Vascular Research Initiatives Conference

Vascular Research Initiatives Conference

VRIC 2020 ONLINE: From Discovery to Translation

DAY 1

November 5th, 2020, 1:00-3:00 PM Central Time

ABSTRACT SESSION I: ARTERIAL REMODELING AND DISCOVERY SCIENCE FOR VENOUS DISEASE

ABSTRACT SESSION II: ATHEROSCLEROSIS AND ROLE OF THE IMMUNE SYSTEM

DAY 2

November 12th, 2020, 7:00-9:00 PM Central Time

ABSTRACT SESSION III: AORTOPATHIES AND NOVEL VASCULAR DEVICES

ABSTRACT SESSION IV: VASCULAR REGENERATION, STEM CELLS AND WOUND HEALING

Program Overview

The 34th annual Vascular Research Initiatives Conference (VRIC), presented by the Society for

Vascular Surgery®, is designed to encourage interaction and collaboration between vascular

surgeon investigators and scientists from other vascular biology‐related disciplines. An additional

objective is to stimulate interest in research among trainees who are aspiring academic vascular

surgeons. The VRIC 2020 ONLINE is taking place on November 5th and 12th as virtual online events

instead of the traditional in-person meeting in response to the 2020 COVID-19 pandemic.

2020-2021 SVS Basic and Translational Research Committee:

Luke Brewster – Chair, Emory University, Atlanta, GA

Jayer Chung, MD, University of Texas Southwestern Medical Center, Houston, TX

Scott Damrauer, MD, University of Pennsylvania School of Medicine, Philadelphia, PA

Sean English, MD, Washington University‐Barnes Hospital, St. Louis, MO

Katherine Gallagher, MD, University of Michigan, Ann Arbor, MI

Caitlin Hicks, MD, MS, Johns Hopkins University Program, Baltimore, MD

Karen Ho, MD, Northwestern University, Chicago, IL

Jason MacTaggart, MD, University of Nebraska Medical Center, Omaha, NE

Firas Mussa, MD, MS, University of South Carolina School of Medicine, New Hyde Park, NY

Ryan McEnaney, MD, University of Pittsburgh Medical Center, Pittsburgh, PA

Nicholas Osborne, MD, University of Michigan Hospitals, Ann Arbor, MI

Elsie Ross, MD, Stanford Hospital and Clinics, Stanford, CA

Ulka Sachdev, MD, University of Pittsburgh, Pittsburgh, PA

Kevin Southerland, MD, Duke University, Durham, NC

Bryan Tillman, MD, PhD, University of Pittsburg, Pittsburgh, PA

Shirling Tsai, MD, University of Texas Southwestern Medical Center, Houston, TX

Areck Ucuzian, MD, PhD, University of Maryland School of Medicine, Baltimore, MD

Dai Yamanouchi, MD, University of Wisconsin, Madison, WI

AGENDA

Thursday, November 5, 2020 (All times are Central Time US)

Luke Brewster, MD, Chair, Basic and Translational Research Committee

Moderator: Ulka Sachdev, MD

Moderator: Firas Mussa, MD

1:00 PM INTRODUCTORY REMARKS

VRIC 2020 ONLINE Day 1

ABSTRACT SESSION I: ARTERIAL REMODELING AND DISCOVERY SCIENCE FOR VENOUS DISEASE

1:05 PM

^* Elastic Fibers Of The Internal Elastic Lamina Are Unraveled But Not Created

With Expanding Arterial Diameter In Arteriogenesis

Derek Afflu, Univ of Pittsburgh Medical Ctr, PITTSBURGH, PA; Dylan D McCreary,

Nolan Skirtich, VA PHS, Pittsburgh, PA; Kathy Gonzalez, UPMC, Pittsburgh, PA;

Edith Tzeng, UNIVERSITY PITTSBURGH, Pittsburgh, PA; Ryan M McEnaney

VA PHS, Pittsburgh, PA

1:12 PM

A Synthetic Resolvin Analogue (Benzo-Rvd1) Attenuates Vascular Smooth

Muscle Cell (VSMC) Migration And Neointimal Hyperplasia

Alexander Kim, SMITH CARDIOVASCULAR RESEARCH BLDG, San Francisco, CA;

Hideo Kagaya, UCSF, San Francisco, CA; Mian Chen, SMITH CARDIOVASCULAR

RESEARCH BLDG, San Francisco, CA; Bian Wu, Giorgio Mottola, UCSF, San

Francisco, CA; Matthew R Spite, HARVARD MEDICAL SCHOOL, Boston, MA;

Brian Sansbury, Brigham and Women's Hosp, Boston, MA; Michael S Conte, UC

SAN FRANCISCO, San Francisco, CA

1:19 PM

Evaluation Of A Targeted Drug Eluting Intravascular Nanotherapy To Prevent

Neointimal Hyperplasia In A Novel Atherosclerotic Rat Model

Emily R Newton, Univ of North Carolina, Chapel Hill, NC; Brooke Dandurand,

UNC Sch of Med, Chapel Hill, NC; David C Gillis, Kui Sun, Robin Siletzky, Univ of

North Carolina, Chapel Hill, NC; Mark R Karver, Samuel I Stupp, Northwestern

Univ, Chicago, IL; NICK D TSIHLIS, UNC-CHAPEL HILL, Chapel Hill, NC; Melina R

KIBBE, Univ of North Carolina, Chapel Hill, NC

1:26 PM

Programmed Death Ligand-1 Regulates T-cells And M2 Macrophages To

Control Wall Thickening During Arteriovenous Fistula Maturation

Yutaka Matsubara, Luis Gonzalez, Jia Liu, Arash Fereydooni, John Langford,

Shin-rong Lee, Jolanta Gorecka, Mingjie Gao, Xixiang Gao, Ryosuke Taniguchi,

Bogdan Yatsula, Alan Dardik, Dept of Surgery, Yale Univ Sch of Med, New

Haven, CT

1:33 PM

1:40 PM

TRPC6 Depletion Results In Loss Of Myocardin And Phenotypic Modulation In

Vascular Smooth Muscle Cells

Andrew H. Smith, Priya Putta, Erin Driscoll, Pinaki Chaudhuri, Cleveland Clinic,

Cleveland, OH; Michael Rosenbaum, Louis Stokes Cleveland VAMC,

Cleveland, OH; Linda M. Graham, Cleveland Clinic, Cleveland, OH

Periadventitial Delivery Of Simvastatin From Microparticles Attenuates

Arteriovenous Fistula Outflow Vein Neointimal Hyperplasia

Chenglei Zhao, Mayo Clinic, Rochester, MN; Sean T Zuckerman, Affinity

Therapeutics, LLC, Cleveland, OH; Chuanqi Cai, Michael L Simeon, Avishek

Singh, Sreenivasulu Kilari, Mayo Clinic, Rochester, MN; Julius N Korley, Affinity

Therapeutics, LLC, Cleveland, OH; Sanjay Misra, MAYO CLINIC, Rochester, MN

1:50-2:00 Pause and Reset Presenters

Moderator: Ryan McEnaney, MD

Moderator: Caitlin Hicks, MD

2:00 PM ^ Absence Of Cpla2 In Lrp1 Smooth Muscle Cell Deficient Mice Promotes

Severe Aortic Atherosclerotic Disease

Roberto Ivan Mota Alvidrez, Shirling Tsai, Joachim Herz, Univ of Texas

Southwestern Medical Ctr, Dallas, TX

2:07 PM Serum Circulating Fatty Acid Synthase As A Diagnostic Biomarker For

Chronic Limb-Threatening Ischemia

Shirli Tay, Amanda Penrose, Gayan S De Silva, Yan Yan, Washington Univ Sch of

Med, St. Louis, MO; Clay F Semenkovich, WASHINGTON UNIV SCH OF MED, Saint

Louis, MO; Mohamed A Zayed, WASHINGTON UNIVERSITY SCHOOL M, Saint

Louis, MO

2:14 PM ZEB2 Regulates Activation And Exhaustion Programming Of CD8 T Cells In

Atherosclerosis

Dawn Fernandez, Nicolas F Fernandez, adeeb Rahman, Christopher Hill, Mount

Sinai, New York, NY; Roza Shamailova, Icahn Sch of Med, New York, NY;

Seunghee Kim-schulze, J Mocco, Peter Faries, Miriam Merad, Chiara

Giannarelli, Mount Sinai, New York, NY

2:21 PM The Role Of Mast Cells In Atherosclerotic Plaque Calcification

Nikolaos-Taxiarchis Skenteris, Malin Kronqvist, Mariette Lengquist, Karolinska

Inst, Solna, Sweden; Ulf Hedin, Karolinska Inst, Stockholm, Sweden; Erik L Biessen,

Maastricht Univ Medical Ctr, Maastricht, Netherlands; Ljubica Matic, Karolinska

Inst, Stockholm, Sweden

ABSTRACT SESSION II: ATHEROSCLEROSIS AND ROLE OF THE IMMUNE SYSTEM

2:28 PM

2:35 PM

2:42 PM

+ Inflammatory Activity of Human Perivascular Adipose Tissue in Abdominal

Aortic Aneurysms

Jorn Meekel, Marina Dias-Neto, Natalija Bogunovic, Gloria Conceigao, Claudia

Sousa-Mendes, Gawin R. Stoll, Adelino Leite-Moreira, Jennifer Huynh, Dimitra

Micha, Etto C. Eringa, Ron Balm, Jan D. Blankensteijn, Kak K. Yeung

Polygenic Risk Score Identifies Patients At Increased Risk For Abdominal Aortic

Aneurysm And May Benefit From Ultrasound Screening

Derek Klarin, Univ of Florida Coll of Med, Gainesville, FL; Ozan Dikilitas, Mayo

Clinic, Rochester, MN; Brooke Wolford, University of Michigan, Ann Arbor, MI;

Michael Levin, Univ of Pennsylvania Sch of Med, Philadelphia, PA; Ishan

Paranjpe, Mount Sinai Sch of Med, New York, NY; Renae Judy, Univ of

Pennsylvania Sch of Med, Philadelphia, PA; Julie Lynch, Dept of Veterans

Affairs, Salt Lake City Health Care System, Salt Lake City, UT; Themistocles L

Assimes, Stanford Univ Sch of Med, Palo Alto, CA; Yan Sun, Emory Univ, Atlanta,

GA; Daniel Rader, Univ of Pennsylvania Sch of Med, Philadelphia, PA; Peter W

Wilson, Emory Univ Sch of Med, Atlanta, GA; Salvatore Scali, Scott Berceli, Univ

of Florida Coll of Med, Gainesville, FL; Sekar Kathiresan, Verve Therapeutics,

Cambridge, MA; Pradeep Natarajan, Massachusetts General Hosp, Boston,

MA; Girish Nadkarni, Mount Sinai Sch of Med, New York, NY; Cristen Willer, Univ

of Michigan, Ann Arbor, MI; Iftikhar Kullo, Mayo Clinic, Rochester, MN; Scott M

Damrauer, University of Pennsylvania Sch of Med, Philadelphia, PA; Philip Tsao,

Stanford Univ Sch of Med, Palo Alto, CA

Vascular Surgery and the K Award: An Abundance of Successes: Presentation

will highlight the K award recipients, SVSF winners, and SVSF commitment to

helping young surgeons succeed in their academic pursuits.

