pyrazole-­10 (PYR10).
Methods: Cumulative stimulus-­
response curves to ACh
(1 nmol/L–10 μmol/L) or ILF (0-­20 μL/min) were performed in pres-
surized (60 mm Hg), phenylephrine (3 μM/L) constricted mesenteric
resistance arteries isolated from anesthetized (sodium thiopen-
tone, 100 mg/kg i.p.) male Sprague-­Dawley rats (8 weeks/300 g).
Protocols were approved by the UNSW Animal Care and Ethics
Committee.
Results: ILF caused flow-­
m ediated vasodilation (FMD), with
maximum FMD at 14 μL/min (17.2 ± 3.2%, n = 6). Inhibition of ni-
tric oxide (NO) using L-­N G-­Nitroarginine methyl ester (L-­N AME;
100 μmol/L) and 1H-­[1,2,4]oxadiazolo[4,3-­a]quinoxalin-­1-­one
(ODQ; 10 μmol/L) did not alter FMD. In the presence of PYR10
(1 μmol/L), FMD persisted at low flow rates (<10 μL/min), but
flow-­induced constriction was observed at ILF ≥ 12 μL/min
(max −21.8 ± 10.5% P < 0.05, n = 4). ACh caused concentration-­
dependent dilation of mesenteric arteries (pEC50 = 7.63 ± 0.09,
max 95.1 ± 2.6%, n = 4), which was inhibited by PYR10 (max
51.0 ± 1.5%, P 
< 0.05, n 
= 4). The combination of L-­
N AME,
ODQ and PYR10 further reduced ACh-­
induced dilation (max
10.0 ± 1.1%, P < 0.05, n = 4). PYR10 did not alter phenylephrine-­
induced vasoconstriction.
Conclusion: TRPC3 is involved in mediating both agonist-­and
flow-­induced EDD of rat mesenteric arteries. TRPC3 appears to be
coupled to non-­NO-­dependent signalling pathways, presumably in-
volving endothelium-­derived hyperpolarization of vascular smooth
muscle.
TUPE051 | Casein kinase 1 is a regulator of
membrane-­bound TNF reverse signalling and
myogenic responsiveness
Jeff Kroetsch; Darcy Lidington; Steffen-Sebastian Bolz
University of Toronto, Department of Physiology, Toronto, ON, Canada
Myogenic responsiveness, the ability of small resistance arteries
to match resistance to perfusion pressure, transduces mechanical
stimuli (e.g., stretch) into intracellular biochemical signals (e.g., va-
soconstriction pathways). Recently, membrane-­bound TNF (mTNF)
has been identified as a proximal element that initiates the serial
phosphorylation of key elements in the myogenic signalling cascade
(e.g., ERK1/2, Sphk1). mTNF does not contain intrinsic kinase ac-
tivity; however, it can be phosphorylated by casein kinase 1 (CK1),
which could provide a docking site for the assembly of signalling
complexes. We therefore hypothesized that CK1 is a modulator of
mTNF reverse signalling and myogenic tone.
In cultured microvascular smooth muscle cells, mTNF reverse
signalling (induced by the intrinsically active TNF type I receptor, sT-
NFR1-­Fc) increases phosphorylation of ERK1/2. sTNFR1-­Fc-­induced
ERK1/2 phosphorylation is abolished by both the pan CK1 inhibitor
CKI-­7 and the specific CKIδ inhibitor PF-­670462. These findings were
verified in cremaster skeletal muscle resistance arteries assessed by
pressure myography. In vitro application of CKI-­7 abrogates sTNFR1-­
Fc-­induced mTNF reverse signalling and reduces myogenic respon-
siveness. CK1 inhibition did not affect myogenic responsiveness in
TNF−/− arteries. In vitro PF-­670462 also reduces myogenic respon-
siveness and when applied in vivo, PF-­67062 reduced myogenic re-
sponsiveness in isolated cremaster arteries. Importantly, none of the
above treatments affected agonist-­induced vasoconstriction.
We suggest that CK1 acts as a regulator of mTNF reverse sig-
nalling and hence, myogenic responsiveness. The ability of CK1
inhibitors to reduce myogenic responsiveness without affecting
agonist-­induced vasoconstriction suggests a substantial safety mar-
gin for potential clinical application to diseases where microvascular
tone is exaggerated.
TUPE052 | Large-­conductance Ca2+-­
activated potassium channels are implicated in
arteriolar constriction in response to spreading
depolarization
Ferenc Bari1; Attila E. Farkas2; Dániel P. Varga1; Réka Tóth1;
Armand R. Bálint1; Péter Makra1; István A. Krizbai2; Eszter
Farkas1; Ákos Menyhárt1
1
Department of Medical Physics and Informatics, Faculty of Medicine and
Faculty of Science and Informatics, University of Szeged; Szeged, Hungary;
2
Physiology and Pathology of the Blood-­Brain Barrier Research Group, Molecular
Neurobiology Research Unit, Institute of Biophysics, Biological Research Centre,
Hungarian Academy of Sciences; Szeged, Hungary
Modest increases in astrocytic Ca2+ lead to moderate perivascular
K+ accumulation and induce vasodilation in the brain. In contrast,
during spreading depolarization (SD), larger astrocytic Ca2+ concen-
trations drive perivascular K+ concentration above 20 mmol/L and
could switch dilation to constriction.
Our research objectives were to explore; (i) whether the im-
mediate vasoconstriction in response to SD is coincident with the
massive extracellular accumulation of K+, and (ii) if the SD-­caused
K+ elevation occurs via large-­conductance Ca2+-­activated potassium
(BK) channels.
Applying two-­photon microscopy in combination with a K+ indi-
cator fluorescent dye, we demonstrated in anesthetized mice (n = 8)
(ethical permission nr. XXXII./3128/2017), that initial vasoconstric-
tion in response to SD was coincident in space and time with the large
extracellular accumulation of K+. The selective BK channel blocker
paxilline (500 nmol/L) or its vehicle was applied topically on the
brain surface in other experiments (n = 15), involving the recording
of cerebrocortical local field potential, extracellular K+ concentration
and local cerebral blood flow (CBF) with laser-­Doppler flowmetry.
Paxilline completely diminished SD-­related hypoperfusion in 6 of 7
mice. Further, paxilline hampered the extracellular accumulation of
ABSTRACTS |
      81 of 96

K+ with SDs, which was reflected by the smaller relative amplitude
(12.7 ± 7.5 vs. 22.2 ± 2.7 mmol/L, paxilline vs. control) and absolute
amplitude (29.7 ± 5.4 vs. 37.0 ± 3.5 mmol/L, paxilline vs. control),
and slower rate of SD-­related K+ surge (i.e. slope; 0.54 ± 0.27 vs.
2.57 ± 0.89 mmol/L/s, paxilline vs. control).
We propose that potassium efflux through BK channels is a cen-
tral component in the devastating neurovascular effects of recurrent
spreading depolarizations in tissue at risk.
Funding: NKFIH (K111923, K120358 and K116158), the
Hungarian Academy of Sciences (BO/00023/17/8 to AEF); GINOP-­
2.3.2-­15-­2016-­0 0048; EFOP-­3.6.1-­16-­2016-­0 0008.
TUPE053 | Central role of connexin 43
TUPE55 | The effect on pathological and
ultrastructural organization of gastric mucosa of
rats with the therapy of dual anti-­platelet after
AMI
Jiangang Liu1; Lei Zhang1; Yuyang Liu2; Qingxiang Zhang3;
Dazhuo Shi1
1
Xiyuan Hospital of China Academy of Chinese Medical Sciences, Institute of
Cardiovascular Disease, Beijing; 2University of British Columbia, Vancouver,
Canada; 3Taiwan Traditional Chinese Medicine Hospital of Shandong Province,
Taiwan
Objective: To observe the changes of Pathological morphology and
ultrastructural organization after gastric mucosa injury caused by
aspirin and clopidogrel on rats with AMI (acute myocardial infarc-

hemichannels and pannexin-­1 channels in the tion, AMI) which was induced by ligating the left anterior descending
coronary.
PAF-­activated endothelial cell Ca2+ signal
Methods: 60 SD rats with model establishment were randomly di-
Nelson Barrera; Xavier Figueroa; Camilo Navarrete; Ines vided into 5 groups, The 5 groups were: (1) model group which distilled
Poblete; Pia Burboa
water was given 2 mL/(kg d); (2) aspirin group of which aspirin was
Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia
given 9 mg/(kg d); (3) clopidogrel group which clopidogrel was given
Universidad Católica de Chile, Santiago, Chile
6.75 mg/(kg d); (4) aspirin+clopidogrel group of which both of aspi-
rin and clopidogrel were given; (5) sham group (the suture was pen-
Inflammatory agents induce a Ca2+-­
dependent increase of en-
etrated around the left anterior descending coronary artery, but not
dothelial cell barrier permeability to macromolecule (hyperperme-
tied) of which distilled water was given 2 mL/(kg d). After continuous
ability) in post-­c apillary venules. We used rat mesenteric vascular
intragastric administration of 4 weeks. The changes of pathological
beds and primary cultures of mesenteric endothelial cells of venules
morphology and ultrastructural organization of gastric mucosa were
(EC-­V ) to analyze the participation of Cx43-­based hemichannels
observed with optical microscope and scanning electron microscope.
and Pannexin-­1-­formed channels in the Ca2+ signaling associated
2+ Results: Compared with sham group, the Guth and Whittle score
to PAF-­activated hyperpermeability. Changes in [Ca ]i were de-
increased significantly in aspirin group, clopidogrel group and
tected using the Ca2+ indicator Fluo-­4 and the interendothelial cell
aspirin+clopidogrel group (P < 0.01). With optical microscope, we
pore formation was assessed using Atomic Force Microscopy. In
observed that the hyperemia phenomenon and edema were obvi-
addition, hemichannel and pannexin-­1 channel activation was eval-
ous on gastric mucosa surface. With scanning electron microscope,
uated by ethidium uptake, ATP release by luciferin-­luciferase lu-
general mucosa epithelial cell detachment and basement membrane
minescent assay and protein S-­nitrosylation by Proximity Ligation
exposure were observed, superficial epithelium was shown like favi-
Assay. The Institutional Bioethics Committee approved all the pro-
form and most basement membrane disappeared in severe cases in
tocols. PAF induced an increase in ethidium uptake in intact ven-
2+ aspirin group and aspirin+clopidogrel group.
ules and in EC-­V, which was associated with a rapid rise of [Ca ]i.
Conclusion: In the process of gastric mucosa injury caused by aspirin
Both the hemichannel activation and Ca2+ signaling were abolished
plays the most roles, and clopidogrel mainly acts on damaged gastric
by treatment with connexin blocking peptides 37,43Gap27 or
mucosa. The damage degree and mechanism on gastric mucosa of
43Gap26 and attenuated by application of the pannexin-­1 block-
both medicines were different.
ing peptide 10Panx or the purinergic receptor blocker PPADS. PAF
also induced a 43Gap26-­and 10Panx-­sensitive ATP release and
evoked a NO-­mediated S-­nitrosylation of Cx43. Consistent with
this, inhibition of NO production with NG-­nitro-­L-­arginine blunted TUPE056 | Correlation clinical research
the PAF-­induced ethidium uptake and Ca2+ signaling. Furthermore, of blood flow visualization and erythrocyte
37,43Gap27 blocked the PAF-­initiated pore formation. These re- rheology with the elderly hypertension
sults indicate that Cx43 hemichannel activation by NO-­mediated
Wei Xiong1; Jiangang Liu2; Hao Li1
S-­nitrosylation and subsequent pannexin-­1 channel opening is es-
1
Xiyuan Hospital of China Academy of Chinese Medical Sciences, Department
sential for the increase in [Ca2+]i associated to PAF-­induced hyper-
of Geriatrics Disease, Beijing; 2Xiyuan Hospital of China Academy of Chinese
permeability. These channels may allow direct Ca2+ influx or lead Medical Sciences, Institute of Cardiovascular Disease, Beijing
to purinergic receptor activation through ATP release.
