TABLE OF CONTENTS

page

Statement of ISACB Mission; Executive Committee and Advisory Board Members...... 2

Letters from the ISACB Program and Local Arrangements Chair and ISACB President...... 3

General Information And Program Summary University College, London Map...... 4-5

ISACB 13th Biennial Meeting Schedule at a Glance...... 6

ISACB 13th Biennial Meeting Satellite Symposium...... 7

ISACB 13th Biennial Meeting Program...... 8-12

Young Investigator Awards...... 13

Oral Presentation Abstracts: Session 1...... 14-16

Oral Presentation Abstracts: Session 2...... 17-19

Oral Presentation Abstracts: Session 3...... 20-21

Oral Presentation Abstracts: Session 4...... 22-24

Oral Presentation Abstracts: Session 5...... 25

Posters Presentation Abstracts...... 26-55

ISACB New Membership...... 56

Notes...... 57

ISACB’S 2012 BIENNIAL MEETING 1 THE INTERNATIONAL SOCIETY FOR APPLIED CARDIOVASCULAR BIOLOGY

Provides a forum for communicating issues related to the translation of innovative cardiovascular research from the laboratory to the bedside; — Uniquely nurtures strong face-to-face communication and is a most effective alliance among academic scientists, clinicians and industry-based scientists; — Serves as a key forum for discussion of solutions to difficult clinical, scientific and technical challenges engendered by the validation of the efficacy and safety of diagnostic imaging and laboratory tests, and therapeutic procedures and devices, including advanced biomaterials, endovascular techniques, stents, and vascular grafts, valves, and cardiac assist and other innovative devices.

International Society for Applied Cardiovascular Biology Executive Committee – 2010/2012

OFFICERS

President Secretary-Treasurer Howard P. Greisler, M.D. David Vorp, Ph.D. Maywood, Illinois, USA Pittsburgh, Pennsylvania, USA —

Elena Aikawa, M.D., Ph.D. Steven Schmidt, Ph.D. Boston, Massachusetts, USA Akron, Ohio, USA

Masanori Aikawa, M.D., Ph.D. Frederick J. Schoen, M.D., Ph.D. Boston, Massachusetts, USA Boston, Massachusetts, USA

Julie Campbell, M.D. David Williams, Ph.D. Brisbane, Australia Liverpool, U.K.

Elliot Chaikof, M.D., Ph.D. ADVISORY BOARD Boston, Massachusetts, USA Teddy Fishlein, M.D. Adrian Chester, M.D. Nuremberg, Germany Harefield, England, U.K. Manfred Deutsch, M.D. Arthur Coury, Ph.D. Vienna, Austria Boston, Massachusetts, USA Roland Fasol, M.D. Simon Hoerstrup, M.D., Ph.D. Gobelsburg, Austria Zurich, Switzerland Bo Risberg, M.D., M.B.A. Robert M. Nerem, Ph.D. Gothenburg, Sweden Atlanta, Georgia, USA Bauer Sumpio, M.D., Ph.D. Teruo Okano, Ph.D. New Haven, Connecticut, USA Tokyo, Japan Magdi Yacoub, M.D. Harefield, Middlesex, United Kingdon

2 ISACB.ORG MESSAGES FROM ISACB

Dear Members Dear Colleagues, and Friends of Welcome to the International Society for Applied the International Cardiovascular Biology’s (ISACB) 13th Biennial Society for Applied Meeting in London, England. I would like to Cardiovascular Biology, acknowledge the ISACB Program Committee and Welcome to the ISACB’s 13th Local Arrangement Committee for their hard work in Biennial Meeting in organizing this extraordinary meeting. Without their post-Olympic London! efforts, this event would not have been possible. The meeting is situated The conference will focus on five critically important entirely within Bloomsbury, areas of active and evolving research in translational an historic and elegant area of central London. cardiovascular biology, with a full session devoted to each. The session Bloomsbury is characterized by its Georgian and themes are 1) Molecular and Cardiovascular Imaging, 2) Developmental Victorian architecture arranged around beautiful Cardiovascular Biology in Children and Adults, 3) Cell-Cell and Cell- mature city gardens, and an ‘intellectual’ past and Matrix Interactions in Cardiovascular Biology, 4) Emerging Medical present, for example containing University College Technologies in Cardiovascular Repair and Therapeutics – Proteomics London, the British Museum, and the houses of and Epigenetics and 5) Critical Issues in Tissue Engineering: Construct Charles Dickens and Virginia Woolf. Vascularization and Scaffold-Generated Signaling. Each of the invited On Wednesday afternoon there will be a Satellite speakers in the scientific program, 3-4 per session, is an expert in his/ Meeting hosted by the Royal Free London Hospital her field and the invited talks are accompanied by 21 papers selected in the Anatomy Department of UCL with the Keynote by the Program Committee from proffered abstracts detailing innovative Lecture “Self- Assembled ex-vivo Engineered research. Additionally, two exciting panel discussions have been Cardiovascular Tissues to Study Mechanisms of organized – one on Friday addressing Regulatory Issues and Evaluation Disease and Drug Toxicity” given by Kevin Parker at Strategies for the Next Generation of Cardiovascular Devices and the 6:00 p.m. Directly following this the Reception begins other on Saturday focused on Grand Challenges in Applied Cardiovascular a short stroll away in UCL’s historic Portico. Biology. A dedicated and manned poster session on Saturday morning will include poster presentations based on proffered abstracts accepted The main ISACB Meeting starts on Thursday by the Program Committee based on scientific merit and is an important morning a short distance away in the Kennedy component of the program. I strongly encourage all participants to view Lecture Theatre complex within UCL’s Institute of the posters, which will be up throughout the meeting and to address Child Health. The scientific focus of the meeting discussion of the posters to the authors at the dedicated poster session. will be on Molecular and Cardiovascular Imaging, An exciting Satellite Meeting on Wednesday entitled Genetics, Metabolism Developmental Cardiovascular Biology in Children and Repair in Cardiovascular Disease has been organized by Janice Tsui and Adults, Lifetime Management of Arterial and George Hamilton and colleagues from the University College London Disease, Cell-Cell and Cell-Matrix Interactions in in coordination with the ISACB. Cardiovascular Biology, Critical Issues in Tissue Engineering, Biomaterials in the Development of ISACB and its Biennial Meetings are organized to bring together experts Small Diameter Vascular Conduits and Heart Valves in applied cardiovascular biology from wide and often seemingly Emerging Medical Technologies in Cardiovascular disparate backgrounds including academia, industry and government. Repair and Therapeutics – Proteomics and We strive to promote dialogue and collaborations between clinicians and Epigenetics, and Inflammation, Adaptive Immunity scientists, recognized scientific leaders and trainees, and among cell and and Hypertension. There will a mix of invited molecular biologists, pathologists, engineers, biomaterials researchers, internationally recognized experts, scientific surgeons, radiologists and physicians and others whose work addresses presentations and a Dedicated Poster Session. fundamental aspects of applied cardiovascular biology. While each of these groups makes important contributions to the field, ISACB believes As always the ISACB aims to foster knowledge that only the integration of these individual scientific arenas can ultimately transfer by keynote lectures and updates, cross- generate profound advances in our understanding of the pathobiology and fertilisation of ideas and disciplines, scientific the development of novel efficacious diagnostic therapeutic modalities. and clinical interaction to the advancement of While the ambitious program is built around lectures from carefully international research in cardiovascular disease. selected speakers, the success of the meeting is dependent upon free The faculty is truly international and leading edge flowing thought-provoking discussion and debate among all in attendance in their fields. The Society’s aim is also to foster and the generation of cross fertilizing new collaborations and new friendship and the venue is particularly well adapted synergies that generate improved therapies for heart and vascular disease. to facilitate this. All of the venues and hotels are situated within Bloomsbury and within walking London is a dynamic city and Bloomsbury is centrally situated making distance. There are many beautiful gardens, places exploration of the city quite accessible. Bloomsbury has a remarkable of interest, old pubs and a host of affordable literary heritage and the British Museum is a short walk away from the restaurants reflecting almost every cuisine. conference venue. The facilities of the recently concluded Olympic games are a short tube ride away. I hope you will find time before or after the We look forward to welcoming you to Bloomsbury, conference to explore this exciting city. and thank you for your continued support for ISACB in its mission to enhance the discovery Thank you for participating in this exciting conference. The ISACB and development of cardiovascular research has many new programs in development and I strongly encourage all and technology. participants to remain active in this extraordinary Society. George Hamilton Howard P. Greisler, M.D. ISACB LOCAL PRESIDENT ARRANGEMENTS CHAIR

