May 4, 2016, Nashville, TN

Vascular Research Initiatives Conference

The 30th Annual Vascular Research Initiatives Conference (VRIC) is presented by the Society for Vascular Surgery (SVS).

This program is supported by an educational grant from Medtronic and Gore.

Outside-In: Paradigm Shifts in Vascular Disease

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May 4, 2016 I Omni Nashville Hotel I Nashville, TN Program Overview The 30th 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 is a one day conference preceding the main Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB) Scientific Sessions on May 5-7, 2016. Learning Objectives At the end of the following sessions, participants should be able to:

Session I: Stem Cells and Tissue Engineering 1. Discuss the current use of animal, cellular, and mathematical models elucidating the

pathophysiology of stem cells and tissue engineering. 2. Identify new areas of basic and methodological research in stem cells and tissue engineering. 3. Describe new venues of current clinical and translational research in stem cells and tissue

engineering. Session II: Vascular Endothelium and Thrombosis 1. Discuss the current use of animal models elucidating the pathophysiology of thrombosis in both

arterial and venous disease. 2. Identify new areas of basic and methodological research in thrombosis in both arterial and

venous disease. 3. Describe new venues of current clinical and translational research in thrombosis in both arterial and

venous disease. Session III: Aneurysms 1. Discuss the current use of animal models elucidating the pathophysiology of aneurysmal disease. 2. Identify new areas of basic and methodological research in aneurysmal disease. 3. Describe new venues of current clinical and translational research in aneurysmal disease. Session IV: Peripheral Arterial Disease 1. Discuss the current use of animal models elucidating the pathophysiology of peripheral arterial

disease. 2. Identify new areas of basic and methodological research in peripheral arterial disease. 3. Describe new venues of current clinical and translational research in peripheral arterial disease. Target Audience The Vascular Research Initiative Conference brings together vascular surgeons, vascular biologists, physicians with an interest in vascular problems, vascular surgery trainees, research trainees in vascular surgery and vascular biology, and industry personnel with an interest in vascular disease. Accreditation The Society for Vascular Surgery is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Designation of Credit The Society for Vascular Surgery designates this live activity for a maximum of 7 AMA PRA Category 1 Credits™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

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VASCULAR RESEARCH INITIATIVES CONFERENCE OUTSIDE-IN: PARADIGM SHIFTS IN VASCULAR DISEASE

WEDNESDAY, MAY 4, 2016

8:00AM - 5:25PM

*Draft Program Schedule, subject to change

7:00 AM REGISTRATION AND CONTINENTAL BREAKFAST

8:00 AM INTRODUCTORY REMARKS

John A. Curci MD, FACS – Chair, Research and Education Committee

Bruce A. Perler, MD – President, Society for Vascular Surgery

Alan Dardik, MD, FACS –Chair, SVS Research Council

ATVB Chair, Program Committee

ABSTRACT SESSION I: STEM CELLS AND TISSUE ENGINEERING

Moderator: Edith Tzeng, MD

Moderator: David A. Vorp, MD

8:15 am Angiogenesis is Triggered by Nutrient Deprivation via GCN2/ATF4-dependent

Regulation of VEGF and H2S Production Alban Longchamp1, Teodelinda Mirabella2, Christopher Hine1, Lear E. Brace1, Nelson Knudsen1, J. Humberto Treviño-Villarreal1, Pedro Mejia1, Ming Tao3, Gaurav Sharma3, Rui Wang4, Jean-Marc Corpataux5, Jacques-Antoine Haefliger5, Kyo Han Ahn6, Chih-Hao Lee1, Christopher Chen2, Charles Keith Ozaki3, James R. Mitchell1 1Harvard Sch of Public Health, Boston, MA; 2Boston Univ, Boston, MA; 3Brigham and Women’s Hospital, Boston, MA; 4Laurentian Univ, Sudbury, ON, Canada; 5Ctr Hospier Univire Vaudois, Lausanne, Switzerland; 6Ctr for Electro-Photo Behaviors in Advanced Molecular Systems, Nam-Gu, Korea

8:27 am Nitric Oxide Favors Sca-1+ Vascular Progenitor Cell Differentiation toward Endothelial Cells Adam J. Schuldt, Rommel C. Morales, Zheng Wang, Vera P. Shively, Melina R. Kibbe Northwestern Univ, Chicago, IL

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8:39 am Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies Cindy Huynh1, Ting-Yu Shih2, Amruta Samant1, Saif G. Pathan, David W. Nelson3, David J. Mooney2, Leena Pradhan-Nabzdyk1, Frank W. LoGerfo1 1Beth Israel Deaconess Medical Ctr, Harvard Medical Sch, Boston, MA; 2Harvard Univ, Cambridge, MA; 3BioSurfaces, Inc., Ashland, MA

8:51 am Epigenetically Altered TLR4 Expression May Contribute to Increased Inflammation and Impaired Wound Healing in a Murine Model of Diabetes Andrew Kimball, Amrita Joshi, Matthew Schaller, Beau Carson, Kanakadurga Singer, Jennifer Bermick, Shreyas Ramani, Peter Henke, Steven Kunkel, Katherine Gallagher Univ of Michigan, Ann Arbor, MI

9:03 am N-Acetylcysteine Enhances Amputation Stump Healing and Perfusion in Diabetes Mohamed A. Zayed1, Xiachao Wei2, Larisa Belaygorod3, Uzma Naim3, Kyoung-mi Park3, Clay F. Semenkovich3 1Washington Univ Sch of Med & St. Louis Veterans Affairs Health Care System, St. Louis, MO; 2Washington Univ Sch of Med, Div of Endocrinology, Metabolism, and Lipid Res, St. Louis, MO; 3Washington Univ Sch of Med, St. Louis, MO

9:15 am Wound Application of Nitrite Enhances Healing in Diabetic Mice Ankur Aggarwal1, Michael C. Madigan1, Ellen Cody2, Edith Tzeng1 1VA Pittsburgh Healthcare System and Univ of Pittsburgh, Pittsburgh, PA; 2Univ of Pittsburgh, Pittsburgh, PA

SPECIAL SESSION

9:30 am SVS FOUNDATION MENTORED CLINICAL SCIENTIST RESEARCH CAREER DEVELOPMENT AWARD (K08) - 2014 RECIPIENT

Luke P. Brewster, MD, PhD Emory University School of Medicine Project Title: Molecular Mechanism of Disturbed Flow in Arterial Stiffening

SVS FOUNDATION MENTORED PATIENT-ORIENTED RESEARCH CAREER DEVELOPMENT AWARD (K23) - 2014 RECIPIENT

Marlene Grenon, MD University of California, San Francisco Project Title: Effects of Fish Oil on Inflammation and Vascular Function in Claudicants

9:50 AM COFFEE BREAK

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ABSTRACT SESSION I I : VASCULAR ENDOTHELIUM AND THROMBOSIS

Moderator: Joseph D. Raffetto, MD

10:10 am P2X7R Antagonism Alleviates Traction Injury Induced Extracellular ATP Release in Vascular Tissues Weifeng Luo, Olukemi J McWilliam, Colleen Brophy, Joyce Cheung-Flynn Vanderbilt Univ Medical Ctr, Nashville, TN

10:22 am Periadventitial or Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia after Arterial Injury Alex Helkin1, David Bruch1, David R Wilson2, Rebecca Bader2, Kristopher G Maier1, Vivian Gahtan3 1SUNY Upstate Medical Ctr and Dept of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY; 2Syracuse Univ, Syracuse, NY; 3SUNY Upstate Medical Ctr, Dept of Veterans Affairs Healthcare Network Upstate New York at Syracuse, and Syracuse Biomaterials Inst, Syracuse, NY

10:34 am A Single Nucleotide Polymorphism of p27Kip1 Associated with Vein Graft Patency Regulates Expression of p27Kip1 in Both Venous Adventitial Cells and Smooth Muscle Cells but Selectively Regulates Proliferation of Adventitial Cells Richard D. Kenagy1, Shinsuke Kikuchi2, Lihua Chen1, Errol S. Wijelath3, Alexander W. Clowes1, Michael Sobel3 1Univ of Washington, Seattle, WA; 2Asahikawa Medical Univ, Asahikawa, Japan; 3VA Puget Sound Health Care System and Univ of Washingto, Seattle, WA

10:46 am Arteriovenous Fistula Adaptation Requires Caveolin1 Signaling Takuya Hashimoto, Trenton R. Foster, Hualong Bai, Haidi Hu, Jeans M. Santana, Jesse Hanisch, Alan Dardik Yale Sch of Med, New Haven, CT

10:58 am Platelet and Monocyte Activity in Carotid Artery Stenosis Treated With Carotid Endarterectomy Arvind Reddy Devanabanda, Caron Rockman, Nicole Allen, Maya Rubin, Binita Shah, Mark Adelman, Jeffrey Berger New York Univ, New York, NY

11:10 am Induction of miR-21 Increases Fibrous Cap Stability in Vulnerable Atherosclerotic Lesions Hong Jin1, Yuhuang Li1, Alexandra Bäcklund1 Albert Busch1, Suzanne M Eken1, Ekaterina Chernogubova1, Peter Gustafsson1, Ljubica Perisic1, Isabel Schellinger2, Uwe Raaz2, Philip S Tsao3, Göran K Hansson1, Gabrielle Paulsson- Berne1, Ulf Hedin1, Lars Maegdefessel1 1Karolinska Inst, Solna, Sweden; 2Georg-August-Univ Göttingen, Göttingen, Germany; 3Stanford Univ, Palo Alto, CA

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11:22 am Il-6 Mediates Vein Wall Response to Thrombosis in a Model Dependent Manner Andrea T. Obi, Andrew Kimball, Megan Elfline, Catherine Luke, Jose Diaz, Thomas W. Wakefield, Peter K. Henke Univ of Michigan, Ann Arbor, MI

11:34 am Anti-thrombin Perfluorocarbon Nanoparticles Decrease Clot Burden in a Murine Model of Venous Thrombosis Chandu Vemuri, Batool Arif, Susannah A. Grathwohl, John S. Allen, Peter K. Henke, Samuel A. Wickline Washington Univ in St. Louis, St. Louis, MO

NOON LUNCH

1:00 PM AWARDEE RECOGNITION AND REPORTS

Peter F. Lawrence, MD

TRANSLATIONAL PANEL:

MANIPULATING LARGE DATA SETS TO ANSWER BIG QUESTIONS

1:15 pm Moderator: Bruce A. Perler, MD

Moderator: Scott A. Berceli, MD, PhD

• Speaker: Matthew R. Spite, PhD: “Inflammation-Resolving Lipid Mediators and Vascular Disease”

• Speaker: J. Jeffrey Carr, MD, MSc

• Speaker: Scott A. Berceli, MD, PhD: “Systems Biology: Extracting a Signature from the Noise”

ABSTRACT SESSION I II : ANEURYSMS

Moderator: Luke P. Brewster, MD, PhD

Moderator: Peter F. Lawrence, MD

2:20 pm An XY Sex Chromosome Complement in Females Confers Susceptibility and

Rupture of Angiotensin II-induced Abdominal Aortic Aneurysms in Hypercholesterolemic Female Mice Yasir Alsiraj, Sean Thatcher, Eric Blalock, Kuey Chen, Richard Charnigo, Alan Daugherty, Lisa Cassis Univ of Kentucky, Lexington, KY

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2:32 pm Depletion of Plasmacytoid Dendritic Cells Inhibits Experimental Abdominal Aortic

Aneu Baohui Xu, Haojun Xuan, Naoki Fujimura, Sara A. Michie, Ronald L. Dalman Stanford Univ Sch of Med, Stanford, CA

2:44 pm Collagen type V Contributes to the Development of Aortic Dissections and

Aneurysms Noel M. Phan, Arick Park, Ting Zhou, David Scott, Qiwei Wang, Paul Tang, Daniel S. Greenspan, Bo Liu Univ of Wisconsin-Madison, Madison, WI

2:56 pm Number of Reentry Tears Influences Flap Motion and Flow Reversal in an In Vitro Model of Type B Aortic Dissection Joav Birjiniuk1, Mark Young2, Lucas H. Timmins1, Bradley G. Leshnower1, John N. Oshinski1, David N. Ku3, Ravi K. Veeraswamy1 1Emory Univ Sch of Med, Atlanta, GA; 2Medtronic, Inc., Santa Rosa, CA; 3Georgia Inst of Technology, Atlanta, GA

3:10 PM COFFEE BREAK

SPECIAL SESSION: DISTINGUISHED INVITED SPEAKER

3:30 pm VASCULAR RESPONSE TO INJURY: A NEW PARADIGM

Colleen M. Brophy, MD

ABSTRACT SESSION IV: PERIPHERAL ARTERIAL DISEASE

Moderator: Gale L. Tang, MD, FACS

Moderator: David G. Harrison, MD

4:00 pm Revascularization but not Supervised Exercise Therapy Prevents Progression of