Grants: FONDECYT 1150530, CONICYT 21141023 Objective: Apply micro channel array flow analyzer (MC-­FAN) to
observe blood flow characteristics of elderly hypertension patients
 |
82 of 96       ABSTRACTS
and test the correlation between them and red blood cell rheological
property. Methods Selected 109 elderly hypertension patients, and
there is an elderly healthy group (21 cases). Using MC-­FAN to do the
hemorheology visualization testing of elderly hypertension patients
and observing the erythrocyte deformation indexes, erythrocyte
aggregation indexes and erythrocyte related plasma ATPase activity
and do the relevant analysis between blood flow property and red
blood cell rheological indexes.
Results: (1) Micro channel array flow transiting time comparison:
Compared with elderly healthy group, the micro channel array flow
transiting times of elderly healthy group have significantly extended
(P < 0.05). (2) Plasma Na+-­K+-­ATPase activity and of Plasma Ca2+-­
Mg2+-­ATPase activity of elderly hypertension group have signifi-
cant reduced (P < 0.01). (3) The elderly hypertension patients’ micro
channel array flow transiting times (10, 30, 60 and 100 μL) were re-
spectively significant negative correlated with erythrocyte deforma-
tion index. 100 μL transiting time was significant positive correlated
with erythrocyte aggregation index.
Conclusion: Micro channel array flow transiting times of elderly hy-
pertension patients have significantly extended. And it has the inti-
mate correlation with erythrocyte rheological indexes and plasma
ATPase activity.
Keywords: Elderly hypertension; Erythrocyte deformability;
Erythrocyte aggregation; ATPase activity; Blood flow visualization
P OS TE R PR E S E NTATI O N S – W E D N E S DAY,
S E P TE M B E R 12 , 2 018
WEPE001 | Influence of sex and age
on cerebrovascular function: role of large
conductance potassium channel subunits
Mallikarjuna Pabbidi
University of Mississippi Medical Center, Jackson, MS 39216
Although adult females are protected, the cerebrovascular disease is
higher in elderly post-­menopause women than in age-­matched men.
We hypothesize that “compared to adult males, cerebral vasculature
of intact adult female rats displays an attenuated myogenic response
and greater intrinsic tone due to differential expression and func-
tion of BK channel subunits. Aging exaggerates myogenic response,
diminishes intrinsic tone and thus, vasodilatory capacity in females
more than males due to a reduction in the BK subunit function.” Our
results suggest that middle cerebral arteries (MCA) of intact adult fe-
males had attenuated myogenic response compared to adult males.
Development of intrinsic tone was higher in intact adult females than
adult males. The ratio of BK β1 to α subunits was less in intact adult
females than adult males. Spontaneous transient outward currents
(STOC) were higher in smooth muscle cells of intact adult females
compared to adult males. While aging exaggerated the myogenic re-
sponse in females, it did not affect males. However, intrinsic tone
was decreased in aged females but increased in aged males. Aging
reduced the amplitude of STOCs but with a greater magnitude of ef-
fect in females. While aging increased BKα subunit, it decreased β1
subunit protein. Acute activation of BKβ1 by estradiol while relaxed
MCA of adult females and males, it had less effect in aged females.
In conclusion, a higher incidence of cerebrovascular disease in aged
female may be due to sex-­specific changes in vascular properties
and BK channels that could result in diminished estradiol-­mediated
vasodilation.
WEPE002 | Mediators of inflammation in
blood-­brain barrier hyperpermeability following
traumatic brain injury
Bobby Robinson1; Chinchusha Anasooya Shaji1; Claire
Isbell1; Xu Peng2; Jason Huang3; Binu Tharakan1
1
Department of Surgery, Baylor Scott and White Health and Texas A&M
University Health Science Center College of Medicine, Temple, Texas, USA;
2
Department of Medical Physiology, Texas A&M University Health Science Center
College of Medicine, Temple, Texas, USA; 3Department of Neurosurgery, Baylor
Scott and White Health and Texas A&M University Health Science Center College
of Medicine, Temple, Texas, USA
Microvascular hyperpermeability that occurs via the blood-­b rain
barrier (BBB) is critical to vasogenic cerebral edema following
traumatic brain injury (TBI). Our recent studies have demonstrated
the involvement pro-­inflammatory cytokine IL-­1β in mediating this
hyperpermeability. NLRP3 inflammasome activation is critical to
IL-­1β secretion. We hypothesized that NLRP3 inflammasome sign-
aling regulated by extracellular ATP is critical to IL-­1β-­m ediated
BBB breakdown/hyperpermeability following TBI. Rat brain micro-
vascular endothelial cell (RBMEC) monolayers were treated with
BzATP following treatment with an NLRP3 inhibitor, parthenolide
in vitro. Transwell permeability assay and immunofluorescence
localization of zonula occludens-­1 (ZO-­1) was performed. Mild-­
moderate TBI was inflicted in C57BL/6 or NLRP3 gene knock-
out mice (NLRP3−/−) using a controlled cortical impactor (n = 5/
group). Brain IL-­1β levels were measured by ELISA. The C57BL/6
mice were treated with various NLRP3 pathway inhibitors (Z-­VAD,
a pancaspase inhibitor, Ac-­Y VAD, a caspase-­1 inhibitor and CP-­
456573, an NLRP3 specific inhibitor). Evans blue assay or intravital
microscopic imaging of pial venules was performed for evaluat-
ing BBB permeability. BzATP induced monolayer hyperpermeabil-
ity and ZO-­1 displacement at the tight junctions. These effects
were attenuated by parthenolide. In C57BL/6 mice TBI induced
a significant increase in IL-­1β levels compared to sham (P < 0.05).
Z-­VAD, Ac-­Y VAD and CP-­456573 attenuated TBI-­induced BBB
hyperpermeability significantly compared to control groups
(P < 0.05). NLRP3−/− mice showed a decrease in TBI-­induced BBB
hyperpermeability compared to TBI in wild-­t ype (P < 0.05). NLRP3
inflammasome activation leads to BBB breakdown/microvascular
ABSTRACTS
hyperpermeability regulated by extracellular ATP. Genetic and
pharmacological inhibition of this pathway provides protection
against TBI-­induced BBB hyperpermeability.
WEPE003 | N-­acetylcysteine attenuates the
platelet activation and thrombosis responses in
the brain during type 2 diabetes
Xin Fang; Tak Yee Aw; Karen Y. Stokes
Department of Molecular and Cellular Physiology, & the Center for
Cardiovascular Disease and Sciences, LSU Health Sciences Center, Shreveport,
Louisiana, USA
Type 2 diabetes mellitus (T2D) is a well-­established risk factor for
stroke and small vessel disease. Both oxidative and dicarbonyl stresses
have been implicated in the pathologies of diabetes. Glutathione
(GSH) is an antioxidant, and a rate-­limiting factor in the elimination
of methylglyoxal, a functionally important dicarbonyl species. GSH
levels are decreased in diabetes. Here we tested if NAC, which was
effective in Type 1 diabetes, offered protection against brain ves-
sel thrombosis and systemic pro-­thrombotic changes in a T2D model
(Db/Db mice). Wild type (WT) and Db/Db mice were maintained
on normal drinking water or treated with 2 mmol/L NAC in drinking
water for 3 weeks (as approved by our IACUC). Flow cytometry was
used for assessment of platelet-­leukocyte aggregation (PLA), ELISA
for coagulation factors and the light-­dye method with intravital mi-
croscopy for thrombosis. While the absolute numbers of PLAs per ul
blood were unchanged by T2D, the # circulating leukocytes, specifi-
cally lymphocytes, dropped by almost half in Db/Db mice, and the %
of monocytes, neutrophils and lymphocytes forming platelet aggre-
gates increased by almost 50% in Db/Db, compared with WT. NAC
decreased the PLAs. Thrombosis was accelerated in Db/Db mice,
particularly arterioles, compared with WT mice. NAC offered modest
protection against the thrombosis. Preliminary data showed elevated
coagulation factors in Db/Db mice and NAC decreased their levels.
Taken together, we have characterized pro-­thrombotic responses in
the Db/Db mice and shown NAC affords partial protection. Further
work will include longer NAC treatment and investigating the role for
methylglyoxal in T2D-­induced cerebral thrombosis.
WEPE005 | Imaging single cell blood flow in
the anesthetized and awake, behaving mouse
retina
Jesse Schallek1,2,3
1
Flaum Eye Institute, University of Rochester, Rochester, New York, NY, USA;
2
Center for Visual Science, University of Rochester, Rochester, New York, NY,
USA; 3Department of Neuroscience, University of Rochester, Rochester, New
York, NY, USA
Objective: Adaptive optics ophthalmoscopy corrects for the optical
blur of the eye to achieve single cell resolution. Combined with fast
|
      83 of 96
camera acquisition of over 15 kHz, we image and measure the veloc-
ity of single blood cells in the living mouse retina with and without
anesthesia.
Methods: A custom adaptive optics scanning light ophthalmoscope
(AOSLO) was built to image the retina in confocal, phase-­contrast
and fluorescence modalities. Healthy C57BL6/J mice were im-
aged awake or with isoflurane anesthesia. Awake, head-­fixed mice
were positioned above a running wheel allowing voluntary move-
ment. Protocol approved by University of Rochester (AAALAC
accreditation).
Results: We were able to resolve the smallest through largest reti-
nal vessels with 1 micrometer resolution. Combined with 15 kHz
line-­scan imaging, we imaged single blood cells without motion blur.
Four constituents of blood were imaged including, plasma, red blood
cells, white blood cells and platelets. This approach provided the first
measurements of single blood cell flux, velocity and flow in the full
spectrum of vessels of the living mouse eye. Anesthesia reduced
erythrocyte speed by 2.8x (25.4 vs 9.1 mm/s) in an 18 μm diame-
ter vessel. Blood cell pulsatility of 481-­555 beats/min in the awake
mouse dropped to 181-­300 beats/min under anesthesia.
Conclusion: We show the first demonstrations of AOSLO blood cell
imaging of the living and awake, behaving mouse. AOSLO opens new
possibilities to study particulate blood flow in the full spectrum of
retinal vessels to reveal microvascular physiology or dysfunction.
Support NIH EY028293 EY001319, Dana Foundation, Research to
Prevent Blindness, Hoffmann-­L a Roche.
Disclosure: Jesse Schallek: Commercial Relationship(s); University of
Rochester: Code P (Patent); Hoffmann-­L aRoche: Code F (Financial
Support)
WEPE006 | The alterations in rho-­kinase
contribution to contractile responses of rat
skeletal muscle arteries resulted from maternal
and chronic adult hypothyroidism
Olga S. Tarasova1,2; Dina K. Gaynullina1,2; Svetlana I.
Sofronova1,2; Anastasia A. Shetsova1,2; Ekaterina K.