Luke Brewster, MD, and Katherine Gallagher, MD

Adjourn (day 1)

Thursday, November 12, 2020 (All times are Central Time US)

Luke Brewster, MD, Chair, Basic and Translational Research Committee

Moderator: Jason MacTaggart, MD

Moderator: Elsie Ross, MD

7:05 PM

^* Epigenetic Modifications Influence Macrophage-mediated Inflammation

In abdominal Aortic Aneurysms

Frank Michael Davis, Aaron Dendekker, Amrita Joshi, Sonya Wolf, Christopher

Audu, William J Melvin, UNIVERSITY OF MICHIGAN, Ann Arbor, MI; Hong Lu, UNIV

KENTUCKY, Lexington, KY; Alan Daugherty, UNIVERSITY OF KENTUCKY, Lexington,

KY; Johann Gudjonsson, UNIVERSITY OF MICHIGAN, Ann Arbor, MI; Katherine A

Gallagher, UNIVERSITY MICHIGAN, Ann Arbor, MI

7:12 PM The Perfuse Dual Chamber Stent Improves Donor Organ Recovery In A Porcine

Model

Bryan W. Tillman, Catherine Go, Brian Frenz, UNIV OF PITTSBURGH MED CTR,

Pittsburgh, PA; Moataz Elsisy, UNIV OF PITTSBURGH, Pittsburgh, PA; Youngjae

Chun, UNIVERSITY OF PITTSBURGH, Pittsburgh, PA

7:19 PM Endovascular Repair Of Blunt Thoracic Aortic Trauma Is Associated With

Increased Left Ventricular Mass, Hypertension, And Off-target Aortic

Remodeling

Alexey Kamenskiy, Univ of Nebraska Omaha, Omaha, NE; Paul Aylward, Univ

of Nebraska Medical Cent, Omaha, NE; Anastasia Desyatova, Univ of

Nebraska Omaha, Omaha, NE; Matthew DeVries, Christopher Wichman, Univ

of Nebraska Medical Ctr, Omaha, NE; Jason N Mactaggart, Univ of Nebraska

Medical Cent, Omaha, NE

7:26 PM

Maresin 1 Attenuates Murine Abdominal Aortic Aneurysms Via Vascular

Smooth Muscle Cell Dependent TGF-β Signaling

Craig T Elder, Univ of Florida, Gainesville, FL; Gang Su, Gainesville, FL; Amanda

Filiberto, Univ of Florida Coll of Me, GAINESVILLE, FL; Guanyi Lu, Univ of Florida

Coll of Me, GAINESVILLE, FL, Gainesville, FL; Zhihua Jiang, UNIVERSITY OF

FLORIDA COM, Gainesville, FL; Ashish Sharma, UNIVERSITY OF FLORIDA,

Gainesville, FL; Gilbert R UPCHURCH Jr., Univ of FL Dept of Surgery, Gainesville,

FL

7:00 PM INTRODUCTORY REMARKS

VRIC 2020 ONLINE Day 2

ABSTRACT SESSION III: AORTOPATHIES AND NOVEL VASCULAR DEVICES

7:33 PM

7:40 PM

7:47 PM

7:54 PM

A Theranostic Role For Ccr2 In Rodent Abdominal Aortic Aneurysm

Development And Rupture

Sergio Sastriques Dunlop, Washington Univ Sch Med, Saint Louis, MO; Batool

Arif, Sean English, Washington Univ Sch Med, SAINT LOUIS, MO

Pharmacological Inhibition Of Bruton's Tyrosine Kinase Attenuates

Experimental Abdominal Aortic Aneurysms

Baohui Xu, STANFORD UNIVERSITY SCHOOL OF MEDIC, Stanford, CA; yankui li,

Stanford Univ Sch of Med, Stanford, CA; Gang Li, STANFORD UNIVERSITY

SCHOOL OF MEDIC, Stanford, CA; Yingbin Ge, Nanjing Medical Univ, Nanjing,

China; Jia Guo, Stanford Univ Sch of Med, Stanford, CA; Weirong Fang, China

Pharmaceutic Univ, Nanjing, China; Wei Wang, Xiangya Hosp, Central South

Univ, Chnagsha, China; Fanru Shen, STANFORD UNIVERSITY SCHOOL OF MEDIC,

Stanford, CA; Takahiro Shoji, Vascular Surgery, Stanford, Stanford, CA; Toru

Ikezoe, Xiaoya Zheng, STANFORD UNIVERSITY SCHOOL OF MEDIC, Stanford, CA;

Sihai Zhao, Stanford Univ Sch of Med, Stanford, CA; Xiaofeng Chen, TAIZHOU

HOSPITAL, Taizhou; Masaaki MIYATA, KAGOSHIMA CITY HOSPITAL, Kagoshima,

Japan; Alan Daugherty, UNIVERSITY OF KENTUCKY, Lexington, KY; Hong Lu, Univ

of Kentucky Sch of Med, Lexington, KY; Ronald L DALMAN, STANFORD

UNIVERSITY MEDICAL CE, Stanford, CA

Landscape Of Human Secretome In Abdominal Aortic Aneurysm Profiled

By Single-cell RNA Sequencing.

Bhama Ramkhelawon, NYU MEDICAL CENTER, New York, NY; Tarik Hadi, NYU

Langone Medical Ctr, New York, NY; Michele Silvestro, NYU Sch of Med, New

York, NY

Pause and Reset Presenters

Moderator: Jayer Chung, MD

Moderator: Kevin Southerland, MD

8:00 PM ^* Autophagy Remodels Mitochondria During Differentiation And Enhances

Longevity Through Ulk1 Kinase Signaling Of Induced Pluripotent Stem Cell-

derived Endothelial Cells

Katherine E Hekman, Kyle Koss, David Z Ivancic, Northwestern Univ, Chicago, IL;

Congcong He, Chicago, IL; Jason A Wertheim, Northwestern Univ, Chicago, IL

ABSTRACT SESSION IV: VASCULAR REGENERATION, STEM CELLS AND WOUND HEALING

8:07 PM

* Downregulation Of Inflammation And A Cascade Of Pro-angiogenic Signals

Mediate The Beneficial Effects Of Gene-modified Stem Cell Therapy In Hindlimb

Ischemia

Hallie J Quiroz, Univ of Miami, Miami, FL; Hongwei Shao, Yan Li, Samantha F

Valencia, Punam Parikh, Univ of Miami, Miami, FL; Yulexi Ortiz, Univ of Miami Miller

Sch of Med, Miami, FL; Roberta Lassance-Soares, Univ of Miami, Miami, FL; Zhao-

Jun Liu, Univ of Miami Miller, Miami, FL; Omaida Caridad Velazquez, Univ of Miami

Miller Sch of Med, Miami, FL

8:14 PM

Different Outcomes after Revascularization or Standard Supervised Exercise

Treadmill Training of Claudicating Patients with Peripheral Artery Disease

Shuai Li, Jonathan Robert Thompson, Univ of Nebraska Med Ctr, Omaha, NE;

Sara A Myers, Univ of Nebraska at Omaha, Omaha, NE; Julian K Kim, UNIV

NEBRASKA MEDICAL CENTER, Omaha, NE; Panagiotis Koutakis, Florida State Univ,

Tallahassee, FL; Mark Williams, Creighton Cardiac Ctr, Omaha, NE; Zhen Zhu, Univ

of Nebraska Med Ctr, Omaha, NE; Molly Schieber, Univ of Nebraska Medical

Cent, Omaha, NE; Timothy Lackner, Gregory Willcockson, Christina Shields,

Katyarina Brunette, Holly Despiegelaere, Univ of Nebraska Med Ctr, Omaha, NE;

Iraklis I Pipinos, UNIV NEBRASKA MEDICAL CTR, Omaha, NE; George Casale, Univ

of Nebraska Medical Cent, Omaha, NE

8:21 PM Differential Expression Of Canonical Mitosis And Dna-damage Repair Pathways

Characterize Muscle Satellite Cells Affected By Pad

Ricardo Ferrari, Guangzhi Cong, Bryan Thompson, Debbie Hollingshead,

Janette Lamb, Xiangdong Cui, Ulka Sachdev, UPMC, Pittsburgh, PA

8:28 PM Fluvastatin And Vegf Containing Resins Promote Angiogenesis And

Arteriogenesis

Furqan Muqri, SUNY Upstate Medical Ctr & Syracuse VA Medical Ctr, Syracuse,

NY; Dandan Guo, SUNY Upstate Medical Univ, Syracuse, NY; Mohammed

Kassem, Mary DaCosta, SUNY Upstate Medical Ctr & Syracuse VA Medical Ctr,

Syracuse, NY; David Bruch, SUNY Upstate Medical Univ, Syracuse, NY; Kristopher

Maier, SUNY Upstate Medical Ctr & Syracuse VA Medical Ctr, Syracuse, NY;

Juntao Luo, SUNY Upstate Medical Ctr, Syracuse, NY; Vivian Gahtan, SUNY

Upstate Medical Ctr & Syracuse VA Medical Ctr, Syracuse, NY

8:35 PM

8:42 PM

8:49 PM

8:56 PM

9:03 PM

9:10 PM

Increased Neutrophil Elastase Expression In Regions Of High Intraluminal

Thrombus Deposition In Human Abdominal Aortic Aneurysms.

Matthew Levesque, Lauren Bath, Max Rady Coll of Med, Winnipeg, MB, Canada;

Annie Ducas, Royal Victoria Regional Health Ctr, Barrie, ON, Canada; April Boyd,

Health Sciences Ctr, Winnipeg, MB, Canada

Adult and Pediatric Fibromuscular Dysplasia Are Genetically Distinct Dysplasia-

associated Arterial Diseases

Yu Wang, Dawn M. Coleman, Univ of Michigan Medical Sch, Ann Arbor, MI; Min-

Lee Yang, Issaquah, WA; Susan Blackburn, Issaquah, WA, Ann Arbor, MI; Kristina

L. Hunker, Univ of Michigan Medical Sch, Ann Arbor, MI; Heather L Gornik, Univ

Hosp Cleveland, Cleveland, OH; Jun Li, Univ of Michigan, Ann Arbor, MI; James

C Stanley, 2210 Taubman Ctr, Ann Arbor, MI; Santhi Ganesh, 2210 Taubman Ctr,

Ann Arbor, MI, Ann Arbor, MI

Impact Of Dietary Tungsten And Topical Nitrite In Diabetic Wound Healing And

The Composition Of The Wound Microbiome

Kathy Gonzalez, Karim M Salem, UPMC, Pittsburgh, PA; Barbara Methé, Kelvin

Li, Guiying Hong, Univ of Pittsburgh, Pittsburgh, PA; Edith Tzeng, UNIVERSITY

PITTSBURGH, Pittsburgh, PA

MicroRNA-181b Regulates Critical Limb Ischemia In Diabetic Mice

Henry S Cheng, Brigham and Women's Hosp, Boston, MA; Daniel Perez-

Cremades, BWH, Boston, MA; Marc P Bonaca, CPC Clinical Res, Aurora, CO;

Mark Feinberg, Brigham and Women's Hosp, Boston, MA

Nitric Oxide Bioavailability Is Reduced In Peripheral Artery Disease In Association

With Increased Oxidative Stress And An Altered Biopterin System

Ahmed Ismaeel, Evlampia Papoutsi, Florida State Univ, Tallahassee, FL; William T

Bohannon, Robert Smith, Baylor Scott and White Heath, Temple, TX; Robert

Brumberg, Vascular Surgery Associates, Tallahassee, FL; Carlos H Castro, Jeffrey S

Kirk, Capital Regional Medical Ctr, Tallahassee, FL; Iraklis I Pipinos, UNIV NEBRASKA

MEDICAL CTR, Omaha, NE; Panagiotis Koutakis, Florida State Univ, Tallahassee, FL

Adjourn (day 2)

* SVS Foundation VRIC Trainee Awardee

^ Best Abstract in Category

+ 2020 ESVS Young Researcher Prize winner

701

There are no unlabeled/unapproved uses of drugs or products

Knowledge Strategy:

With a better understanding of the mechanisms governing arteriogenesis, we may find pharmacological

targets that could enhance collateral artery formation. This could lead to a pharmacological therapy that

effectively increases blood flow to an ischemic tissue which would revolutionize management of

patients with arterial occlusive diseases.

Professional Practice Gap:

Patients suffering from arterial occlusive diseases would benefit tremendously if there were an effective

medical therapy that could revascularize ischemic tissues. Collateral arteries develop (in a process

termed arteriogenesis) as a spontaneous adaptation when a large conductance artery becomes

occluded. The importance of collateral arteries for maintaining organ and limb perfusion has long been

recognized, but their development is incompletely understood. If enhanced collateral artery

development could be achieved thorough pharmacological means, it would offer a promising means to

noninvasively enhance perfusion for these patients.

Elastic Fibers of The Internal Elastic Lamina Are Unraveled But Not Created With Expanding Arterial

Diameter In Arteriogenesis

Derek Afflu, Univ of Pittsburgh Medical Ctr, PITTSBURGH, PA; Dylan D McCreary, Nolan Skirtich, VA PHS,

Pittsburgh, PA; Kathy Gonzalez, UPMC, Pittsburgh, PA; Edith Tzeng, UNIVERSITY PITTSBURGH,

Pittsburgh, PA; Ryan M McEnaney, VA PHS, Pittsburgh, PA

Objective: Outward remodeling of the arterial wall is essential during arteriogenesis to grow small

arterioles into conductance vessels. Prior reports suggest that internal elastic laminae (IEL) are degraded

and rebuilt in arteriogenesis. However, it is unclear whether new elastic fibers can be synthesized in

adult arteries. We sought to understand the IEL changes that occur in arteriogenesis. Methods: Rats age

8-12 wks had femoral artery ligation (FAL) with distal arteriovenous fistula (AVF) placement to enhance

collateral growth. Rats were fed β-aminopropionitrile (BAPN) in drinking water to inhibit lysyl

oxidoreductase (LOX). Rats were euthanized at 2 days to 12 wks and arteries were harvested (N=5/time

point) for multiphoton microscopy and quantitative measurement of desmosine content. Human

collateral arteries from amputation specimens were also analyzed for comparison. Results: At 2 days,

FAL+AVF treated profunda femoral arteries (PFA) showed maximal vasodilation, but IEL retained normal

structure. At 4 wks, PFA diameters increased over contralateral sham-operated PFAs (mean 255 +/-

18.5%) and IEL unraveled into a loose web of elastic fibers which persisted at 12 wks. This IEL pattern

was also seen in human collaterals. Despite PFA size increase, elastin content trended downward (Sham,

0.41 ± 0.07ng; AVF, 0.31 ± 0.06ng, p=0.058). LOX inhibition resulted in severe fragmentation of IEL in

PFAs and abnormally thickened elastic fibers in 3rd order collaterals. Conclusion: In arteriogenesis, the

IEL unravels and remains web like for up to 12 wks after FAL+AVF, a pattern also observed in human

collaterals. Despite the growth in arterial diameter, elastin content was not increased, suggesting new

elastic fibers are not created. Stabilization of IEL changes appeared to be mediated by LOX. Further

dissection of the mechanisms of arterial remodeling in arteriogenesis will allow us to harness this

adaptive process for therapeutic purposes.