Fibrosis in the Gastrocnemius of Patients with Peripheral Artery Disease while Improving Limb Function Duy Ha, George Casale, Alicia Luis, Kevin Harkins, Reagan Huber, Tanmayee Chengalasetty, Ruby Hickman, Mina Hanna, Stanley Swanson, Holly DeSpiegelaere, Iraklis Pipinos Univ of Nebraska Medical Ctr, Omaha, NE

4:12 pm Statins Have a Dose-dependent Effect on Amputation Risk and Survival in

Peripheral Arterial Disease Patients Shipra Arya1, Anjali Khakharia1, Zachary O. Binney1, Randall R Demartino2, Luke P. Brewster1, Philip P. Goodney3, Peter W. Wilson1 1Emory Univ, Atlanta, GA; 2Mayo Clinic, Rochester, MN; 3Geisel Sch of Med Dartmouth-Hitchcock, Lebanon, NH

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4:24 pm Proprotein Convertase Subtilisin/Kexin Type 6 is a Key Protease in the Control of

Smooth Muscle Cell Function in Vascular Disease Ulf Hedin1, Ljubica Perisic1, Urszula Rykaczewska1, Anton Razuvaev1, Mariette Lengquist1, Maria Sabater-Lleal1, Ida Eriksson1, Samuel Röhl1, Malin Kronqvist1, Chi-Nan Tseng1, Patricia Rodriguez1, Lasse Folkersen2, Lei Du1, Maria Gonzalez Diez1, Cecilia Osterholm1, Joy Roy1, Jaakko Patrakka1, Gabrielle Paulsson-Berne1, Göran Hansson1, Jacob Odeberg1, Anders Hamsten1, Per Eriksson1 1Karolinska Inst, Stockholm, Sweden; 2Technical Univ of Denmark, Kongens Lyngby, Denmark

4:36 pm Smooth Muscle Specific Knock-Out of the Zinc-Finger Protein 148 (ZFP148)

Attenuates Atherosclerotic Lesion Formation via Regulation of Apoptotic Signaling Pathways Morgan Salmon1, Anna Z. Fashandi1, Michael D. Spinosa1, Ashish K. Sharma1, Gary K. Owens1, Juanita L. Merchant2, Zendra E. Zehner3, Gilbert R. Upchurch Jr. 1, Gorav Ailawadi1 1Univ of Virginia Medical Ctr, Charlottesville, VA; 2Univ of Michigan at Ann Arbor, Ann Arbor, MI; 3Virginia Commonwealth Univ Medical Coll of Virginia Campus, Richmond, VA;

4:48 pm Vitamin D3-Induced Arterial Calcification Decreases Functional Recovery and

Limb Perfusion in a Murine Model of Hindlimb Ischemia Sara L Zettervall, Stephanie Monk, Xue-Lin Wang, Yujun Cai, Tonghui Lin, Raul J. Guzman Beth Israel Deaconess Medical Ctr, Boston, MA

5:00 pm The Myristolated Alanine-Rich C Kinase Substrate (MARCKS) Differentially

Regulates Proliferation of Vascular Smooth Muscle Cells and Endothelial Cells through Affecting Protein Stability and Proteolytic Processing of the Kinase Interacting with Stathmin (KIS) Thomas S Monahan1, Dan Yu1, Charles Drucker2, Rajabrata Sarkar2, Dudley K. Strickland2 1Baltimore VA Medical Ctr, Baltimore, MD; 2University of Maryland Sch of Med, Baltimore, MD;

5:15 PM RECEPTION AND POSTERS

7:00 PM VRIC CLOSE

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Course Director John A. Curci, MD, FACS Vanderbilt University Medical Center, Nashville, TN Faculty Scott A. Berceli, MD, PhD University of Florida, Gainesville, FL Luke P. Brewster, MD, PhD Emory University School of Medicine Colleen M. Brophy, MD Vanderbilt University Medical Center, Nashville, TN J. Jeffrey Carr, MD, MSc Vanderbilt University Medical Center, Nashville, TN Alan Dardik, MD, FACS Yale University, New Haven, CT Marlene Grenon, MD University of California, San Francisco, San Francisco, CA David G. Harrison, MD Vanderbilt University Medical Center, Nashville, TN Peter F. Lawrence, MD UCLA Health System, Los Angeles, CA Bruce A. Perler, MD John Hopkins Hospital, Baltimore, MD Joseph D. Raffetto, MD VA Boston Health Care System Vascular Surgery, West Roxbury, MA Matthew R. Spite, PhD Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Gale L. Tang, MD, FACS VA Puget Sound Health Care System, Seattle, WA Edith Tzeng, MD University of Pittsburgh, Pittsburgh, PA David A. Vorp, MD University of Pittsburgh, Pittsburgh, PA

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2016 SVS – Vascular Research Initiatives Conference

FINANCIAL DISCLOSURES As an accredited sponsor of the Accreditation Council for Continuing Medical Education (ACCME), SVS must ensure balance, independence, objectivity and scientific rigor in all its individually sponsored educational activities. In accordance with ACCME guidelines and standards, all faculty participating in an accredited activities must disclose to the audience any significant financial interest or other relationship with (1) the manufacturer(s) of any commercial product(s) and/or provider(s) of commercial services discussed in an educational presentation and (2) any commercial supporters of the activity. (Significant financial interest or other relationships can include such things as grants or research support, employment, consultant, major stockholder, member of speakers’ bureau, etc.) Listed below are the disclosures provided by the faculty for this meeting.

Faculty Role Financial Relationship

Company/Organization

Scott Berceli, MD, PhD Speaker None Luke P. Brewster, MD, PhD

Moderator, Speaker, Content Reviewer

None

Colleen M. Brophy, MD Speaker Recipient of Intellectual Property Rights/Patent Holder

Vein Harvest Kit, Polyplex

J. Jeffrey Carr, MD, MSc Speaker None John A. Curci, MD, FACS Course

Director, Planner, Moderator, Committee Reviewer

None

Marlene Grenon, MD Speaker None David G. Harrison, MD Moderator,

Content Reviewer

None

Peter F. Lawrence Moderator, Content Reviewer

None

Bruce A. Perler, MD Moderator, Content Reviewer

None

Joseph D. Raffetto, MD Moderator, Content Reviewer

None

Matthew R. Spite, PhD Speaker None Gale L. Tang, MD Moderator,

Content Reviewer

None

Edith Tzeng, MD Moderator, Content Reviewer

None

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David A. Vorp, MD Moderator, Content Reviewer

None

Research and Education Committee

Role Financial Relationship

Company/Organization

Luke P. Brewster, MD, PhD

Committee Reviewer, Moderator

None

John A. Curci, MD, FACS Course Director, Planner, Moderator, Committee Reviewer

None

Katherine A. Gallagher, MD

Committee Reviewer

None

Benjamin M. Jackson, MD, MS

Committee Reviewer

None

Joseph D. Raffetto, MD Moderator, Committee Reviewer

None

Michael P. Murphy, MD Committee Reviewer

None

Kathleen G. Raman, MD Committee Reviewer

None

Ulka Sachdev, MD Committee Reviewer

None

Gale L. Tang, MD Moderator, Committee Reviewer

None

Shirling Tsai, MD Committee Reviewer

None

Edith Tzeng, MD Committee Reviewer

None

Chandu Vemuri, MD Moderator, Committee Reviewer

None

The Education Council has oversight of all SVS programs for continuing education for practicing vascular surgeons. Education Council Financial

Relationship Company/Organization Daniel Clair, MD Consultant Endologix, Bard Advisory Board Medtronic, Boston Scientific Kellie Brown, MD None Rabih Chaer, MD None Ronald Dalman, MD None Clement Darling, MD Investigator W.L. Gore, Medtronic, Endologix, Harvest

Technologies, New England Research Institute Mark Davies, MD, PhD None Audra Duncan, MD None Matthew Eagleton, MD Consultant Cook Medical, Bolton Medical Linda Harris, MD

Investigator DCB

Vikram Kashyap, MD Research Grant National Institutes of Health/NIA Data and

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Safety Monitoring Board Ashraf Mansour, MD None Joseph Raffetto, MD Investigator BSN Jobst Ulka Sachdev, MD None Murray Shames, MD Consultant Medtronic, Cook, W.L.Gore Staff Role Financial

Relationship Company/Organization

Joanna Bronson CME Staff None

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Society for Vascular Surgery Research and Education Committee

2015-2016 Chair John A. Curci, MD, FACS Vanderbilt University Medical Center, Nashville, TN Members Luke P. Brewster, MD, PhD Emory University School of Medicine, Atlanta, GA Katherine A. Gallagher, MD University of Michigan, Ann Arbor, MI Benjamin M. Jackson, MD, MS University of Pennsylvania Health System, Pennsylvania, PA Michael P. Murphy, MD Indiana University School of Medicine, Indianapolis, IN Joseph D. Raffetto, MD VA Boston Health Care System Vascular Surgery, West Roxbury, MA Kathleen G. Raman, MD Washington University School of Medicine, St. Louis, MO Ulka Sachdev, MD University of Pittsburgh, Pittsburgh, PA Gale L. Tang, MD, FACS University of Washington School of Medicine, Seattle, WA Shirling Tsai, MD University of Texas Southwestern Medical Center, Dallas, TX Edith Tzeng, MD University of Pittsburgh, Pittsburgh, PA Chandu Vemuri, MD Washington University School of Medicine, St. Louis, MO

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Abstract Session I: Stem Cells and

Tissue Engineering

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8:15 am

Angiogenesis is Triggered by Nutrient Deprivation via Gcn2/atf4-dependent Regulation of Vegf and H2s Production

Alban Longchamp1, Teodelinda Mirabella2, Christopher Hine1, Lear E. Brace1, Nelson Knudsen1, J. Humberto Treviño-Villarreal1, Pedro Mejia1, Ming Tao3, Gaurav Sharma3, Rui Wang4, Jean-Marc Corpataux5, Jacques-Antoine Haefliger5, Kyo Han Ahn6, Chih-Hao Lee1, Christopher Chen2, Charles Keith Ozaki3, James R. Mitchell1 1Harvard Sch of Public Health, Boston, MA; 2Boston Univ, Boston, MA; 3Brigham and Women’s Hospital, Boston, MA; 4Laurentian Univ, Sudbury, ON, Canada; 5Ctr Hospier Univire Vaudois, Lausanne, Switzerland; 6Ctr for Electro-Photo Behaviors in Advanced Molecular Systems, Nam-Gu, Korea

Introduction: Angiogenesis is crucial to maintain tissue homeostasis under nutrient and oxygen deprivation (ischemia). Although considerable evidence supports that angiogenesis is regulated by hypoxia-HIF1α induction of vascular endothelial growth factor (VEGF), the role of nutrient deprivation in angiogenesis is poorly defined.

Approach and Results: We report that nutrient deprivation in the form of dietary sulfur amino acid restriction (Methionine/cysteine Restriction; MR) promotes VEGF expression and functional growth of new capillaries in skeletal muscle of mice (Fig.1 A, B). This occurred independently of hypoxia or HIF1α, but instead required the amino acid-sensing eIF2α kinase GCN2 and the transcription factor ATF4 (Fig. 1C). In addition to increased VEGF, nutrient deprivation boosted production of the pro-angiogenic gas hydrogen sulfide (H2S) via increased GCN2/ATF4-dependent expression of the H2S-generating enzyme cystathionine-gamma-lyase (CGL). The genetic requirement for CGL in angiogenesis triggered by nutrient deprivation, exercise or local VEGF overexpression, as well as the ability of local CGL overexpression to promote angiogenesis in vivo, revealed the critical importance of CGL-derived H2S in angiogenesis (Fig. 1D). Finally, plasma H2S was reduced in patients with vascular disease (versus non-diseased age-matched controls), and correlated with 2-year survival following vascular surgery (Fig.1 E, F). Conclusions: These data reveal a nutrient-sensing pathway targetable by diet as a previously unrecognized central regulator of VEGF expression and angiogenesis independent of canonical hypoxic signaling. This discovery points to novel dietary interventions and GCN2/ATF4/CGL/H2S-based strategies to manipulate angiogenesis.

Figure 1

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Author Disclosure Block: A. Longchamp: Research Grant; Significant; SNF P1LAP3_158895. T. Mirabella: None. C. Hine: None. L. Brace: None. N. Knudsen: None. J. Treviño-Villarreal: None. P. Mejia: None. M. Tao: None. G. Sharma: None. R. Wang: None. J. Corpataux: None. J. Haefliger: None. K. Ahn: None. C. Lee: None. C. Chen: None. C. Ozaki: None. J. Mitchell: Research Grant; Significant; NIH (AG036712, DK090629).