Selivanova1,2; Anna A. Borzykh1; Andrey A. Martyanov1,2
1
Institute for Biomedical Problems, Russian Academy of Sciences, Moscow,
Russia; 2Lomonosov Moscow State University, Moscow, Russia
Recently we demonstrated that maternal hypothyroidism (MH)
increases Rho-­
kinase pro-­
contractile influence in arteries of
2-­week-­old progeny. Here we hypothesized that (1) augmented role
of Rho-­kinase persists in arteries from adult progeny of hypothy-
roid dams and (2) chronic hypothyroidism (CH) can induce similar
vasoregulatory alterations in adult rats. Dams were treated with
6-­propyl-­2-­thiouracil (PTU) in drinking water (7 ppm) during preg-
nancy and 2 weeks postpartum and their male progeny was grown to
the age of 12-­14 weeks. To induce CH, mature male Wistar rats were
treated with PTU (250 ppm) for 14 weeks. Gastrocnemius muscle
arteries were studied by wire myography, Western blotting and
 |
84 of 96       ABSTRACTS
qPCR. All procedures were approved by the Institutional Committee
on animal welfare. Serum T3/T4 levels (ELISA) were not changed in
12-­week-­old MH rats, but CH rats demonstrated prominent and sta-
ble reductions in T4/T3. Arteries of MH rats compared to control
demonstrated augmented responses to α1-­adrenoceptor agonist
methoxamine, increased contents of RhoA mRNA and phospho-­
MYPT1-­Thr855. Intergroup differences in contractile responses and
MYPT1 phosphorylation were eliminated by Y27632 (3 μmol/L). CH
rats also demonstrated augmented Rho-­kinase contribution to con-
tractile responses. In both hypothyroidism models, similar results
were obtained after endothelium denudation or pharmacological
blockade. Our data show, for the first time, that MH can program
Rho-­kinase activity in skeletal muscle arteries during early develop-
ment. Similar vasoregulatory alterations can be induced by long-­term
hypothyroidism in adult age. Of note, augmented Rho-­kinase activity
represents a risk factor for cardiovascular dysfunction. Supported
by the Russian Science Foundation (Grant N14-­15-­0 0704).
WEPE008 | Investigating local effects of
microvascular strokes
Eva Erlebach1,2; Jillian L. Stobart1,2,3; Matthew Barrett1,2;
Chaim Glück1,2; Sergei Vinogradov3,4; Bruno Weber1,2
1
Institute of Pharmacology and Toxicology, University of Zurich, Switzerland;
2 and two-­photon microscopy. We found that modulating the activity
Neuroscience Center Zurich, University and ETH Zurich, Switzerland;
3
Department of Biochemistry and Biophysics, University of Pennsylvania, USA;
4
Department of Chemistry, University of Pennsylvania, USA
Ischemic lesions resulting from small vessel ruptures or occlusions
are called microvascular strokes. Because of their small size, single
microstrokes do not produce symptoms in patients. However, the
accumulation of these small strokes is associated with dementia and
cognitive impairment. Despite their common occurrence in the aging
population, they are poorly studied and not well understood due to
the detection limitations of available clinical techniques (e.g. MRI).
Here, we present a microvascular occlusion model established for
in vivo induction and imaging of hemorrhagic and ischemic strokes
of capillary size in mice. An intravenously injected porphyrin-­based
probe leads to singlet-­oxygen production upon two-­photon exci-
tation, which resulted in the occlusion of the vessel. Simultaneous
two-­photon imaging allowed us to study the cellular and vascular
reaction upon such an occlusion in detail. Using this protocol, we
were able to record local microglia activation and we aim to study
alterations in calcium signals of nearby cells upon stroke induction.
This model enables the targeted induction of ischemic/hem-
orrhagic strokes in single capillary microvessels and also provides
novel insights in post-­stroke cellular events and hemodynamics that
will help us to better understand these small lesions.
Procedures were approved by the local veterinary authorities
and conformed to the guidelines of the Swiss Animal Protection
Law, Veterinary Office, Canton Zurich (Act of Animal Protection 16
December 2005 and Animal Protection Ordinance 23 April 2008).
This project was supported by the Swiss National Science
Foundation (Project: 31003A_156965)
WEPE010 | Dissecting the neural control of
baseline and evoked vascular tone in the brain
Christina Echagarruga1,2,3; Kyle Gheres1,2,3; Patrick J.
Drew1,2,3
1
Department of Biomedical Engineering; 2Molecular, Cellular, and Integrative
Biology Graduate Program; 3Center for Neural Engineering, Department
of Engineering Science and Mechanics, The Pennsylvania State University,
University Park, PA, USA
Changes in local neural activity drive vasodilation and vasoconstriction
of the cerebral vasculature, and these changes in the cerebral blood
flow are essential for brain tissue health. While previous work has sug-
gested that activity of interneurons is the primary drivers of arteriole
diameter changes, the signaling mechanisms that control blood flow
and arterial diameter are not completely understood. To test the role
of neural activity in manipulating vascular tone, we used chemoge-
netics and pharmacology in the somatosensory cortex of awake mice,
allowing us to bi-­directionally manipulate neural activity in specific
cellular subpopulations. We assayed changes in neural activity and
vascular responses to voluntary locomotion using electrophysiology
of all neurons produced corresponding changes in resting and evoked
arteriole diameters. However, while modulation of excitatory neurons
produced changes in the electrophysiologically-­recorded population
activity, there were no corresponding changes in resting or evoked ar-
terial tone. Decreasing the activity of nNOS-­expressing neurons or in-
hibition of NO production both blocked the hemodynamic response to
locomotion and impacted resting tone similarly. Due to the changes in
vessel tone, ratiometric imaging of hemodynamic signals can give mis-
leading results when measured across conditions where background
neural activity may change. Our results show that both baseline and
evoked hemodynamics signals reflect the activity of a small set of neu-
rons, not the average activity of entire neural population.
WEPE011 | Quantifying the neural and non-­
neuronal components of inter-­hemispheric
hemodynamic correlations
Kevin Turner1; Aaron Winder1; Patrick Drew1,2
1
Department of Engineering Science and Mechanics, Pennsylvania State
University, University Park, PA, USA; 2Department of Neurosurgery and
Department of Biomedical Engineering, Pennsylvania State University, University
Park, PA, USA
Hemodynamic signals in the brain are used to infer neural activ-
ity, and strong correlations in hemodynamic signals between bi-
lateral cortical areas have been observed in the absence of a task
(‘functional connectivity during the resting-­s tate’). Understanding
ABSTRACTS |
      85 of 96

the relationship between these bilateral hemodynamic signals and WEPE013 | Characterizing long-­term microglial
the underlying neural activity is important for the interpretation
responses and microvessel repair following
of these hemodynamic signals. Previous work has proposed that
cerebral microbleeds in a type 1 model of
hemodynamic signals are a sum of a neurally-­evoked component
and a putatively non-­neuronal component. Importantly, the rela-
diabetes mellitus
tive contributions of the neural and non-­neuronal components de- Eslam Mehina1; Stephanie Taylor2; Sun Eui Choi1; Craig
pend on the behavioral state of the animal. Here we investigate Brown1
1
the role of behavior and neural activity in sculpting bilateral hemo- Division of Medical Sciences, University of Victoria, Victoria, British Columbia,
Canada; 2Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn, North
dynamic signals. We used intrinsic optical signal imaging through
Rhine-­Westphalia, Germany
chronically implanted thinned-­skull windows to measure bilateral
changes in cerebral blood volume in parallel with electrophysiolog-
Type 1 diabetes mellitus is a risk factor for vascular complications,
ical recordings in the somatosensory cortices of awake mice. We
including cerebral microbleeds (CMBs), and impairs wound healing.
continuously monitored both animal motion and whisking behav-
When CMBs occur, microglia rapidly respond to envelop the site of
ior to classify behavioral state. We also stimulated the whiskers to
damage and help repair the injury. Our previous work has shown that
compare the strength of neurovascular coupling in the evoked con-
microglial responses to injury are perturbed in the diabetic brain.
dition with neurovascular coupling in the resting state. To test the
However, since microglial-­
dependent vascular repair evolves over
influence of the neural activity in driving bilateral hemodynamic
days to weeks, it is unclear how diabetes impacts this process. Here
correlations, we suppressed local neural activity in the somatosen-
we longitudinally imaged microglial responses and vascular repair in
sory cortex of one hemisphere, allowing us to directly measure
vivo in unregulated and insulin-­treated type 1 diabetic mice follow-
the strength of non-­neuronal correlations between hemispheres.
ing induction of CMBs (certified by local ACC). Microglial aggregation
These experiments will elucidate the neural contribution to bilat-
around the CMB peaked 1-­3 days after CMB and declined thereafter
eral functional connectivity across behavioral states.
(measured as area, % vessel coverage, # of cells either through migra-
Support: National Institutes of Health Grants R01NS078168 and
tion or infiltration), and did not differ significantly between groups.
R01NS079737
However, in contrast to non-­diabetic mice where 100% (28/28 vessels)
of ablated vessels restored flow and remained patent over 3 weeks,
~20% of vessels from insulin-­
treated and untreated diabetic mice
WEPE012 | The contribution of cortical NK1R were pruned within 3 days and were not compensated for by angio-
interneurons in neurovascular coupling genesis. By depleting microglia/macrophages with colony stimulating
1 2 2 factor 1 receptor antagonist, we find that 20% of ablated vessels are
Catherine F. Ruff ; Jonathan J. Couey ; Bryan M. Hooks ;
Alberto L. Vazquez3,4; Sarah E. Ross1 eliminated in non-­diabetic mice, suggesting that microglial responses
1
Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA; indeed help repair damaged microvessels. Since we have previously
2
Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA; shown that interferon gamma (IFNγ) signalling is abnormally upregu-
3
Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA; lated in diabetic mice, we are currently investigating the role of IFNγ in
4
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
modulating microvessel repair and fate following CMB. NSERC, Vanier
CGS, CIHR, and the Heart and Stroke Foundation funded this work.
Neurovascular coupling (NVC) is a regional increase in blood flow
to a brain area triggered by local neural activity. Although NVC is
critical to normal brain function and its dysfunction is reported in
many neuropathologies, the underlying neural basis remains un- WEPE014 | Anatomical basis for cerebrospinal
clear. Here, we report the generation of a neurokinin-­1 receptor fluid and interstitial fluid transport through the
(NK1R)-­creER mouse that selectively labels a subset of cortical cribriform plate in mice
inhibitory neurons that are ideally situated to regulate NVC. Using
J. N. Norwood1; A. Craine2; P. J. Drew2,3,4
this genetic tool, with a combination of imaging, electrophysiol- 1
Cell and Developmental Biology, Pennsylvania State University, University
ogy, optogenetic techniques and in vivo laser Doppler flowmetry, Park, PA, USA; 2Biomedical Engineering, Pennsylvania State University,
we found that NK1R cortical interneurons receive local excita- University Park, PA, USA; 3Engineering Science and Mechanics, Pennsylvania
State University, University Park, PA, USA; 4Neurosurgery, Pennsylvania State
tory input and their activation is sufficient for local vasodilation.
University, University Park, PA, USA
Together, these findings suggest that NK1R cortical inhibitory
interneurons act as local integrators of neural activity to medi-
The normal flow of cerebrospinal fluid (CSF) plays an important role
ate neurovascular coupling, providing important insight into the
in transporting chemical signals to neurons and removing waste
neural circuitry of NVC.
from the brain. CSF is thought to be transported into and out of
Research reported in this abstract was supported by NINDS of the
the brain, and exchanged with interstitial fluid (ISF), via the glym-
National Institutes of Health under award number F31NS106724.
phatic system. Decreased turnover of CSF in the brain is thought
86 of 96      |
to play a role in neurodegenerative disorders. However, the ana-
tomical pathways of CSF and ISF clearance from the skull are poorly
understood, and elucidating these pathways is important as they
play a role in controlling CSF turnover. There is evidence that CSF
and ISF exits into the nasal epithelium via the cribriform plate, the
perforated bone through which olfactory sensory neurons (OSNs)
enter the brain. To better understand the CSF outflow pathway in
mice, we used histological techniques and vascular fills to exam-
ine the nasal turbinates, cribriform plate, olfactory bulbs and the
microscopic anatomy (interstitial space between nerves and blood
vessels). OSN axons and blood vessels were observed traversing
the cribriform plate. Using transgenic mice expressing GFP under a
lymphatic promoter (LYVE1-­GFP), the relationship of the olfactory
nerve and lymphatic vessels was investigated. We visualize the flow
of CSF and ISF through the cribriform with dye injections, confirm-
ing that it is a conduit for both CSF and ISF. To visualize the role
of lymphatic vessels in ISF and CSF drainage, dye injections were
repeated in LYVE1-­GFP mice. Our results show that the cribriform
plate plays an important role in the CSF and ISF outflow pathways.