ISACB’S 2012 BIENNIAL MEETING 3 GENERAL INFORMATION AND PROGRAM SUMMARY

Biennial Meeting Objectives Registration ISACB works to create a network for scientists and Registration for the 13th Biennial Meeting of the clinicians in academia, industry and government who International Society for Applied Cardiovascular Biology wish to share their discoveries among each other, will take place in front of the lecture theatre (please see as well as with other industry professionals and map below). government regulatory officials. ISACB’s meetings foster Registration Hours: these connections through thought-provoking speakers and lectures on groundbreaking topics. Biennial Thursday, September 13 08:00 – 17:00 meetings provide an unusual opportunity for interaction Friday, September 14 08:00 – 16:00 between internationally renowned leaders in applied Saturday, September 15 08:00 – 14:00 cardiovascular biology and industry leaders in the fields of tissue engineering, implantable cardiovascular Registration for this meeting includes: devices, endovascular technologies, diagnostic technologies, biomaterials, cell and molecular biology, • Welcome reception pathology, pharmacology and non-invasive and • Entrance to scientific sessions and exhibits histologic and ultrastructural imaging. • Coffee Breaks on Thursday, Friday, and Saturday • Lunch on Thursday, Friday, and Saturday Venue and Official Language Lectures for this meeting will be held at the Kennedy Tickets for the Friday evening banquet may be Lecture Theatre at the University College, London, purchased, subject to availability, at the Registration United Kingdom. The official language of the meeting is desk. Payment for registration may be made in English. U.S. Dollars (cash or check) or via credit card — MasterCard, Visa, or American Express by September 14, 2012 at 11:00.

Lost and Found The Lost and Found for this meeting will be located at Registration.

MAP OF INSTITUTE OF CHILD HEALTH

4 ISACB.ORG MAP OF UCL CAMPUS

ISACB’S 2012 BIENNIAL MEETING 5 ISACB 13TH BIENNIAL MEETING SCHEDULE AT A GLANCE

September 12-15, 2012

12:10 - 13:10 15:15 - 15:40 Wednesday, September 12 Luncheon Coffee Break Winter Garden and Balcony Winter Garden and Balcony 10:00 - 12:30 Executive Council Meeting 13:10 - 17:00 Session 2: 19:00 - 22:30 Cruciform B.01 Developmental Cardiovascular Banquet University College, London Biology in Children and Adults Goodenough Club Session Cosponsored by the 23 Mecklenburgh Square 13:30 - 17:00 Society for Cardiovascular Pathology London WC1 N 2AD Satellite Meeting JZ Young Lecture Theatre University College, London University College, London Kennedy Lecture Theatre Saturday, September 15 Anatomy Building, Bloomsbury Campus, London 15:00 - 15:25 Coffee break 08:00 – 14:00 Winter Garden and Balcony Registration 18:00 – 19:00 Second Floor Foyer ISACB Meeting 17:00 Institute for Child Health Keynote Lecture Dinner on your own 30 Guilford Street JZ Young Lecture Theatre London, WC1N 1EH Friday, September 14 19:00 – 21:00 08:00 - 10:00 Welcome Reception 08:00 – 16:00 Dedicated Poster Session and University College, London Continental Breakfast Wilkins Building Portico Registration Second Floor Foyer Levinsky Room and Institute for Child Health Philip Ullman Room Thursday, September 13 30 Guilford Street London, WC1N 1EH 10:00 - 10:30 08:00 – 17:00 08:15 - 12:45 ISACB Business Meeting Registration Session 3: Cell-Cell and University College, London Second Floor Foyer Cell-Matrix Interactions in Kennedy Lecture Theatre Institute for Child Health Cardiovascular Biology 30 Guilford Street University College, London 10:30 - 14:30 London, WC1N 1EH Kennedy Lecture Theatre Session 5: Critical Issues in Tissue 10:05 - 10:30 Engineering: Construct 08:00 – 12:00 Coffee Break Vascularization and Poster Set Up Winter Garden and Balcony Levinsky Room and Scaffold-Generated Signalling Philip Ullman Room 11:35 - 12:45 University College, London Keynote Lectures Kennedy Lecture Theatre 08:30 - 8:35 and discussion Welcome and Introduction 12:05 - 12:30 University College, London 12:45 - 13:45 Boxed Lunch Kennedy Lecture Theatre Luncheon Winter Garden and Balcony Winter Garden and Balcony 08:35 - 12:10 14:45 Session 1: Molecular and 13:45 - 17:05 Meeting Adjourned Cardiovascular Imaging Session 4: Emerging Medical University College, London Technologies in Cardiovascular Kennedy Lecture Theatre Repair and Therapeutics – Proteomics and Epigenetics 10:25 - 10:50 University College, London Coffee Break Kennedy Lecture Theatre Winter Garden and Balcony

6 ISACB.ORG ISACB 13TH BIENNIAL MEETING SATELLITE SYMPOSIUM

4th UCL-Royal Free International Cardiovascular Diseases Workshop ‘Genetics, metabolism & repair in cardiovascular disease’ September 12, 2012 • UCL Anatomy Building, Bloomsbury Campus, London Hosted by the Royal Free Vascular Unit

Wednesday, September 12, 2012 Session 2 13:30 J Z Young Lecture Theatre Registration & Coffee Gavin de Beer Lecture Theatre 16:00 The pathogenesis of abdominal aortic aneurysms 13:50 Dr Gillian Cockerill, Welcome St George’s London Professor Mark Emberton, J Z Young Lecture Theatre 16:30 Unexpected roles of reactive oxygen Session 1 species in the stressed and failing heart J Z Young Lecture Theatre Professor Ajay Shah, King’s College London 14:00 Translational opportunities in 17:00 cardiovascular genomics Close of satellite meeting Professor Aroon Hingorani, Professor George Hamilton University College London

14:30 Cardiac progenitor cells in the adult myocardium Professor Michael Schneider, Imperial College London

15:00 Protecting the ischaemic myocardium Professor Michael Marber, King’s College London