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8:27 am

Nitric Oxide Favors Sca-1+ Vascular Progenitor Cell Differentiation toward Endothelial Cells

Adam J. Schuldt, Rommel C. Morales, Zheng Wang, Vera P. Shively, Melina R. Kibbe Northwestern Univ, Chicago, IL Introduction: Treatment outcomes for severe atherosclerosis are limited by neointimal hyperplasia and restenosis. Nitric oxide (NO) is a potent inhibitor of neointimal hyperplasia, but its effects on vascular progenitor cells (VPC) in this process are unknown. Stem cell antigen-1-positive (Sca-1+) VPC can differentiate into both vascular smooth muscle cells (VSMC) and endothelial cells, which potentially contribute to or prevent the development of neointimal hyperplasia and restenosis, respectively. We hypothesize that NO favors differentiation of Sca-1+ VPC into endothelial cells rather than VSMC, thereby limiting the development of neointimal hyperplasia. Methods: Aortas from WT C57BL/6 male mice were digested and analyzed by FACS for Sca-1 and hematopoietic lineage (Lin) markers CD3e, CD11b, CD45, Ly-76, and Ly-6C/G. Aortic Sca-1+ cells were enriched by magnetic activated cell sorting and cultured at passage 2-3 for 72 hours in stem cell media or smooth muscle cell differentiation media containing PDGF-BB, with the NO donor DETA/NO (0-100 μM). CD31+ cells were assessed by FACS. Results: Sca-1+/Lin- cells comprised 9.0±1.6% of total aortic cells (n=4 replicates, 8 mice each). Aortic Sca-1+ cells in stem cell media showed an increase in CD31+ cells from 5.0±1.9% in controls to 6.8±2.3% and 11.2±1.7% with 50 and 100 μM DETA/NO, respectively (p<0.05). Aortic Sca-1+ cells in smooth muscle cell differentiation media showed an increase in CD31+ cells from 4.3±1.2% in controls to 5.0±1.3% and 7.2 ±1.9% with 50 and 100 μM DETA/NO, respectively (p<0.05).

Conclusion: The arterial wall contains Sca-1+ VPC with the ability to differentiate into multiple lineages. In support of our hypothesis, exposure to NO in vitro increased differentiation of Sca-1+ VPC to endothelial-like cells, even when the VSMC differentiation stimulus of PDGF-BB is present. This shift toward endothelial differentiation with NO may act to decrease neointimal hyperplasia in vivo.

Author Disclosure Block: A.J.T. Schuldt: None. R.C. Morales: None. Z. Wang: None. V.P. Shively: None. M.R. Kibbe: None.

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8:39 am

Development of a Hybrid Cryogel-coated Prosthetic Vascular Graft for Delivery of Targeted Gene Therapies

Cindy Huynh1, Ting-Yu Shih2, Amruta Samant1, Saif G. Pathan, David W. Nelson3, David J. Mooney2, Leena Pradhan-Nabzdyk1, Frank W. LoGerfo1 1Beth Israel Deaconess Medical Ctr, Harvard Medical Sch, Boston, MA; 2Harvard Univ, Cambridge, MA; 3BioSurfaces, Inc., Ashland, MA Introduction: Anastomotic neo-intimal hyperplasia (ANIH) remains a limiting factor in the long-term success of prosthetic vascular grafts. Understanding of the molecular mechanisms involved in the host response to prosthetic material has set the stage for using this material to deliver siRNA and other modulators of the ANIH-associated transcriptome response, along with antithrombotic agents. To create practical and effective drug delivery from a prosthetic material, we have combined a platform of polyethylene terephthalate (PET) fabric with an applied cryogel carrying the biologic agents, resulting in a bioactive prosthetic graft system (BPGS). Methods: Hybrid grafts were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun, knitted, or woven PET. Arg-Gly-Asp peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Human aortic endothelial cell (HAoEC) adherence and proliferation were assessed with a resazurin-based assay, and confocal microscopy was used to visualize cell interaction with the graft material. Heparin activity of the material in buffer solution was measured using an anti-Xa assay. Results: SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. HAoEC incorporated, adhered, and proliferated over 7 days for all materials (Figure 1). Anti-Xa assay confirmed continued activity of heparin from all grafts over 7 days. Conclusion: We have created a BPGS with a biocompatible cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity. This system allows application of the gel and biologic agents to PET prior to implantation, and provides flexibility in combining bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving strategies.

Author Disclosure Block: C. Huynh: None. T. Shih: None. A. Samant: None. S.G. Pathan: None. D.W. Nelson: None. D.J. Mooney: None. L. Pradhan-Nabzdyk: None. F.W. LoGerfo: None.

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8:51 am

Epigenetically Altered TLR4 Expression May Contribute to Increased Inflammation and Impaired Wound Healing in a Murine Model of Diabetes

Andrew Kimball, Amrita Joshi, Matthew Schaller, Beau Carson, Kanakadurga Singer, Jennifer Bermick, Shreyas Ramani, Peter Henke, Steven Kunkel, Katherine Gallagher Univ of Michigan, Ann Arbor, MI Introduction: Wound healing is impaired in diabetes due to dysregulated inflammation. Previous studies by our group and others have demonstrated that inflammatory mediators are significantly increased in diabetic wounds, however, the mechanisms behind this hyper-inflammatory response remain largely unknown. As there is known to be high levels of endotoxin in type 2 diabetic (T2D) wounds, we aimed to assess the role of high fat diet (HFD) on Toll-Like Receptor 4 (TLR4) expression. We hypothesized that HFD/T2D would alter histone methylation, leading to increased receptor expression and enhanced inflammation. Methods: C57/BL6 mice were subjected to either normal or HFD for 12-16 weeks to induce the diet-induced obese (DIO) model of physiologic T2D. 4mm hindlimb wounds were created and healing was monitored daily using NIH ImageJ software. Bone marrow-derived macrophages (BMDM) were cultured and TLR4 expression measured by qPCR. Myeloid TLR4 protein expression was assessed in peripheral blood and wound cell isolates by analytical flow cytometry. Macrophages (NK1.1-/Ly6G-/CD11b+) were isolated using magnetic sorting and inflammatory cytokine expression determined with qPCR and bioplex. Chromatin Immunoprecipitation (ChIP) analysis at the NF-kB binding site on the TLR4 promoter was performed using antibodies to trimethylated Histone 3 Lysine 4 (H3K4me3). (N=~5 mice per group for all studies). Results: DIO mice demonstrated significantly delayed wound healing as compared to littermate controls and BMDM isolated from these mice revealed increased TLR4 gene expression. Correspondingly, peripheral blood and wound monocyte/macrophages showed increased TLR4 receptor levels by flow cytometry. ChIP analysis at the TLR4 promoter revealed significantly increased H3K4me3, a gene activating mark, in the HFD BMDM. Finally, macrophage wound isolates from DIO mice had elevated levels of inflammatory gene expression and cytokines. Conclusion: Enhanced H3K4me3 at the TLR4 promoter in diabetic macrophages may increase receptor expression and contribute to the hyper-inflammatory response seen in T2D. Further mechanistic studies are needed in order to examine the role of TLR4 in wound healing. Author Disclosure Block: A. Kimball: None. A. Joshi: None. M. Schaller: None. B. Carson: None. K. Singer: None. J. Bermick: None. S. Ramani: None. P. Henke: None. S. Kunkel: None. K. Gallagher: None.

19

9:03 am

N-Acetylcysteine Enhances Amputation Stump Healing and Perfusion in Diabetes

Mohamed A. Zayed1, Xiachao Wei2, Larisa Belaygorod3, Uzma Naim3, Kyoung-mi Park3, Clay F. Semenkovich3 1Washington Univ Sch of Med & St. Louis Veterans Affairs Health Care System, St. Louis, MO; 2Washington Univ Sch of Med, Div of Endocrinology, Metabolism, and Lipid Res, St. Louis, MO; 3Washington Univ Sch of Med, St. Louis, MO

Introduction: Over 150,000 extremity amputations are performed in the US per year. More than 60% of these are performed in patients with diabetes and peripheral arterial disease, and 25% require subsequent re-amputation due to poor surgical site healing.

Methods: The mechanisms underlying poor amputation stump healing in the setting of diabetes are not understood, which greatly limits management options. N-acetylcysteine (NAC) is a well-known thiol that regulates intracellular glutathione levels, promotes endothelial cell function and angiogenesis, and is proposed to have therapeutic benefits in the setting of diabetes.

Results: To test the hypothesis that NAC can improve surgical site healing in chronically ischemic amputation stumps in the setting of diabetes, we developed a novel in vivo murine hindlimb ischemia-amputation model. In this model, streptozotocin-treated C57B6 mice are allowed to develop hyperglycemia for 1 month, followed by unilateral hindlimb femoral artery ligation. After 1 week, the recovered yet still mal-perfused hindlimb undergoes an infrageniculate amputation. Compared to sham control, mice receiving a daily intraperitoneal administration of NAC 150mg/kg demonstrated a 31% improvement in stump healing (P=0.06), 160% increase in hindlimb stump laser-Doppler perfusion at 3 days post-amputation (P=0.03), as well as 179% increase in hindlimb adductor muscle neovascularization (P=0.01). Adductor muscle segments from ischemic amputated hindlimbs also demonstrated less muscle damage, and expressed higher levels of de-palmitoylated activated Gaq (P<0.05), which is essential for ischemia-induced neovascularization. Similarly, HUVECs treated with NAC 3mM demonstrated a transient 19% increase in de-palmitoylated Gaq (P=0.03), suggesting a novel mechanism of action for NAC.

Conclusion: These findings demonstrate that NAC may have important therapeutic benefits in amputation stump healing in the setting of diabetes, which may translate into a valuable treatment option in vulnerable patient populations.

Author Disclosure Block: M.A. Zayed: None. X. Wei: None. L. Belaygorod: None. U. Naim: None. K. Park: None. C.F. Semenkovich: None.

20

9:15 am

Wound Application of Nitrite Enhances Healing in Diabetic Mice

Ankur Aggarwal1, Michael C. Madigan1, Ellen Cody2, Edith Tzeng1 1VA Pittsburgh Healthcare System and Univ of Pittsburgh, Pittsburgh, PA; 2Univ of Pittsburgh, Pittsburgh, PA

Introduction: Impaired diabetic wound healing, a source of significant disability, has been attributed to the reduced nitric oxide (NO) bioavailability and increased oxidative stress. We have previously reported that xanthine oxidoreductase (XOR), important in purine metabolism and reactive oxygen species synthesis, is highly expressed in regenerating skin and is required for normal healing. XOR is also expressed in diabetic wounds at similar or greater levels. Recently, XOR has been identified as a strong nitrite reductase and can generate NO from nitrite. Based on these findings, we hypothesize that topical nitrite applied to diabetic wounds may improve healing.

Methods: Anesthetized diabetic db/db mice underwent excisional wounding. The wounds were treated with either Aquaphor with 2% sodium nitrite or Aquaphor alone and covered with a bioocclusive dressing. Wounds were photographed every other day for digital planimetry until day 28. Wounds were collected on day 28 and fixed, sectioned, and examined for proliferation (Ki67) and angiogenesis (CD31) by immunostaining.

Results: Wounds in db/db mice treated with topical Aquaphor + nitrite displayed significantly accelerated healing compared to those treated with Aquaphor alone (Table). Aquaphor + nitrite also increased wound angiogenesis (29.3 ± 4.7 vs 13.2 ± 4.8, N=6-8/group; p<0.038) and keratinocyte proliferation (20.4 ± 5.1 vs 3.9 ± 2.4; p<0.034) compared with those treated with Aquaphor alone.

Conclusion: Topical nitrite significantly accelerated wound healing in db/db mice. Nitrite treatment enhanced angiogenesis and keratinocyte proliferation in the wounds. These prohealing effects are likely mediated by XOR mediated conversion of nitrite to NO which can potentially divert XOR away from ROS production. Future studies will define the ability of nitrite to alter wound oxidative stress as well as the translation of this simple and safe therapy for human application.

Author Disclosure Block: A. Aggarwal: None. M.C. Madigan: None. E. Cody: None. E. Tzeng: None.

21

Abstract Session II: Vascular Endothelium

and Thrombosis

22

10:10 am

P2X7R Antagonism Alleviates Traction Injury Induced Extracellular ATP Release in Vascular Tissues

Weifeng Luo, Olukemi J McWilliam, Colleen Brophy, Joyce Cheung-Flynn Vanderbilt Univ Medical Ctr, Nashville, TN

Introduction: Mechanical injury during intraoperative preparation of human saphenous vein graft during vascular bypass procedures leads to impaired physiologic functions and reduced viability of the conduit. We hypothesized that vein graft injury leads to release of ATP which activates the purinergic P2X7 receptor (P2X7R) signaling pathways.