WEPE015 | Angiogenesis in the cerebral
cortex: transition from two-­dimensional to three-­
dimensional microvascular networks
1 1,2
Jonathan P. Alberding ; Timothy W. Secomb
1
ARL Microcirculation Division, University of Arizona, Tucson, AZ, USA;
2
Department of Physiology, University of Arizona, Tucson, AZ, USA
During the early development of the cerebral cortex, an effectively
two-­dimensional (2D) network of pial vessels forms on the surface.
Sprouts from this network penetrate into the expanding cortical tis-
sue, eventually forming a dense three-­dimensional (3D) microvascu-
lar structure that supports the metabolic needs of the tissue, while
the surface network partially regresses. In previous theoretical stud-
ies of angiogenesis and structural adaptation in the mesentery, we
showed that functionally adequate and efficient networks can be
formed in 2D through over-­abundant stochastic generation of ves-
sels in response to a growth factor generated in hypoxic tissue re-
gions, in parallel with network refinement by structural adaptation
and pruning. Here, we extend these simulations of vascular network
growth to 3D in a region of cortex in the form of a hexagonal prism
150 μm on a side and 500 μm deep, with periodic boundary condi-
tions. A single pial vessel with two sprouts extending into the cor-
tex is assumed to be present initially. The sprouts elongate, branch
and connect, forming a three-­dimensional mesh of microvessels, and
undergo structural adaptation. Regression of pial vessels is a criti-
cal part of this process, because surface vessels can potentially form
functional shunts, diverting flow from the deeper vasculature. The
simulations show that regression of pial vessels depends on informa-
tion transfer mechanisms from distal to more proximal vessels that
influence structural adaptation of vessel diameters. In particular,
upstream conducted responses play an essential role in generating
ABSTRACTS
functionally adequate network structures. Supported by NIH grants
HL034555 and HL133362.
WEPE017 | Development of a novel
microfluidic device for the manipulation of local
microvascular blood flow in vivo
Gaylene Russell McEvoy; Graham Fraser
Division of BioMedical Sciences, Faculty of Medicine, Memorial University of
Newfoundland
Objective: Develop a microfluidic system capable of maintaining
fixed solute concentration, temperature, and physiologic gas con-
ditions in a microscale region of tissue while simultaneously allow-
ing for visualization and quantification of microvascular blood flow.
Methods: Microfluidic devices were fabricated using standard soft li-
thography techniques, molded in polydimethylsiloxane, and coupled
with a laser cut 600 × 300 μm micro-­outlet. Ten Sprague-­Dawley rats
were anaesthetized and instrumented to monitor systemic parameters.
The extensor digitorum longus was externalized and reflected across
the micro-­outlet on the stage of an inverted microscope. The microcir-
culation overlying the micro-­outlet was visualized and recorded under
baseline conditions while the device was constantly perfused with
buffer. Sequential logarithmic concentrations (10−⁸ to 10−³ mol/L) of
acetylcholine, ATP, and phenylephrine were introduced via the perfu-
sion fluid. Under each condition capillaries overlying the micro-­outlet
at various depths were recorded. Animal procedures were approved
by Memorial University's Institutional Animal Care Committee.
Results: Graded flow increases were observed in regions directly over-
lying the micro-­outlet for varying concentrations of acetylcholine and
ATP; and graded decreases for phenylephrine. Flow changes appeared
to be limited to vessels within 300 μm of the micro-­outlet at the high-
est concentrations, with normal perfusion present elsewhere. Offline
analysis of capillaries was conducted to characterize changes to velocity,
lineal density, hematocrit, and supply rate at each concentration.
Conclusions: Our novel approach allows for a controlled delivery of
dissolved substances to constrained microscale regions of micro-
vasculature while simultaneously measuring changes to perfusion
within discrete vessels and networks.
Project funded through a NSERC Discovery Grant to GM Fraser.
WEPE018 | Bioengineering blood vessels to
study Alzheimer's Disease
Jerome Robert1,2
1
Pathology and Laboratory Medicine, University of British Columbia, Vancouver,
BC, Canada; 2Djavad Mowafaghian Centre for Brain Health, University of British
Columbia, Vancouver, BC, Canada
Alzheimer's Disease (AD) is the leading cause of senile dementia with
over 44 million affected persons and an economic burden of over
ABSTRACTS |
      87 of 96

$600 billion. Amyloid plaques, consisting of deposited beta-­amyloid
(Aβ), and neurofibrillary tangles consisting of hyperphosphoryl-
ated tau proteins are neuropathological hallmarks of Alzheimer's
Disease (AD). As cardiovascular risk factors increase dementia risk,
major pathways that regulate Aβ clearance from the brain involve
the cerebrovasculature, and most AD patients have vascular amy-
loid deposition (cerebral amyloid angiopathy (CAA)), it is now clear
that cerebral vessels play a major role in AD pathogenesis. Here we
present a novel human experimental platform to investigate the
cerebrovascular contribution to AD, in which three dimensional per-
fusible cerebral blood vessels were engineered in a scaffold-­directed
flow bioreactor system from primary human endothelial cells (EC)
and smooth muscle cells (SMC), with or without astrocytes and neu-
rons. As proof-­of-­principle relevant to Alzheimer's disease, we dem-
onstrated that circulating high-­density lipoprotein (HDL), a major
player in cardiovascular health, reduces Aβ-­mediated endothelial
activation and also reduces Aβ accumulation in the vascular wall of
our bioengineered tissues, thereby helping to explain more about
the associations between cardiovascular disease and Alzheimer dis-
ease. Taken together, these results establish the utility of human en-
gineered cerebral vessels as a highly innovative in vitro platform to
study key mechanistic questions relevant to AD.
WEPE019 | Reversing beta-­one adrenergic
NO production during shear stress increased in the YC group only.
Immunofluorescence revealed higher beta-­one and beta-­t wo stain-
ing in YC and O+SVF compared to OC.
Conclusion: i.v. injected SVF cells are capable of incorporating into
the cardiac peri-­vascular tissue and were shown to transiently im-
prove beta-­receptor dependent adrenergic microvascular function
in a model of advanced age.
WEPE022 | Preserved cardiovascular
homeostasis despite blunted acetylcholine-­
induced dilation in mice with endothelial
muscarinic M3 receptor deletion
Cor de Wit1,2; Alexandra Rhoden2,3; Jakob Speiser1,2; Birgit
Geertz2,3; June Uebeler2,3; Kjestine Schmidt1,2; Thomas
Eschenhagen2,3
1
Department of Physiology, University Lübeck, Lübeck, Germany; 2DZHK
(German Centre for Cardiovascular Research), partner site Hamburg/Kiel/
Lübeck, Germany; 3Department for Experimental Pharmacology and Toxicology,
University Medical Centre Hamburg-­Eppendorf, Hamburg, Germany
Muscarinic acetylcholine receptors (AChMR1-­
5) mediate cellular
responses upon release of acetylcholine (ACh) from parasympa-
thetic nerves. In addition, ACh is the prototypical agonist stimulat-
ing endothelium-­dependent dilation, but most blood vessels lack

dysfunction in coronary arterioles in advancing parasympathetic innervation, raising the question as to the physi-
ologic function of endothelial AChMR. Global deletion of AChM3R
age
revealed a role in ACh-­induced vasodilation in vitro as well as food
Amanda Leblanc1,2; Evan Tracy1; Fangping Yuan1; Jason uptake, but overall cardiovascular homeostasis has not been exam-
Beare1; Natia Kelm1
ined thoroughly. We hypothesized that ACh exerts through these re-
1
Cardiovascular Innovation Institute, University of Louisville, Louisville KY;
2 ceptors a continuous dilator influence in resistance vessels, thereby
Department of Physiology, University of Louisville, Louisville KY
affecting blood pressure and a long-­term defect herein impacts car-
diac function. To characterize the function of endothelial AChM3R
Our past research has shown that tail vein injection of adipose-­
in vivo, we deleted AChM3R specifically in endothelial cells using an
derived stromal vascular fractions (SVF) was associated with im-
inducible or a non-­inducible Cre-­loxP system driven by the promot-
proved coronary flow reserve when stimulated with dobutamine
ers VE-­cadherin (indEC-­M3R−/−) or TIE2 (EC-­M3R−/−). In both EC-­
in vivo, but we did not observe improvements in endothelial and
M3R−/−, ACh-­induced dilation was strongly impaired in arterioles in
smooth-­muscle dependent vasoreactivity. We hypothesized that i.v.
vivo (at 10 μmol/L from 76 ± 2 to 40 ± 4 or 24 ± 4%, indEC-­M3R−/−
injection of SVF was improving coronary microvascular function via
and EC-­M3R−/−, respectively) while responses to other dilators were
alterations in adrenergic signaling.
mostly preserved. However, mean arterial pressure was not altered
Methods: Groups: Young (3 months, n = 7), old (24 months, n = 9),
in ndEC-­M3R−/− (99 ± 4 vs 104 ± 2 mm Hg in controls) and, ad-
and old rats which received 1x10(7) green fluorescent protein
ditionally, arteriolar tone was also comparable in EC-­M3R−/− mice
(GFP+) SVF cells (n = 8) four weeks prior female F344 were used.
and respective controls. Aged EC-­M3R−/− mice (74-­78 weeks) did
Coronary arterioles from each group were isolated for microvessel
not differ in body weight, heart weight, cardiac structure or contrac-
reactivity studies, reactive oxygen species (ROS) fluorescent signal-
tile function from controls. We conclude that AChM3R elicits the
ing, and immunofluorescence.
endothelium-­dependent dilation upon ACh also in arterioles in vivo.
Results: There was a decrease in vasorelaxation to dobutamine
Despite this prominent role, the endothelial deletion of AChM3R
(beta-­one receptor agonist) and norepinephrine (alpha and beta re-
does not affect overall cardiovascular homeostasis. Thus, their phys-
ceptor agonist) in OC (vs YC) that was rescued in old+SVF. Atenolol,
iologic function in endothelial cells remains obscure.
a beta-­one receptor antagonist, decreased the vasodilation to nor-
epinephrine in YC more than O+SVF, indicating greater beta-­two
sensitivity in the latter. Mitochondrial H2O2 production during
shear stress (25 ul/min) was increased in OC vs. YC and O+SVF.
 |
88 of 96       ABSTRACTS
WEPE023 | Regulation of coronary collateral
growth
Weiguo Wan1; Anurag Jamaiyar1,2; Cody Juguilon1,3; Devan
Cumpston1,3; James Gadd1; Molly Enrick1; Chris Kolz1;
Vahagn Ohanyan1; William M. Chilian1; Liya Yin1
1 micity is lost in ClockΔ19/Δ19 mice and myogenic tone is locked at
Department of Integrative Medical Sciences, Northeast Ohio Medical University,
Rootstown, OH, USA; 2School of Biomedical Sciences, Kent State University,
Kent, OH, USA; 3College of Graduate Studies, Northeast Ohio Medical
University, Rootstown, OH, USA
Cardiovascular disease is the leading cause of death in the United
States, Ischemic Heart Disease (IHD) accounts for one in seven car-
diovascular related deaths. Patients with poorly developed coronary
collateral networks have a poorer prognosis after a cardiovascular
event than those with well-­developed collaterals. Furthermore, cor-
onary collateral growth is impaired in metabolic syndrome such as
diabetes, and obesity. The underlying mechanism(s) is (are) that are
causal for growth are unknown. In an attempt to elucidate a mecha-
nistic understanding, we developed a murine model that allows us
to use genetically modified mice to study the coronary collateral
growth (CCG) spatially and temporally. Coronary collateral growth
(CCG) was measured by myocardial blood flow with contrast echo-
cardiography and by collateral numbers by micro-­C T. We are able to
study some key genes that regulate the CCG in physiological condi-
tion and pathological conditions (diabetes) and use lineage tracing
to understand which cell type is important for CCG in a temporal
manner.