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Knowledge Strategy:

This research is demonstrating the efficacy of a novel compound that may help to reduce neointimal

hyperplasia following vascular intervention


The analogue discussed in this abstract may provide durable patency following vascular interventions

A Synthetic Resolvin Analogue (Benzo-Rvd1) Attenuates Vascular Smooth Muscle Cell (VSMC)

Migration And Neointimal Hyperplasia

Alexander Kim, Evan C Werlin, SMITH CARDIOVASCULAR RESEARCH BLDG, San Francisco, CA; Hideo

Kagaya, UCSF, San Francisco, CA; Mian Chen, SMITH CARDIOVASCULAR RESEARCH BLDG, San Francisco,

CA; Bian Wu, Giorgio Mottola, UCSF, San Francisco, CA; Matthew R Spite, HARVARD MEDICAL SCHOOL,

Boston, MA; Brian Sansbury, Brigham and Women's Hosp, Boston, MA; Michael S Conte, UC SAN

FRANCISCO, San Francisco, CA

Background: Persistent inflammation following vascular injury drives neointimal hyperplasia (NIH).

Specialized lipid mediators (SPM) play a critical role in the process of resolution. We investigated the

effects of a novel synthetic SPM on vascular cells and in a model of rat carotid angioplasty.

Methods: Human venous VSMC and endothelial cells (EC) were employed in toxicity, migration,

proliferation and NF-κB activation assays. A model of rat carotid angioplasty was used to evaluate the

effects of Benzo-RvD1 (BRvD) applied externally via 25% Pluronic gel. Drug concentration was measured

at 3 days after injury via LC-MS/MS, and effects on vessel morphometry were examined at 14 days after

injury.

Results: BRvD demonstrated no significant cytotoxicity, and modest anti-proliferative activity on VSMC

at 100 nM. BRvD significantly attenuated VSMC migration across a range of concentrations (0.1-100 nM)

that was equivalent or better than 17R-RvD1, a naturally-occurring SPM (Figure 1). BRvD (10-100nM)

inhibited NF-κB translocation in cytokine-stimulated EC by 12-21% (p<0.01), similar to 17R-RvD1.

Following external delivery, BRvD was detectable in rat carotid tissue at 3 days (mean 0.17 pg/mg; n=3)

after injury. Periadventitial treatment with BRvD reduced carotid neointimal thickness at 14 days

compared to controls, with similar efficacy to 17R-RvD1 (Figure 2).Conclusions: Benzo-RvD1 is a

synthetic analogue of the SPM 17R-RvD1 that demonstrates similar in vitro and in-vivo efficacy to inhibit

NIH as its naturally occurring cognate. The enhanced stability of BRvD may provide therapeutic

advantages for anti-restenosis strategies.

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Knowledge Strategy:

The results of this project, and subsequent projects it leads to, can lead to a paradigm shifting

technology that can completely alter how we treat vascular diseases, improving outcomes and the

overall cost of cardiovascular disease.


Current vascular therapies often fail from restenosis due to neointimal hyperplasia development. Drug

eluting stents developed to combat this problem inhibit healing of the endothelial lining and lead to

catastrophic late in-stent thrombosis and now many of these products are being cautioned against by

the FDA for increasing mortality. This research is developing a new therapeutic to prevent restenosis

after vascular intervention, to make these procedures safer and more durable.

Evaluation of A Targeted Drug Eluting Intravascular Nanotherapy To Prevent Neointimal Hyperplasia

In A Novel Atherosclerotic Rat Model

Emily R Newton, Univ of North Carolina, Chapel Hill, NC; Brooke Dandurand, UNC Sch of Med, Chapel

Hill, NC; David C Gillis, Kui Sun, Robin Siletzky, Univ of North Carolina, Chapel Hill, NC; Mark R Karver,

Samuel I Stupp, Northwestern Univ, Chicago, IL; NICK D TSIHLIS, UNC-CHAPEL HILL, Chapel Hill, NC;

Melina R KIBBE, Univ of North Carolina, Chapel Hill, NC

Introduction: Vascular interventions often fail due to neointimal hyperplasia. We developed a

biocompatible nanotherapeutic that, when given systemically, targets sites of arterial injury and inhibits

neointimal hyperplasia in healthy arteries. Given the oxidative environment of atherosclerosis, the aim

of this study was to evaluate targeting and efficacy of our nanotherapy in a novel atherosclerotic rat

model. We hypothesize that our targeted drug-eluting nanofiber will bind sites of injury and inhibit

neointimal hyperplasia in an atherosclerotic environment. Methods: Knockout status, hyperlipidemia,

and plaque development were assessed in Sprague Dawley ApoE knockout (ApoE-/-) rats using Western

blot analysis, blood work, and Oil Red O (ORO) staining. Fluorescently labelled collagen-targeted, nitric

oxide-releasing peptide amphiphiles (CBP-NO PA) were synthesized by solid-phase peptide synthesis and

purified by HPLC. PA nanofiber formation was assessed by transmission electron microscopy (TEM).

Male 16-week-old rats underwent carotid balloon injury and tail vein injection of nanofiber (5 mg).

Targeting was quantified using fluorescent microscopy and efficacy was assessed via morphometric

analysis. Controls included non-targeted nanofibers and uninjured carotid arteries. Results: Western

blot analysis confirmed ApoE-/- status and blood work showed a 3-fold increase in cholesterol levels in

ApoE-/- vs. wild type rats (292±22 vs. 1050±4 mg/dL, p<0.05). ORO staining showed lipid-rich lesions in

ApoE-/- aortas. PAs were >95% pure and formed nanofibers on TEM. After carotid artery injury and

injection of the CBP-NO nanofiber, specific targeting to the site of arterial injury was seen after 20

minutes on fluorescent microscopy, while uninjured carotid and non-targeted NO nanofibers had

minimal fluorescence (3445±282, 11±2, and 4514±94 AU, p<0.05, n=4/group). Two weeks after injury,

the CBP-NO nanofiber resulted in 67% inhibition of neointimal area and 68% reduction in the

intima/media area ratio vs. injury alone (p<0.0001). Conclusion: We have shown our targeted, drug-

eluting nanofiber localizes to sites of vascular injury and significantly decreases neointimal hyperplasia

after 2 weeks in a novel atherosclerotic rat model.

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Knowledge Strategy:

PDL1 antibody increased T-helper cells and decreased Treg, consistent with increase of inflammation.

Moreover, PDL1 antibody reduced vascular wall thickening during AVF maturation. These results

indicate that PDL1 have a critical role to cause inflammation during AVF maturation and might be a

therapeutic target to improve AVF success.


T-cells are essential for arteriovenous fistula (AVF) maturation, however, little is known about regulation

of T-cells (Treg) during AVF maturation. Programed death ligand-1 (PDL1) can induce regulatory T-cells

and reduce inflammation. We show that how PDL1 control T-cell population and AVF maturation.

Programmed Death Ligand-1 Regulates T-cells And M2 Macrophages to Control Wall Thickening

During Arteriovenous Fistula Maturation

Yutaka Matsubara, Luis Gonzalez, Jia Liu, Arash Fereydooni, John Langford, Shin-rong Lee, Jolanta

Gorecka, Mingjie Gao, Xixiang Gao, Ryosuke Taniguchi, Bogdan Yatsula, Alan Dardik, Dept of Surgery,

Yale Univ Sch of Med, New Haven, CT

Introduction: Vascular remodeling during arteriovenous fistula (AVF) maturation is characterized by

infiltration of T-cells and macrophages. We have previously shown that M2 macrophages play an

important role in AVF maturation to reduce inflammation and promote wall thickening. Although T-cells

can activate macrophages, little is known about T-cell regulation of macrophage function during AVF

maturation. Programed death ligand 1 (PD-L1) induces regulatory T-cells (Treg) to suppress other T-cells

and is expressed in endothelial cells. We hypothesized that PD-L1 induces Treg accumulation in the AVF

to promote AVF maturation. Methods: We used the mouse aortocaval AVF model. Intraperitoneal

injection of PD-L1 antibody (3x/wk) was used to inhibit PD-L1; control was matched IgG2 isotype

antibody. Helper T-cells (Th)-1, Th2, Treg, macrophage accumulation in the AVF was assessed by

immunofluorescence with their specific markers. Vascular wall thickening was assessed by elastin van

Gieson stain. Results: Inhibition of PD-L1 significantly increased accumulation of Th1 (15.9 vs 8.6

cells/hpf; p<0.05) and Th2 (18.0 vs 9.7 cells/hpf; p<0.05), but decreased Treg (3.8 vs 11.2 cells/hpf;

p<0.05) cells compared with control. PD-L1 significantly inhibited accumulation of TGM2+ (M2)

macrophages (5.8 vs 15.7 cells/hpf; p<0.05) and CD206+ (M2) macrophages (2.6 vs 13.2 cells/hpf;

p<0.05), but not iNOS+ (M1) macrophages (8.8 vs 10.0 cells/hpf; p=0.23) or TNF-α+ (M1) macrophages

(9.8 vs 12.3; p=0.29). There was less wall thickening in AVF treated with PD-L1 antibody compared with

control (8.2 µm vs. 17.55 µm; p<0.01) as well as reduced AVF maturation (p=0.03; n=16-17;

Figure). Conclusions: Inhibition of PD-L1 is associated with reduced vascular wall thickening as well as

less Treg and M2 macrophage accumulation in the maturing AVF. These results suggest that PD-L1

induces Treg cells to promote M2 macrophage accumulation during AVF maturation.

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702


Knowledge Strategy:

This research describes a novel, TRPC6-dependent pathway that regulates vascular smooth muscle cells

phenotype. The results of this study may lead to mechanism-based therapies to reduce phenotypic

switching, and thus mitigate neointimal hyperplasia, following vascular intervention.


The durability of interventions for peripheral and coronary arterial occlusive disease is currently limited.

Neointimal hyperplasia is the major cause of early restenosis and failure of vascular bypass grafting,

angioplasty, and stenting. Strategies that combat neointimal hyperplasia therefore have the potential to

improve outcomes of vascular intervention.

TRPC6 Depletion Results in Loss of Myocardin And Phenotypic Modulation In Vascular Smooth Muscle

Cells

Andrew H. Smith, Priya Putta, Erin Driscoll, Pinaki Chaudhuri, Cleveland Clinic, Cleveland, OH; Michael

Rosenbaum, Louis Stokes Cleveland VAMC, Cleveland, OH; Linda M. Graham, Cleveland Clinic, Cleveland,

OH

Objective:The contractile phenotype of vascular smooth muscle cells (VSMC) is regulated by expression

of the master transcription factor, myocardin. Loss of myocardin is observed during VSMC phenotypic

switching and within the hyperplastic neointima after vascular intervention. We have shown that

depletion of the nonvoltage gated calcium-permeable channel, canonical transient receptor potential 6

(TRPC6) channel, promotes VSMC phenotypic modulation and more severe carotid stenosis following

wire injury. The goal of this study was to determine if TRPC6 regulates VSMC phenotype by altering

expression of myocardin.

Methods: Myocardin expression was assessed in primary aortic wild type (WT) and TRPC6-/-VSMC as well

as common carotid artery (CCA) explants from WT and TRPC6-/-mice. Acute TRPC6 knockdown was

performed in immortalized mouse VSMC (MOVAS cells) using siRNA. Functional alteration of VSMC was

assessed in vitro using a wound healing assay.

Results: Myocardin mRNA levels were significantly reduced in primary aortic TRPC6-/-VSMC compared to

primary aortic WT VSMC (mean 5.53x107fold reduction, n=4, p<0.001). Myocardin protein was also

reduced in TRPC6-/-primary aortic VSMC compared to WT, though not to the same degree (mean 2.03

fold reduction, n=3, p=0.001). Myocardin protein was significantly decreased in whole tissue explants of

TRPC6-/- CCA vs WT CCA (55.5% reduction, n=4 mice per genotype, p=0.007), demonstrating that

significant differences in myocardin expression was present in vivo. In MOVAS cells, acute siRNA-

mediated TRPC6 knockdown induced a 44.7% decrease in myocardin protein (n=3, p=0.044). Wound

healing assays showed a significant increase in the number of migrating TRPC6-/-VSMC compared WT

VSMC in response to growth factor stimulation, supporting our previous findings that TRPC6-/-VSMC are

modulated from a contractile to a proliferative phenotype.

Conclusions: TRPC6 depletion is associated with decreased myocardin and the emergence of pathogenic

VSMC behavior. TRPC6-dependent signaling may therefore be a therapeutic target to promote

myocardin expression and stabilize the VSMC contractile phenotype after arterial injury.

707


Knowledge Strategy:

Periadventitial delivery of simvastatin from microparticles attenuates arteriovenous fistula outflow vein

neointimal hyperplasia. It provides a new option to prevent arteriovenous fistula failure.


In the present study, there was no assembled marker on simvastatin or microparticles, which could

illustrate migration or residual dose in the outflow veins.