Method: A stretch injury model of rat inferior vena cava (RIVC) was developed. Isolated RIVC were stretched to 2 times resting length and treated with P2X7R antagonists. Physiological function was determined in a muscle bath. Release of extracellular ATP (eATP) into the buffer was measure using an ATP bioluminescent assay.

Results: Stretch injury led to decreased vasomotor function that was associated with increased eATP levels. P2X7R blockade by A437809 (A43; Panel A) or brilliant blue FCF (FCF; Panel B) after stretch injury restored contractile function. Treatment with the ATP hydrolyzing enzyme apyrase (Apy) or P2X7R antagonists inhibited stretch-induced increases in eATP (Panel C).

Conclusion: These data provide evidence for a new mechanism wherein surgical injury during harvest leads to release of extracellular ATP, activation of the P2X7R, further release of ATP, and impaired functional responses of the vascular smooth muscle. Thus, P2X7R antagonism represents a prime candidate in preventing tissue injury during autologous vein bypass procedures.

Author Disclosure Block: W. Luo: None. O.J. McWilliam: None. C. Brophy: None. J. Cheung-Flynn: None.

23

10:22 am

Periadventitial or Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia after Arterial Injury

Alex Helkin1, David Bruch1, David R. Wilson2, Rebecca Bader2, Kristopher G. Maier1, Vivian Gahtan3 1SUNY Upstate Medical Ctr and Dept of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY; 2Syracuse Univ, Syracuse, NY; 3SUNY Upstate Medical Ctr, Dept of Veterans Affairs Healthcare Network Upstate New York at Syracuse, and Syracuse Biomaterials Inst, Syracuse, NY Introduction: Oral statin administration reduces intimal hyperplasia (IH) after arterial injury by only ~25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid tagged polysialic acid-polycaprolactone micelles) in vitro on vascular smooth muscle cell (VSMC) migration and in vivo after arterial injury. We hypothesized both free and micelles containing statins would inhibit VSMC chemotaxis and local statin treatment would reduce IH in Sprague-Dawley (SD) rats following carotid balloon injury greater than oral delivery. Methods: VSMCs pretreated with free simvastatin (1 μM, 20 min or 20 hrs) or simvastatin-loaded micelles underwent chemotaxis to thrombospondin-1 (20 µg/mL) or serum-free media using a modified Boyden chamber. Next, SD rats (13-15 wks) who underwent balloon injury of the common carotid artery (5 atm/5 min) received statin therapy – either intraluminal simvastatin loaded micelles (50 μM, pressurized for 5 min) prior to injury or periadventitial pluronic gel (50 μM simvastatin) following injury. Arteries were harvested 14 days postoperatively. Morphometric analysis was performed using the intimal/media area ratio defined as intima/(intima + media). Findings were compared to our historic animals which received oral simvastatin (10 mg/kg/day) or controls without statin therapy. Statistical analysis was by ANOVA and p<0.05 was significant. Results: Simvastatin loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%, p<0.05). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively, p<0.05). However, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03, p>0.05). Conclusions: Intraluminally and periadventially delivered statins were effective in reducing IH. The efficacy of single dose, locally delivered statins alone may lead to novel treatment modalities for the prevention of IH. The different routes of administration allow for treatment at the time of both open and endovascular procedures, without the need for systemic therapy. Author Disclosure Block: A. Helkin: None. D. Bruch: None. D.R. Wilson: None. R. Bader: None. K.G. Maier: None. V. Gahtan: None.

24

10:34 am

A Single Nucleotide Polymorphism of p27Kip1 Associated with Vein Graft Patency Regulates Expression of p27Kip1 in Both Venous Adventitial Cells and Smooth Muscle Cells but Selectively Regulates Proliferation of Adventitial Cells

Richard D. Kenagy1, Shinsuke Kikuchi2, Lihua Chen1, Errol S. Wijelath3, Alexander W. Clowes1, Michael Sobel3 1Univ of Washington, Seattle, WA; 2Asahikawa Medical Univ, Asahikawa, Japan; 3VA Puget Sound Health Care System and Univ of Washingto, Seattle, WA

Introduction: p27Kip1 (p27) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (SNP) accounts for ~40% of the risk of peripheral vein graft failure. However, whether this SNP is functional has not been definitely established.

Methods: To determine functionality, we investigated paired adventitial cells and smooth muscle cells (SMC) derived from fresh human saphenous veins (N≥7 lines per group). After growth arrest in 2% serum followed by stimulation with 10 ng/ml PDGF-BB, we measured p27 mRNA, p27 protein, and cell proliferation using qRT-PCR, Western blotting, and cell counts, respectively.

Results: The SNP genotype was associated with 72 hour adventitial cell growth, but not SMC growth. Adventitial AA cells grew 33% slower than those with the CC genotype (Figure 1A, B, p=.004). We expected AA adventitial cells to produce more p27 mRNA, but paradoxically, p27 mRNA was lower in both cell types of the AA genotype (Figure 1C, D, P<.01). However, levels of p27 protein (a single ~27 kD band in both cell types) were ~2 fold higher in AA adventitial cells and SMCs compared to the CC cells at both 0 and 72 hours (Figure 1E, 72 hours; P<.001 for genotype; 0 h data not shown). Adventitial cells and SMCs made comparable amounts of p27 protein.

Conclusion: The p27Kip1-838C>A SNP regulates levels of p27 in both venous adventitial cells and SMCs. There is an effect of genotype on the growth of adventitial cells, but not on SMCs. AA cells produce ~2 fold higher p27 protein than CC cells, despite AA cells having ~2 fold lower levels of p27 mRNA. These data demonstrate that the p27 SNP is functional, that different cell types within the vein wall respond uniquely to this genetic polymorphism, and suggest an important role in graft failure for the precursor of the cultured adventitial cell.

Author Disclosure Block: R.D. Kenagy: None. S. Kikuchi: None. L. Chen: None. E.S. Wijelath: None. A.W. Clowes: None. M. Sobel: None.

25

10:46 am

Arteriovenous Fistula Adaptation Requires Caveolin1 Signaling

Takuya Hashimoto, Trenton R. Foster, Hualong Bai, Haidi Hu, Jeans M. Santana, Jesse Hanisch, Alan Dardik Yale Sch of Med, New Haven, CT Introduction: Arteriovenous fistula (AVF) is the preferred access for hemodialysis. However, many AVFs fail to mature and require intervention, showing a need to improve AVF maturation. Caveolin1 (Cav1) is the major scaffolding protein of caveolae, a distinct microdomain that serves as a flow activated mechanosensor at the membrane of endothelial cells. We have previously shown that stimulation of embryonic venous determinant EphB4 associates with Cav1, mediating vessel remodeling in the murine vein graft model. Since Cav1 is a mechanism of EphB4 mediated vein graft adaptation, we examined the role of Cav1 in mediating AVF maturation, a different and distinct hemodynamic and molecular conditions from vein graft. We hypothesized that Cav1 function is critical for AVF maturation. Methods: We used a mouse aortocaval fistula model; the venous AVF limb and control (sham) inferior vena cava of wild type (WT) C57BL/6 mice were analyzed and compared for Cav1 expression. Vessel remodeling was assessed in WT and Cav1 knockout (KO) mice by serial ultrasound measurements of the IVC/AVF diameter. AVF were harvested at day 21 and examined with histology; IVC wall thickness was measured using computerized morphometry. Results: Both Cav1 mRNA and protein were increased in the fistula veins compared to control veins (Figure A; mRNA: *p=0.019, ANOVA, n=8; protein: *p=0.005, t-test, n=5). Cav1 KO mice had increased fistula vein diameter compared with WT mice (Figure B; *p<0.0001, ANOVA, n=7-10). The fistula vein wall thickness was increased in Cav1 KO mice as compared with WT mice (Figure B; 15.6 vs 30.9μm, *p=0.0005, t-test, n=7, bar 50μm). Conclusions: Cav1 expression is increased during AVF maturation, and loss of Cav1 leads to increased fistula vein diameter and wall thickness, similar to the function of Cav1 during vein graft adaptation. These results suggest that Cav1 is a regulator of AVF maturation and manipulation of Cav1 function may enhance venous adaptation to the arterial environment.

Author Disclosure Block: T. Hashimoto: None. T.R. Foster: None. H. Bai: None. H. Hu: None. J.M. Santana: None. J. Hanisch: None. A. Dardik: None.

26

10:58 am

Platelet and Monocyte Activity in Carotid Artery Stenosis Treated with Carotid Endarterectomy

Arvind Reddy Devanabanda, Caron Rockman, Nicole Allen, Maya Rubin, Binita Shah, Mark Adelman, Jeffrey Berger New York Univ, New York, NY Introduction: Carotid artery stenosis (CAS) is a marker of atherosclerosis, a disease mediated by abnormalities in platelet and monocyte function, and a significant cause of stroke. Moreover, the effect of carotid artery revascularization via carotid endarterectomy (CEA) on platelet and monocyte markers is unknown. Objective: This study aims to investigate platelet activity, monocyte subsets and monocyte platelet aggregates (MPA) in CAS and changes with CEA. Methods: This prospective cohort study evaluated 48 patients who underwent non emergent CEA. Peripheral venous blood samples were obtained before, immediately postoperative and at 24 hours postoperative. Twenty healthy subjects served as controls. Platelet surface expression of P-selectin and PAC-1, monocyte subsets, and MPA were assessed using flow cytometry. Three distinct monocyte subsets were measured: anti-inflammatory (i.e. classical CD14++CD16-) and pro-inflammatory (i.e. intermediate CD14++CD16+ and nonclassical CD14+CD16++) monocytes. Differences between two matched samples and between the study and control groups were statistically analyzed. Results: Compared to healthy subjects, CAS subjects had significantly greater markers of platelet activity (P-selectin [p=0.003] and PAC-1 [p=0.01]), pro-inflammatory monocytes (intermediate [p<0.0001] and nonclassical [p=0.009]) and MPA (p=0.0002). Following CEA, anti-inflammatory monocytes increased and pro-inflammatory monocytes decreased (Figure 1A). Platelet expression of P-selectin and MPA did not change, while PAC-1 transiently increased but then returned to baseline by 24 hours postoperative (Figure 1B &C). Conclusions: Subjects with CAS have elevated markers of thrombosis, inflammation, and their interface. However, only the pro-inflammatory monocytes are significantly reduced following CEA. Future studies investigating the clinical consequences of this reduction are warranted.

27

Author Disclosure Block: A. Devanabanda: None. C. Rockman: None. N. Allen: None. M. Rubin: None. B. Shah: None. M. Adelman: None. J. Berger: None.

28

11:10 am

Induction of miR-21 Increases Fibrous Cap Stability in Vulnerable Atherosclerotic Lesions

Hong Jin1, Yuhuang Li1, Alexandra Bäcklund1 Albert Busch1, Suzanne M. Eken1, Ekaterina Chernogubova1, Peter Gustafsson1, Ljubica Perisic1, Isabel Schellinger2, Uwe Raaz2, Philip S. Tsao3, Göran K. Hansson1, Gabrielle Paulsson- Berne1, Ulf Hedin1, Lars Maegdefessel1 1Karolinska Inst, Solna, Sweden; 2Georg-August-Univ Göttingen, Göttingen, Germany; 3Stanford Univ, Palo Alto, CA Introduction: The aim of the present study was to explore the role of miRNAs as potential regulators in patients with carotid artery stenosis and concordant vulnerable atherosclerotic plaques. A pre-determined miRNA-array of laser captured micro-dissected (LCM) tissue specimen from fibrous caps of 10 symptomatic patients (stroke or TIA within > 14 days and histo-morphologically identified ruptured lesion) compared to fibrous caps from 10 asymptomatic patients (stable lesions; high grade stenosis) discovered miR-21 as one of the two miRNAs (miRs-21 and -210) being substantially down-regulated in symptomatic patients. Methods: To functionally evaluate the contribution of miR-21 to plaque vulnerability, we created miR21-/-

/ApoE-/- mice on a C57BL/6 background. We explored the phenotype of these newly developed miR21-/-

/ApoE-/- mice in experimental models of vascular remodelling and plaque vulnerability. Results: First, miR21-/- mice revealed a complete lack of SMC proliferation in response to carotid ligation injury. In the second inducible plaque rupture model, using incomplete ligation of the common carotid artery with consecutive cuff placement proximal to the ligated region indicated that all miR21-/-/ApoE-/- mice (n=10) presented atherothombotic events and signs of severe plaque instability. The rupture rate in control miR21+/+/ApoE-/- mice was significantly lower at 56% (n=9). In addition, miR21-/-/ApoE-/- showed an increase in lesion area in the aortic root and substantially higher levels of macrophage infiltration in injured carotid arteries. Dynamic live cell imaging, using isolated aortic SMCs and peritoneal macrophages from miR21-/-/ApoE-/- vs. miR2+/+/ApoE-/- mice displayed substantial lower cellular proliferation and survival rates (for SMCs), and distinct advanced inflammatory activity upon oxidized LDL (oxLDL) stimulation of macrophages. Conclusions: In the present study we identified miR-21 as a key modulator of pathologic processes in atherosclerosis-related vascular remodelling. Targeted, lesion-site specific overexpression of miR-21 could be a novel and powerful future strategy to stabilize vulnerable plaques by inducing pro-proliferative mechanisms in SMC-enriched fibrous caps. Author Disclosure Block: H. Jin: None. Y. Li: None. A. Bäcklund: None. A. Busch: None. S.M. Eken: None. E. Chernogubova: None. P. Gustafsson: None. L. Perisic: None. I. Schellinger: None. U. Raaz: None. P.S. Tsao: None. G.K. Hansson: None. G. Paulsson- Berne: None. U. Hedin: None. L. Maegdefessel: None.