WEPE024 | Disrupting a circadian clock
mechanism that regulates myogenic reactivity
mitigates cardiac injury in heart failure
1 2
Jeff Kroetsch ; Faisal Alibhai ; Darcy Lidington ; Cristine 1
Reitz2; Hangjun Zhang1; Danny Dinh1; Scott Heximer1; Tami
Martino2; Steffen-Sebastian Bolz1
1 2
University of Toronto, Department of Physiology, Toronto, ON, Canada; Centre
for Cardiovascular Investigations in the Department of Biomedical Sciences,
University of Guelph, Guelph, ON, Canada
The coordination of circadian rhythms in the cardiovascular sys-
tem is crucial for maintaining proper function. Circadian rhythms in
microvascular myogenic reactivity, particularly in beds that promi-
nently regulate total peripheral resistance (TPR), are poorly under-
stood. This investigation assessed myogenic rhythmicity in skeletal
muscle resistance arteries and its contribution to cardiac decline in
heart failure (HF).
Cremaster skeletal muscle resistance arteries were isolated and
assessed with standard pressure myography techniques. Circadian
rhythmicity was genetically disrupted in mice via the ClockΔ19/Δ19
mutation. HF was induced by ligation of the left anterior descending
coronary artery. Cardiac function was determined by echocardio-
graphic, hemodynamic, and histological assessments.
Wild-­type cremaster muscle resistance arteries demonstrate a
pronounced circadian rhythm in myogenic responsiveness; agonist-­
induced vasoconstriction is not rhythmic. In HF, myogenic respon-
siveness is locked at the circadian maximum, although circadian
molecular clock gene expression cycles normally. Myogenic rhyth-
the circadian minimum, even in HF mice. The reduction in TPR in
ClockΔ19/Δ19 HF mice associates with improved cardiac function
and reduced infarct expansion.
Our study reveals that the circadian molecular clock regulates
myogenic responsiveness. HF augments myogenic responsiveness,
putatively by targeting the link utilized by the circadian clock to os-
cillate myogenic reactivity. Our data show that the circadian regula-
tion of myogenic responsiveness is a novel target for the treatment
of HF-­induced microvascular dysfunction and may significantly im-
prove cardiac function.
WEPE025 | NADPH oxidase 4 mediates
beneficial effects of exercise on vascular function
Heike Langbein; Amna Shahid; Jennifer Mittag; Anja
Hofmann; Henning Morawietz; Coy Brunssen
Division of Vascular Endothelium and Microcirculation, Department of Medicine
III, University Hospital Carl Gustav Carus, Dresden, Germany
Physical activity is one of the most potent strategies to prevent en-
dothelial dysfunction. Recent evidence indicates vaso-­
protective
properties of H2O2 produced by main endothelial NADPH oxidase
isoform Nox4 in the vasculature. Therefore, we hypothesized that
Nox4 contributes to the vaso-­protective effects of physical activity.
Ex vivo analysis of endothelial function by Mulvany myograph
showed endothelial dysfunction in Wildtype as well as Nox4−/− mice
after 20 weeks on high-­fat diet. Access to voluntary running wheels
during 20 weeks high-­fat diet prevented endothelial dysfunction in
Wildtype, but not in Nox4−/− mice. Exercise led to increased H2O2
release in the aorta of Wildtype mice, as well as increased phosphor-
ylation of AKT1, which was diminished in aortas of Nox4−/− mice.
Deletion of Nox4 also led to decreased capacity for intracellular
calcium release indicated by reduced phenylephrine-­mediated con-
traction, whereas potassium-­induced contraction was unaffected.
H2O2 scavenger catalase reduced phenylephrine-­
contraction in
Wildtype mice. Adding H2O2 significantly increased phenylephrine-­
induced contraction in Nox4−/− mice.
Monitoring of the voluntary running of the mice indicated re-
duced activity and running distance of the Nox4−/− mice compared
with wildtype mice throughout the 20 weeks study period. In the
skeletal muscle Nox4−/− mice revealed no exercise induced eleva-
tion of PGC-­1-­α . Furthermore, exercise induced citrate synthase ac-
tivity and mitochondria were reduced in the absence of Nox4.
We concluded that Nox4 derived H2O2 plays a role in physical
activity induced adaptations by modifying intracellular calcium re-
lease and subsequently PGC-­1α signaling.
ABSTRACTS
WEPE026 | Racial disparities in coherence
between fluctuations in bloodflow and
oxygenation dynamics
Yunus A. Abdulhameed; Gemma Lancaster; Aneta
Stefanovska
Department of Physics, Lancaster University, UK
Prevalence of ethnic disparities in cardiovascular diseases means
investigating its effects on the key features of cardiovascular dy-
namics is crucial for understanding cardiovascular pathophysiology.
One such feature is the oxygenation dynamics (OD), whose function
is oxygen transportation across the body. This process occurs in an
oscillatory fashion and had extensively been studied. Research has
shown that haemoglobin A1c levels differ in black and white persons
independent of glucose level. Considering the race dependent dif-
ferences in cardiovascular risk, it is plausible that rhythmic coordi-
nation between blood flow (BF) and oxygenation oscillations may
better elucidate these differences. Therefore, we investigate effects
of biracial disparity on the fluctuations in OD.
BF, oxygen saturation (OXY), oxygenated hemoglobin (oxyHb)
and deoxygenated hemoglobin (deoxyHb) were simultaneously re-
corded for 30 minutes from 16 black Africans (BA) and 16 Caucasian
white (CW) healthy subjects (19-­35 years). Time-­varying oscillations
of the signals and their instantaneous phases were extracted using
wavelet analysis. Rhythmic coordination between BF and oxygen-
ation dynamics were investigated using wavelet phase coherence.
BF and OXY coherence were significantly higher in CW at 0.05-­
0.15 Hz interval (P < 0.05). BA exhibit significantly (P < 0.05) lower co-
herences between BF – oxyHb at 0.05-­0.15 Hz and BF – deoxyHb at
0.08-­0.1 Hz. However, at 0.3-­0.6 Hz and 0.6-­2 Hz intervals, coherence
between oxyHb and deoxyHb were significantly (P < 0.05) higher in BA.
Similar to previous findings that reported racial effects on hemo-
globin A1c, this study points to potential effects of racial disparities
on causal connections between BF and OD.
WEPE027 | Gas exchange platform for the
investigations of oxygen-­dependent regulation in
the microcirculation
Richard Sové1; Stephanie Milkovich1,2; Hristo Nikolov1,2;
David Holdsworth1,2; Graham Fraser3; Christopher Ellis1,2
1
Department of Medical Biophysics, University of Western Ontario, London,
Canada; 2Robarts Research Institute, University of Western Ontario, London,
Canada; 3Biomedical Sciences, Memorial University, St. Johns, Canada
The local control of oxygen (O₂) supply to tissue has long been sus-
pected to depend on local O₂, however, the location of the O₂ sensor
remains unknown. Intravital video microscopy in conjunction with a
system for altering surface oxygen tension have been used to study
regulation in the microcirculation. The objective of this study is to
develop an O₂ delivery platform with a sufficiently small exchange
|
      89 of 96
area that is thin enough to allow us to focus on capillaries through-
out the depth of the muscle that is normally visualized without the
gas exchange platform. A GPU-­accelerated, 3D O₂ transport model
of diffusion through the tissue was developed to estimate the ex-
tent of the change in tissue oxygenation. The experimental proto-
cols were approved by the University of Western Ontario's Animal
Care and Use Committee. The O₂ exchange platform was able to
cause changes in RBC SO₂ of capillaries close to the window in male
Sprague-­
Dawley rats. Further, these changes resulted in corre-
sponding changes in RBC supply rate. The computational model pre-
dicted that the O₂ delivery is confined within 80 μm from the edge of
the window. Overall, we have developed an O₂ delivery system that
is capable of causing localized changes in SO₂ which can be used to
further investigate the location of the O₂ sensor in the microvascular
regulation of O₂. Supported by NSERC Discovery Grant.
WEPE028 | Microfluidic device for the rapid
deoxygenation of red blood cells in vitro
Richard Sové1; Daniel Lorusso2,3; Hristo Nikolov1,3; David
Holdsworth1,3; Graham Fraser4; Christopher Ellis1,3
1
Department of Medical Biophysics, University of Western Ontario, London,
Canada; 2Department of Physiology & Pharmacology, University of Western
Ontario, London, Canada; 3Robarts Research Institute, University of Western
Ontario, London, Canada; 4Biomedical Sciences, Memorial University, St. Johns,
Canada
Red blood cells (RBCs) are thought to be involved in the local regula-
tion of oxygen (O₂) supply through the O₂-­dependent release of ATP.
In this proposed mechanism, ATP is released from RBCs in a hemo-
globin O₂ desaturation-­dependent manner to cause the vasodilation
of supplying upstream arterioles. Though there has been compelling
evidence for O₂-­dependent ATP release, there has been no direct
quantification of the dynamics of (e.g. the time for ATP to be re-
leased following desaturation). Quantifying the dynamics would not
only give insight into the efficacy of the control system, it would di-
rectly support the existence of this proposed mechanism. However,
the quantification of ATP release time requires a rapid temporal de-
saturation of RBCs; a difficult task to achieve. The objective of this
study is to develop a microfluidic device to cause a rapid spatial de-
oxygenation of RBCs to facilitate the measurement of the dynamics
of ATP release. The device consists of two parts: a micro-­delivery
gas exchange platform and an O₂ permeable microfluidic channel.
RBCs suspended in physiological buffer, collected from healthy male
Sprague-­Dawley rats, were passed through the microfluidic device;
experimental protocols were approved by the University of Western
Ontario's Animal Care and Use Committee. Hemoglobin oxygen satu-
ration (SO₂) was measured using dual-­wavelength video microscopy.
The microfluidic device caused a rapid spatial change in RBC SO₂.
In summary, we have developed an experimental system to control
the spatial deoxygenation of RBCs in a steady flow microfluidic de-
vice to study the dynamics of O₂-­dependent ATP release from RBCs.
Supported by NSERC Discovery Grant.
 |
90 of 96       ABSTRACTS
WEPE029 | The microvascular lattice: an
updated paradigm of flow distribution through
capillary networks in skeletal muscle
Asher Mendelson1,2; Edward Ho1; Stephanie Milkovich1,2;
Christopher Ellis1,2; Daniel Goldman1
1
Department of Medical Biophysics, University of Western Ontario, London,
Canada; 2Robarts Research Institute, University of Western Ontario, London,
Canada
Background: Classical microvascular flow modelling uses linear
branching networks such that each capillary bundle (CB) corre-
sponds to a unique arteriolar-­venular pair. However, it has long been
observed in vivo that terminal arterioles supply multiple CBs and
similarly, post-­capillary venules collect from multiple CBs. Such a
network forms a lattice-­like structure with an interconnecting pat-
tern of arterioles, capillaries, and venules.
Objective: To develop a model to represent the lattice physiology
we observe in vivo. We hypothesize that rheologic properties in the
microcirculation support the even distribution of RBC flow for capil-
laries in a bundle.
Methods: Microvascular lattice networks are analyzed from rodent
intravital video-­microscopy data and stylized geometries are con-
structed. A dual-­phase steady-­state blood flow model is then ap-
plied. RBC and plasma flow in the lattice are compared to a linear
branching network using inflow hematocrits of 0.1, 0.2, and 0.3.
Results: Plasma flow is skimmed preferentially to upstream capil-
laries along a bundle; inflow hematocrit affects the magnitude of
plasma flow without affecting relative distribution. RBC flow is sig-
nificantly less variable than plasma flow for capillaries in a bundle;
increases of inflow hematocrit affect relative RBC distribution and
reduce flow variability between capillaries.
Conclusion: The Microvascular Lattice represents an updated para-
digm for describing blood flow through capillary networks. This lat-
tice structure raises important questions regarding mechanisms of
flow regulation in this component of the microcirculation; further
research will examine the impact of spatial heterogeneity on linear
and lattice flow properties.