Periadventitial Delivery of Simvastatin From Microparticles Attenuates Arteriovenous Fistula Outflow

Vein Neointimal Hyperplasia

Chenglei Zhao, Mayo Clinic, Rochester, MN; Sean T Zuckerman, Affinity Therapeutics, LLC, Cleveland,

OH; Chuanqi Cai, Michael L Simeon, Avishek Singh, Sreenivasulu Kilari, Mayo Clinic, Rochester, MN;

Julius N Korley, Affinity Therapeutics, LLC, Cleveland, OH; Sanjay Misra, MAYO CLINIC, Rochester, MN

Background: Venous neointimal hyperplasia (VNH) is vexing problem to maintain arteriovenous fistula

(AVF) patency in end-stage renal disease patients. The available drug delivery systems to prevent VNH

formation are limited. VNH is characterized with increased expression of transforming growth factor- β1

(TGF- β1), vascular endothelial growth factor-A (VEGF-A) and monocyte chemoattractant protein-1

(MCP-1). Hypothesis: Periadventitial delivery of microparticles coated with simvastatin (MP-SIM) could

prevent VNH formation via inhibition of gene expression of TGF- β1, VEGF-A and MCP-1 in a murine AVF

model with chronic kidney disease (CKD). Methods: At day -28, eight-week C57BL/6J male mice were

randomly grouped into control group (MP alone) or MP-SIM group and nephrectomy was used to induce

CKD. At day 0 AVF was created. A volume of 20uL of PBS with 16.6mg/ml of either MP or MP-SIM was

applied to the periadventitia of the proximal AVF outflow vein at the time of AVF creation. Fistula

patency was assessed weekly using Doppler ultrasound. Mice were euthanized at day 3 and 28 for gene

expression and immunohistochemistry staining respectively. Results: At day 3, the gene expression of

TGF- β1, VEGF-A and MCP-1 was significantly decreased in MP-SIM group. At day 28, there was a

significant increase in peak systolic velocity and decrease in the average neointimal area and cell density

in MP-SIM group. At day 28, as assessed using Immunohistochemistry staining, there was a significant

increase in apoptosis and a decrease in the smooth muscle cell, fibroblasts, macrophages, fibrosis and

cellular proliferation in MP-SIM group.Conclusion: Our study indicates that periadventitial delivery of

MP-SIM attenuates VNH 4 weeks after AVF creation. Further studies using a porcine animal model to

confirm these findings are recommended. The potential clinical applicability of controlled-release

simvastatin to decrease expression of TGF-β1, VEGF-A and MCP-1 while increasing apoptosis and

decreasing cellular proliferation is encouraging.

832


Knowledge Strategy:

Results from our studies will identify molecular mechanisms in the process of underpinning novel

therapeutic targets for the devastating consequences of treating atherosclerotic disease plaque

vulnerability and rupture by studying bioactive lipid dysregulation.


Our studies aim to understand the molecular mechanisms of atherosclerotic plaque vulnerability and

rupture that continue to cause life-threatening complications affecting millions of people around the

world. Our studies aim to understand atherosclerosis disease development via the abrogation of the

inflammatory process by altered activation of the bioactive lipid machinery.

Absence Of Cpla2 In Lrp1 Smooth Muscle Cell Deficient Mice Promotes Severe Aortic Atherosclerotic

Disease

Roberto Ivan Mota Alvidrez, Shirling TSAI, Joachim Herz, Univ of Texas Southwestern Medical Ctr,

Dallas, TX

Smooth muscle cell targeted deficiency of LDL Receptor related protein 1 (LRP1) in a mouse model

(smLRP1-/-) results in accelerated aortic atherosclerosis through activation of cytoplasmic phospholipase

A2 (cPLA2), leading to reduced ABCA1 expression in vascular smooth muscle cells (VSMCs), and

increased intracellular cholesterol accumulation. We therefore hypothesized that deficiency of cPLA2

would impede atherogenesis in the smLRP1-/- mouse model. Methods: Adult male smLRP1-/-;cPLA2-/-

;LDLR-/- (Triple knockout) mice were placed on a high cholesterol diet (HCD) for 16 weeks and compared

to age- and diet- matched sibling control smLRP1+/+;cPLA2-/-;LDLR-/- mice. Histologic analysis was

performed using en face whole aorta Oil red O (ORO) staining, as well as cross-sectional analysis of the

aortic root with ORO, Picro Sirius Red, Alizarin Red and immunofluorescence. Immunoblot protein

analysis was performed using lysed whole aortas. Data is represented as mean±SEM. Statistical analysis

was performed using one and two way ANOVA with Tukey’s correction. Results: En face ORO analysis

revealed increased lipid accumulation in Triple Knockout mice as compared to controls (60+/-3% vs

13+/-2%, p<0.001) (Figure 1). Uniquely, Triple knockout mice develop extensive necrotic cores and thin

fibrous caps in atherosclerotic lesions in the aortic root (Figure 1). ABCA1 is paradoxically increased both

in whole aorta lysate as well as in immunofluorescence staining of the aortic

root. Conclusions: Deficiency of cPLA2 in the smLRP1-/- mouse model rescued ABCA1 expression, but

unexpectedly increased lipid accumulation within the plaque and generated more vulnerable plaque.

Future studies will underpin the mechanisms that guide severe disease development in our Triple

knockout mice via regulation of ABCA1. Funding: NHLBI R37 HL063762 and NINDS/NIA R01NS108115.

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841


Knowledge Strategy:

This study showed that circulating fatty acid synthase (cFAS) is independently associated with CLTI but

not DM. Future studies may explore using cFAS as biomarker for severity of PAD to guide clinical

management, or as a target for development of medical therapeutics.


This study evaluates the potential association between serum biomarkers and clinical severity in

patients with diabetes and peripheral artery disease.

Serum Circulating Fatty Acid Synthase As A Diagnostic Biomarker For Chronic Limb-Threatening

Ischemia

Shirli Tay, Amanda Penrose, Gayan S De Silva, Yan Yan, Washington Univ Sch of Med, St. Louis, MO; Clay

F Semenkovich, WASHINGTON UNIV SCH OF MED, Saint Louis, MO; Mohamed A Zayed, WASHINGTON

UNIVERSITY SCHOOL M, Saint Louis, MO

Objectives: Circulating fatty acid synthase (cFAS), a de novo lipogenesis enzyme, is elevated in the serum

of patients with DM and carotid artery stenosis. However, it is unknown whether cFAS is similarly

elevated in patients with DM and advanced PAD. This study aims to evaluate whether cFAS content and

enzyme activity are biomarkers for clinical severity in patients with DM and CLTI. Methods: Serum

samples were prospectively collected from patients undergoing arterial revascularization procedures

and maintained in an IRB-approved institutional biobank. cFAS content and enzyme activity were

evaluated using colorimetric ELISA assays. Multivariable logistic regression was used to evaluate DM and

CLTI outcomes while adjusting for patient clinical characteristics. Hosmer-Lemeshow tests and C-index

assessed goodness-of-fit and classification accuracy. All tests were two-sided and P<0.05 was considered

significant. Results: A total of 86 patients underwent cFAS analysis (67 content; 63 activity). Mean age

was 65 (±8.5) with 67.4% male, 46.5% DM and 47.7% CLTI. Bivariable analyses demonstrated association

of CLTI with cFAS content (P<.01), DM (P=.01) and insulin use (P=.01); DM was associated with BMI

(P=.001), CKD (P=.001) and current smokers (P<.05). On multivariable analysis, CLTI was associated with

cFAS content (OR 1.16 [1.02-1.32] P=.02) and closely with DM (OR 2.72 [0.94-7.89] P=.066); while DM

was associated with BMI (OR 1.13 [1.05-1.23] P<.01) and CKD (OR 4.86 [1.72-13.73] P<.01). No

interactions were observed to be significant. Both models showed good fit (P>.15) and classification.

(Fig. 1) Conclusions: Serum cFAS content is associated with an increased risk of CLTI but not DM. Each

unit increase in cFAS content increases the odds of CLTI by 16%. Future analysis with a larger sample and

statistical bootstrapping will determine whether cFAS content is a clinically-relevant biomarker of CLTI

severity in patients with advanced atherosclerosis.

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840


Knowledge Strategy: N/A

Professional Practice Gap: N/A

ZEB2 Regulates Activation And Exhaustion Programming Of CD8+ T Cells In Atherosclerosis

Dawn Fernandez, Nicolas F Fernandez, adeeb Rahman, Christopher Hill, Mount Sinai, New York, NY;

Roza Shamailova, Icahn Sch of Med, New York, NY; Seunghee Kim-schulze, J Mocco, Peter Faries, Miriam

Merad, Chiara Giannarelli, Mount Sinai, New York, NY

T cells are among the most prevalent immune cells found in human atherosclerotic lesions, yet their role

remains obscure. In previous single-cell immuno-phenotyping studies we found that CD8+ T cells of

carotid atherosclerotic plaques display a spectrum of functionally heterogeneous states that vary based

on differentiation, activation, and exhaustion. Furthermore, CD8+ T cell profiles varied between patients

without (Asymptomatic, ASYM) and with (Symptomatic, SYM) recent cardiovascular (CV) events (i.e.

transient ischemic attack and stroke), suggesting that T cells might contribute to adverse outcomes. The

transcriptional regulator ZEB2 has dual roles in T cell differentiation and cardiovascular disease, as

genome wide association studies have reported ZEB2 polymorphisms as independent risk alleles for

coronary artery disease and myocardial infarction. Our independent analysis identified ZEB2 as a key

driver of CD8+ T cell alterations in atherosclerotic lesions. We found that ZEB2 was differentially

regulated between patient types, with ASYM patients expressing higher ZEB2 and GZMB levels

compared to SYM (Fig.1A). ZEB2high CD8+ T cells upregulated genes involved in cytotoxic functions, and

conversely ZEB2low CD8+ T cells upregulated PD-1 Signaling in T cell exhaustion (Fig.1B.). To probe the

mechanistic implications of ZEB2, we performed in vitro chronic stimulation assays of human primary

CD8+ T cells using depleted of ZEB2 using CRISPR/CAS9. ZEB2 knockout cells had reduced cytotoxic

function and had a dampened activation state upon acute stimulation. Persistent stimulation induce

exhaustion in these cells showed that ZEB2 knockout increased PD-1 expression levels, a protein marker

that is critical for T cell exhaustion (Fig.1C). Collectively these experiments suggest that ZEB2 may

contribute to the regulation of T cell activation and exhaustion states which is different between clinical

phenotypes.

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693


Knowledge Strategy:

Mast cell phenotype and secreting mediators could be detected in the plasma of patients with carotid

atherosclerotic plaque and predict the clinical outcome of the patient.


The aim of this project is to associate mast cell activation with particular processes in human

atherosclerotic plaques and patient clinical data in a mean of associating the mast cell phenotype with

patient clinical outcome.

The Role Of Mast Cells In Atherosclerotic Plaque Calcification

Nikolaos-Taxiarchis Skenteris, Malin Kronqvist, Mariette Lengquist, Karolinska Inst, Solna, Sweden; Ulf

Hedin, Karolinska Inst, Stockholm, Sweden; Erik L Biessen, Maastricht Univ Medical Ctr, Maastricht,

Netherlands; Ljubica Matic, Karolinska Inst, Stockholm, Sweden

Background: Vascular calcification is a key feature of atherosclerosis and has been associated with

major adverse cardiovascular events. Unstable carotid atherosclerotic plaques cause stroke and lesions

from those patients are abundant with activated mast cells at the sites of rupture. Recent data from our

group showed a statistically significant upregulation of activated mast cells in low calcified whereas

resting mast cells were upregulated in high calcified plaques, indicating that mast cells fractions may

associate with various aspects of plaque pathology. Hypothesis: Our hypothesis is that mast cell

fractions associate with key features of plaque vulnerability such as calcification, intraplaque

hemorrhage and other immune cell fractions. Resources: Biobank of Karolinska Endarterectomies (BiKE)

prospectively enrolls patients (n=1300) treated for carotid atherosclerosis in Stockholm, comprising

BioBank with paraffin-embedded plaque tissues for histology, ImageBank with quantified diagnostic CT

images using VascuCap software and DataBank of 100 clinical variables as well as transcriptomics and

proteomics large-scale datasets. Results: Histological stainings of plaque tissue microarrays confirmed

the presence of mast cells in atheromatous lesions and revealed that mast cells were systematically

found in Perls+ regions. The average total number of mast cells per mm2 per patient correlated

negatively with the calcification content. In addition, immunohistochemical analysis demonstrated that

mast cells correlate positively with CD3+ cells while they did not correlate with markers of other

immune cells. By stratifying the results according to patient symptoms, we found that activated mast

cells were elevated in both symptomatic and asymptomatic patients and increased with severe

symptoms of plaque instability. However, patients’ medication does not impact mast cell

regulation. Conclusions: Systematic enumeration of mast cell fractions in human plaques indicates that

activated mast cells associate with increased vulnerability, both when it comes to clinical patient

symptoms and morphological plaque features.

792


Knowledge Strategy:

Through the use of low cost genotyping, we are able to identify a subset of the population at higher risk

for AAA and who may benefit from screening beyond the current USPSTF recommendations.


Currently, AAA screening guidelines as presented by the USPSTF recommend a single ultrasound

screening for AAA in male individuals over the age of 65. These recommendations miss a large portion of

the population at risk for AAA.