29

11:22 am

Il-6 Mediates Vein Wall Response to Thrombosis in a Model Dependent Manner

Andrea T. Obi, Andrew Kimball, Megan Elfline, Catherine Luke, Jose Diaz, Thomas W. Wakefield, Peter K. Henke Univ of Michigan, Ann Arbor, MI

Introduction: Venous thrombosis (VT) and the vein wall responses may be related to interleukin 6 (IL-6) signaling in humans and mice. Inflammatory and fibrotic effects of IL-6 largely signal through the trans pathway: IL-6/soluble IL-6Rα complex associates with ubiquitously expressed signal transducing protein gp130. We hypothesized that IL-6 signaling would differ depending upon thrombosis mechanism (complete v. partial stasis), and disruption of IL-6/IL-6Rα/gp130 axis would alter vein wall response to VT.

Methods: Wild type C57BL/6 or IL-6-/- mice underwent induction of VT via inferior vena cava (IVC) stenosis (partial stasis) or ligation (complete stasis). Select WT mice received sgp130Fc chimera to inhibit trans pathway signaling. Vein wall, thrombus, and plasma were harvested at various time points and analyzed by PCR, western blot, ELISA or immunohistochemistry.

Results: Compared to sham animals, those undergoing VT (complete or partial stasis) exhibited elevated vein wall IL-6 on post-VT day 4 (n=6-9, p<0.0001) but only complete stasis also had significantly elevated IL-6Rα (n=6-9, p<0.0001) and downstream intracellular signaling pathway molecule pSTAT3 (n=6-9, p=0.0002). Complete stasis VT in IL-6-/- mice was characterized by decreased circulating gp130 (n=4-6 p=0.0257), similar size thrombi to WT controls (0.020±0.005 v. 0.023±0.006, p=ns), and decreased CD68+ macrophage recruitment to the vein wall (n=3-5, p=0.0279). The IL-6-/- mice were protected from endothelial to mesenchymal-like transformation, as reflected by decreased FSP-1 (n=3-6, p<0.0001) and increased VE cadherin (n=3-6, p=0.0421) as compared with WT. Disruption of trans-signaling pathway via gp130Fc chimera resulted in less vein IL-6 (n=6-7, p=0.0027), TGF-β (n=6-7, p=0.0094), and decreased CD68 cell recruitment (n=3, p=0.0299).

Conclusions: Experimental VT stimulates IL-6 and downstream signaling processes and is dependent on the mechanism of thrombosis. Genetic deletion of IL-6 and selective disruption of trans-signaling pathway by sgp130 decreases vein wall inflammation and influences vein wall remodeling. Targeting of IL-6 trans-signaling pathway may represent a therapeutic option to treat vein wall inflammation post thrombosis.

Author Disclosure Block: A.T. Obi: None. A. Kimball: None. M. Elfline: None. C. Luke: None. J. Diaz: Consultant/Advisory Board; Modest; Research Council Chair, American Venous Forum. T.W. Wakefield: None. P.K. Henke: None.

30

11:34 am

Anti-thrombin Perfluorocarbon Nanoparticles Decrease Clot Burden in a Murine Model of Venous Thrombosis

Chandu Vemuri, Batool Arif, Susannah A. Grathwohl, John S. Allen, Peter K. Henke, Samuel A. Wickline Washington Univ in St. Louis, St. Louis, MO Introduction: Venous thromboembolism (VTE) afflicts nearly an million Americans with significant mortality and long-term morbidity. Current medical treatment regimens pose significant bleeding risks and recurrence risks. The purpose of this work is to determine if anti-thrombin perfluorocarbon nanoparticles (NP-PPACK) can attenuate clot progression after vascular injury in a murine model of venous thrombosis. Methods: Male, C57 black-6 mice underwent inferior vena cava (IVC) ligation through an institutionally approved protocol. Following ligation, groups of ten mice were randomized to receive intravenous, weight-based (1 ml/kg) tail vein injections of saline, plain nanoparticles, NP-PPACK or heparin (80 units/kg). After 6 hours the animals were sacrificed, IVC with clot excised and weight and length recorded. Clot integrity (N=4) analysis was then performed by incubating clots with 750 units of streptokinase for 90 minutes at 37 degrees Celsius, removing liquid clot and recording the percent change in clot weight. Results: There was a significant difference in clot burden between NP-PPACK and the control group (0.59 mg/mm ± 0.063 vs. 1.26mg/mm ± 0.85, p=.0001). Immunofluorescent histology performed on a subgroup of animals verified nanoparticle tracking to venous thrombus. Additionally, using exogenous clot lysis as a surrogate for clot integrity, NP-PPACK treated animals exhibited a trend towards enhanced lysis over saline treatments (change in clot weight over 90 minutes: 57.8 ±14.4 vs. 21.5 ± 7.49, NP PPACK vs saline p=.067). Conclusions: This initial work demonstrates that NP-PPACK significantly decreases clot burden by local targeting and reduces clot strength in this model of VTE. We have shown previously that the system is locally active for hours against thrombosis yet produces no sustained systemic anticoagulant effect beyond 60 minutes, indicative of its significant safety margin for clinical application.

Author Disclosure Block:

C. Vemuri: None. B. Arif: None. S.A. Grathwohl: None. J.S. Allen: None. P.K. Henke: None. S.A. Wickline: Ownership Interest; Significant; AcuPlaq, LLC.

31

Abstract Session III:

Aneurysms

32

2:20 pm

An XY Sex Chromosome Complement in Females Confers Susceptibility and Rupture of Angiotensin II-induced Abdominal Aortic Aneurysms in Hypercholesterolemic Female Mice

Yasir Alsiraj, Sean Thatcher, Eric Blalock, Kuey Chen, Richard Charnigo, Alan Daugherty, Lisa Cassis Univ of Kentucky, Lexington, KY

Introduction: We previously demonstrated that female mice are less susceptible to angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) compared to males, a sex difference present in humans. Sex chromosome abnormalities, such as Turner’s syndrome (monosomy X), are associated with aortic vascular disease. In this study, we tested the hypothesis that an XY sex chromosome complement in females promotes AngII-induced AAAs. In addition, as previous studies demonstrated that testosterone promotes AngII-induced AAAs in male mice, we determined if testosterone would augment AAA severity in XY females.

Methods and Results: Transgenic male mice with deletion of Sry from the Y-chromosome expressing Sry on autosomes (8-12 weeks of age) were bred to female Ldlr-/- mice to generate female mice with an XY or an XX sex chromosome complement. Female mice (XX and XY) were fed a Western diet and segregated into sham and ovariectomized (OVX) groups. Two weeks later, mice were implanted with osmotic minipumps to infuse AngII (1,000 ng/kg/min) for 28 days. The AAA incidence (XX sham, 40%; XX OVX 29%; XY sham, 71%; XY OVX, 57%, p=0.031) and rupture rate (XX sham, 0%; XX OVX 0%; XY sham, 35%; XY OVX, 29%, p=0.003) were significantly increased in XY compared to XX females. Internal abdominal aortic lumen diameters were significantly increased in XY OVX versus XX OVX female mice at day 27 (XY, 2.31 ± 0.14; XX, 1.58 ± 0.2, p= 0.009). Moreover, AAA external diameters were significantly increased in XY OVX versus XX OVX females (XY, 2.34 ± 0.15; XX, 1.71 ± 0.18, p=0.0004). Administration of testosterone to adult female XY mice as well as neonatal female mice markedly enhanced AAA rupture (maximum of 73%). DNA microarrays of abdominal aortas revealed that male specific genes on the Y chromosome and inflammatory genes were enriched in aortas from XY females, while genes that escape X-inactivation were enriched in aortas from XX females.

Conclusion: These results demonstrate that an XY sex chromosome complement is sufficient to promote a high AAA incidence, and markedly increase AAA severity in female mice. Moreover, testosterone augmented AAA ruptures in XY females. Future studies will identify gene targets influenced by sex chromosome complement and/or testosterone.

Author Disclosure Block: Y. Alsiraj: None. S. Thatcher: None. E. Blalock: None. K. Chen: None. R. Charnigo: None. A. Daugherty: None. L. Cassis: None.

33

2:32 pm

Depletion of Plasmacytoid Dendritic Cells Inhibits Experimental Abdominal Aortic Aneurysms

Baohui Xu, Haojun Xuan, Naoki Fujimura, Sara A. Michie, Ronald L. Dalman Stanford Univ Sch of Med, Stanford, CA

Introduction: Abdominal aortic aneurysms (AAA) manifest histologic features consistent with other chronic inflammatory diseases. Infiltrating mural myeloid cells (e.g. macrophages) are already recognized as important contributors to aneurysm pathogenesis, however, the role of plasmacytoid dendritic cells (pDC), major type 1 interferon-producing myeloid cells involving in autoimmune diseases and atherosclerosis, has not been previously investigated in this context.

Methods and Results: AAAs were created in 12 week old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase (PPE). AAA development and progression were assessed via serial ultrasound determination of aortic diameter in vivo, and histology at sacrifice. The fraction of circulating leukocytes identified as pDCs was significantly increased immediately following PPE infusion (aneurysm initiation). Treatment with mPDCA-1 mAb (400 μg i.p. q.o.d.), beginning one day prior to PPE infusion, depleted more than 90% of bone marrow, spleen and peripheral blood pDCs (data not shown) and suppressed subsequent aneurysm development and progression compared to that noted in PPE-infused mice treated with control mAb. mPDCA-1 treatment promoted aortic medial elastin and smooth muscle preservation, while limiting mural macrophage accumulation and neocapillary formation.

Conclusion: These findings suggest a role for plasmacytoid dendritic cells in promoting the initiation and progression of experimental abdominal aortic aneurysms.

Author Disclosure Block: B. Xu: None. H. Xuan: None. N. Fujimura: None. S.A. Michie: None. R.L. Dalman: None.

34

2:44 pm

Collagen Type V (Col V) Contributes to the Development of Aortic Dissections and Aneurysms

Noel M. Phan, Arick Park, Ting Zhou, David Scott, Qiwei Wang, Paul Tang, Daniel S. Greenspan, Bo Liu Univ of Wisconsin-Madison, Madison, WI

Introduction: Negative remodeling of extracellular matrix (ECM) is a key pathological feature associated with aortic dissections and aneurysms. Col V is a minor fibrillar collagen that incorporates into growing fibrils with Col I, and is involved in regulating the geometry and tensile strength of ECM. Col V normally exists as α1(V)2α2(V)1 heterotrimers but may present as α1(V)3 homotrimers in pathological tissues. To experimentally increase the existence of α1(V)3 homotrimer, we deleted the Col5a2 gene, which encodes the α2(V) chain. Col5a2+/- mice showed increased compliance and reduced tensile strength in the skin and aorta. In the present study, we tested whether Col5a2 deficiency affects the pathogenesis of aortic dissections and aneurysms.

Methods and Results: Six-month-old male Col5a2+/- and Col5a2+/+ mice on an Ldlr null background were infused with Angiontensin II (AngII). Mortality for Col5a2+/- mice within the first 7 days was 45% vs 0% for WT (P<0.045, n=9), with 60% due to aortic arch rupture and 40% due to dissection. Among those that survived the 28 day treatment, 7/ 7 Col5a2+/- mice developed abdominal aortic aneurysm, defined as greater than or equal to 50% increase in the maximal external suprarenal diameter over infrarenal aorta, whereas 5/9 WT showed aneurysm dilatation. Compared to WT, aneurysm dilatations were significantly increased in Col5a2+/- mice (132.1 ± 8.5% vs 90.12 ± 9.6%, P<0.017). Histological analyses revealed marked disarray and loss of elastic fibers in Col5a2+/- aortas and no difference in levels of fragmentation. Moreover, medial layer integrity was deteriorated in Col5a2+/- mice and was associated with increased infiltration of macrophages.