Supported by NSERC Discovery Grants to Drs Ellis and Goldman.
WEPE030 | Mechanism inducing longitudinal
capillary recruitment
1,2
Jacques Huyghe
1
Bernal Institute, University of Limerick, Limerick, Ireland; 2Darcy Centre,
Department of Mechanical Engineering, Eindhoven University of Technology,
Eindhoven, The Netherlands
Numerous researchers have found that capillary vessel haematocrit
depends on the vasodilatory state of the arterioles. At rest, ves-
sel haematocrit is down to 15%, suggesting a red blood cell veloc-
ity three times higher than the plasma velocity. The same capillary
vessel haematocrit increases to 35-­40% in vasodilation. Classical
insights in the context of the Fahraeus-­Lindqvist effect cannot jus-
tify this. Some suggest that this shift of haematocrit can be inter-
preted as longitudinal capillary recruitment [Poole et al. PNAS, 2014).
Therefore, capillary haematocrit is analysed in the context of present
understanding of propulsion of red blood cells (RBCs) and plasma by
means of the arteriovenous pressure gradient, as well as in the light
of recent developments in interfacial forces in microfluidic systems.
Interfacial forces between red blood cells and plasma are proposed
as an explanation of the observed red blood cell velocities. While
the arteriovenous pressure gradient across the capillaries propels red
blood cell and plasma jointly, interfacial forces along the red blood
cell membrane can propel RBCs at the cost of the plasma. Options
are explored for the origin of these interfacial forces. Oxygen gra-
dients are found to be the most probable source. Indeed, propul-
sion of microbeads by means of self-­generated oxygen gradients
are among the most common propulsion methods in colloidal sys-
tems, while oxygen gradient around RBCs are very steep at capillary
entry. Microfluidic experiments clearly confirm that oxygenated red
blood cells move towards low oxygen gradients. Funding from Dutch
Science Foundation and Irish Research Council is acknowledged.
WEPE031 | Relationship between total
peripheral vascular resistance and single vascular
resistance in skeletal muscle arteriole
Tomohiro Komine; Morisuke Asai; Yuna Takagi; Rina Suzuki;
Nobuo Watanabe; Masahiro Shibata
Department of Bioscience and Engineering, Shibaura Institute of Technology,
Tokyo, Japan
The total peripheral vascular resistance (TPR)is essential index in the
cardiovascular system, since both the systemic blood pressure (BP)
and blood flow (BF)could be determined by the changes of TPR. Such
important index, the TPR cannot be measured directly, so Darcy's law
is applied to determine. On the other hand, Single vascular resistance
(R)is represented as R = 8 μL/πr⁴, and mainly be controlled by the con-
traction or dilation of arteriole. In addition, it is considered the major
contribution of TPR would be caused by skeletal muscle arterioles,
since the BF in skeletal muscle dramatically changes from resting to
excise state, approximately 20 times increases. These facts suggest
the TPR would be determined by the levels of contraction and dilation
in skeletal muscle arterioles. In the present study we tried to investi-
gate whether the TPR can be estimated from the diameter changes of
single arteriole in skeletal muscle. The TPR was quantified by direct
measurements of BP and BF in rat carotid artery. Whereas R is de-
termined by direct observation of microcirculation in cremaster mus-
cle. During Phenylephrine induced vasoconstriction, BP increased
from 106 ± 6 to 153 ± 151 mm Hg, and BF decreased from 8 ± 1
to 5 ± 1 mL/min, while arterial diameter decreased from 97 ± 16 to
76 ± 13 μm. The changes in TPR and R during vasoconstriction were
no significant differences. These results confirmed TPR would mainly
be controlled by the diameter changes of arterioles in skeletal muscle.
ABSTRACTS
WEPE032 | Modeling of the spatial
inhomogeneous degradation of nitric oxide
shows a key role of anatomically localized NO
production
1 2 2,3
W. Davis Haselden ; Ravi Teja ; Patrick J. Drew
1
Neuroscience Graduate Program, MD/PhD Medical Scientist Training Program,
The Pennsylvania State University; 2Department of Engineering Science and
Mechanics, The Pennsylvania State University; 3Departments of Biomedical
Engineering and Neurosurgery, The Pennsylvania State University
The interaction between neural activity and the hemodynamic re-
sponse is known as neurovascular coupling (NVC). Understanding
how changes in neural activity drive blood flow is vital for the accurate
interpretation of hemodynamic signals. While neurovascular coupling
is thought to be mediated by many signaling molecules, here we focus
on Nitric oxide (NO). NO is a potent vasodilator and neural activity
modulator that freely diffuses across cell membranes. NO released
from neurons dilates arteries and is also rapidly degraded by interac-
tions with hemoglobin (Hb) in the blood. We used a computational
model to simulate NO production, diffusion and degradation in a cor-
tical column. The maximum NO production rate was upper bound NO
concentrations that cause a toxic inhibition of aerobic respiration in
cytochrome c oxidase (CcO). We show that in order to generate physi-
ologically plausible dilations without toxic inhibition of CcO, proximal
production of NO is required. Additionally, we show that vessel size is
an important factor in determining arteriole sensitivity to NO, as the
amount of Hb present in the vessel impacts the degradation rate of
NO and can account for larger dilations in smaller arteries observed
experimentally. We also investigate how NO-­mediated NVC can be
altered during hypoxia and when free Hb levels in the plasma are
pathologically elevated. Lastly, we investigate how vascular changes
could impact perivascular NO concentrations. Our simulations show
that the spatial arrangement of NO production plays a key role in de-
termining the efficacy of NO on arteries and other tissues.
Supported by R01EB021703
WEPE033 | Study of cell-­cell interactions and
cytokine release profile in a co-­culture of human
monocytes with SMCs differentiated from ASCs
1,2 1,2,3,4
Xiaoqing Zhang ; Craig A. Simmons ; J. Paul
Santerre1,2,4
1
Institute of Biomaterials and Biomedical Engineering, University of Toronto,
Toronto, Ontario, Canada; 2Translational Biology and Engineering Program,
Ted Rogers Centre for Heart Research, University of Toronto, Toronto, Ontario,
Canada; 3Department of Mechanical and Industrial Engineering, University of
Toronto, Toronto, Ontario, Canada; 4Faculty of Dentistry, University of Toronto,
Toronto, Ontario, Canada
Arteriogenesis is critical for restoring perfusion in ischemic tissues.
Monocytes, which can adopt inflammatory or wound-­healing phe-
notype, are among the first cell types present after tissue injuries.
|
      91 of 96
Both monocytes and smooth muscle cells (SMCs) are involved in tis-
sue regeneration and arteriogenesis. While monocytes can interact
with SMCs during tissue repair, the mechanisms by which monocytes
stimulate SMC proliferation/migration during arteriole regeneration
are still not fully explored. Adipose derived stromal cells (ASCs) can
be differentiated into SMC-­like cells (ASC-­SMCs), which can be used
as a replacement for tissue-­isolated human SMCs in tissue engineer-
ing, to address the limited supply and expansion ability of mature
SMCs.
Objective: Establishing an ASC-­SMC/monocyte co-­culture system
in vitro, to investigate cell interactions/cytokine release within the
system. Hypothesis: Cytokines that can induce SMC proliferation/
migration (e.g. MCP-­
1, FGF-­
2, GM-­
C SF) would be preferentially
released when ASC-­
SMCs were co-­
cultured with monocytes vs.
without. ASC-­SMCs and monocytes (300,000 ASC-­VSMCs/75,000
monocytes) were co-­cultured on D-­PHI scaffolds for 24 hours and
4-­week. It was found that at 4 weeks, co-­cultures supported an in-
crease in cell number and cell migration in the scaffold vs. mono-­
culture (P < 0.05). This observation corresponded with an increased
release of MCP-­1, FGF-­2, GM-­C SF and accumulation of elastin and
glycosaminoglycan in co-­culture vs. mono-­culture at 4 weeks (all
P < 0.05). This study suggests monocytes can interact with ASC-­
SMCs to increase the release of MCP-­1, FGF-­2, GM-­C SF, which have
been reported to be mediators of cell proliferation/migration during
arteriogenesis.
Acknowledgement: CIHR grant #230762, OGS and NSERC PGS-­D
scholarships.
WEPE034 | Objective Assessment of Skin
Microcirculation Using a Smartphone Camera
Erik Tesselaar1,2
1
Department of Clinical and Experimental Medicine, Faculty of Health Sciences,
Linköping University, Linköping, Sweden; 2Department of Medical Radiation
Physics, Department of Medical and Health Sciences, Linköping University,
Linköping, Sweden
Background: Existing microcirculation imaging techniques are bulky,
expensive and not so easy to use. In recent years, great improve-
ments have been made in smartphone camera technology. We de-
veloped SkinSight, an app for iPhone and iPad, to measure changes
in skin microcirculation during physiological provocations. The app
uses the phone's camera to estimate changes in the concentration of
hemoglobin in the skin by analyzing the reflected light emitted from
the built-­in LED flash.
Methods: The relative hemoglobin (Hb) index was measured during
a 5-­minute arterial occlusion, post-­occlusive reactive hyperemia and
a 5-­minute venous occlusion in 10 healthy subjects, on two separate
days.
Results: During arterial occlusion, relative Hb index was unchanged
compared to baseline (P = 0.40). After release of the cuff, a sudden
60-­75% increase in Hb index was observed (P < 0.001) followed by
 |
92 of 96       ABSTRACTS
a gradual return to baseline. During venous occlusion, Hb index in-
creased by 80% (P < 0.001) followed by a gradual decrease to base-
line after reperfusion. Day-­to-­day reproducibility of the relative Hb
index was excellent (ICC: 0.92, r = 0.94), although relative Hb index
was consistently higher during the second day, possibly as a result of
changed lighting conditions or calibration issues.
Conclusion: Microvascular responses to physiological provocations
in the skin can be accurately and reproducibly measured using a
smartphone application. Although the system offers a handheld,
easy to use and flexible technique for skin microvascular assess-
ment, the effects of lighting on the measured values and need for
calibration need to be further investigated.
WEPE036 | Altered sensory neurotransmitter
content, release and receptor expression underlie
impaired vasodilation in mesenteric arteries with
inflammatory bowel disease
Elizabeth Grunz-Borgmann; Erika Boerman
Medical Pharmacology and Physiology, University of Missouri, Columbia, MO,
USA
Perivascular sensory nerves of mesenteric arteries (MAs) medi-
ate intestinal blood flow, and their function appears impaired with
Inflammatory Bowel Disease (IBD). We hypothesized that altered
content, release and receptor signaling of sensory neurotransmitters
calcitonin gene-­related peptide (CGRP) and substance P (SP) underlie
impaired MA function with IBD. With institutional ethics approval,
IL10−/− mice were inoculated with H. hepaticus after weaning and
developed IBD over 90 days; non-­inoculated littermates served as
controls. Sensory vasodilation was assessed via electrical field stim-
ulation (EFS) in pre-­constricted (phenylephrine, 1 μmol/L) MAs with
sympathetic nerves blocked (guanethidine, 10 μmol/L). Dilations
were greater in Control MAs at 8 Hz (35 ± 2%) and 16 Hz (55 ± 6%)
vs IBD (8 Hz: 18 ± 1%, 16 Hz: 25 + 2%). Blocking CGRP receptors
(BIBN 4096, 1 μmol/L) attenuated dilation in Control (8 Hz: 12 ± 4%;
16 Hz: 20 ± 4%) and IBD (8 Hz: 10 ± 1%; 16 Hz: 19 ± 3%) MAs.
Addition of a SP receptor (NK1) antagonist (CP99994, 1 μmol/L) fur-
ther attenuated dilation only in IBD MAs at 16 Hz (8 ± 3%). Reversing
antagonist order had no effect in Controls. However, NK1 blockade
in IBD augmented sensory dilation [8 Hz, 35 ± 5%; 16 Hz, 48 ± 7%].