15:30 Coffee Break Gavin de Beer Lecture Theatre

ISACB’S 2012 BIENNIAL MEETING 7 ISACB 13TH BIENNIAL MEETING PROGRAM

September 12-September 15, 2012

08:45 - 09:20 Wednesday, September 12, 2012 Molecular Targets for Cardiovascular Imaging Thomas F. Lüscher, M.D., Professor and Chairman of 10:00 - 12:30 Executive Council Meeting Cardiology, University Hospital Zurich Cruciform B.01 09:20 - 09:35 ABSTRACT 13:30 - 17:00 Satellite Meeting Visualization of Flow-Induced ATP Release and J.Z. Young Lecture Theatre Ca2+ Waves In Vascular Endothelial Cells University of College, London Joji Ando, Laboratory of Biomedical Engineering, Dokkyo Anatomy Building, Bloomsbury Campus, London Medical University, Tochigi, Japan; Kimiko Yamamoto, Department of Biomedical Engineering, University of Tokyo, 18:00– 19:00 Keynote Lecture Tokyo, Japan Micro Models of Cardiovascular Function J.Z. Young Lecture Theatre 09:35 – 09:50 ABSTRACT University College, London Spatio-Temporal Mapping of Elastin Remodeling Anatomy Building, Bloomsbury Campus, London in Experimental Abdominal Aortic Aneurysms Kevin Parker, Ph.D., Tarr Family Professor of Bioengineering (AAAs) Towards Induced Regenerative Elastic and Applied Physics; Wyss Institute for Biologically-Inspired Matrix Repair Utilizing Resident Elastogenic Cells Engineering; Harvard Stem Cell Institute Partha Deb, Department of Biomedical Engineering, The Cleveland Clinic, Cleveland, OH, USA; Anand Ramamurthi, 19:00 – 21:00 Welcome Reception Department of Biomedical Engineering, The Cleveland Clinic, University College, London Cleveland, OH, USA Wilkins Building Portico 09:50 - 10:25 Imaging Macrophage Biology in Cardiovascular Disease Thursday, September 13, 2012 Masanori Aikawa, M.D., Ph.D., Principal Investigator, Center for Excellence in Vascular Biology, Brigham and Women’s 08:00 – 17:00 Registration Hospital, Harvard Medical School Second Floor Foyer 10:25 - 10:50 Coffee break 08:00 – 12:00 Poster set-up Winter Garden and Balcony Levinsky Room and Philip Ullman Room 10:50 - 11:25 08:30 - 08:35 Welcome and Introduction Optical Coherence Tomography (OCT) University College, London for Cardiovascular Diagnosis Kennedy Lecture Theatre Guillermo Tearney M.D., Ph.D., Professor of Pathology, Harvard Remarks provided by Howard Greisler, M.D., Professor, Medical School Department of Surgery and Department of Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, 11:25 - 11:40 ABSTRACT President, ISACB Inflammation-dependent Mechanism of Calcification: A New Role for Macrophage 08:35 - 12:10 SESSION 1 Matrix Vesicles Molecular and Cardiovascular Imaging Sophie New, Claudia Goettsch, Masanori Aikawa, Julio University College, London Meirelles Marchini, Brigham and Women’s Hospital, Harvard Kennedy Lecture Theatre Medical School; Catherine Shanahan, King’s College, London, UK; Kevin Croce, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; Elena Aikawa, Brigham and 08:35 - 08:45 Session Introduction Women’s Hospital, Harvard Medical School, Boston, MA, USA Session Chairs: Masanori Aikawa, Ph.D., Thomas F. Lüscher, M.D.

8 ISACB.ORG ISACB 13TH BIENNIAL MEETING PROGRAM

11:40 – 11:55 ABSTRACT 14:10 – 14:25 ABSTRACT Polyethyieneimine (PEI) Sirna Complexes The Role of Dll4-Notch Signaling in Shared Released From Coated Electrospun Polyethylene Mechanisms for Atherosclerosis and Terephthalate (PET) Bypass Graft Materials Metabolic Disorders Facilitate Gene Silencing in Infiltrating Primary Daiju Fukuda, Cardiovascular Division, Department of Human Aortic Smooth Muscle Cells Medicine, Brigham and Women’s Hospital, Harvard Medical Christoph S Nabzdyk, Tufts Medical Center, Department of School; Tetsuro Miyazaki, Cardiovascular Division, Department Surgery, Tufts University School of Medicine, Boston, MA, Beth of Medicine, Brigham and Women’s Hospital, Harvard Medical Israel Deaconess Medical Center, Harvard Medical School, School; Kunio Morishige, Cardiovascular Division, Department Boston, MA; Maggie Chun, Beth Israel Deaconess Medical of Medicine, Brigham and Women’s Hospital, Harvard Medical Center, Harvard Medical School, Boston, MA; Hunter Oliver- School; Elena Aikawa, Cardiovascular Division, Department Allen, Beth Israel Deaconess Medical Center, Harvard Medical of Medicine, Brigham and Women’s Hospital, Harvard School, Boston, MA; Matthew D. Phaneuf, Biosurfaces Inc., Medical School; Masanori Aikawa, Cardiovascular Division, Ashland, MA; Saif G Pathan, Biosurfaces Inc., Ashland, MA; Department of Medicine, Brigham and Women’s Hospital, Jin-Oh You, Harvard University, School of Engineering and Harvard Medical School Applied Sciences, Boston, MA; Leena Pradhan, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 14:25 - 15:00 MA; Frank W LoGerfo, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Notch Signaling in Cardiovascular Development and Pathology Holger Gerhardt, Ph.D., VIB Vesalius Research Center, 11:55 - 12:10 ABSTRACT Katholieke Universiteit Leuven, Belguim In-Situ Capture Technology of Endothelial Progenitor Cells (EPCs) 15:00 - 15:25 Coffee break Takehisa Matsuda, Kanazawa Institute of Technology, Hakusan, Japan; Shu Takabatake, Kanazawa University Medical School, Winter Garden and Balcony Kanazawa, Japan; Masakazu Yamagishi, Kanazawa University Medical School, Kanazawa, Japan 15:25 - 15:40 ABSTRACT Engraftment of Human MSCs as Perivascular 12:10 - 13:10 Luncheon Cells of Bioengineered Microvessels Enhances Winter Garden and Balcony Mesenchymal Tissue Formation Ruei-Zeng Lin, Department of Cardiac Surgery, Children’s Hospital Boston, and Harvard Medical School, Boston, 13:10 - 17:00 SESSION 2 MA; Arin K. Greene, Department of Plastic & Oral Surgery, Developmental Cardiovascular Children’s Hospital Boston, Boston, MA; Juan M. Melero- Biology in Children and Adults Martin, Department of Cardiac Surgery, Children’s Hospital University College, London Boston, and Harvard Medical School, Boston, MA Kennedy Lecture Theatre Session Cosponsored by the Society for 15:40 - 15:55 ABSTRACT Cardiovascular Pathology The Roles of Various Metabolic Enzymes in Diabetic Cardiomyopathy – In Vivo and In Vitro 13:10 - 13:20 Session Introduction Approach Session Chairs: Thomas Larrew, Clemson University, Clemson, SC; James Diego Franco, Ph.D., Fred Schoen, M.D., Ph.D. Chow, Clemson University, Clemson, SC; Jason Schulte, Clemson University, Clemson, SC; Dan Simionescu, Clemson University, Clemson, SC; Agneta Simionescu, Clemson 13:20 - 13:55 University, Clemson, SC Genetics of Cardiovascular Development Diego Franco, Ph.D., Department of Experimental Biology, 15:55 – 16:10 ABSTRACT Cardiovascular Development Group, University of Jaen, Spain Human Calcified Valves Demonstrate an Atypical Expression of Smooth Muscle Cells and their 13:55 - 14:10 ABSTRACT Transcription Factors Biochemical and Spatial Control of Human Najma Latif, Harefield Heart Science Centre, Imperial College, Pluripotent Stem Cell Mesoderm Formation Harefield, Middx, UK; Padmini Sarathchandra, Harefield Heart Oscar J. Abilez, Stanford University, Stanford, CA; Frank Science Centre, Imperial College, Harefield, Middx, UK; Magdi Myers, University of California, Berkeley, Berkeley, CA; Jason H. Yacoub, Harefield Heart Science Centre, Imperial College, Silver, University of California, Berkeley, Berkeley, CA; Luke Harefield, Middx, UK; Adrian H Chester, Harefield Heart P. Lee, University of California, Berkeley, Berkeley, CA; Science Centre, Harefield, Imperial College, Middx, UK Christopher K. Zarins, Stanford University, Stanford, CA

ISACB’S 2012 BIENNIAL MEETING 9 ISACB 13TH BIENNIAL MEETING PROGRAM

16:10 – 16:25 ABSTRACT and Emory University, Biomedical Engineering, Atlanta, GA; Hemodynamics of the Transitional Aortic Arch Rudy L. Gleason, Georgia Institute of Technology, School of Patterning at a Key Embryonic Stage Mechanical Engineering, Atlanta, GA William J. Kowalski, Carnegie Mellon University, Pittsburgh, PA; Onur Dur, Carnegie Mellon University, Pittsburgh, PA; Yajuan 09:15 – 09:30 ABSTRACT Wang, Carnegie Mellon University, Pittsburgh, PA; Joseph Varying Effects of Different Types of P. Tinney, Cardiovascular Innovation Institute, University of Hemodynamic Forces on Tissue Factor (TF) Louisville, Louisville, KY; Bradley B. Keller, Cardiovascular Expression in Endothelial Cells (EC) Innovation Institute, University of Louisville, Louisville, KY; Ryuzo Abe, Yale University School of Medicine, New Haven, Kerem Pekkan, Carnegie Mellon University, Pittsburgh, PA CT; Norio Yamashita, Yale University School of Medicine, New Haven, CT; Adrienne Rochier, Yale University School of 16:25 - 17:00 Medicine, New Haven, CT; Alexander Nixon, Yale University Role of Developmental Programs in Heart Valve School of Medicine, New Haven, CT; Rei Abe, Yale University Growth, Aging, Disease and Tissue Engineering School of Medicine, New Haven, CT; Takeshi Moriguchi, Yale University School of Medicine, New Haven, CT; Bauer E. Frederick J. Schoen, M.D., Ph.D., Pathologist, Brigham Sumpio, Yale University School of Medicine, New Haven, CT; and Women’s Hospital; Professor of Pathology and Health Shigeto Oda, Chiba University Graduate School of Medicine, Sciences and Technology, Harvard Medical School; Executive Chiba, Japan; Hiroyuki Hirasawa, Chiba University Graduate Vice Chairman, Department of Pathology, Brigham and School of Medicine, Chiba, Japan Women’s Hospital, Boston, Massachusetts