Polygenic Risk Score Identifies Patients At Increased Risk For Abdominal Aortic Aneurysm And May

Benefit From Ultrasound Screening

Derek Klarin, Univ of Florida Coll of Med, Gainesville, FL; Ozan Dikilitas, Mayo Clinic, Rochester, MN;

Brooke Wolford, Univeristy of Michigan, Ann Arbor, MI; Michael Levin, Univ of Pennsylvania Sch of Med,

Philadelphia, PA; Ishan Paranjpe, Mount Sinai Sch of Med, New York, NY; Renae Judy, Univ of

Pennsylvania Sch of Med, Philadelphia, PA; Julie Lynch, Dept of Veterans Affairs, Salt Lake City Health

Care System, Salt Lake City, UT; Themistocles L Assimes, Stanford Univ Sch of Med, Palo Alto, CA; Yan

Sun, Emory Univ, Atlanta, GA; Daniel Rader, Univ of Pennsylvania Sch of Med, Philadelphia, PA; Peter W

Wilson, Emory Univ Sch of Med, Atlanta, GA; Salvatore Scali, Scott Berceli, Univ of Florida Coll of Med,

Gainesville, FL; Sekar Kathiresan, Verve Therapeutics, Cambridge, MA; Pradeep Natarajan,

Massachusetts General Hosp, Boston, MA; Girish Nadkarni, Mount Sinai Sch of Med, New York, NY;

Cristen Willer, Univ of Michigan, Ann Arbor, MI; Iftikhar Kullo, Mayo Clinic, Rochester, MN; Scott M

Damrauer, Universtiy of Pennsylvania Sch of Med, Philadelphia, PA; Philip Tsao, Stanford Univ Sch of

Med, Palo Alto, CA

Introduction: Abdominal aortic aneurysm (AAA) is a significant heritable cause of cardiovascular-related

mortality, yet published GWAS have only identified 10 genome-wide significant (P<5x10-8) risk loci to

date. In addition, current AAA screening recommendations remain limited to men age 65-75 with a

history of smoking. Hypothesis: Genetic variants affecting multiple biological pathways are associated

with AAA risk and may help identify asymptomatic individuals at higher risk for disease. Methods: Using

EHR data, we identified individuals with and without clinical AAA in Million Veteran Program (MVP)

participants. Individuals were genotyped on a customized Affymetrix array, and we tested 18 million

genotyped and imputed DNA variants for association with AAA using logistic regression models adjusting

for age, sex and population structure. We then performed replication in external datasets and set a

P<5x10-8 for statistical significance. In downstream analyses, we tested and validated a series of AAA

polygenic risk scores (PRS) and assessed the associated AAA risk per standard deviation (SD) increase in

PRS using prevalent data from an independent set of MVP participants (1,656 AAA cases; 44,908

controls). We set P < 0.05 for statistical significance. Results: We identified 7,642 AAA cases and 172,172

controls. Following replication, we identified 14 novel AAA loci implicating known risk factors including

lipids (LPA, PCSK9) and smoking (CHRNA3). We generated a 29 variant PRS and observed that a 1 SD

increase in the AAA PRS was associated with a 32% increased risk of AAA (OR = 1.32, PPRS =1.7x10-34).

Men in MVP with the 5% highest PRS and over 50 years of age had a disease prevalence of 7.8%

(142/1680), higher than that observed in AAA screening trials informing current guidelines. Conclusions:

Here, we identify novel AAA genetic associations with therapeutic implications and identify a subset of

the population at significantly increased risk of AAA. Our data suggest that extending current screening

guidelines to include testing for those with high polygenic AAA risk would significantly increase the yield

of current screening.

755


Knowledge Strategy:

The results of this study allow us to better understand vascular inflammation driving AAA formation and

potential intervene medically.


There is a lack of proven medical therapy to prevent AAA growth. This abstract demonstrates that

macrophage epigenetic modifications regulate inflammation in AAAs and highlights a new mechanism to

target

Epigenetic Modifications Influence Macrophage-mediated Inflammation Inabdominal Aortic

Aneurysms

Frank Michael Davis, Aaron Dendekker, Amrita Joshi, Sonya Wolf, Christopher Audu, William J Melvin,

UNIVERSITY OF MICHIGAN, Ann Arbor, MI; Hong Lu, UNIV KENTUCKY, Lexington, KY; Alan Daugherty,

UNIVERSITY OF KENTUCKY, Lexington, KY; Johann Gudjonsson, UNIVERSITY OF MICHIGAN, Ann Arbor,

MI; Katherine A Gallagher, UNIVERSITY MICHIGAN, Ann Arbor, MI

Introduction: Abdominal aortic aneurysms (AAA) are characterized by inflammatory macrophage (Mφ)

infiltration and pathological vascular remodeling. The mechanisms regulating Mφ polarization during

AAA development remain unknown. There is increasing evidence that epigenetic enzymes, specifically

the histone demethylase JMJD3, direct Mφ polarization. The purpose of this study was to investigate if

JMJD3-mediated epigenetic modifications regulate Mφ phenotype and hence inflammation during AAA

formation.

Methods: Single-cell sequencing was conducted on human AAA and control tissue samples. For our

murine model, male C57BL/6 mice (n=60) were intraperitoneally injected with an AAV encoding a mouse

PCSK9 gain-of-function mutation (D377Y). Mice were then fed a saturated fat-enriched diet and infused

with AngII (1 µg/kg/min) or saline for 4 weeks. AAA maximum diameters were quantified and Mφs

(CD11b+,CD3-,CD19-,Ly6G-) were sorted. Jmjd3 and NFκB mediated inflammatory gene expression was

examined. Chromatin immunoprecipitation (ChIP) was used to evaluate histone 3 lysine 27

trimethylation (H3K27me3). Statistical significance was determined using Student’s t-test or ANOVA.

Results: Single-cell transcriptome analysis of human AAAs demonstrated a significant upregulation

of JMJD3 and inflammatory cytokines in myeloid subsets compared to controls. Congruently, Mφs from

AngII-induced AAAs displayed increased Jmjd3 and NFκB-mediated cytokine expression (i.e. IL-1β, IL-12)

(p<0.05). Histone methylation was evaluated by ChIP in AngII-induced AAA Mφs and demonstrated

decreased levels of the Jmjd3-mediated repressive histone methylation mark, H3K27me3, on

inflammatory gene promoters. In vitro inhibition of JMJD3 using siRNA significantly reduced Mφ-

mediated inflammatory cytokine expression (p<0.05). Finally, pharmacological inhibition of JMJD3 (GSK-

J4) in the AngII-induced murine model resulted in a significant reduction in AngII-induced AAA formation

and inflammatory cytokines.

Conclusions: These results suggest an important role for JMJD3 in regulating Mφ-mediated

inflammation in human and murine AAAs as well as identify a potential target for the treatment of

chronic inflammation.

759


Knowledge Strategy:

Use of a dual chamber stent to improve organ recovery and reduce ischemic injury.


Perfusion of abdominal organs during organ recovery.

The Perfuse Dual Chamber Stent Improves Donor Organ Recovery In A Porcine Model

Bryan W. Tillman, Catherine Go, Brian Frenz, UNIV OF PITTSBURGH MED CTR, Pittsburgh, PA; Moataz

Elsisy, UNIV OF PITTSBURGH, Pittsburgh, PA; Youngjae Chun, UNIVERSITY OF PITTSBURGH, Pittsburgh,

PA

Background: Amidst a critical shortage of organs for transplantation, ischemic injury from malperfusion

during the agonal period remains a prohibitive barrier. We hypothesized that a dual chamber stent graft

could isolate visceral perfusion from the agonal systemic circulation, while respecting the ethics of organ

donation. Methods: A retrievable dual chamber stent graft was welded from nitinol and covered with

polymer. A central lumen maintained aortic flow, with an outer visceral chamber perfused by an

oxygenator. Anesthetized pigs were assigned to either control (n=7) or the dual chamber stent (n=6). A

one hour agonal phase of hypoxia (saturations < 60-70%) and hypotension (MAP < 25) was simulated

both medically and with partial balloon occlusion. The Perfuse stent visceral flow totaled 500 ml/min

during the agonal phase followed by stent recapture and resuscitation to an endpoint of 2

days. Results: Study groups had comparable agonal O2 saturations, HR and MAP. Cardiac output and

right ventricular end diastolic volume did not change during stent graft deployment. Compared to the

low pO2 of controls (48 mm Hg) and systemic stent animals (49 mm Hg), the visceral pO2 averaged 413

mm Hg and visceral flow was significantly higher in stent animals. 5/7 controls were euthanized from

acute renal failure and volume overload while all stent animals survived without renal impairment.

Transaminases were between 1.8 to 3 fold elevated in control as compared to stented

animals. Conclusions: During a simulation of the agonal period, a dual chamber stent provided

endovascular separation and marked improvement in perfusion and organ outcome. This was

accomplished without significant impact on cardiac function, respecting current ethical considerations

of the DCD donor. The ability to separate the perfusion of the abdominal organs from the agonal

systemic circulation without the need for open surgery might significantly improve the availability of

donor organs for transplant.

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795


Knowledge Strategy:

Trauma TEVAR is associated with LV mass increase, development of hypertension, and off-target

accelerated expansile ascending aortic remodeling. Our findings call for better follow-up of young

patients receiving stent-grafts and for improved endovascular devices capable of preserving optimal

ventricular-arterial coupling.


Thoracic endovascular aortic repair (TEVAR) is first-line therapy for blunt thoracic aortic trauma, but its

effects on the left ventricle (LV) and off-target aortic zones are poorly understood. We have measured

changes in LV mass, LV mass index, and diameters and lengths of the ascending thoracic aorta in trauma

TEVAR patients, and compared these patients with similarly-aged control subjects without aortic repair

evaluated at similar follow-ups.

Endovascular Repair Of Blunt Thoracic Aortic Trauma Is Associated With Increased Left Ventricular

Mass, Hypertension, And Off-target Aortic Remodeling

Alexey Kamenskiy, Univ of Nebraska Omaha, Omaha, NE; Paul Aylward, Univ of Nebraska Medical Cent,

Omaha, NE; Anastasia Desyatova, Univ of Nebraska Omaha, Omaha, NE; Matthew DeVries, Christopher

Wichman, Univ of Nebraska Medical Ctr, Omaha, NE; Jason N Mactaggart, Univ of Nebraska Medical

Cent, Omaha, NE

Introduction: Aortic elasticity creates a cushion that protects the heart from pressure injury, and a recoil

that helps perfuse the coronary arteries. Thoracic endovascular aortic repair (TEVAR) has become first-

line therapy for many aortic pathologies including trauma, but stent-grafts stiffen the aorta and likely

increase left ventricular (LV) afterload. We hypothesized that trauma TEVAR is associated with LV mass

increase and adverse off-target aortic remodeling.

Methods: CTA scans of 20 trauma TEVAR patients (17M/3F) at baseline (age 34.9±18.5[11.4-71.5] years)

and 5.1±3.1[1.1-12.3] years after repair were used to measure changes in LV mass, LV mass index

(LVMi), and diameters and lengths of the ascending thoracic aorta (ATA). Measurements were

compared with similarly-aged control patients without aortic repair (21M/21F) evaluated at similar

follow-ups.

Results: LV mass and LVMi of TEVAR patients increased from 138.5±39.6g and 72.35±15.17g/m2 to

173.5±50.1g and 85.48±18.34g/m2 at the rate of 10.03±12.79g/year and 6.25±10.28g/m2/year, while in

control patients LV characteristics did not change (Figure). ATA diameters of TEVAR patients increased at

a rate of 0.60±0.80mm/year, which was 2.4-fold faster than in controls. ATA length in both TEVAR and

control patients increased at 0.58mm/year. Half of TEVAR patients had hypertension at follow-up

compared to only 5% at baseline.

Conclusions: TEVAR is associated with LV mass increase, development of hypertension, and accelerated

expansile remodeling of the ascending aorta. While younger trauma patients may adapt to these effects,

these changes may be even more important in older patients with other aortic pathologies and

diminished baseline cardiac function.

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809


Knowledge Strategy:

There currently exists no successful medical management of abdominal aortic aneurysms. This research

helps to identify potential therapeutic targets and mechanisms in the treatment of this disease.


This research addresses potential medical therapies in the treatment of AAA as currently operative

intervention remains the only definitive treatment.

Maresin 1 Attenuates Murine Abdominal Aortic Aneurysms Via Vascular Smooth Muscle Cell

Dependent TGF-β Signaling

Craig T Elder, Univ of Florida, Gainesville, FL; Gang Su, Gainesville, FL; Amanda Filiberto, Univ of Florida

Coll of Me, GAINESVILLE, FL; Guanyi Lu, Univ of Florida Coll of Me, GAINESVILLE, FL, Gainesville, FL;

Zhihua Jiang, UNIVERSITY OF FLORIDA COM, Gainesville, FL; Ashish Sharma, UNIVERSITY OF FLORIDA,

Gainesville, FL; Gilbert R UPCHURCH Jr., Univ of FL Dept of Surgery, Gainesville, FL

Introduction: The endogenous pro-resolving lipid mediator Maresin 1 (MaR1) is derived from the ω-3

polyunsaturated fatty acid docosahexanoic acid and is involved in the resolution phase of inflammation.

Specifically, MaR1 has been shown to attenuate inflammatory signaling in smooth muscle cells. It was

hypothesized that exogenous administration of MaR1 would attenuate AAA growth via smooth muscle

cell dependent TGF-β signaling.