Thus, CGRP plays a major role in sensory dilation across groups, but
the role of SP shifts with IBD, where NK1 activation inhibits sensory
dilation. ELISA of CGRP and SP content in MA homogenate and re-
lease into the bath during EFS each decreased ~35-­60% with IBD vs
Control. Confocal fluorescence imaging of CGRP and SP receptors in
MAs indicates elevated endothelial NK1 expression with IBD. These
data support a role for SP and NK1 as mediators of MA dysfunction
in IBD. R00HL129196.
WEPE038 | Carbon monoxide-­releasing
molecule-­3 (CORM-­3) diminishes inflammation,
oxidative stress and vascular endothelial
monolayer breakdown in human model of
compartment syndrome
Aurelia Bihari1,2; Gediminas Cepinskas2,3; Akira Chung2;
Abdel-Rahman Lawendy1,2,3
1
Div. Orthopaedic Surgery, Dept. Surgery, London Health Sciences Centre,
London (ON), Canada; 2Centre for Critical Illness Research, Lawson Health
Research Institute, London (ON), Canada; 3Department of Medical Biophysics,
Western University, London (ON), Canada
Acute limb compartment syndrome (CS), a devastating complication
of musculoskeletal trauma, results in muscle necrosis and cell death.
Oxidative stress due to ischemia and inflammation both appear to
contribute to the microvascular dysfunction and parenchymal injury.
Previously, administration of the carbon monoxide releasing mole-
cule-­3 (CORM-­3) has been shown to protect microvascular perfu-
sion and reduce inflammation in animal models of CS.
The purpose of the study was to test the effect of CORM-­3 in
human in vitro model of CS, allowing exploration of the mecha-
nism(s) of CO protection. This would lead to potential development
of a rational pharmacologic treatment for CS.
Human vascular endothelial cells (HUVECs), grown to conflu-
ency, were stimulated for 6 hours with serum (40%) isolated from
CS patients. Levels of intracellular oxidative stress (production of re-
active oxygen species (ROS)) apoptosis, trans-­endothelial resistance
(TEER), polymorphonuclear leukocyte (PMN) activation (rolling and
adhesion to HUVECs under the conditions of flow), and transmigra-
tion across the monolayer in response to the CS stimulus were as-
sessed. All experiments were performed in the presence of CORM-­3
(100 μmol/L), or its inactive form, iCORM-­3.
CS serum induced a significant increase in the production of ROS,
apoptosis and endothelial monolayer breakdown; it also increased
PMN superoxide production, leukocyte rolling and adhesion/transmi-
gration. CORM-­3 treatment completely prevented CS serum-­induced
ROS production, apoptosis, PMN adhesion, rolling and transmigration,
while improving monolayer integrity. The data indicate that CORM-­3
offers potent anti-­oxidant and anti-­inflammatory effects, and thus
may have a potential application to patients at risk of developing CS.
WEPE039 | Ambient ultra fine particle exposure
impairs gut vascular barrier via notch signaling
Chih-Chiang Chang1; Kyung In Baek1; Yichen Ding2;
Constantinos Sioutas3; Rongsong Li2; Tzung Hsiai1,2
1
Department of Bioengineering, University of California Los Angeles; 2Division
of Cardiology, Department of Medicine, University of California, Los Angeles;
3
Department of Civil and Environmental Engineering, University of Southern
California
Ultrafine particles (UFP, d < 0.1 μm), a major sub-­fraction of par-
ticulate matter (PM2.5, d < 2.5 μm) in air pollutant, have been
ABSTRACTS
reported to increase cardiovascular morbidity. UFP promote
endothelial dysfunction/permeability associated with Notch in-
hibition and degradation of tight junction protein. Gut vascular
barrier (GVB) is a distinct protective barrier preventing infections
by oral digestions. We hypothesized UFP impair GVB by attenu-
ating Notch signaling and disrupting tight junction protein. The
transgenic Tg(flk1:mCherry) zebrafish embryos were immobilized
and performed micro-­g avage at 5 days post-­fertilizations (5 dpf).
A mixture of 10 kD FITC conjugated dextran and phenol red dye
was micro-­g avaged on the intestinal bulb with or without UFP at
25 μg/mL. Anterior trunk and posterior cardinal vein (PCV) post
micro-­g avage were imaged under a confocal microscope. Notch
signaling related genes including the Notch ligand, Dll4, and the
target, HES1, and the level of tight junction protein genes zonula
occludens-­1 (ZO-­1) and Occludin (OCLN) mRNA expression follow-
ing different courses of exposure to UFP were assessed in Human
Aortic Endothelial Cells (HAEC) with quantitative RT-­P CR. In the
control group, most of FITC-­d extran remained inside the gastro-
intestinal system. In contrast, UFP exposure developed a signifi-
cant accumulation of FITC-­d extran in the intersomatic spaces (ISS)
between the dorsal aorta (DA) and the PCV. Moreover, the ex-
pression of HES1, DLL4, ZO-­1, and OCLN in HAEC were down-
regulated in concentration and time-­course dependent manner by
the exposure to UFP. These findings support that UFP exposure
disrupted both the epithelial boundary and the endothelial vascu-
lar layer of the gut.
WEPE041 | A novel proangiogenic function
for interferon-­lambda through upregulation of
VEGF, bFGF and IL-­8 from cocultured human
endothelial cells with fibroblasts
Haiyan Jia1; Parvathy Harikumar1; Craig Thelwell2; Chris
Bird1; Sarah Daniels2; Paula Dilger1; Meenu Wadhwa1
1
Section of Cytokines and Growth Factors, Division of Biotherapeutics, National
Institute for Biological Standards and Control, UK; 2Section of Haemostasis,
Division of Biotherapeutics, National Institute for Biological Standards and
Control, UK
Interferon-­lambda (IFN-­λ) has been extensively studied for its anti-
viral and anti-­tumour activities, however, the role of IFN-­λ in human
vascular endothelial cells and fibroblasts is not defined. We there-
fore aimed to investigate the effects of IFN-­λ on (1) proliferation of
human umbilical vein endothelial cells (HUVECs) and human dermal
fibroblasts (HDFs), (2) angiogenesis in a co-­culture model and 3) en-
dothelial cell-­mediated blood haemostasis.
Cell surface expression of IFN-­
λ receptor was detected by
flow cytometry. Proliferation was measured by alamarBlue assay.
Angiogenesis was evaluated in a co-­culture assay of HUVECs and
HDFs. Secreted molecules in cell culture supernatants were deter-
mined using ELISA.
|
      93 of 96
HUVECs and HDFs showed comparable expression levels of the
distinct IFN-­λ receptor subunits on the surface. IFN-­λ1 inhibited
growth of HUVECs, but not HDFs, in a concentration-­dependent
manner mainly mediated by induction of cell cycle arrest at the G0/
G1 phase. Furthermore, VEGF-­
induced survival of HUVECs was
dose-­dependently suppressed by IFN-­λ1. Surprisingly, IFN-­λ1 pro-
moted angiogenesis as evidenced by an increase in the development
of capillary-­like structures during the co-­culture. This proangiogenic
activity of IFN-­λ1 was associated with its significant upregulation of
secretion of VEGF, bFGF and IL-­8, but not IP-­10 during the period
of angiogenesis assay. Additionally, IFN-­λ1-­treated HUVECs showed
no alteration on the surface expression of von Willebrand factor, but
markedly reduced release of urokinase-­t ype plasminogen activator
and greatly elevated secretion of plasminogen activator inhibitor-­1
compared with tissue-­type plasminogen activator, suggesting de-
creased fibrinolytic activity. In conclusion, we have shown that IFN-­
λ1 is a novel regulator of angiogenesis and fibrinolysis.
WEPE042 | Comparison of renal perfusion
assessed by vascularity index, measured
by supermicro vascular imaging, and tissue
perfusion, measured by laser Doppler
Naoya Iguchi1,2; Yugeesh Lankadeva1; Roger Evans3; Clive
May1
1
Florey Institute of Neuroscience and Mental Health, Melbourne, VIC, Australia;
3
Department of Anesthesiology and Intensive Care Medicine, Graduate School
of Medicine, Osaka University, Osaka, Japan; 2Cardiovascular Disease Program,
Biomedicine Discovery Institute and Department of Physiology, Monash
University, Melbourne, VIC, Australia
Introduction: At present, using ultrasound it is difficult to meas-
ure the changes in renal perfusion that precede the development
of acute kidney injury (AKI). Recently, a new ultrasound technique,
Superb Micro Vascular Imaging (SMI), which allows imaging of the
renal microvascular has become available. To validate the use of
SMI to measure renal microvascular perfusion, we have compared
measurement of renal perfusion using SMI with that measured by
implanted laser Doppler probes.
Objectives: To compare microvascular perfusion in the renal cortex
determined using SMI with that measured with surgically implanted
fibre-­optic probes in sheep on cardiopulmonary bypass (CPB).
Methods: Sheep (N = 4) were instrumented with a renal artery flow
probe and fibre-­optic probes in the renal cortex, to measure tis-
sue perfusion. CPB was established under general anaesthesia and
pump flow was held at 60, 80 and 100 mL/kg/min in random order.
We compared vascularity index (%), blood perfusion determined by
SMI in a set area of the renal cortex, with renal cortical perfusion
measured by fibre-­optic probes.
Results: Changes in pump flow from 60 to 80 and 100 mL/kg/min
resulted in increases in mean renal cortical perfusion from 748 ± 103
to 1083 ± 252 and 1302 ± 353 blood perfusion units with changes in
 |
94 of 96       ABSTRACTS
simultaneously measured mean vascularity index from 20.3 ± 0.6 to
24.9 ± 1.4 and 30.6 ± 1.0%, respectively.
Conclusion: In sheep on CPB, changes in pump flow resulted in par-
allel changes in vascularity index, determined by SMI, and renal cor-
tical perfusion, measured by laser Doppler.
WEPE044 | A novel catecholamine-­
sparing strategy towards protecting the renal
microcirculation in septic acute kidney injury
Yugeesh R. Lankadeva1; Marianne Tare2,3; Helena C.
Parkington2; Roger G. Evans2; Shaui Ma1,4; Naoya Iguchi1,5;
Rinaldo Bellomo6; Clive N. May1
1
Florey Institute of Neuroscience and Mental Health, Melbourne, VIC,
Australia; 2Cardiovascular Disease Program, Biomedicine Discovery Institute
and Department of Physiology, Monash University, Melbourne, VIC, Australia;
3
Monash Rural Health, Monash University, Melbourne, VIC, Australia; 4Division
of Nephrology & Unit of Critical Nephrology, Shanghai Ninth People's Hospital,
School of Medicine, Shanghai Jiaotong University, Shanghai, China; 5.
Department of Anesthesiology and Intensive Care Medicine, Graduate School of
Medicine, Osaka University, Osaka, Japan; 6. School of Medicine, University of
Melbourne, Melbourne, VIC, Australia
Objectives: In septic shock, excessive sympathetic activity can lead
to catecholamine-­refractory hypotension and acute kidney injury
(AKI). Consequently, high doses of norepinephrine are required to
attain target blood pressure. We have examined (1) the effects of
norepinephrine on the renal macro-­and microcirculation in ovine
septic AKI, and (2) determined the effects of the α2-­adrenoreceptor
agonist, clonidine, on renal sympathetic nerve activity (RSNA) and
pressor responsiveness to phenylephrine.
Methods: We implanted a renal artery flow probe, nerve recording
electrodes and laser-­Doppler/oxygen-­sensing probes in the renal
cortex and medulla in sheep. We infused Escherichia coli to induce
septic AKI in conscious sheep. Norepinephrine (0.4-­0.8 μg/kg/min),
clonidine (1 μg/kg/h) or vehicle-­saline were infused (24-­32 hours of
sepsis) (all/N = 6). Pressor responses to phenylephrine were meas-
ured at baseline, and 24 and 32 hours of sepsis.