17:00 Dinner on your own 09:30 – 10:05 Role of TGFβ Receptors in Cardiovascular Development and Disease Friday, September 14, 2012 Helen Arthur, Ph.D., Senior Lecturer, Institute of Human Genetics, Newcastle University, Tyne and Wear, United Kingdom 08:00 – 16:00 Registration Second Floor Foyer Institute for Child Health 10:05 - 10:30 Coffee break 30 Guilford Street Winter Garden and Balcony London WC1N 1EH, United Kingdom 10:30 - 11:05 08:15 - 12:45 SESSION 3 Microniche Control of Stem Cell Behavior Cell-Cell and Cell-Matrix Interactions in Samy Gobaa, Ph.D., Assistant Professor, Ecole Polytechnique Cardiovascular Biology Fédérale de Lausanne, Lausanne, Switzerland University College, London Kennedy Lecture Theatre 11:05 - 11:20 ABSTRACT A Novel Tissue-Engineered Devitalized Vascular 08:15– 08:25 Session Introduction Prosthesis Session Chairs: Sara Zanivan, Ph.D., Howard Greisler, M.D. Maxime Tondreau, LOEX/Laval University; Jean-Michel Bourget,LOEX/Laval University, Quebec, CN; Michel Fortin, 08:25 - 09:00 LOEX/Laval University, Quebec, CN; Carl Bouchard, LOEX/ Laval University, Quebec, CN; Dan Lacroix, LOEX/Laval Angiogenesis and Inosculation in Tissue University, Quebec, CN; François A. Auger, LOEX/Laval Engineering and Cardiovascular Pathobiology University, Quebec, CN Sara Zanivan, Ph.D., Vascular Proteomics Lab, Beatson Institute for Cancer Research, Glascow United Kingdom 11:20 - 11:35 ABSTRACT The Function of Shear-responsive and Side- 09:00 - 09:15 ABSTRACT dependent microRNA-486-5p in Aortic Valve Restrictive Remodeling in Response to Disturbed Endothelium Flow in Stiffened Carotid Arteries is Related to Casey J. Holliday-Ankeny, Georgia Institute of Technology Elastin Dysfunction in Fibulin V Knockout Mice and Emory University, Atlanta, GA; Randall F. Ankeny, Georgia Luke P. Brewster, Emory University School of Medicine. Institute of Technology, Atlanta, GA; Zannatul Ferdous, Georgia Institute of Technology, Institute of Bioengineering and University of Tennessee, Knoxville, TN; Robert M. Nerem, Bioscience, Atlanta, GA; Laura Hansen, Georgia Institute of Georgia Institute of Technology, Atlanta, GA; Hanjoong Jo, Technology, Department of Biomedical Engineering, Atlanta, Georgia Institute of Technology and Emory University, GA; Chanwoo Kim, Emory University, Biomedical Engineering, Atlanta, GA Atlanta, GA; William Wan, Georgia Institute of Technology, Mechanical Engineering, Atlanta, GA; Hiromi Yanagisawa, University of Texas Southwestern, Molecular Biology, Atlanta, GA; Hanjoong Jo, Georgia Institute of Technology

10 ISACB.ORG ISACB 13TH BIENNIAL MEETING PROGRAM

11:35 - 12:45 15:15 - 15:40 Coffee break Keynote Lectures and Discussion – Regulatory Winter Garden and Balcony Issues and Evaluation Strategies for the Next Generation of Cardiovascular Devices 15:40 - 16:15 Nicola Lennard, M.D., Deputy Medical Director, Transcatheter Treatment of Aortic Stenosis - Devices Directorate, MHRA and Bruce Campbell, M.S., Who benefits? Professor and Chairman, Interventional Procedures Advisory Michael Mullen, M.D., Consultant Cardiologist, the Heart Committee, National Institute for Health and Clinical Excellence, London, United Kingdom Hospital, London, United Kingdom

12:45 - 13:45 16:15 - 16:30 ABSTRACT Luncheon Sustained Release of Rapamycin from Winter Garden and Balcony Electrospun Polyurethane Vascular Grafts Jingjia Han, Drexel University, School of Biomedical Engineering, Science and Health Systems, Philadelphia, PA; 13:45 - 17:05 SESSION 4 Shady Farah, School of Pharmacy, Faculty of Medicine, The Emerging Medical Technologies in Cardiovascular Hebrew University of Jerusalem, Jerusalem, Israel; Abraham J. Repair and Therapeutics – Proteomics and Domb, School of Pharmacy, Faculty of Medicine, The Hebrew Epigenetics University of Jerusalem, Jerusalem, Israel; Peter I. Lelkes, University College, London Department of Bioengineering, Temple University, Philadelphia, PA Kennedy Lecture Theatre 16:30 – 17:05 13:45 - 13:55 Session Introduction Is Integration of Postgenomic Technologies Session chairs: Mark Lythgoe, Ph.D., David Vorp, Ph.D. a way Forward? Manuel Mayr, M.D., Ph.D., Professor of Cardiovascular Proteomics, British Heart Foundation Centre of Research 13:55 - 14:30 Excellence, King’s College London, London, United Kingdom Magnetic Targeting and Imaging of Stem Cells Using Nanoparticles Mark Lythgoe, Ph.D., Director of the Centre for Advanced 19:00 - 22:30 Banquet Biomedical Imaging, University College London Goodenough Club 23 Mecklenburgh Square London WC1 N 2AD 14:30 – 14:45 ABSTRACT Platelet-derived Deoxyribose-1-Phosphate Promotes Endothelial Cell Motility in vitro and Saturday, September 15, 2012 Angiogenesis in vivo Dina S. Vara, University of Bath, UK; Tim Woodman, University of Bath, UK; Warwick B Dunn, University of Manchester, 08:00 – 14:00 Registration open UK; Elena Garonna, Royal Veterinary College London, UK; Second Floor Foyer Michelangelo Campanella, Royal Veterinary College London Institute for Child Health and University College London, UK; Caroline PD Wheeler- 30 Guilford Street Jones, Royal Veterinary College London, UK; Giordano Pula, University of Bath, UK London WC1N 1EH, United Kingdom

14:45 - 15:00 ABSTRACT 08:00 - 10:00 Cardioprotective Role of Myocardial Dedicated Poster Session and Continental Ischemia-Induced Hepatic FGF21 Breakfast Shu Q. Liu, Northwestern University, Evanston, IL, USA; Alexei Levinsky Room and Philip Ullman Room Kharitonenkov, Lilly Res Labs, Indianapolis, IN, USA; Li-Qun Zhang, Northwestern University, Evanston, IL, USA; Yu H. Wu, 10:00 - 10:30 Northwestern University, Evanston, IL, USA ISACB Business Meeting University College, London 15:00 - 15:15 ABSTRACT Kennedy Lecture Theatre Impact of Electrospun Scaffold Topography, Composition, and Biofunctionalization on 10:30 - 14:45 SESSION 5 Intraperitoneally-Grown Vascular Constructs Critical Issues in Tissue Engineering: Chris A Bashur, Department of Biomedical Engineering, Construct Vascularization and Scaffold-Generated Cleveland Clinic, Cleveland, OH; Matthew J. Eagleton, Signalling Department of Vascular Surgery, Cleveland Clinic, Cleveland, OH; Anand Ramamurthi, Department of Biomedical University College, London Engineering, Cleveland Clinic, Cleveland, OH Kennedy Lecture Theatre