Methods: AAAs were induced in C57BL/6 wild-type mice (n=9 elastase + MaR1 [4ng/g] or n=10 elastase

+ vehicle) using an established topical elastase AAA model. Mice were treated with MaR1 or vehicle via

IP injection on days 7, 9, 11, and 13 post AAA induction. Abdominal aortas were harvested on day 14 for

phenotypic evaluation of aortic diameter. Histological analysis of smooth muscle actin (n=3-4/group)

and western blot of TGF-β1 expression (n=6-7/group) were performed. Additionally, AAAs were induced

in smooth muscle cell specific TGF-β2 receptor knockout mice and treated with MaR1 (n=7) vs vehicle

(n=10) and harvested on day 14 for phenotypic evaluation of aortic diameter. Groups were analyzed

using one-way ANOVA with post hoc Tukey’s test and data presented as mean ± SEM.

Results: MaR1 treatment significantly attenuated AAA growth compared to vehicle (121.4% ± 9.4 vs.

165.3% ± 9.4; p<0.01). A significant increase in aortic wall smooth muscle cell actin was identified in

MaR1 treated mice compared to vehicle (27.1% ± 3.4 vs 14.2% ± 2.4; p=0.03). TGF-β1 expression was

also significantly higher in MaR1 treated mice compared to vehicle (2.95 x106 ± 11.1 x105 vs 1.63 x106 ±

2.6 x105 densitometry units; p=0.02). Finally, smooth muscle cell specific TGF-β2 receptor knockout mice

showed no difference in AAA diameter in MaR1 treated mice compared to vehicle (133.9% ± 8.0 vs

123.8 % ± 7.9; p=0.4).

Conclusion: These results demonstrate that MaR1, important in the resolution of inflammation,

attenuates murine AAA growth, preserves aortic wall smooth muscle cell actin, and increases TGF-β1

expression. This attenuation in AAA growth is lost in smooth muscle cell specific TGF-β2 receptor

knockout mice further suggesting that MaR1 modulates TGF-β signaling in aortic smooth muscle cells.

787


Knowledge Strategy:

Develop new diagnostic and treatment modalities for AAA


Experimental AAA models are subacute.

A Theranostic Role For Ccr2 In Rodent Abdominal Aortic Aneurysm Development And Rupture

Sergio Sastriques Dunlop, Washington Univ Sch Med, Saint Louis, MO; Batool Arif, Sean English,

Washington Univ Sch Med, SAINT LOUIS, MO

Introduction: Abdominal aortic aneurysm (AAA) is common in the aging population, and its rupture

carries a high mortality risk. AAA is characterized by mononuclear phagocyte destructive aortic extra

cellular matrix remodeling. Monocyte chemoattractant protein (MCP-1)/C-C chemokine receptor type 2

(CCR2) axis plays an important role in AAA development. We sought to evaluate if increased murine

AAA 64Cu-DOTA-ECL1i uptake, a radiolabeled CCR2 binding peptide, by positron emission tomography

(PET), is predictive of rupture, as well as if CCR2 inhibition prevents rupture.

Methods: Sprague-Dawley rats and C57BL/6 (wild type, WT) mice underwent intraluminal aortic

exposure to porcine pancreatic elastase (PPE). Four groups were considered: 1. Active PPE (AAA), 2.

Heat-inactivated PPE (Sham), 3. APPE + β-aminopropionitrile (BAPN, a lysyl oxidase inhibitor to stimulate

rupture) (RAAA), and 4. APPE + BAPN + RS-504393 (CCR2 inhibitor, CCR2i). WT and CCR2-/- mice were

also exposed to Angiotensin II. 64Cu-DOTA-ECL1i PET imaging was performed at 7 and 14 days post AAA

induction.

Results: RATS: AAA demonstrated significantly greater 64Cu-DOTA-ECL1i uptake by PET and

autoradiaography compared to Sham (P<0.05). RAAA that subsequently ruptured demonstrated

significantly greater uptake compared to those animals that did not rupture (P<0.001). RAAA and CCR2i

rupture rates were 56% and 0%, with mean aortic diameter % increases of 353 ± 43% and 171 ± 26 %,

respectively (P<0.0001). CCR2i PET uptake decreased by 60%. MICE: WT, CCR2i and CCR2 -/- rupture

rates were 53, 33 and 6%, with mean aortic diameter % increases of 330 ± 62, 191 ± 67 and 101 ± 16%,

respectively (P<0.01). Quantification of tracer uptake by PET was significantly greater in AAA compared

to Sham and CCR2-/- (p<0.0001).

Conclusion: CCR2 targeted PET imaging demonstrated inflammation associated with rodent AAA

development. Increased radiotracer uptake by AAA that subsequently ruptured may aid in assessing

AAA rupture potential. CCR2 inhibition with our model prevents AAA rupture and inhibits AAA

associated inflammation, suggesting a theranostic role for CCR2 in the management of AAA patients.

762


Knowledge Strategy:

This study demonstrated that BTK inhibitor ibrutinib, FDA-approved drug, attenuated the formation of,

and further progression of established, experimental AAAs in two mechanistically distinct but

complementary AAA model systems. Our results suggest that pharmacological inhibition of BTK may

represent a novel translational strategy for AAA disease suppression.


Macrophages are critical for abdominal aortic aneurysm (AAA) pathogenesis. Bruton’s tyrosine kinase

(BTK) contributes to macrophage-driven diseases s. It is not known whether BTK plays a role in AAA

disease. Additionally, currently there is no approved drugs for treating aortic aneurysm disease.

Pharmacological Inhibition Of Bruton's Tyrosine Kinase Attenuates Experimental Abdominal Aortic

Aneurysms

Baohui Xu, STANFORD UNIVERSITY SCHOOL OF MEDIC, Stanford, CA; yankui li, Stanford Univ Sch of

Med, Stanford, CA; Gang Li, STANFORD UNIVERSITY SCHOOL OF MEDIC, Stanford, CA; Yingbin Ge,

Nanjing Medical Univ, Nanjing, China; Jia Guo, Stanford Univ Sch of Med, Stanford, CA; Weirong Fang,

China Pharmaceutic Univ, Nanjing, China; Wei Wang, Xiangya Hosp, Central South Univ, Chnagsha,

China; Fanru Shen, STANFORD UNIVERSITY SCHOOL OF MEDIC, Stanford, CA; Takahiro Shoji, Vascular

Surgery, Stanford, Stanford, CA; Toru Ikezoe, Xiaoya Zheng, STANFORD UNIVERSITY SCHOOL OF MEDIC,

Stanford, CA; Sihai Zhao, Stanford Univ Sch of Med, Stanford, CA; Xiaofeng Chen, TAIZHOU HOSPITAL,

Taizhou; Masaaki MIYATA, KAGOSHIMA CITY HOSPITAL, Kagoshima, Japan; Alan Daugherty, UNIVERSITY

OF KENTUCKY, Lexington, KY; Hong Lu, Univ of Kentuky Sch of Med, Lexington, KY; Ronald L DALMAN,

STANFORD UNIVERSITY MEDICAL CE, Stanford, CA

Objective: Macrophages are critical for abdominal aortic aneurysm (AAA) pathogenesis. Bruton’s

tyrosine kinase (BTK) contributes to macrophage driven diseases such as atherosclerosis and ischemic

stroke by modulating proinflammatory macrophage activation. It is not known whether BTK plays a role

in AAA disease. This study was to investigate the influence of the FDA-approved BTK inhibitor ibrutinib

on the formation and progression of experimental AAAs. Methods: AAAs were generated via intra-aortic

elastase infusion in male normolipidemic C57BL/6J mice or subcutaneous angiotensin II infusion in male

hyperlipidemic C57BL/6J mice (inoculated an adeno-associated virus expressing a gain-of-functional

mutation of PCSK9 and fed high fat diet). Ibrutinib administration (30 mg/kg) was initiated one day prior

to, or indicated days after, AAA creation. Influence on AAAs was evaluated via ultrasonography and

histology. The effect of ibrutinib on macrophage mediator mRNAs was assessed via quantitative RT-PCR

assay. Results: By ultrasonography, ibrutinib pretreatment remarkably attenuated elastase infusion-

induced aortic enlargement, in association with delayed onset and reduced incidence of AAAs, as

compared to vehicle treatment. In established AAA mice, ibrutinib treatment limited further aneurysmal

enlargement. On histology, ibrutinib treatment preserved medial elastin and smooth muscle cell

cellularity and reduced mural macrophage, lymphocyte and neocapillary density. Ibrutinib

administration also reduced the incidence and severity of angiotensin II-induced AAAs in hyperlipidemic

mice. Additionally, BTK treatment attenuated CCL2 and TNF-alpha mRNA expression, but accentuated IL-

10 mRNA, in classically or alternatively activated macrophages. Conclusion: BTK mediates AAA

progression in two complementary experimental AAA models. Pharmacological inhibition of BTK may

represent a novel translational strategy for AAA disease suppression.

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694




Landscape Of Human Secretome In Abdominal Aortic Aneurysm Profiled By Single-cell RNA

Sequencing.

Bhama Ramkhelawon, NYU MEDICAL CENTER, New York, NY; Tarik Hadi, NYU Langone Medical Ctr,

New York, NY; Michele Silvestro, NYU Sch of Med, New York, NY

Introduction: Abdominal aortic aneurysms (AAA) is a lethal vascular disease upon aortic rupture.

Evidence suggests pathological cellular crosstalks that fuel extracellular matrix (ECM) degradation in

AAA. However, the comprehensive ligand-receptor intra-aortic signaling networks that persists in

human AAA is undefined. Hypothesis: Heterogeneous cell-cell communication via specific ligand-

receptor signaling contributes to human AAA.

Methods: Human AAA, control organ donor, mouse control and AAA modeled by angiotensin II infusion,

samples were processed to for single cell RNA Sequencing (scRNA seq) libraries. Reads were aligned to

reference genome and analyzed for significant differential expression profiles. Pathway analysis were

used to outline biologically relevant networks. Human secretome of ligand-receptor interactions were

applied to human and mouse scRNA seq libraries. Results: Analysis of human scRNA seq showed 16 cell

clusters (9 immune and 7 vascular) expressing 923 significantly different genes in AAA. Secretome

analysis revealed a highly connected network of 262 transcripts enriched in ECM remodeling,

metabolites, leukocyte migration, Wnt signaling and cytokines. Comparison between mouse and human

showed that only macrophages displayed similar gene profile to that of human AAA. Amongst the 2005

increased genes in murine macrophages, about 10% was common in human, 20 of which were in the

secretome analysis. We observed an emergence of a novel population of IL7 expressing B cells only in

human AAA. CD4 and CD8, but not naïve B cells and dendritic cells expressed

elevated IL7 receptor suggesting active recruitment and differentiation of lymphoid progenitors of T cell

lineage in AAA sac. Further studies are needed to delineate the role of IL7+ B cells and its crosstalk with

T cells in AAA. Circulating IL7 in serum might be of reliable predictive value to predict AAA. Conclusion:

We uncovered novel insights in the landscape of intercellular crosstalk highlighting the similarity

between murine and human transmural macrophages and the distinct role of lymphoid signaling in

human AAA. These candidates that directly stem from AAA could be used as valuable biomarkers much

needed to predict clinical AAA.

788


Knowledge Strategy:

This research represents a significant step in overcoming prior major limitations for patient-specific

induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) regarding longevity and function in

culture that have been a barrier for clinical applications. These findings open the door for future

investigations into clinical applications for iPSC-ECs such as therapeutic injections for critical limb

ischemia, or for engineering patient-specific bypass conduits for peripheral arterial disease.


To date, clinical applications for patient-specific stem cell-derived endothelial cells have been limited by

poor longevity in culture and loss of mature cellular phenotype and function due to premature

senescence.

Autophagy Remodels Mitochondria During Differentiation And Enhances Longevity Through Ulk1

Kinase Signaling Of Induced Pluripotent Stem Cell-derived Endothelial Cells

Katherine E Hekman, Kyle Koss, David Z Ivancic, Northwestern Univ, Chicago, IL; Congcong He, Chicago,

IL; Jason A Wertheim, Northwestern Univ, Chicago, IL

Background Stem cells hold great promise for the future of peripheral arterial disease therapy, and

patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic

modality. However, ECs derived from patient-specific induced pluripotent stem cells (iPSCs) suffer from

premature replicative senescence with rapid loss of mature cellular function in culture currently limiting

clinical applications. In the present study we characterize autophagy during differentiation and

maintenance of iPSC-ECs as a target to modulate longevity.

Methods and Results Autophagy, measured by autophagosome number in TEM samples and by

quantitation of the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3),

demonstrates multiple significant increases during differentiation suggesting an active role in

remodeling. Mitotracker staining shows increasing mitochondrial membrane potential and TEM

demonstrates that maturation continues through serial passages; however, these stagnate, coinciding

with decreased cellular proliferation as measured with Edu labeling. Plant-based small molecule Rg2

significantly improved proliferative capacity of iPSC-ECs over multiple passages (Figure 1). This benefit

was not observed in cells treated with either rapamycin or the autophagy inducer ML246, or with

mitophagy inducers Antimycin A or oligomycin. The benefit of Rg2 was blocked by treatment with ULK1

kinase inhibitor SBI-0206965 in a dose-dependent manner.