Conclusions: Collectively, our data suggest that haploinsufficiency for the α2(V) chain leads to media layer changes and increased susceptibility to aortic arch ruptures, dissections, and larger aortic aneurysms. The α2(V) chain may thus represent a novel candidate marker/target for the identification and treatment of aortic dissection and aneurysm. <!--EndFragment-->

Author Disclosure Block: N.M. Phan: None. A. Park: None. T. Zhou: None. D. Scott: None. Q. Wang: None. P. Tang: None. D.S. Greenspan: None. B. Liu: None.

35

2:56 pm

Number of Reentry Tears Influences Flap Motion and Flow Reversal in an in vitro Model of Type B Aortic Dissection

Joav Birjiniuk1, Mark Young2, Lucas H. Timmins1, Bradley G. Leshnower1, John N. Oshinski1, David N. Ku3, Ravi K. Veeraswamy1 1Emory Univ Sch of Med, Atlanta, GA; 2Medtronic, Inc., Santa Rosa, CA; 3Georgia Inst of Technology, Atlanta, GA

Introduction: Aortic remodeling after dissection is poorly understood. Thus, optimal patient-specific recommendations for treatment are lacking. An in vitro aortic model of Type B dissection was used to interrogate local aortic hemodynamic parameters implicated in thrombosis and aneurysm formation. We hypothesize that dissections with multiple reentry tears will exhibit decreased flap motion, and, as a result, reduce flow reversal. Methods Anatomic models of aortic dissection with fidelity to patient CT images were fabricated out of silicone. Models with primary entry and single fenestration (Figure 1A), two fenestrations (Figure 1B), and three fenestrations (Figure 1C) were installed in a flow loop. Physiologic flow was established at a cardiac index of 4 L/min. Flow velocities were acquired using phase contrast magnetic resonance (PCMR) imaging. Flow rates and flap motion were quantified using custom made software.

Results: Relative true lumen area (RTLA) varied along the dissection (entry: 55% +/- 3, middle: 34% +/-7, exit: 91%+/-3, p<0.00001 pair-wise for 2-tear model). At mid-dissection, RTLA was lower in dissections with fewer tears (p<0.01). Total flow was nearly identical in all cases, while true and false lumen flow rates differed significantly across tear configurations and along the dissection (p<0.01). Secondary tears allowed for flow communication within the dissected portion of the aortic model. Flow reversal was seen in the false lumen at the mid-dissection plane in the absence of secondary tears (Figure 1D). However, as secondary tears were added, the flow reversal in the false lumen decreased, with concomitant flow reversal in the true lumen (Figure 1E,F).

Conclusions: Anatomic characteristics of dissection, such as number of tears, affect blood flow and motion of the dissection flap, as shown quantitatively. This compliant aorta model illustrates alterations in flow reversal in both true and false lumina that may lead to aneurysmal degeneration.

Author Disclosure Block: J. Birjiniuk: Research Grant; Significant; Medtronic, Inc. M. Young: Employment; Significant; Medtronic, Inc. L.H. Timmins: None. B.G. Leshnower: None. J.N. Oshinski: None. D.N. Ku: None. R.K. Veeraswamy: Research Grant; Significant; Medtronic, Inc. Consultant/Advisory Board; Significant; Cook Inc., Lombard Inc.

36

Abstract Session IV: Peripheral Arterial

Disease

37

4:00 pm Revascularization but Not Supervised Exercise Therapy Prevents Progression of Fibrosis in the Gastrocnemius of Patients with Peripheral Artery Disease While Improving Limb Function Duy Ha, George Casale, Alicia Luis, Kevin Harkins, Reagan Huber, Tanmayee Chengalasetty, Ruby Hickman, Mina Hanna, Stanley Swanson, Holly DeSpiegelaere, Iraklis Pipinos Univ of Nebraska Medical Ctr, Omaha, NE

Introduction: Patients with peripheral artery disease (PAD) develop myofiber degeneration and fibrosis in their ischemic lower extremities, along with limb dysfunction. Walking performance improves with revascularization and exercise therapy, but effects on the myopathy are unknown. We previously showed fibrosis progresses with PAD and positively correlates with expression of vascular transforming growth factor-beta 1 (TGF-β1), a cytokine that stimulates collagen deposition.

Hypothesis: We hypothesize that revascularization (RVS) and supervised exercise therapy (EXE) improve limb function in association with improved TGF-β1 dependent fibrosis.

Methods: Gastrocnemius biopsies were collected from PAD patients (Fontaine Stage II; N=56) at baseline and 6 months after RVS (N=20), EXE (N=19), or no intervention (CTL; N=17). TGF-β1 expression was measured as grey scale units (gsu) by quantitative fluorescence microscopy of paraffin-embedded gastrocnemius sections. Collagen abundance was measured as optical density by quantitative multi-spectral bright-field microscopy of Masson Trichrome stained paraffin sections. Six Minute Walking Distance (SMWD), in meters, and Peak Walking Time (PWT), in seconds, were determined at baseline and 6 months. Relationships among TGF-β1, collagen, and limb function were assessed.

Results: TGF-β1 expression and collagen density increased in CTL and EXE but not RVS patients. SMWD and PWT increased among RVS patients. PWT but not SMWD increased among EXE patients. SMWD and PWT were unchanged among CTL patients. The data are summarized in Table 1.

Conclusions: RVS and EXE improved walking performance of patients with PAD, but only RVS prevented progression of fibrosis in the gastrocnemius of these patients. Over the same 6-month period, fibrosis increased in CTL muscle but was not sufficient to alter walking performance. The data suggest that benefits to the PAD leg may be greater with RVS compared to EXE. Author Disclosure Block: D. Ha: None. G. Casale: None. A. Luis: None. K. Harkins: None. R. Huber: None. T. Chengalasetty: None. R. Hickman: None. M. Hanna: None. S. Swanson: None. H. DeSpiegelaere: None. I. Pipinos: None.

38

4:12 pm

Statins Have a Dose-dependent Effect on Amputation Risk and Survival in Peripheral Arterial Disease (PAD) Patients

Shipra Arya1, Anjali Khakharia1, Zachary O. Binney1, Randall R. Demartino2, Luke P. Brewster1, Philip P. Goodney3, Peter W. Wilson1 1Emory Univ, Atlanta, GA; 2Mayo Clinic, Rochester, MN; 3Geisel Sch of Med Dartmouth-Hitchcock, Lebanon, NH

Introduction: Statin dose guidelines for PAD patients are based on coronary artery disease and stroke data. The aim of our study was to determine the effect of statin dose (based on 2013 ACC/AHA guidelines) on PAD outcomes of amputation and mortality.

Methods: Patients with PAD in the Veterans Affairs database were identified from 2003-2014. The exposure was highest statin dose usage (none, low-moderate and high intensity) around diagnosis of PAD (within 1 year). The outcomes were risk of incident amputation (below knee or above knee) and mortality at 1, 3 and 5 years. The effect of statin dose on the two outcomes was also analyzed using Cox proportional hazards modeling to adjust for covariates.

Results: In 208,275 patients with PAD [Males: 98.1%; Mean age: 67.4 yrs (SD 9.9)], 17,643 amputations and 99,951 deaths occurred in a median follow up of 5.2 years. Almost a quarter (27.7%) of patients were not on a statin while 30.4% were on simvastatin 80 mg (no longer recommended due to drug toxicity). The risk of incident amputation declined significantly at the high intensity statin dose as seen in Table 1. In Cox models adjusting for age, gender, race, comorbidities, cholesterol levels and creatinine, the high intensity statins were associated with lower amputation risk and mortality as compared to no statin [HR 0.67; 95% CI (0.63, 0.72) and HR 0.71; 95% CI (0.68, 0.73), respectively]. Low-moderate-dose statins also had significant but smaller reductions in risk of amputation and mortality [HR amputation 0.78 (0.75, 0.82), HR death 0.78 (0.77, 0.80)].

Conclusion: This is one of the largest population based studies to examine the effect of statins on long-term PAD outcomes and the first to explore the dose-dependent effect of statins on amputation and mortality. High intensity statins are associated with a significant reduction in limb loss and mortality in PAD patients followed by a smaller risk reduction by low-moderate intensity statins as compared to no statin therapy.

Author Disclosure Block: S. Arya: Research Grant; Modest; 2014 AHA Mentored Clinical and Population Research Award. A. Khakharia: None. Z.O. Binney: None. R.R. Demartino: None. L.P. Brewster: None. P.P. Goodney: None. P.W.F. Wilson: None.

39

4:24 pm

Proprotein Convertase Subtilisin/Kexin Type 6 is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Disease

Ulf Hedin1, Ljubica Perisic1, Urszula Rykaczewska1, Anton Razuvaev1, Mariette Lengquist1, Maria Sabater-Lleal1, Ida Eriksson1, Samuel Röhl1, Malin Kronqvist1, Chi-Nan Tseng1, Patricia Rodriguez1, Lasse Folkersen2, Lei Du1, Maria Gonzalez Diez1, Cecilia Osterholm1, Joy Roy1, Jaakko Patrakka1, Gabrielle Paulsson-Berne1, Göran Hansson1, Jacob Odeberg1, Anders Hamsten1, Per Eriksson1 1Karolinska Inst, Stockholm, Sweden; 2Technical Univ of Denmark, Kongens Lyngby, Denmark

Introduction: Proprotein convertases (PCSKs) process matrix metalloproteases (MMPs) and cytokines. Apart from PCSK9, the role of these enzymes in vascular disease is largely unknown. Previously, we demonstrated upregulation of PCSK6 in carotid atherosclerosis, primarily localized to smooth muscle cells (SMCs) and positively correlated to inflammation, extracellular matrix remodeling and cytokines. Here, we extended these findings to determine the role of PCSK6 in vascular development and disease.

Methods and Results: Increased expression of PCSK6 in vascular disease was validated by microarrays from two non-overlapping cohorts of carotid plaques vs. non-atherosclerotic arteries (n=50 patients and n=32 patients, p<0.0001), as well as abdominal (AAA, n=14, p<0.0001) and thoracic aortic aneurysms (TAA, n=244, p=0.012). By eQTL, variants in the PCSK6 gene were found to influence it’s expression in both plaques and aneurysms. Among these, rs6598465 also showed association with maximum progression of carotid intima-media thickness in high-risk coronary artery disease subjects (n=3388, p=0.037). By IHC, PCSK6 localized mainly to SMCs in the fibrous cap and neovessels in atherosclerotic, AAA and TAA tissues. In mouse-, rat-, and human intimal hyperplasia, PCSK6 was expressed in proliferating SMCs. By microarrays, after rat carotid balloon injury there was an early downregulation of PCSK6 followed by an upregulation in later phases during SMC activation, as well as positive correlation to PDGFB and IGF1 (Spearman r>0.7, p<0.0001) and to MMP2 and MMP14 (r>0.5, p<0.0001). In zebrafish embryos, PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. PCSK6-/- mice did not present an obvious vascular phenotype but showed reduced intimal hyperplasia compared to wild-type mice after carotid artery ligation (p=0.015). In vitro, PCSK6 overexpression markedly increased SMC migration upon PDGFBB stimulation (p<0.0001).

Conclusion: The present study establishes PCSK6 as a key modulator of SMC function in vascular disease and demonstrates a functional link between PCSK6 expression and SMC migration in vascular remodeling.

Author Disclosure Block: U. Hedin: None. L. Perisic: None. U. Rykaczewska: None. A. Razuvaev: None. M. Lengquist: None. M. Sabater-Lleal: None. I. Eriksson: None. S. Röhl: None. M. Kronqvist: None. C. Tseng: None. P. Rodriguez: None. L. Folkersen: None. L. Du: None. M. Gonzalez Diez: None. C. Osterholm: None. J. Roy: None. J. Patrakka: None. G. Paulsson-Berne: None. G. Hansson: None. J. Odeberg: None. A. Hamsten: None. P. Eriksson: None.