ISACB’S 2012 BIENNIAL MEETING 11 ISACB 13TH BIENNIAL MEETING PROGRAM

10:30 - 10:40 13:20– 14:45 Session Introduction Grand Challenges in Applied Cardiovascular Session chairs: Kevin Parker, Ph.D., Biology Simon Hoerstrup, M.D., Ph.D. Moderator: Howard Greisler, M.D. 10:40 - 11:15 Biomaterials in the Development of Small • John Deanfield, Ph.D., Faculty of Population Health Diameter Vascular Conduits and Heart Valves Sciences, University College London Alexander Seifalian, Ph.D., Professor of Nanotechnology and Regenerative Medicine, University College, London • Kevin Kit Parker, Ph.D., Tarr Family Professor of Bioengineering and Applied Physics; Wyss Institute for Biologically-Inspired Engineering; Harvard Stem Cell Institute 11:15 - 11:30 ABSTRACT Comparison of Synthetic Small-calibre • Frederick J. Schoen, M.D., Ph.D., Pathologist, Brigham Biodegradable vs. Stable ePTFE Vascular and Women’s Hospital; Professor of Pathology and Health Prosthesis after Long-term Implantation in the Sciences and Technology, Harvard Medical School; Rat Aorta Executive Vice Chairman, Department of Pathology, Brigham Beat H. Walpoth, Department of Cardiovascular Surgery, and Women’s Hospital University Hospital of Geneva; Damiano Mugnai, Department of Cardiovascular Surgery, University Hospital of Geneva; Sarra • William R. Wagner, Ph.D., Interim Director, McGowan de Valence, Department of Pharmaceutics & Biopharmaceutics Institute for Regenerative Medicine, University of Pittsburgh EPGL, University of Geneva; Wojciech Mrowczynski, Department of Cardiovascular Surgery, University Hospital of Geneva; Jean-Christophe Tille, Department of Clinical Pathology, University Hospital of Geneva; Xavier 14:45 Meeting Adjourned Montet, Department of Radiology, University Hospital of Geneva; Robert Gurny, Department of Pharmaceutics and Biopharmaceutics EPGL, University of Geneva; Afksendiyos Kalangos, Department of Cardiovascular Surgery, University Hospital of Geneva; Michael Moeller, Department of Pharmaceutics & Biopharmaceutics EPGL, University of Geneva, Switzerland

11:30 - 12:05 Cell-Free Biomaterials - Rescue of Myocardium Post-MI William R. Wagner, Ph.D., Interim Director, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

12:05 – 12:30 Lunch Winter Garden and Balcony

12:30 - 12:45 ABSTRACT Vitalize Synthetic Vascular Grafts in vivo Yadong Wang, University of Pittsburgh, PA; Wei Wu, University of Pittsburgh, PA; Robert Allen, University of Pittsburgh, PA

12:45– 13:20 Lifetime Management of Arterial Disease John Deanfield, Ph.D., Faculty of Population Health Sciences, University College London

12 ISACB.ORG YOUNG INVESTIGATOR AWARDS

ISACB encourages promising research by Young Investigators and awards ISACB Young Investigators Awards at each of the Biennial Meetings. To qualify for an ISACB Young Investigator Award, the presenting author must be responsible for the scientific content of the abstract and be in a “training program” (Post-doctoral fellow, resident, graduate student). ISACB Young Investigators receive a stipend to offset their travel and registration expenses for the ISACB meeting and recognition at the ISACB Banquet of their achievement. The ISACB Young Investigators for the 12th Biennial meeting of ISACB are:

The Function of Shear-responsive and A Novel Tissue-Engineered Side-dependent microRNA-486-5p in Devitalized Vascular Prosthesis Aortic Valve Endothelium

Maxime Tondreau Casey J. Holliday-Ankeny LOEX/Laval University Georgia Institute of Technology and Emory University, Quebec, Canada Atlanta, GA

ISACB’S 2012 BIENNIAL MEETING 13 ORAL PRESENTATION ABSTRACTS: SESSION 1

1.1 1.2 Visualization Of Flow-Induced ATP Release Spatio-Temporal Mapping of Elastin And Ca2+ Waves In Vascular Endothelial Remodeling in Experimental Abdominal Cells Aortic Aneurysms (AAAs) Towards Induced Joji Ando, Laboratory of Biomedical Engineering, Regenerative Elastic Matrix Repair Utilizing Dokkyo Medical University, Tochigi, Japan; Kimiko Resident Elastogenic Cells Yamamoto, Department of Biomedical Engineering, Partha Deb, Department of Biomedical Engineering, University of Tokyo, Tokyo, Japan The Cleveland Clinic, Cleveland, OH, USA and Anand Purpose: ATP is not only the major source of energy Ramamurthi, Department of Biomedical Engineering, in the cell but also functions as an autocrine and The Cleveland Clinic, Cleveland, OH, USA paracrine signaling molecule. Its release in response Purpose: Although growing AAAs are known to to mechanical and biochemical stimuli controls cellular be stabilized by positive collagen remodeling, function by activating purinoceptors, such as ion compensatory elastogenesis has not been investigated. channel P2X receptors and G protein-coupled P2Y Accordingly, we sought to investigate the quantity receptors. For example, vascular endothelial cells and quality of compensatory de novo elastic matrix (ECs) release ATP in response to shear stress, which assembly within induced rat AAAs and to identify cell results in a Ca2+ influx and increased Ca2+-induced types involved in elastin remodeling. signal transduction. This process is important for the regulation of vascular function, but the mechanism Methods: Rat AAAs at 7,14, & 21d post-induction behind the ATP release remained unclear. by elastase-infusion (n=3rats/time), were divided into proximal(below lumbar), mid(maximal-expansion) Methods: To assess the release of ATP in real time, we and distal(above iliac-bifurcation) regions, sectioned, developed a novel chemiluminescence imaging method histologically stained for elastin, collagen, and GAGs, by using cell-surface attached firefly luciferase and a and IF-labeled for elastin, fibrillin, hyaluronan (HA), SM- CCD camera. α-actin, and nuclei. Morphometric analysis performed Results: Upon shear stress stimulation, cultured human using custom algorithms run on Image Pro (n=6regions/ pulmonary artery ECs simultaneously released ATP in section/region/treatment) determined relative two different ways: diffusely and in a highly localized abundance of the above ECM components, aortic wall/ pattern. The localized ATP release occurred at caveolin- medial thickness, media/adventitia thickness ratio, no of 1-rich regions of the membrane and knockdown elastic lamellae, mean elastic fiber length, fragmented of caveolin-1 expression using siRNA inhibited this fibers and, variations in fiber diameter. process without affecting the more diffuse release of Results: Distinct spatio-temporal differences in elastic ATP, indicating involvement of caveolae in the localized matrix amounts, fiber length, density, and lamellar ATP release. Ca2+ imaging with Fluo-4 combined architecture, and amounts and distribution of collagen, with ATP imaging revealed that shear stress evoked and GAGs were observed. HA accumulated in regions an increase in intracellular Ca2+ concentration and of medial elastin fragmentation, and of early neointimal subsequent Ca2+ wave that originated from the same remodeling by α-SMA+cells, though it was replaced by sites as the localized ATP release. collagen and non-lamellar elastin in the neointima of Conclusions: ATP release at caveolae triggers shear longer-term AAAs. Though fibrillar, neointimal elastic stress Ca2+ signaling in ECs. This imaging method matrix was deficient in fibrillin. should prove useful for the study of ATP release Conclusions: Our study indicates significant de novo mechanisms and the functional roles of ATP in various elastin deposition in the neointima of AAAs, but not cell types. in the elastin-disrupted media, and that neointimal elastic fiber formation is deficient, likely due to poor fibrillin production by neointimal SMCs. Future work will investigate specific deficiencies/aberrations in elastic matrix assembly by these cells, and investigate feasibility of elastogenic induction to overcome these defects towards reinstating AAA-wall elasticity.