Conclusion In summary, stimulating mTOR-independent ULK1-mediated autophagy with Rg2 improved

the longevity of iPSC-ECs in culture. This represents a novel approach to overcome a major barrier to the

use of iPSC-ECs for clinical applications.

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828


Knowledge Strategy:

We attempt to increase stem cell's inherent therapeutic potential with a novel vector to increase cell's

homing capabilities and overall potency.


Patients with critical limb ischemia often have progressed on current medical therapies and have either

failure surgical interventions or cannot undergo a surgical procedure. These no-option patients currently

have no other option other than amputation, our pre-clinical study attempts to discovery new

therapeutics for CLI patients in hopes of future limb salvage

Downregulation Of Inflammation And A Cascade Of Pro-angiogenic Signals Mediate The Beneficial

Effects Of Gene-modified Stem Cell Therapy In Hindlimb Ischemia

Hallie J Quiroz, Univ of Miami, Miami, FL; Hongwei Shao, Yan Li, Samantha F Valencia, Punam Parikh,

Univ of Miami, Miami, FL; Yulexi Ortiz, Univ of Miami Miller Sch of Med, Miami, FL; Roberta Lassance-

Soares, Univ of Miami, Miami, FL; Zhao-Jun Liu, Univ of Miami Miller, Miami, FL; Omaida Caridad

Velazquez, Univ of Miami Miller Sch of Med, Miami, FL

Introduction: Novel therapies to afford limb salvage in patients with imminent amputation due to

critical limb ischemia are needed. We have previously shown E-selectin, a cell adhesion molecule, as an

essential participant in neovascularization. Thus, we hypothesized E-selectin supercharged

mesenchymal stem cells (E-selectin+/MSC) would augment limb reperfusion, tissue regeneration, and

functionality.

Methods: C57Bl6 mice underwent femoral artery ligation and received either vehicle (PBS, n=9) or

syngeneic donor MSCs, transduced ex vivo to express either GFP+ (control, n=20) or E-selectin-

GFP+ (treatment, n=18). Laser doppler imaging (LDI), confocal laser microscopy, and treadmill

exhaustion test were utilized to determine neovascularization and limb function. Extent of ischemic

skeletal muscle atrophy (mean myocyte size,µm²) was assessed via leg muscle histology. RT2 Profiler PCR

Array analysis of 84 genes involved in angiogenesis assessed therapeutic mechanism of action. Student’s

t-test or ANOVA was utilized to compare means and significance set at p<0.05.

Results: Compared with GFP+/MSC and PBS, treatment with E-selectin-GFP+/MSC increased ischemic leg

LDI reperfusion (55% vs. 39% vs. 24%, p<0.001), ischemic mouse footpad vessel density (23% vs. 14% vs.

14%, p<0.01) and treadmill distance traversed (162m vs. 111m vs. 110m, p<0.01). The ischemic limb in

mice treated with E-selectin-GFP+/MSC were less atrophic than controls (793µm² vs. 556µm² vs.

546µm², p<0.001). Seven pro-angiogenic genes were upregulated in E-selectin-GFP+/MSC treated

ischemic leg tissue while tumor necrosis factor (TNF) was downregulated, when compared with

GFP+/MSC treated tissues.

Conclusion: This innovative E-selectin supercharged stem cell therapy confers increased limb

reperfusion, function, and decreased atrophy, likely via upregulation of pro-angiogenic cytokines and

downregulated TNF.

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717




Prediction Of Walking Performance After Revascularization Of Arteries Supplying The Lower

Extremities Of Claudicating Patients With Peripheral Artery Disease

Shuai Li, Jonathan Robert Thompson, Univ of Nebraska Med Ctr, Omaha, NE; Sara A Myers, Univ of

Nebraska at Omaha, Omaha, NE; Julian K Kim, UNIV NEBRASKA MEDICAL CENTER, Omaha, NE;

Panagiotis Koutakis, Florida State Univ, Tallahassee, FL; Mark Williams, Creighton Cardiac Ctr, Omaha,

NE; Zhen Zhu, Univ of Nebraska Med Ctr, Omaha, NE; Molly Schieber, Univ of Nebraska Medical Cent,

Omaha, NE; Timothy Lackner, Gregory Willcockson, Christina Shields, Katyarina Brunette, Holly

Despiegelaere, Univ of Nebraska Med Ctr, Omaha, NE; Iraklis I Pipinos, UNIV NEBRASKA MEDICAL CTR,

Omaha, NE; George Casale, Univ of Nebraska Medical Cent, Omaha, NE

Rationale and Objectives: Peripheral Artery Disease (PAD) affects over 200 million patients globally.

Revascularization is a mainline treatment for PAD, however, change in walking performance and

Quality-of-Life (QoL) varies substantially among patients, ranging from no to marked improvement. We

evaluated biochemical and histopathological measurements of calf muscle and leg hemodynamics,

before revascularization, as potential predictors of change in walking performance and QoL, in response

to revascularization.

Methods and Results: We measured, at baseline and 6-months post-revascularization, Ankle-Brachial

Index (ABI), Systolic Pedal Pressure at 15 seconds after Post-occlusive Reactive Hyperemia (SPP15sec),

Six-Minute Walking Distance (SMWD), Initial Claudication Time (ICT), Peak Walking Time (PWT) and

Quality of Life (QoL) survey scores of forty claudicating PAD patients. In addition, we determined a)

activities of mitochondrial Citrate Synthase (CS), Complexes 1-4 and MnSOD, b) myofiber morphology

and oxidative damage (carbonyl adducts), and c) fibrosis, in calf muscle. Prediction of walking

performance and QoL was evaluated by Spearman Rho and ROC curve. Baseline SPP15sec and change in

SMWD were most strongly correlated (Rs = -0.72 at P < 0.0001). A ROC curve identified patients whose

SMWD improved > 20 m at a baseline SPP15sec cutoff of 50 mmHg, with sensitivity of 89% and

specificity of 100%. In addition, we detected significant improvements (P < 0.05) of hemodynamics (ABI,

SPP15sec), walking performance (SMWD, ICT and PWT), QoL survey scores, and calf muscle pathology

(CS, Complex I and II and MnSOD activities, myofiber shape and carbonyl adducts).

Conclusions: Our study shows that revascularization improves hemodynamics, walking performance,

QoL and calf muscle pathology in claudicating PAD patients. Baseline SPP15sec (cutoff = 50 mmHg)

strongly predicts which patients will experience > 20 m improvement of SMWD.

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695


Knowledge Strategy:

We may opt to treat patients with claudication more aggressively


Resting perfusion to muscle in PAD may cause long term effects beyond schema and may alter how

disease should be managed

Differential Expression Of Canonical Mitosis And Dna-damage Repair Pathways Characterize Muscle

Satellite Cells Affected By Pad

Ricardo Ferrari, Guangzhi Cong, Bryan Thompson, Debbie Hollingshead, Janette Lamb, Xiangdong Cui,

Ulka Sachdev, UPMC, Pittsburgh, PA

Introduction: Peripheral arterial disease (PAD) is associated with diminished muscle strength, even in

claudicants with adequate resting perfusion. Muscle Satellite Cells (MuSC) regeneration may be

deterred by PAD. We propose that MuSC in PAD have transcriptomic differences causing ischemia-

induced myopathy. Methods: Anterior tibialissegments were harvested during amputations for critical

limb ischemia from viable (proximal; P) and ischemic (distal; D) muscle. Pressure indices documented

perfusion. MuSC were isolated by antibody-mediated magnetic cell sorting. Control myocytes were

purchased. Both were measured for myocyte fusion index (MFI; 1&5d). TruSeq stranded mRNA kits

facilitated library creation for next-generation sequencing. Ingenuity Pathway Analysis (IPA) was

performed on differentially expressed genes with a false discovery rate p-value of <0.05 for pathway

enrichment. Results: MFI in PAD (N=8, P and D) MuSC was half that of controls (p<0.03; N=4, ANOVA).

Pressure indices confirmed a perfusion gradient along the limb. The # of differentially expressed genes

among PAD and controls are shown (Fig.1). MuSC in PAD vs. controls had significant differences in

canonical pathway expression including mitotic roles of polo-like kinase (PLK; z score = 7.5 distal, 13.5

proximal), and G2/M DNA damage checkpoint regulation (z score = 6.5 distal, 9.0 proximal). Genetic

expression did not differ within PAD muscle as a function of perfusion. Conclusion: Compared to

controls, MuSC in PAD have significant differences in gene expression even in better perfused muscle.

Pathways affected included those essential to mitosis and DNA-damage repair, both critical to cell

survival and differentiation. These findings suggest perfusion-independent PAD effects on muscle that

persist despite optimization. Adjunctive treatments beyond reperfusion may be required to mitigate

muscle damage from arterial insufficiency.

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781


Knowledge Strategy:

Through nanotechnology created sustained release of growth factors or statin drugs in the stimulation

of angiogenesis.


Potential intervention in the treatment of critical limb ischemia.

Fluvastatin And Vegf Containing Resins Promote Angiogenesis And Arteriogenesis

Furqan Muqri, SUNY Upstate Medical Ctr & Syracuse VA Medical Ctr, Syracuse, NY; Dandan Guo, SUNY

Upstate Medical Univ, Syracuse, NY; Mohammed Kassem, Mary DaCosta, SUNY Upstate Medical Ctr &

Syracuse VA Medical Ctr, Syracuse, NY; David Bruch, SUNY Upstate Medical Univ, Syracuse, NY;

Kristopher Maier, SUNY Upstate Medical Ctr & Syracuse VA Medical Ctr, Syracuse, NY; Juntao Luo, SUNY

Upstate Medical Ctr, Syracuse, NY; Vivian Gahtan, SUNY Upstate Medical Ctr & Syracuse VA Medical Ctr,

Syracuse, NY

Select patients with critical limb ischemia who are not candidates for surgical revascularization

rely on angiogenesis to ameliorate symptoms. Statin drugs and vascular endothelial growth factor

(VEGF) stimulate angiogenesis. Hypothesis: resins used for local statin delivery to the ischemic

bed would increase angiogenesis and be augmented by dual delivery with VEGF.

Functionalized hydrogel resins were loaded with fluvastatin or VEGF-165A. Matrigel or

proliferation assays were performed on endothelial cells (ECs) treated with free drug, resin, or

resin bound with drugs. Free or bound resins, or a combination of both resins were injected into

male rat hindlimbs after femoral artery ligation. Doppler flow was measured pre and postoperative

day 1, 3, 7 and 14 and compared to the contralateral uninjured leg. On day 14, the ischemic

vastus and gastrocnemius muscles were stained with alpha smooth muscle actin (arteriogenesis)

and CD34 (angiogenesis). In vitro findings: statin release rate from resin was 10% per 24 hrs;

unloaded resin no EC toxicity and tubule formation was unaffected; both free and resin statin or

VEGF promoted tubule formation (p<0.05); tubule formation was the same for free statin and resin

statin; resin VEGF or VEGF plus resin statin had less tubule formation compared to its respective

free form (p<0.05); free and resin statin and VEGF equally increased EC proliferation. Doppler

blood flow studies (day 14) showed: untreated control and empty resin had an 80% blood flow

recovery; resin statin returned flow to preoperative levels (p<0.001); the combination group

increased blood flow more than statin alone (p<0.001); and resin VEGF had the greatest effect

increasing flow above preoperative levels (p<0.05). Resin statin, resin VEGF or both increased

angiogenesis and arteriogenesis (p<0.05); however, the empty resin had no effect. In conclusion,

in vitro, free fluvastatin and free VEGF were more effective than the respective resin, likely due

to slow release of the drugs from the resin. Both angiogenesis and arteriogenesis improved with

VEGF or fluvastatin bound resins. Resin VEGF alone was strongest in vivo. Single dosing of

resin VEGF effectively promoted angiogenesis, which may make this revascularization method

more feasible clinically.

726


Knowledge Strategy:

Understanding of the potential role of intraluminal thrombus in aortic aneurysm development, growth,

and rupture.


Understanding mechanisms that lead to aortic aneursym expansion and rupture.

Increased Neutrophil Elastase Expression in Regions of High Intraluminal Thrombus Deposition In

Human Abdominal Aortic Aneurysms.

Matthew Levesque, Lauren Bath, Max Rady Coll of Med, Winnipeg, MB, Canada; Annie Ducas, Royal

Victoria Regional Health Ctr, Barrie, ON, Canada; April Boyd, Health Sciences Ctr, Winnipeg, MB, Canada

INTRODUCTION

Abdominal aortic aneurysms (AAA) have previously been shown to rupture at sites of high intraluminal

thrombus (ILT) deposition. Neutrophil and macrophage activity have also been associated with AAA

development, but their precise roles are not known. The purpose of this study was to evaluate the

possible differential expression of macrophage chemoattractant protein 1 (MCP1), macrophage-derived

protease (CD68), neutrophil chemoattractant factor (IL8), and neutrophil elastase (MMP8) in regions of

human AAA with high and low ILT deposition.

HYPOTHESIS

We hypothesized that higher ILT deposition would be associated with increased MCP1, CD68, IL8, and

MMP8 levels compared with low ILT deposition and control aorta.