40

4:36 pm Smooth Muscle Specific Knock-Out of the Zinc-Finger Protein 148 (ZFP148) Attenuates Atherosclerotic Lesion Formation via Regulation of Apoptotic Signaling Pathways Morgan Salmon1, Anna Z. Fashandi1, Michael D. Spinosa1, Ashish K. Sharma1, Gary K. Owens1, Juanita L. Merchant2, Zendra E. Zehner3, Gilbert R. Upchurch Jr. 1, Gorav Ailawadi1 1Univ of Virginia Medical Ctr, Charlottesville, VA; 2Univ of Michigan at Ann Arbor, Ann Arbor, MI; 3Virginia Commonwealth Univ Medical Coll of Virginia Campus, Richmond, VA; Introduction: Zinc-finger protein 148 (ZFP148) plays a profound role in the modulation of aortic aneurysm formation in part via modulation of smooth muscle (SMC) genes. The current study objective was to determine whether smooth muscle specific knock-out of ZFP148 is critical in atherosclerotic lesion formation. Methods: ZFP148 was examined via immunohistochemistry and confocal microscopy in human atherosclerotic lesion samples (n=12/group). 6-8 week male (n=12/group) ZFP flx/flx Myh11 Cre+ ApoE-/-(SMC tamoxifen ZFP148 KO), Myh11 ZFP148 flx/wt Cre+ ApoE-/- and Myh11 ZFP wt/wt Cre+ ApoE-/- underwent tamoxifen injections followed by western diet feeding for either 13 or 25 weeks. A separate set of mice were fed western diet for 18 weeks and then administered tamoxifen injections. Aortic samples were evaluated with histology for α-actin, macrophages, neutrophils, TER119, caspase3, Ki67, picosirus red and movat staining. In vitro ZFP148 was knocked down using siRNA in smooth muscle cells and stimulated with the oxidized phospholipid POVPC. Results: ZFP148 expression was elevated in human atherosclerotic lesion samples and localized to smooth muscle cells. Lesion size was significantly reduced in SMC ZFP148 KO mice compared with controls in 25 week western diet fed mice(p<0.0357). SMC ZFP148 KO demonstrated reduced macrophage, Caspase3, and TER119 staining. Conversely, SMC ZFP148 KO increased SMα-actin coverage. Lesion size was also decreased in mice that were administered tamoxifen injections following 18 weeks of western diet feeding(p<0.0415). There were no significant changes in lesion size at 13 weeks of western diet feeding; however, macrophage staining was decreased. Knock-down of ZFP148 followed by treatment with POVPC attenuated the down-regulation of SM22α, SM-MHC, and SMαA. Knock-down of ZFP148 followed by POVPC treatment also prevented the up-regulation of Bax and BAD in vascular smooth muscle cells. Conclusions: While earlier studies documented a role for ZFP148 in aneurysm disease, the present study suggests that SMC ZFP148 KO attenuates atherosclerotic lesion formation in early and late atherosclerotic disease. ZFP148 represents a key regulator of multiple types of vascular disease. Author Disclosure Block: M. Salmon: None. A.Z. Fashandi: None. M.D. Spinosa: None. A.K. Sharma: None. G.K. Owens: None. J.L. Merchant: None. Z.E. Zehner: None. G.R. Upchurch: None. G. Ailawadi: None.

41

4:48 pm

Vitamin D3-Induced Arterial Calcification Decreases Functional Recovery and Limb Perfusion in a Murine Model of Hindlimb Ischemia

Sara L. Zettervall, Stephanie Monk, Xue-Lin Wang, Yujun Cai, Tonghui Lin, Raul J. Guzman Beth Israel Deaconess Medical Ctr, Boston, MA Introduction: Clinical studies demonstrate associations between arterial calcification and adverse outcomes in patients with peripheral arterial disease. The causal relationship between calcification and ischemia, however, remains unclear. This study aims to evaluate the effects of arterial calcification on functional recovery and limb perfusion following acute limb ischemia in a murine model. Methods: Arterial calcification was induced in 64 Sprague-Dawley rats using Vitamin D3. A control group of 32 rats received vehicle only. After 2 weeks, to allow for calcification, unilateral hindlimb ischemia was induced by ligation and excision of the superficial femoral artery. At weekly intervals, functional and ischemia status were evaluated in a blinded manner and laser Doppler perfusion imaging was performed. Results: Calcium assay demonstrated greater arterial calcification in the superficial femoral arteries (0.1 vs. 17.0, P < .01) of Vitamin D3 injected animals. Rats injected with Vitamin D3 showed significantly worse Tarlov, Ischemia, Modified Ischemia, and Total Ischemia scores at each post-operative time point (Week 1: 16.6 vs. 14.5 P <.01, Week 2: 17.1 vs. 14.5 P <.01, Week 3 17.5 vs. 15.0 P < .01). The control group regained normal functional status by week 4; however Vitamin D3-injected rats did not return to baseline by the conclusion of the study (17.9 vs. 16.6, P < .01). Laser Doppler perfusion revealed decreased raw perfusion values pre-operatively (616 vs. 428, P < .01) and at weekly intervals beginning at week 2 in both the ischemic and non-ischemic limbs among animals with calcified arteries as compared to controls (Table 1). Conclusions: Vitamin D3 induced calcification is associated with decreased perfusion and worse functional recovery in a rat model of hindlimb ischemia. Future research aimed at understanding the relationship between vitamin D3, arterial calcification, and lower extremity ischemia may help to improve outcomes in patients with PAD.

Author Disclosure Block: S.L. Zettervall: None. S. Monk: None. X. Wang: None. Y. Cai: None. T. Lin: None. R.J. Guzman: None.

42

5:00 pm

The Myristolated Alanine-Rich C Kinase Substrate (MARCKS) Differentially Regulates Proliferation of Vascular Smooth Muscle Cells and Endothelial Cells through Affecting Protein Stability and Proteolytic Processing of the Kinase Interacting with Stathmin (KIS)

Thomas S. Monahan1, Dan Yu1, Charles Drucker2, Rajabrata Sarkar2, Dudley K. Strickland2 1Baltimore VA Medical Ctr, Baltimore, MD; 2University of Maryland Sch of Med, Baltimore, MD Introduction: Presently, the antiproliferative agents used in drug eluting stents and drug coated balloons inhibit both VSMC and endothelial cell (EC) proliferation, and thus these patients require dual antiplatelet therapy indefinitely. Identification of a VSMC-specific target to prevent proliferation represents a significant unmet clinical need. Previously we found that knockdown of MARCKS arrests VSMC proliferation through a p27kip1-dependent mechanism. Interestingly MARCKS knockdown increases EC proliferation. p27kip1 is phosphorylated by KIS allowing it to exit the nucleus and be degraded. Here we seek to understand how MARCKS influences KIS protein expression in these two cell types. Approach and Results: We performed siRNA-mediated knock down of MARCKS in human coronary artery endothelial cells (CAECs) and human coronary artery smooth muscle cells (CASMCs). MARCKS knockdown did not affect KIS mRNA expression as determined with RT-PCR in either cell type. KIS protein stability was evaluated in the presence of cyclohexamide with Western blot. In CAECs, MARCKS knockdown increased KIS stability, however, in CASMCs, MARCKS knockdown significantly decreased KIS protein stability. In CASMCs, MARCKS knockdown significantly increased KIS ubiquitinization where as in CAECs, MARCKS knockdown decreased KIS ubiquitinization. Interestingly, the well-studied functional domain of MARCKS(ED domain) is not directly involved in KIS regulation. MARCKS mutants (S4G and S4D) rescued proliferation in VSMCs. MARCKS knockdown in vivo in the murine femoral wire injury model resulted in decreased medial bromodeoxyuridine (BrdU) integration and neointima formation. MARCKS knockdown enhanced endothelial barrier function recovery four days after injury as assessed by Evans Blue integration. Conclusions: MARCKS differentially regulates the protein stability and proteolytic processing of KIS in VSMCs and ECs. The differential interaction of MARCKS and KIS likely explains the observed difference in proliferation observed with MARCKS knockdown in these two cell types. Author Disclosure Block: D. Yu: None. C. Drucker: None. R. Sarkar: None. D.K. Strickland: None. T.S. Monahan: None.

43

Poster Session

44

Human Umbilical Vein Endothelial Cell Conditioned Medium in the Presence of High Glucose Increases in vitro Adipose-derived Stem Cells Proliferation and in vivo Vascularization

Spencer Brown, Francis Caputo, Marc Fromer, Ping Zhang, Shauhoa Chang, Ashleigh Hagaman, Kiavash Koko, Ryan Nolan, Jeffrey Carpenter Cooper Univ Hosp, Camden, NJ Introduction: Diabetes type 1 and 2 cause hyperglycemia and result in endothelial dysfunction with endothelial vessel and poor wound healing. Adipose-derived stem cells (ASCs), progenitor cells in wound healing, show decreased function under hyperglycemic conditions in vitro and in vivo. We hypothesized that exposing ASCs in the presence of high glucose with the human umbilical vein endothelial cell (HUVEC) secretome will reverse the deleterious effects of glucose on ASCs and subsequently enhance angiogenesis and wound healing. Methods: Human umbilical vein endothelial cells (HUVEC) were treated with glucose (30mM) and the conditioned media (CM) were collected every 3 days. ASCs were then co-cultured with EC/CM for 2 weeks. To produce thermal denaturation of protein, EC/CM was heated at 950C for 30 mins. Cell activity, proliferation, and endothelial-like properties of ASCs were determined by MTT assays, growth curves, and real-time RT-PCR, respectively. EC/CM treated ASC were injected into a normal or diabetic murine left thigh muscle at three different points with hindlimb ischemia. After 4 weeks injection, animals were sacrificed. H & E and double immunostaining for CD31 and anti-human nuclei were used to determine if the ASCs primed with EC/CM underwent neovascularization. Results: In fact, ASCs increased in proliferation when co-cultured with HUVEC/CM (1.4 fold) when compared with controls. This promoting effect was lost in heated HUVEC/CM, indicating that the active molecules are of protein origin. After 10 days stimulated with EC/CM an increase in mRNA expression levels of EC markers were also observed in high glucose (30mM) EC/CM environment including CD31 (2-fold), vWF (1.1-fold), and eNOS (3.2-fold) when compared to ASCs cultured in M199. H & E and immunohistochemical staining results showed elevated vessel density and CD31+ cell levels in HUVEC-primed ASC injection sites of diabetic mice when compared with the control animals. Conclusions: HUVEC secrete protein factors that increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC-primed ASCs leads to improved angiogenesis. Author Disclosure Block: S. Brown: None. F. Caputo: None. M. Fromer: None. P. Zhang: None. S. Chang: None. A. Hagaman: None. K. Koko: None. R. Nolan: None. J. Carpenter: None.

45

High Glucose Pre-treatment Suppresses Rankl-induced Macrophage Activation via Down-regulation of Glucose Uptake through Decreased Insulin Receptor and Insulin Receptor Substrate 1 Expression

Chitaru Kurihara, Teruyoshi Tanaka, Dai Yamanouchi Univ of Wisconsin, Madison, WI

Introduction: Previous studies have suggested that the pathogenesis of abdominal aortic aneurysm (AAA) is associated with the local proteinases activation and the degradation of matrix proteins by matrix metalloproteinases (MMPs) produced from activated macrophages. One of the major features of diabetes mellitus (DM)-induced vascular pathology is severe arterial calcification. Although recent large epidemiological studies have shown that DM is an independent negative risk factor for AAA, the effects of hyperglycemia on macrophages are still controversial. We have hypothesized that hyperglycemia suppresses macrophage activation through altered glucose transportation.

Methods and Results: RAW264.7 cells, a murine macrophage cell line were cultured under high glucose conditions (HG group, 15.5 mM glucose) or normal glucose conditions (NG group, 5.5 mM glucose) for 7days. Cells from both groups were then transferred to normal glucose condition and stimulated with recombinant murine sRANKL. Macrophage activation, confirmed by TRAP staining positive cells, and MMP-9 expression were induced in NG group but were significantly suppressed in HG group. Glucose uptake was increased during osteoclastogenesis in NG group but not in HG group. To elucidate the underlying mechanism for this observation, we studied glucose transporters (GLUTs). Although GLUT-1 and GLUT-3 expression were not affected in either groups, the membrane translocation of GLUT-1 was significantly increased in NG group during macrophage activation but not in HG group. Insulin receptor and insulin receptor substrate-1 (IRS-1) mRNA, known to stimulate membrane translocation of GLUT, were both decreased in HG group compared to NG group.

Conclusions: Our results showed hyperglycemia suppresses macrophage activation. Our results also indicated that under normal conditions, recombinant murine sRANKL increases glucose uptake during macrophage activation. In contrast, this increase is impaired in high glucose pre-treated cells. We conclude that this impairment is due, in part, to suppressed GLUT-1 membrane translocation through down regulation of insulin receptor and IRS-1

Author Disclosure Block: C. Kurihara: None. T. Tanaka: None. D. Yamanouchi: None.