14 ISACB.ORG ORAL PRESENTATION ABSTRACTS: SESSION 1

1.3 Inflammation-dependent Mechanism of Methods and Results: Near-infrared fluorescence Calcification: A New Role for Macrophage microscopy with a hydroxyapatite-binding imaging Matrix Vesicles agent detected microcalcifications, containing vesicular structures, in human atherosclerotic plaques. Sophie New, Claudia Goettsch, Masanori Aikawa, Julio Immunogold electron microscopy with CD68 labeling Meirelles Marchini, Brigham and Women’s Hospital, demonstrated the release of macrophage-derived Harvard Medical School; Catherine Shanahan, King’s MV (Figure, left panel). S100A9, a novel inducer College, London, UK; Kevin Croce, Elena Aikawa, of inflammation, was co-localized with plaque Brigham and Women’s Hospital, Harvard Medical macrophages and microcalcifications. Annexins were School, Boston, MA previously implicated in smooth muscle cell-derived Purpose: We previously linked macrophages and early MV. Immunogold localized S100A9 and annexin-V calcification/microcalcification, using molecular imaging. in macrophage-MV of human plaques (Figure, right Here we propose a novel mechanism for calcification panel). Western blot showed increased S100A9 and via macrophage-derived matrix vesicles (MV), as annexin-V in stimulated macrophage-MV. Co-IP an alternative pathway to a commonly accepted indicated the S100A9-annexin-V interaction. S100A9 mechanism of osteogenic cell transition. siRNA reduced the number and calcification potential of MV derived from human primary macrophages (p<0.05). ApoE-/-S100A9-/- mice had less MV in the plasma, and their macrophage-derived MV had reduced calcification potential compared to apoE-/- mice (n=5). Stimulation with 2 mM calcium/3 mM phosphate increased calcification potential of MV from RAW 264.7 macrophage cell line (p<0.01; n=3). These conditions did not induce apoptosis and thus this effect is not attributed to apoptotic bodies. Conclusions: This study provides the first direct evidence for the role of macrophage-derived MV in microcalcification. S100A9 may play a key role in this process.

ISACB’S 2012 BIENNIAL MEETING 15 ORAL PRESENTATION ABSTRACTS: SESSION 1

1.4 1.5 Polyethyieneimine (PEI) Sirna Complexes In-Situ Capture Technology of Endothelial Released From Coated Electrospun Progenitor Cells (EPCs) Polyethylene Terephthalate (PET) Bypass Takehisa Matsuda, Kanazawa Institute of Technology, Graft Materials Facilitate Gene Silencing in Hakusan, Japan; Shu Takabatake, Kanazawa University Infiltrating Primary Human Aortic Smooth Medical School, Kanazawa, Japan; Masakazu Yamagishi, Muscle Cells Kanazawa University Medical School, Kanazawa, Japan Christoph S Nabzdyk, Tufts Medical Center, Department Purpose: This study aims to develop the surface of Surgery, Tufts University School of Medicine, Boston, architecture enabling selective in-situ capturing of EPCs MA, Beth Israel Deaconess Medical Center, Harvard and subsequent rapid endothelialization on blood- Medical School, Boston, MA; Maggie Chun, Beth Israel contacting surfaces under arterial flow. First, based on Deaconess Medical Center, Harvard Medical School, ex vivo cellular potentials on various surface-bound Boston, MA; Hunter Oliver-Allen, Beth Israel Deaconess candidate ligands, the best-suited pair of cell receptor Medical Center, Harvard Medical School, Boston, MA; exclusively expressed on endothelial lineage cells and Matthew D. Phaneuf, Biosurfaces Inc., Ashland, MA; surface-bound ligand is defined. Second, prolonged Saif G Pathan, Biosurfaces Inc., Ashland, MA; Jin-Oh activation of intracellular signaling transduction You, Harvard University, School of Engineering and pathways of EPCs adhered on surface-bound ligand is Applied Sciences, Boston, MA; Leena Pradhan, Beth examined, and lastly in-situ capture of EPCs and Israel Deaconess Medical Center, Harvard Medical endothelialization in a porcein model is verified. School, Boston, MA; Frank W LoGerfo, Beth Israel Deaconess Medical Center, Harvard Medical School, Methods include 1) covalent bonding of molecules Boston, MA, USA [vascular endothelial growth factor (VEGF) and two VEGF receptor antibodies and Tie-1 and -2 Purpose: Anastomotic Intimal hyperplasia (AIH) remains antibodies] on vinyl alcohol-copolymer. 2) Culture of the leading cause of prosthetic arterial graft failure. human mononuclear cells(MCs) on these protein- Use of electrospun PET (ePET) can be an alternative bound substrates and histocytochemical analyses, 3) to conventional PET grafts. RNAi is a promising implantation of stent in coronaries. tool to silence genes contributing to AIH, including, Thrombospondin-2 (TSP-2) previously shown by us Results showed: Proteins were covalently bound to to be upregulated in AIH. Delivery of silencing RNA the copolymer surface via activation of hydroxyl group. (siRNA) from ePET graft to the anastomotic sites could Among molecules examined, only VEGF exhibited high potentially mitigate AIH. adhesion and proliferation characteristics and a quite high differentiation potential of MCs with culture time. Methods: ePET was dip-coated with control siRNA Day-order continuous activation of intracellular signaling alone or complexed with either, cationic polymer transduction pathways (phosphorylation of VEGF polyethyleneimine (PEI) or the lipophilic transfection receptor, FAK, ERK and Akt) was observed for ECs reagent Lipofectamine RNAiMax for 45 minutes. UV- adhered on VEGF-bound substrate. Implantation study Spectrophotometry was used to quantify siRNA coating using VEGF-bound stents showed that cells expressing on ePET. Control and TSP-2 siRNA-PEI complexes VEGF receptor adhered on stent surface followed by coated ePET samples (5x5mm2) were placed onto endothelialization was observed within several days. a monolayer of human aortic smooth muscle cells (HAoSMCs) for 24 hours. Confocal microscopy and Conclusions: This study concludes that surface- Q-RTPCR were performed to evaluate cellular infiltration bound VEGF enables selective capture of EPCs and into ePET and gene silencing of TSP-2. complete endothelialization in vivo, and simple surface architecture promise to exert non-thrombogenic Results: Significant amount of siRNA from the coating potential to cardiovascular devices. solution was adsorbed to ePET if complexed with PEI compared to RNAiMax or no transfection reagent (62%±3% vs. 1%±0.075% or 1.0%±0.01%), HAoSMCs migrated into and proliferated on ePET coated with siRNA-PEI complexes. Compared to control siRNA, TSP-2 siRNA uptake in HAoSMC resulted in significant gene silencing of TSP-2 from ePET coated with siRNA- PEI. Conclusions: Coating of ePET with siRNA-PEI complexes is feasible, does not impair HAoSMCs in- growth and facilitates TSP-2 gene silencing in HAoSMC in vitro. This clinically feasible approach for delivery of target gene siRNAs may help prevent AIH and subsequent graft failure.