METHODS

Full thickness AAA samples were collected from 15 participants using a systematic map. For each case,

samples were taken from regions with low or high ILT. Infrarenal aortic control tissue was harvested

from 6 participants undergoing aortobifemoral bypass. A cytokine array assay was performed to

measure MMP-8, IL8, and MCP-1. CD68 immunohistochemistry was also performed.

RESULTS

AAA tissue had significantly higher levels of IL8 (145 vs 64 pg/mg protein, p=0.044), MMP8 (42566 vs

3740 pg/mg protein, p=0.015), MCP1 (473 vs 64 pg/mg protein, p=0.003), and CD68 (1.77% vs 0.246%,

p<0.001) compared to control tissue. In AAA patients, the presence of ILT was associated with

significantly higher levels of IL8 (185 vs 99 pg/mg protein, p=0.035), but not MMP8, MCP1, or CD68.

CONCLUSIONS

Human AAA tissue is associated with higher macrophage and neutrophil activity compared to control

tissue as measured by MMP8, IL8, MCP1, and CD68. Within AAA tissue, areas of high ILT deposition were

associated with higher IL8 levels, suggesting that there is increased neutrophil recruitment in high ILT

regions.

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773


Knowledge Strategy:

Our result indicated adult FMD is genetically different with pediatric FMD, suggesting a separate clinical

practice for both adult and pediatric FMD.


We are working on the genetic difference between adult and pediatric fibromuscular dysplasia (FMD),

filling the gap of understanding the mechanism of adult FMD.

Adult and Pediatric Fibromuscular Dysplasia Are Genetically Distinct Dysplasia-associated Arterial

Diseases

Yu Wang, Dawn M. Coleman, Univ of Michigan Medical Sch, Ann Arbor, MI; Min-Lee Yang, Issaquah,

WA; Susan Blackburn, Issaquah, WA, Ann Arbor, MI; Kristina L. Hunker, Univ of Michigan Medical Sch,

Ann Arbor, MI; Heather L Gornik, Univ Hosp Cleveland, Cleveland, OH; Jun Li, Univ of Michigan, Ann

Arbor, MI; James C Stanley, 2210 Taubman Ctr, Ann Arbor, MI; Santhi Ganesh, 2210 Taubman Ctr, Ann

Arbor, MI, Ann Arbor, MI

Introduction: Fibromuscular dysplasia (FMD) is an obstructive, non-atherosclerotic arterial disease with

an estimated population prevalence of 3-5% in adults. FMD occurs primarily in women (> 90%), and its

genetic basis is poorly defined. Pediatric FMD is rare and may represent a more severe FMD phenotype,

with earlier onset hypothesized to be due to high impact deleterious genetic variants. Differences

between adult and pediatric forms of FMD have been defined for angiographic pattern and sex

distribution (with pediatric FMD afflicting males and females equally). Monogenic genes underlying

adult FMD have not yet been defined. Syndromic associations with pediatric FMD are known; for

example genetic variants predicted to cause loss-of-function of the NF1 gene have been identified in up

to 33% of pediatric FMD series. The impact of these known genes have not been systematically

evaluated in adult FMD.

Methods: We performed a whole exome sequencing of 264 unrelated adults with multifocal FMD and

annotated genetic variants in genes described in pediatric renovascular hypertension and FMD: NF1,

JAG1, RNF213, ELN, and GATA6. Pathogenicity criteria included: 1) low population frequency (gnomAD

MAF <0.01%); 2) CADD score >20. Statistical tests of enrichment of variants in FMD cases as compared

to gnomAD were performed using a Fisher's exact test.

Results and Conclusion: For all genes tested, we did not observe any significant enrichment (P>0.05).

Specifically, for the NF1 gene, we observed no loss-of-function variants, and only 3 carriers of

nonsynonymous, predicted deleterious variants (1.1%) in 264 samples. These specific variants were

observed in gnomAD in 2001/251,486 (0.8%) samples. None of these individuals carried any diagnosis of

neurofibromatosis type 1 or associated clinical features. We identified no loss-of-function variants

in JAG1, RNF213, ELN, and GATA6, nor did we find any statistical enrichment of variants predicted to be

deleterious in these genes or NF1, as compared to gnomAD and our lab controls (N=284). In summary,

adult FMD patients lacked pathogenic variations in genes underlying ~30-45% pediatric arterial dysplasia

cases. These genetic findings support that adult and pediatric FMD are likely due to disparate arterial

pathologies.

746


Knowledge Strategy:

The results of this project may lead to the development of new therapies for the treatment of diabetic

wounds, which cause significant morbidity and mortality in a large proportion of the diabetic population.

These therapies will likely include topical nitrite and inhibitors of XOR. Additionally, our research is

providing mechanistic data on the causes of impaired diabetic wound healing and methods to target

these mechanisms.


Our lab has been researching the role of xanthine oxidoreductase (XOR) in diabetic wound healing.

While XOR is necessary for normal wound healing, it has been suggested that it causes excessive

oxidative stress in diabetic wounds and impairs diabetic wound healing. Our prior experiments with oral

and topical febuxostat, an XOR inhibitor, have failed to consistently inhibit XOR and demonstrate a clear

benefit for diabetic wound healing. With this research, we now demonstrate that inhibition of XOR with

dietary tungsten does accelerate diabetic wound healing. Additionally, despite inhibiting XOR, which

also functions as a nitrite reductase, topical nitrite has still been demonstrated to be effective in

accelerating wound healing. Our current results indicate that this may be secondary to changes effected

by nitrite on the wound microbiome.

Impact Of Dietary Tungsten And Topical Nitrite In Diabetic Wound Healing And The Composition Of

The Wound Microbiome

Kathy Gonzalez, Karim M Salem, UPMC, Pittsburgh, PA; Barbara Methé, Kelvin Li, Guiying Hong, Univ of

Pittsburgh, Pittsburgh, PA; Edith Tzeng, UNIVERSITY PITTSBURGH, Pittsburgh, PA

Background:

Impaired diabetic (DM) wound healing is attributed to increased reactive oxygen species (ROS) and

decreased nitric oxide (NO) production. Xanthine oxidoreductase (XOR) produces ROS and can convert

nitrite to NO. XOR is expressed in wounds and necessary for normal healing. In DM wounds, XOR may

contribute to excess ROS and poor healing. We have shown that topical nitrite accelerates DM wound

healing. We hypothesize that XOR inhibition will improve DM wound repair and that concurrent topical

nitrite would not be effective.

Methods:

Diabetic db/db mice were fed a tungsten (W)-enriched diet, to inhibit XOR, or standard chow for 2 wks

before excisional wounding. Wounds were treated with either topical Aquaphor or Aquaphor/2% nitrite

(nitrite). Wounds areas were quantified every other day for 28 days. Healing rates were compared with

ANOVA. Wound swabs were collected on Days 2 and 14 for microbiota analysis using 16S rRNA

sequencing and paired-difference analysis.

Results:

While topical nitrite or W diet improved DM wound healing compared to chow + Aquaphor, W + nitrite

mice healed significantly faster than these groups at all wound sizes (Figure). Microbiota composition

over time was compared between the same taxon in the same mouse. Greater differences between taxa

were found in the nitrite and W + nitrite-treated groups while differences decreased with W diet.

While Staphylococcus (Staph) was the predominant taxon in all groups, nitrite significantly

increased Staph over time in chow fed mice.

Conclusion:

XOR inhibition with W improved DM wound healing. Surprisingly, the combination of W + nitrite further

accelerated healing. This supports the benefit of XOR inhibition to reduce ROS and that nitrite

conversion to NO is supported by other nitrite reductases. W and nitrite both mediate wound

microbiome changes favoring Staph species which may contribute to improved wound repair. The

contribution of the microbiome to nitrite conversion to NO needs to be assessed.

https://files.abstractsonline.com/CTRL/BD/B/820/6CE/D29/454/2B1/916/246/5FB/431/D2/g339_1.gif



727


Knowledge Strategy:

The results from this project may highlight new biomarkers involved with PAD/CLI. Furthermore, RNA

based therapies can be developed from the findings of this project.


The mechanisms involved with the pathogenesis of peripheral artery diseases and critical limb ischemia.

MicroRNA-181b Regulates Critical Limb Ischemia In Diabetic Mice

Henry S Cheng, Brigham and Women's Hosp, Boston, MA; Daniel Perez-Cremades, BWH, Boston, MA;

Marc P Bonaca, CPC Clinical Res, Aurora, CO; Mark Feinberg, Brigham and Women's Hosp, Boston, MA

Despite current therapeutic strategies, human subjects with peripheral artery disease (PAD) suffer from

an increased risk of critical limb ischemia (CLI) and limb amputation. PAD and CLI are associated with

several risk factors, such as aging and diabetes. MicroRNAs are crucial regulators of gene expression

with important therapeutic potential. However, the role of microRNAs in diabetic CLI remains poorly

understood. Due to the high conservation of microRNAs across species, plasma microRNA sequencing

from both human patients with PAD and mouse models were used to identify new microRNAs that

regulate diabetic limb ischemia. Two methods were used to induce limb ischemia in obese diabetic

(db/db) mice: acute limb ischemia model - ligation of the femoral artery; and subacute limb ischemia

model - insertion of ameroid constrictors around the femoral artery to induce slow occlusion. Through

stratification of the microRNA sequencing screens in mouse and human diabetic CLI cohorts, we

identified miR-181b as a top differentially expressed miRNA capable of regulating angiogenesis in vitro.

Furthermore, miR-181b inhibitors can promote proliferation of cultured mouse vascular smooth muscle

cells (VSMC). Systemic miR-181a2b2 knockout (KO) mice demonstrate markedly improved blood flow

recovery (by 3.7-fold at 28 days) after limb ischemia compared to controls (P<0.002). Analysis of skeletal

muscle RNA expression revealed a 2-fold increase of VSMC markers Myosin heavy chain 11 (P<0.011)

and 1.65-fold increase of Myosin light polypeptide 6 (P<0.022) in systemic miR-181a2b2 KO mice. In

contrast, endothelial-specific miR-181a2b2 KO mice showed no difference in blood flow recovery.

Collectively, the stark increase of blood flow recovery and VSMC markers in systemic miR-181a2b2 KO

mice suggests enhancement of arteriogenesis in lieu of angiogenesis. Further investigation of miR-181b

in diabetic CLI may prove to be a key therapeutic target for promoting tissue perfusion in PAD.

817


Knowledge Strategy:

To inform the vascular surgeons that oxidative stress can disrupt the biopterin system impairing vascular

function.


The development of a blood biomarker that can be used for early detection of PAD.

Nitric Oxide Bioavailability Is Reduced In Peripheral Artery Disease In Association With Increased

Oxidative Stress And An Altered Biopterin System

Ahmed Ismaeel, Evlampia Papoutsi, Florida State Univ, Tallahassee, FL; William T Bohannon, Robert

Smith, Baylor Scott and White Heath, Temple, TX; Robert Brumberg, Vascular Surgery Associates,

Tallahassee, FL; Carlos H Castro, Jeffrey S Kirk, Capital Regional Medical Ctr, Tallahassee, FL; Iraklis I

Pipinos, UNIV NEBRASKA MEDICAL CTR, Omaha, NE; Panagiotis Koutakis, Florida State Univ, Tallahassee,

FL

Introduction: Peripheral artery disease (PAD) manifestations are the result of several pathophysiologic

mechanisms including endothelial dysfunction. This process is believed to be mediated by decreased

bioavailability of nitric oxide (NO), and oxidative stress may play a role in inducing endothelial

dysfunction by increasing oxidation of the NO synthase (NOS) co-factor tetrahydrobiopterin (BH4) to

dihydrobiopterin (BH2) as well as by inducing the expression of the endogenous NOS inhibitor

asymmetric dimethylarginine (ADMA). Hypothesis: We hypothesized that the regulators of NO synthesis

and bioavailability are compromised in PAD as a result of elevated oxidative stress. Methods: Blood was

collected from 35 PAD patients with intermittent claudication (IC), 26 patients with critical limb ischemia

(CLI), and 35 non-PAD controls, and serum was analyzed using specific assay kits to determine levels of

BH4, BH2, arginine, ADMA, nitrate/nitrite (NOx, used to quantify NO), and the oxidative stress markers

peroxynitrite, protein carbonyls, 8-OHdG, and 4-HNE. Results: IC and CLI patients demonstrated reduced

serum NOx levels compared to control (control: 84.89 ± 40.81, IC: 50.12 ± 17.48, CLI: 43.96 ± 19.47

μmol/L). Furthermore, BH4 was significantly reduced and BH2 significantly elevated in IC and CLI

compared to control, resulting in a lower BH4/BH2 ratio (control: 5.28 ± 6.71, IC: 1.08 ± 0.64, CLI: 1.09 ±

0.78). The arginine/ADMA ratio was significantly lower in CLI compared to both IC and control.

Furthermore, all the oxidative stress markers were significantly elevated in IC compared to control, with

further elevations in CLI. There was a significant inverse correlation between all the oxidative stress

markers and the BH4/BH2 ratio as well as NOx levels. Conclusions: The NO system is dysregulated in

PAD, which appears to be driven by elevated oxidative stress, worsening as the disease progresses from

IC to CLI.

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Vascular Research Initiatives Conference