Results: Septic AKI was characterized by hypotension (~20%) and
reduced creatinine clearance (~60%). Despite global renal and corti-
cal hyper-­perfusion, medullary perfusion and oxygenation decreased
(both ~50%). Restoring blood pressure with norepinephrine further re-
duced medullary perfusion (~70%) and oxygenation (~80%). Sepsis was
associated with increased RSNA (~75%) and blunted pressor responses
to phenylephrine (~50%). Clonidine-­treatment substantially reduced
RSNA and fully restored pressor responsiveness to phenylephrine.
Conclusions: Renal medullary hypoxia due to intra-­renal shunt-
ing of microvascular perfusion may contribute to septic AKI.
Resuscitation with norepinephrine further worsened the under-
lying medullary hypoxia in septic AKI. Reducing norepinephrine
requirements with α2-­a drenoreceptor agonists may minimize the
harmful effects of excessive catecholamines on the renal microcir-
culation, but the effects on the development of septic AKI require
investigation.
WEPE048 | Impaired endothelial function
and reduced volume compliance in schlager
hypertensive (BPH/2J) mice
C. H. Leo1,2; M. Jelinic2,3; K. O'Sullivan2; K. L. Jackson4; M.
Deo 4; L. I. Parry2; R. H. Ritchie 4; G. A. Head4; C. X. Qin4
1
Science and Math, Singapore University of Technology & Design, Singapore;
2
School of BioSciences, The University of Melbourne, Parkville, Australia;
3
Department of Physiology, Anatomy & Microbiology, La Trobe University, Bundoora,
Australia; 4Baker Heart and Diabetes Research Institute, Melbourne, Australia
Objective: Impaired endothelial function and arterial stiffness are key
hallmarks of hypertension, and major risk factors for cardiovascular
disease. The hypertensive BPH/2J mice are recognized as a genetic
model of hypertension, possibly with sympathetic overdrive. However,
little is known about the vascular pathophysiology of BPH/2J mice and
if there were region-­dependent differences between vascular beds.
The aim of this study is to compare the vascular function and remodel-
ling of BPH/2J mice with their normotensive BPN/3J counterparts.
Methods: Blood pressure was measured in BPH/2J and BPN/3J mice
via pre-­
implanted radiotelemetry probes (AEC#E/1757/2017B).
Wire and pressure myography were used to analyse vascular func-
tion and passive mechanical wall properties of large (aorta and femo-
ral) and resistant (mesenteric) arteries.
Results: BPH/2J mice exhibited elevated mean arterial blood pres-
sure compared to controls (128 ± 3 mm Hg vs. 107 ± 1 mm Hg,
P < 0.05, n = 6). Endothelium-­dependent relaxation to acetylcho-
line was attenuated in the aorta and mesenteric arteries in BPH/2J
mice. Further, the contribution of nitric oxide and endothelium-­
dependent hyperpolarization was impaired in the BPH/2J mesen-
teric arteries, whereas the contribution of prostanoids was reduced
in the BPH/2J aorta compared to BPN/3J. In addition, hypertrophic
inward remodelling was observed in the hypertensive mesenteric
but not femoral arteries. Despite this, both femoral and mesenteric
arteries have reduced volume compliance in the hypertensive mice.
Conclusion: This study demonstrated that hypertensive BPH/2J
mice exhibited endothelial dysfunction and adverse vascular re-
modelling in macro-­and microvasculature, underpinned by distinct
mechanisms. BPH/2J may be a suitable animal model for evaluating
novel therapeutics to treat hypertension.
WEPE049 | Low input microfluidic-­based gene
expression assay reveals differences between
native pial artery and capillary endothelial cells in
the brain
Evan Yamasaki; Harry Pritchard; Scott Earley
Department of Pharmacology, Center for Molecular and Cellular Signaling in the
Cardiovascular System, University of Nevada, Reno School of Medicine, Reno,
Nevada, USA
Endothelial cells (ECs) play a significant role in vascular regulation.
To better understand phenotypic differences among ECs from
ABSTRACTS
different vascular segments, we attempted to develop a single-­cell
method to rapidly profile gene expression in populations of native
cerebral pial artery and capillary ECs (PECs and CECs). Cerebral
pial arteries and capillaries were separately isolated from Tg(Tek-­
GFP)287Sato EC reporter mice using an institutionally-­
approved
animal protocol and were enzymatically dispersed to generate in-
dividual cells. PECs or CECs were identified and sorted into 96-­well
plates at a density of 5 cells/well using fluorescence-­activated cell
sorting and reverse-­transcription/amplification was performed for
a customized gene array encoding cell-­t ype markers, ion channels,
Ca2+-­handling proteins, connexins, and cell surface receptors using
the Fluidigm Biomark HD Delta Gene Assay. From an initial panel of
~30 independent samples/group, the genes with the highest level
of differential expression encode small-­and intermediate-­conduct-
ance Ca2+-­activated K+ channels (SK and IK), which are expressed by
PECs but not CECs; and microtubule-­associated protein 2 (MAP2),
expressed by CECs but not PECs. Using whole cell patch-­clamp elec-
trophysiology, we found that SK/IK currents were present in PECs
but were not detected in CECs. MAP2 was detected using immu-
nofluorescence microscopy in CECs. We conclude that differential
expression of SK/IK channels accounts for functional differences
between PECs and CECs and that MAP2 may be a useful cell-­
type marker for distinguishing PEC and CEC populations. Further,
our findings demonstrate the feasibility of profiling gene expres-
sion patterns in individual ECs. R01HL091905; R01HL137852;
R01HL139585; P30GM110767.
WEPE050 | Endothelial glycocalyx damage
in the early phase of acute respiratory distress
syndrome secondary to respiratory virus
infection
Maíra Benatti; Carlos Henrique Miranda
Ribeirão Preto School of Medicine, São Paulo University, Ribeirão Preto-­SP, Brazil
Influenza viral infection can cause acute respiratory distress syn-
drome (ARDS) in adults, especially in outbreak periods. The mech-
anisms responsible for this injury are not well known yet. We
hypothesized that endothelial glycocalyx damage can contribute to
the development of ARDS after respiratory virus infection.
We included 55 patients with flu-­
like symptoms admitted at
emergency department during influenza A outbreak, divided into
two groups: 22 patients with ARDS, according to Berlin definition
(Group 1), with a median of 5 days of symptoms duration and an
intra-­hospital mortality rate of 45%; and 33 patients without ARDS
(Group 2), with a median of 4 days of symptoms duration and an
intra-­hospital mortality rate of 00%. A control group of 26 healthy
individuals was included for comparison (Group 3).
A blood sample was collected to measured biomarkers of endo-
thelial glycocalyx damage through commercial ELISA Kits, supported
by grant 2016/22147-­1, Sao Paulo Research Foundation (FAPESP).
|
      95 of 96
The hyaluronan levels were significantly higher in the group 1
(34 ± 6 ng/mL) over group 2 (12 ± 4 ng/mL) and group 3 (8 ± 4 ng/
mL); P < 0.0001. The syndecan-­1 levels were higher in the group
1 (80 ± 18 ρg/mL) in comparison to the group 2 (49 ± 11 ρg/mL),
P = 0.003. The thrombomodulin levels were significantly higher
in the group 1 (1687 ± 181 ρg/mL) in comparison to group 3
(175 ± 116 ρg/mL); P < 0.0001.
The elevation of biomarkers of the endothelial glycocalyx dam-
age suggests the possibility of a pivotal role in ARDS development in
this scenario. These biomarkers can be used to select patients with
flu with a higher risk of developing ARDS.
WEPE052 | Effects of chlorinated lipids on
endothelial permeability mediated by apparent
differential effects on transcellular solute
transport pathway activity
Theodore Kalogeris
Department of Medical Pharmacology and Physiology, School of Medicine,
University of Missouri, Columbia, MO, USA
Chlorinating species generated through activity of neutrophil/
macrophage myeloperoxidase produce several species of chlorin-
ated lipids, and these lipids in turn may contribute to the inflam-
matory phenotype seen in sepsis. We previously documented
increases in cortical stiffness and cytoskeletal reorganization in
endothelial cells exposed to chloro-­p almitate. Here we examined
effects of chlorolipids on endothelial barrier function. Human mi-
crovascular endothelial cells (HMEC-­1) on transwell inserts were
incubated with 2-­chlorohexadecanal (chloro-­p almitaldehyde, Cl-­
HDA) or 2-­chlorohexadecanoic acid (chloro-­p almitate, CL-­FA), or
their non-­chlorinated lipid precursors as controls, and the per-
meability of the monolayer to FITC-­albumin was calculated from
measured albumin flux and transwell albumin concentration gradi-
ent. Compared with control treatments, Cl-­FA produced a 3-­4 -­fold
increase in permeability which persisted over at least 4 hours,
while Cl-­HDA's effect was not significantly different from con-
trol. We next examined the effect of the aforementioned treat-
ments on adherens junctional organization. Treatment with either
Cl-­FA or Cl-­HDA induced internalization of surface VE-­c adherin.
Taken together with similar effects of Cl-­FA and Cl-­HDA on actin
stress fiber formation and F-­a ctin polymerization, these results
argue against a differential effect on structural determinants of
the paracellular pathway. To explain the differential effects of
Cl-­FA and Cl-­HDA on endothelial solute permeability, we have
begun to examine the effects of these chlorolipids on transcellu-
lar solute transport activity. We found increased, BSA-­d ependent
phosphorylation (tyr-­114) of caveolin-­1 in response to Cl-­FA but
not Cl-­HDA, a result consistent with a selective effect of Cl-­FA
on endothelial protein permeability via the transcellular transport
pathway. Supported by N.I.H. GM-­115553 & AA-­221081.
 |
96 of 96       ABSTRACTS
WEPE056 | Anti-­angiogenesis triggers
exosomes release from endothelial cells to
promote tumor vasculogenesis
Ye Zeng1; Bingmei M. Fu2
1
Institute of Biomedical Engineering, West China School of Basic Sciences and
Forensic Medicine, Sichuan University, Chengdu, Sichuan, China; 2Department
of Biomedical Engineering, The City College of the City University of New York,
New York, NY, USA
Although anti-­angiogenic therapies (AATs) have some effects against
multiple malignancies, they are limited by subsequent tumor vascu-
logenesis and progression. To investigate the mechanisms by which
tumor vasculogenesis and progression following AATs, we trans-
fected microRNA (miR)-­9 into human umbilical vein endothelial cells
(HUVECs) to mimic the tumor-­associated endothelial cells in hepa-
tocellular carcinoma and simulated the AATs in vitro and in vivo. We
found that administration of the angiogenesis inhibitor vandetanib
completely abolished miR-­
9-­
induced angiogenesis and promoted
autophagy in HUVECs but induced the release of vascular endothe-
lial growth factor (VEGF)-­enriched exosomes. These VEGF-­enriched
exosomes significantly promoted the formation of endothelial ves-
sels and vasculogenic mimicry in hepatocellular carcinoma and its
progression in mice. Anti-­autophagic therapy is proposed to improve
the efficacy of AATs. However, similar effects by AATs were observed
with the application of anti-­autophagy by 3-­methyladenine. Our re-
sults revealed that tumor vasculogenesis and progression after AATs
and anti-­autophagic therapies were due to the cross-­talk between
endothelial and tumor cells via VEGF-­enriched exosomes. This find-
ing provides a critical insight into a new interpretation of why AATs
have not achieved more advantageous outcomes. Our study also
found that anti-­autophagy is as effective as anti-­angiogenesis but
triggers less release of VEGF-­enriched exosomes, suggesting that
anti-­autophagy is an adjuvant or better AAT. Our findings further
suggest that control of exosome release or alteration of exosome
cargo composition to inhibit tumor vasculogenesis may augment the
anti-­angiogenic and anti-­autophagic therapies for tumors.