16 ISACB.ORG ORAL PRESENTATION ABSTRACTS: SESSION 2

2.1 2.2 Biochemical and Spatial Control of Human The Role of Dll4-Notch Signaling in Shared Pluripotent Stem Cell Mesoderm Formation Mechanisms for Atherosclerosis and Oscar J. Abilez, Stanford University, Stanford, CA; Frank Metabolic Disorders Myers, University of California, Berkeley, Berkeley, CA; Daiju Fukuda, Cardiovascular Division, Department of Jason Silver, University of California, Berkeley, Berkeley, Medicine, Brigham and Women’s Hospital, Harvard CA; Luke P. Lee, University of California, Berkeley, Medical School; Tetsuro Miyazaki, Cardiovascular Berkeley, CA; Christopher K. Zarins, Stanford University, Stanford, CA Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School; Kunio Purpose: Various factors including the size, shape, and Morishige, Cardiovascular Division, Department of density of human pluripotent stem cell (hPSC) colonies, Medicine, Brigham and Women’s Hospital, Harvard as well as the spacing between neighboring colonies, play a significant role in the maintenance of pluripotency Medical School; Elena Aikawa, Cardiovascular Division, and in cell fate determination. In order to improve yield Department of Medicine, Brigham and Women’s and repeatability of stem cell differentiation towards a Hospital, Harvard Medical School; Masanori Aikawa, mesoderm fate, we have developed a simple, robust Cardiovascular Division, Department of Medicine, technique for patterning hPSC colonies using thin Brigham and Women’s Hospital, Harvard Medical silicone stencils. School Methods: For biochemical control, we activate and inhibit Purpose: Atherosclerosis and the metabolic syndrome various developmental pathways in various combinations associate with chronic inflammation. Notch signaling to differentiate hPSC towards the mesoderm lineage. participates in various disease mechanisms, but its role To visualize differentiation, we use long-term lapse in inflammation remains obscure. We previous reported microscopy to track the emergence of fluorescent protein expression driven by various mesoderm promoters. For in vitro that the Notch ligand Delta-like 4 (Dll4) and the spatial control, we use thin silicone stencils containing receptor Notch3 promote macrophage activation. The geometric parameters that are varied, such as pitch and present study tested the hypothesis in vivo that the diameter. Dll4-Notch3 axis participates in the pathogenesis of atherosclerosis and metabolic disorders. Results: Efficient directed differentiation is achieved by control of the TGF-/activin, BMP, FGF, VEGF, and Methods and Results: 1) Dll4 In fat-fed LDL-receptor– WNT pathways as demonstrated by gene expression, deficient (Ldlr-/-) mice, a model of metabolic disorders, flow cytometry, immunocytochemistry, and time- we noted increased expression of Dll4 in the aorta lapse microscopy. In addition, spatial patterning of and fat, suggesting its induction by overnutrition. hPSC colonies with defined geometries shows the Administration of neutralizing anti-Dll4 antibody in Ldlr- emergence of mesodermal precursors that give rise to cardiomyocytes in a stereotypical spatial distribution /- mice attenuated the development of atherosclerosis, (Figure 1). arterial and valvular calcification, insulin resistance, fat accumulation, and fatty liver, which were accompanied Conclusions: The stereotypic response arising from by decreased macrophage accumulation, diminished biochemical and spatial control of hPSC differentiation monocyte chemoattractant protein-1 (MCP-1) has implications in serving as an informative and repeatable in vitro system for investigating mesoderm expression, and lower levels of NF-KB activation. In lineage induction and development. vitro experiments revealed that Dll4 regulates MCP-1 via NF-KB, supporting the in vivo data. Dll4 suppression Figure 1. Biochemical and spatial control of hPSC in vivo and in vitro decreased expression of genes differentiation demonstrate the emergence of mesodermal precursors (green) that give rise to representing a pro-inflammatory “M1” phenotype, cardiomyocytes in a stereotypical spatial distribution. osteogenic activity, and matrix degradation. 2) Notch3 Macrophage-selective transgenic mice that express a constitutively-active Notch3, crossed with Ldlr-/- mice (Notch3tg:Ldlr-/-), showed larger atheroma size, more advanced calcification, greater macrophage accumulation in arteries and fat, and greater fat deposition, as compared to control Ldlr-/- mice. Notch3tg:Ldlr-/- macrophages expressed higher levels of M1 genes. Conclusions: These results suggest that Dll4-Notch3 signaling plays a central role in the shared mechanism for the pathogenesis of atherosclerotic vascular diseases and metabolic disorders.

ISACB’S 2012 BIENNIAL MEETING 17 ORAL PRESENTATION ABSTRACTS: SESSION 2

2.3 2.4 Engraftment of Human MSCs as The Roles of Various Metabolic Enzymes in Perivascular Cells of Bioengineered Diabetic Cardiomyopathy – In Vivo and In Microvessels Enhances Mesenchymal Vitro Approach Tissue Formation Thomas Larrew, Clemson University, Clemson, SC; Ruei-Zeng Lin, Department of Cardiac Surgery, James Chow, Clemson University, Clemson, SC; Children’s Hospital Boston, and Harvard Medical Jason Schulte, Clemson University, Clemson, SC; Dan School, Boston, MA; Arin K. Greene, Department of Simionescu, Clemson University, Clemson, SC; Agneta Plastic & Oral Surgery, Children’s Hospital Boston, Simionescu, Clemson University, Clemson, SC, USA Boston, MA; Juan M. Melero-Martin, Department of Cardiac Surgery, Children’s Hospital Boston, and Harvard Medical School, Boston, MA, USA Purpose: to investigate the enzymes involved in the increase of oxidative stress, apoptosis, and fibrosis, processes that contribute to cardiac dysfunction in Purpose: Mesenchymal stem cells (MSCs) can generate diabetes; to create a tissue engineering-based model to multiple end-stage mesenchymal cell types and are a study the pathology of diabetic cardiomyopathy (DCM). promising cell population for regenerative therapies. Methods: Porcine hearts were decellularized and However, controlling the engraftment of MSCs to 3 mm3 punch-outs were used as scaffolds. Cells maximize their differentiation potential in vivo is derived from embryonic rat heart were seeded onto challenging. This study investigates whether implanting the heart extracellular matrix, and the constructs were MSCs as perivascular cells enhances their engraftment incubated for 5 days in a cell culture media containing and the formation of mesenchymal tissue. high glucose levels and oxidized LDL, in a dynamic Methods: Human MSCs were isolated from white setting using the 3DKube perfusion minibioreactor. adipose tissue (watMSCs) and bone marrow (bmMSCs). As control, cell-scaffold constructs were incubated in MSCs were subcutaneously injected into SCID-GFP normal cell culture media. Tissues were then prepared mice in the presence or absence of human endothelial for histological staining and biochemical assays, and colony-forming cells (ECFCs; 2x106 total cells; 40:60 the content of caspase-3, glutathione, and prolyl- ECFC/MSC ratio) using 200 μl of Matrigel. MSC 4-hydroxylase were analyzed. Cell proliferation on engraftment as well as the extent of tissue formation scaffolds and the presence of Receptor for Advanced were evaluated. Glycation End-products were also tested. The in vitro results were compared to in vivo results obtained from Results: Co-implanted ECFCs significantly increased analyzing diabetic rat hearts. MSC survival by reducing early apoptosis, a process that was partially mediated by PDGF-BB prior to Results: Cells were successfully seeded onto the the onset of blood vessel formation. Additionally, decellularized scaffolds in both normal and diabetic the presence of ECFC-lined microvessels enabled conditions. Histological and biochemical assessments specific perivascular engraftment of PDGFR-β+ MSCs, were supportive of current descriptions of the clinical which showed higher clonal growth and multilineage presentation of DCM. There was an elevated level of potential than non-perivascular PDGFR-β- MSCs. apoptosis associated with caspase-3 activity, as well as Thus, implants containing MSCs and ECFCs were able hampered resistance to oxidative stress in both diabetic to generate significantly more tissue at 4 weeks than rat hearts and tissue-engineered constructs. those containing only MSCs, including formation of Conclusion: Oxidative stress coupled with apoptosis adipose tissue (by watMSCs) and BMP-2-induced bone and the heart’s lack of regeneration, seem to largely (bmMSCs). contribute to the formation of DCM. With some minor Conclusion: Survival and functionality of implanted adjustments, the 3DKube bioreactor appears to MSCs were significantly improved by the presence of correctly simulate DCM for future testing. ECFCs, which led to more extensive mesenchymal tissue formation. This two cell based approach has the potential to improve the outcome of future MSC therapies.