46

Androgen Deficiency-induced Hyperplasia Development is Attenuated by Physiological Testosterone Replacement Independent of Metabolite Dihydrotestosterone

Junior Univers, Brian M. Freeman, Deidra J. Mountain, Stacy S. Kirkpatrick, Joshua D. Arnold, Mitchell H. Goldman, Frederick A. Klein, Michael M. McNally, Scott L. Stevens, Paul D. Terry, Michael B. Freeman, Oscar H. Grandas UT Graduate Sch of Med, Knoxville, TN

Introduction: Androgen deficiency (AD) is associated with increased risk of cardio- and peripheral vascular disease, yet the underlying biochemical mechanisms remain unclear. Systemically testosterone (TST) is enzymatically reduced to its more potent metabolite dihydrotestosterone (DHT) or is aromatized to estradiol, which differentially stimulate androgen and estrogen receptor-mediated pathways, respectively. We have previously demonstrated an inverse relationship between TST levels and the cellular processes of intimal hyperplasia (IH) in vitro. Here we investigated TST and DHT replacement in the attenuation of IH in an in vivo model of AD.

Methods: Sub- to high physiologic levels of TST or DHT was administered via pellet implants in aged orchiectomized rats (0.5-5mg). Young intact (YI), aged intact (AI), and orchiectomized placebo (Plac) rats served as controls. After 14d hormone replacement rats underwent balloon angioplasty of the left common carotid. 14d post-injury animals were euthanized, systemic hormone levels were determined by ELISA and comparative weight analysis of androgen sensitive organs (Table 1), and carotid intima:media (I:M) was quantified. Results: I:M was decreased in AI animals and with higher physiological TST replacement compared to YI controls (Fig 1). I:M was higher in Plac, sub- and low-physiological TST animals and at all DHT levels.

Conclusions: Aging and the normal reduction of TST was protective against IH when compared to young animals. However, pathological AD and sub-physiological hormone replacement increased IH. While physiological TST replacement attenuated this effect, equivalent DHT replacement was not protective, but instead exacerbated the hyperplastic response. Future studies will investigate if the protective effect of physiological TST replacement could be via its conversion to estradiol and downstream estrogen receptor signaling and if estrogen therapy attenuates IH in AD males.

Author Disclosure Block: J. Univers: None. B.M. Freeman: None. D.J.H. Mountain: None. S.S. Kirkpatrick: None. J.D. Arnold: None. M.H. Goldman: None. F.A. Klein: None. M.M. McNally: None. S.L. Stevens: None. P.D. Terry: None. M.B. Freeman: None. O.H. Grandas: None.

47

Doxycycline Inhibits Revascularization after Hindlimb Ischemia Galit Ankri- Eliahoo, George Fu, Richard Kenagy, Timothy Cox, Gale L. Tang Univ of Washington, Seattle, Seattle, WA

Objectives: p27kip1, a gene affecting human response to arterial injury, affects collateralization after hindlimb ischemia (femoral artery ligation), possibly by regulating matrix metalloproteinase 2 (MMP2). We hypothesized that MMP2 mRNA expression would increase significantly more in ko mice after hindlimb ischemia and that MMP inhibition would affect p27-/- (ko) collateralization less than p27+/+ (wt).

Methods: Ko and wt mice had their femoral and collateral arteries harvested seven days after hindlimb ischemia for RNA isolation and qRT-PCR. Ko and wt mice were also fed chow without or with doxycycline (a broad spectrum MMP inhibitor) and subjected to hindlimb ischemia. The mice were followed with weekly footpad laser Doppler perfusion imaging for 28 days, and then sacrificed for microCT scanning. Vascular smooth muscle cells (VSMC) isolated from either wt or ko aortae were used in Boyden chamber migration assays without and with an MMP2 specific inhibitor.

Results: MMP2 mRNA expression increased significantly more in ko collaterals than wt collaterals (312% vs. 50%, respectively). However, blood flow recovery in ko and wt mice treated with doxycycline decreased to a similar extent (27±2.5% and 33±1% decrease respectively), although ko mice still revascularized better than wt mice (p < 0.01). Gracilis collateral diameters increased less after doxycycline treatment in both groups compared to mice fed regular chow (78.5±12 and 80±7 vs 98±18 and 106±17 μm, respectively). The bridge collateral diameters increased less in ko mice fed doxycycline (66±32 vs 158±18.3 µm), and no bridge collaterals were detected in wt mice fed doxycycline. Wt mice fed regular chow had rare bridge collaterals. Ko VSMC migration was less affected by MMP2 inhibition than wt (29% less migration vs 48% less migration, p<0.01).

Conclusions: MMP2 mRNA expression increased significantly more in p27 ko collaterals after hindlimb ischemia. The non-specific MMP inhibitor doxycycline inhibited revascularization after hindlimb ischemia, independent of p27 expression. p27 ko mice still revascularized better than wt mice after doxycycline treatment, suggesting that p27 has additional effects other than regulating MMP expression in the collateralization response.

Author Disclosure Block: G. Ankri- Eliahoo: None. G. Fu: None. R. Kenagy: None. T. Cox: None. G. Tang: None.

48

NETosis is Associated with Abdominal Aortic Aneurysm Rupture

Sean J. English1, Hassan Albadawi1, Hyung-Jin Yoo1, Kimberly Martinod2, Jose A. Diaz3, Akshaya Meher4, Gilbert R. Upchurch Jr4, Denisa Wagner2, Michael T. Watkins1 1Massachusetts General Hosp, Boston, MA; 2Boston Children's Hosp, Boston, MA; 3Univ of Michigan Health System, Ann Arbor, MI; 4Univ of Virginia, Charlottesville, VA;

Introduction: Abdominal aortic aneurysm (AAA) rupture (RAAA) is associated with high mortality. Intraluminal thrombus and associated inflammation have been implicated in RAAA. We sought to characterize neutrophil extracellular trap (NET) formation, or NETosis, associated with a rat RAAA model that we previously developed.

Methods: To stimulate AAA formation, infrarenal abdominal aortas of male SD rats were infused with porcine pancreatic elastase (PPE): active (AAA, n=21) and heat-inactivated (control, n=14). Aortas were harvested 3, 6, and 14d post PPE exposure. To stimulate RAAA, a separate group of animals was administered β-aminopropionitrile (BAPN, n=12) daily. Those animals that did not rupture (NRAAA-14d, n=6) were harvested 14d post PPE exposure. Paraffin embedded sections were analyzed using immunofluorescence. To evaluate evidence of primarily neutrophil activity and NET generation, myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4) were quantified utilizing Cy3 and Alexa-488 secondary antibodies, respectively. To determine the presence of NETs, citrullinated histone H3 (H3Cit, Cy3) was visualized. DAPI was utilized to detect nuclei. Aortic sections were imaged with fluorescent microscopy, and quantification of percent area stained (PAS) was performed by threshold analysis.

Results: Greater MPO PAS was observed for AAA-6d compared with control-6d (p=0.03). Greater PAD4 PAS was observed for AAA-3d (p=0.02) and -6d (p=0.05) compared with control-3d and -6d, respectively. Greater H3Cit PAS was observed for AAA-6d (p<0.0001) and -14d (p=0.001) compared with control-6d and -14d, respectively. In the setting of rupture stimulation, greater H3Cit PAS was observed for RAAA compared with AAA-14d (p=0.001) and NRAAA-14d (p=0.0002).

Conclusions: Increased PAD4 expression by AAAs compared with controls at 3 and 6d post PPE exposure, results in increased H3Cit expression at 6 and 14d post PPE exposure. Comparable H3Cit expression by AAA-6d and RAAA demonstrates the acute nature of this rupture model; however, greater H3Cit expression by RAAAs suggests a role for NETosis in RAAA. Further evaluation of NETosis as it pertains to human RAAA is necessary. Therapeutic inhibition of NETosis may slow AAA progression and prevent RAAA.

Author Disclosure Block: S.J. English: None. H. Albadawi: None. H. Yoo: None. K. Martinod: None. J.A. Diaz: None. A. Meher: None. G.R. Upchurch: None. D. Wagner: None. M.T. Watkins: None.

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Low Plasma Mir-155 Levels and RhoA Activation Correlates with Small AAA Expansion

Eugene S. Lee1, Anthony Nguyen1, Paramita Ghosh1, Angelica Rona1, Arash Afkhami2 1Univ of California, Davis, Sacramento, CA; 2Sacramento VA Medical Ctr, Mather, CA Introduction: Given increased abdominal imaging studies, the incidental diagnosis and treatment of small AAAs is problematic. Presently, no specific recommendations for medical management can be made other than serial observation. The ras homolog member A (RhoA) signaling cascade is implicated in vascular diseases due to its ability to modulate monocyte endothelial invasion. We sought to investigate the RhoA signaling pathway in AAA expansion, along with an endogenous inhibitor of RhoA, microRNA-155 (miR-155). Hypothesis: We hypothesize that RhoA is activated to enable AAA expansion, but once expanded, miR-155 is called on to inhibit RhoA activation so that the expanded AAA can be stabilized. Methods: Peripheral blood was collected for monocyte and plasma isolation from stable AAA (< 0.1 cm/year) (n=9) and expanding AAA (≥ 0.1 cm/year) (n=10) patients. Rhotekin-RBD assay and western blots were used to assess relative Rho-GTP expression and quantitative PCR for miR-155 detection in blood plasma. T-test and χ2 analysis were used to determine statistical significance. Results: Given limited monocytes, 6 stable and 10 expanding AAA patients were identified for RhoA analysis. Monocytes from expanding AAA patients contained higher active Rho-GTP protein levels compared to stable AAA monocytes (Figure 1a: 1.60±.075 vs 1.20±.088, Ρ=.0004). Plasma concentrations of miR-155 were significantly lower in expanding AAA plasma compared to stable AAAs (Figure 1b: -2.17±.95, P=.005). Conclusions: Expanding AAA patients have greater monocyte activation of RhoA and decreased plasma expression of miR-155, when compared to stable AAA patients. The RhoA signaling pathway has altered activation in AAA disease and its regulation may reduce expansion rates and inflammation.

Author Disclosure Block: E.S. Lee: Research Grant; Modest; Medtronic Corporation. A. Nguyen: None. P. Ghosh: None. A. Rona: None. A. Afkhami: None.

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Increased Dynamic Mechanical Energy Dissipation in Human Abdominal Aortic Aneurysm Doran S. Mix, Ibrahima Bah, Sandra A. Toth, Michael C. Stoner, Adam J. Doyle, Jennifer L. Ellis, Roan Glocker, Mark R. Buckley, Michael Richards Univ of Rochester Medical Ctr, Rochester, NY Objectives: Individualized patient rupture risk for abdominal aortic aneurysms (AAA) remains elusive due to a limited understanding of the biomechanical events that trigger aneurysm growth and aortic wall failure. To date there has been a paucity of data describing how physiologic pulsatile energy is stored (E') and lost (E'') from AAA tissue. Our hypothesis is that AAA tissue dissipates more cyclic energy at a physiologic frequency, as determined by (E''/ E'), when compared to healthy aortic tissue. Methods: Human healthy aortic and AAA samples were obtained from cadaveric and surgical specimens. Specimens were stored at 4oC in 0.9%NS and mechanically tested within 36 hrs of explant. Uniaxial mechanical testing (ADMET BioTense) was performed in the circumferential orientation with the tissue pre-loaded to an equivalent physiologic stress of a 5 cm at a 110 mmHg mean pressure. A sinusoidal ±5% strain was applied at 1 Hz for 40 cycles with simultaneous force measurements. After mechanical testing, immunohistochemical staining was performed to confirm tissue viability. Results: AAA tissue was significantly stiffer when compared to healthy aorta, as demonstrated by a greater average static modulus (E) 1555.4 ± 384 vs. 970 ± 128 kPa (n=5, p=0.03). Dynamic testing of the AAA tissue noted a significantly greater energy loss (E'') 137.8±36.7 vs. 43.1±21.7 (p<0.01) and loss ratio (E''/ E') 0.090 ± 0.023 vs. 0.044 ± 0.023 (p=0.02), when compared to normal specimens. Figure #1 compares the static modulus (E) to the loss ratio (E''/ E') for the aortic tissue specimens. Histologic analysis confirmed tissue viability during of all specimens. Conclusions: Our data demonstrates that AAA tissue dissipates more energy (E'') and has a greater energy loss ratio (E''/ E'), suggesting that more pulsatile energy is dissipated in diseased tissue. Future work is needed to determine how this energy dissipation influences the biologic pathogenesis of AAA growth and rupture.

Author Disclosure Block: D.S. Mix: None. I. Bah: None. S.A. Toth: None. M.C. Stoner: None. A.J. Doyle: None. J.L. Ellis: None. R. Glocker: None. M.R. Buckley: None. M. Richards: None.