18 ISACB.ORG ORAL PRESENTATION ABSTRACTS: SESSION 2

2.5 2.6 Human Calcified Valves Demonstrate an Hemodynamics of the Transitional Aortic Atypical Expression of Smooth Muscle Cells Arch Patterning at a Key Embryonic Stage and their Transcription Factors William J. Kowalski, Carnegie Mellon University, Najma Latif, Harefield Heart Science Centre, Imperial Pittsburgh, PA; Onur Dur, Carnegie Mellon University, College, Harefield, Middx, UK; Padmini Sarathchandra, Pittsburgh, PA; Yajuan Wang, Carnegie Mellon Harefield Heart Science Centre, Imperial College, University, Pittsburgh, PA; Joseph P. Tinney, Harefield, Middx, UK; Magdi H. Yacoub, Harefield Heart Cardiovascular Innovation Institute, University Science Centre, Imperial College, Harefield, Middx, of Louisville, Louisville, KY; Bradley B. Keller, UK; Adrian H Chester, Harefield Heart Science Centre, Cardiovascular Innovation Institute, University of Harefield, Imperial College, Middx, UK Louisville, Louisville, KY; Kerem Pekkan, Carnegie Mellon University, Pittsburgh, PA, USA

Purpose: We sought to investigate the presence and regulation of the smooth muscle cell-like phenotype Purpose: This study examines the morphologic and during calcification. hemodynamic changes occurring in the early embryonic aortic arch (AA) vessels. Methods: 12 normal and 22 calcified valves were analysed for the expression of smooth muscle Methods: We applied fluorescent dye microinjection (SM) markers and regulators: myocardin and and optical coherence tomography to measure the myocardin-related transcription factors, (MRTFA/B) diameters of the bilateral embryonic chick AAs at by immunocytochemistry. Transmission electron stages 18, 21, and 24 (3, 3.5, and 4 days, respectively). microscopy was used to identify SM cells in calcified Polymeric casts were used to generate 3D models valves. of the AAs at each stage, reconstructed from micro- computed tomography scans. Pulsatile blood flow Results: Normal human valves demonstrate the was simulated using computational fluid dynamics expression of smooth muscle markers localised (CFD), with stage-specific inlet flow based on Doppler specifically to the base of the ventricularis. Smooth ultrasound. muscle myosin, SM1, SM2, calponin, caldesmon and smooth muscle α-actin demonstrated an increased Results: We found that the AAs present at stages 18 and aberrant expression in all the calcified valves. and 24 did not vary from embryo to embryo while the Additionally, surface endothelium and endothelial cells intermediate stage 21 presented with four possible (ECs) lining microvessels demonstrated smooth muscle configurations. This variability was associated with marker expression in 9/16 calcified valves. Normal increased wall shear stress (WSS) levels at stage 21 valves showed no expression of myocardin or MRTFs. compared to stages 18 and 24, as computed from CFD However, myocardin, MRTF-A and MRTF-B–positive results. Diameter measurements showed asymmetric cells were prolific in calcified valves around calcified growth of AA pair IV, where both laterals increased from nodules with some EC-positivity. Importantly, the stage 18 to 21 while only the right lateral increased from expression of MRTFs was nuclear in both VICs and ECs stage 21 to 24 (p<0.05). indicative of activation. TGFβ1 (10ng/ml) was able to CFD revealed that the right lateral flow of AA pair IV was induce and translocate the expression of MRTF-A to the more than 1.8 times that of the left lateral at all stages. nucleus in valvular cells within 4 hours in some isolates. Regression analysis revealed a quadratic relationship TEM identified smooth muscle cells and smooth between a change in WSS and a change in AA diameter muscle-derived foam cells in calcified valves. (p=0.002). Conclusions: Calcified valves harbour a greatly Conclusions: Our study provides the first comparison increased number of smooth muscle marker-positive between quantitative in vivo data and CFD-predicted VICs and endothelial cells. Calcified valves expressed hemodynamics of the early embryonic AA. Changes myocardin and MRTFs, key regulators of smooth muscle in flow distribution and WSS were associated with gene expression. The presence of smooth muscle increased morphogenetic activity of the stage 21 AAs. cell markers and MRTFs in calcified valves suggest a role for this cell phenoptype in the development of the calcification process.

ISACB’S 2012 BIENNIAL MEETING 19 ORAL PRESENTATION ABSTRACTS: SESSION 3

3.1 3.2 Restrictive Remodeling in Response to Varying Effects of Different Types of Disturbed Flow in Stiffened Carotid Arteries Hemodynamic Forces on Tissue Factor (TF) is Related to Elastin Dysfunction in Fibulin V Expression in Endothelial Cells (EC) Knockout Mice Ryuzo Abe,Yale University School of Medicine, New Luke P. Brewster, Emory University School of Haven, CT; Norio Yamashita, Yale University School Medicine. Georgia Institute of Technology, Institute of of Medicine, New Haven, CT; Adrienne Rochier, Yale Bioengineering and Bioscience, GA; Laura Hansen, University School of Medicine, New Haven, CT; Alexander Georgia Institute of Technology, Department of Nixon, Yale University School of Medicine, New Haven, Biomedical Engineering, GA; Chanwoo Kim, Emory CT; Rei Abe, Yale University School of Medicine, New University, Biomedical Engineering, GA; William Haven, CT; Takeshi Moriguchi, Yale University School Wan, Georgia Institute of Technology, Mechanical of Medicine, New Haven, CT; Bauer E. Sumpio, Yale Engineering, GA; Hiromi Yanagisawa, University of Texas University School of Medicine, New Haven, CT; Shigeto Southwestern, Molecular Biology, GA; Hanjoong Jo, Oda, Chiba University Graduate School of Medicine, Georgia Institute of Technology and Emory University, Chiba, Japan; Hiroyuki Hirasawa, Chiba University Biomedical Engineering, GA; Rudy L. Gleason, Graduate School of Medicine, Chiba, Japan Georgia Institute of Technology, School of Mechanical Engineering, GA, USA Purpose: This study investigated EC response to Purpose: Arterial stiffness occurs with aging and varying types of mechanical forces, alone and in is common in patients with cardiovascular disease. combination with chemical stimuli. We assessed It is well established that disturbed flow promotes TF expression, since it is casually associated with atherogenesis. It is not well known how stiffened arteries atherogenesis. remodel under disturbed flow conditions. To determine the effect of disturbed flow on arterial stiffness, we Methods: TF RNA expression in HUVEC exposed exposed dysfunctional elastin from fibulin V knockout to mechanical stress in the presence or absence of (FBVn) mice carotid arteries to disturbed flow (low and chemical stimulation with 4U/ml thrombin (Th) was oscillatory shear) by left carotid artery partial ligation. determined by RT-PCR. The forces examined were: unidirectional laminar flow 14 dyn/cm2 using a parallel Methods: 6 week old FBVn mice underwent partial plate flow chamber (LF), 1 Hz pulsatile unidirectional carotid ligation. At two and four weeks carotid arteries laminar flow (PF), constant oscillatory flow (OF), 1 Hz were harvested and biaxial mechanical tests were pulsatile to-fro flow (TFF) and 1 Hz 10% cyclic strain (CS). performed. Results: LF, PF and TFF for 2 hours induced Results: FBVn mice exposed to disturbed flow became significantly increased TF RNA expressions compared progressively stiffer than contralateral and wild type with static control (8.7±0.7, 4.7±0.9, 8.6±1.7 fold, carotid arteries. (Figure 1) By four weeks the FBVn respectively). Exposure to OF or CS did not result in +disturbed flow arteries were too stiff to test on the significant increase, whereas chemical stimulation system. The contralateral FBVn arteries were stiffer with Th alone led to significant TF expression (4.9±0.3 than the wildtype, but not different than baseline. fold). The combination of mechanical-chemical stimuli Conclusions: Disturbed flow promotes arterial induced significantly higher TF expression than stiffening during remodeling that begins axially but mechanical stresses alone. LF+Th, PF+Th and TFF+Th progresses biaxially in four weeks. This model mimics for 2 hours induced synergistically significant increased advanced peripheral artery disease where elastin TF expression (16.6±1.7, 14.8±2.4, 17.4±1.0 fold, in the arterial media becomes dysfunctional, and it respectively). Furthermore, only TFF demonstrated offers support to the interactive role of fluid and solid significantly higher sustained TF expression after 6 mechanics on progressive arterial disease. hours exposure, both with and without Th. Conclusions: While uniform laminar flow resulted in transient TF expression, TFF, which is analogous to disturbed flow, induced sustained amplification of TF expression even in the presence of chemical stimuli. These results provide important insight into the role of inflammatory reactions in EC, as it relates to localization of atherosclerosis at sites of disturbed flow.

Figure 1

20 ISACB.ORG ORAL PRESENTATION ABSTRACTS: SESSION 3

3.3 3.4 A Novel Tissue-Engineered Devitalized The Function of Shear-responsive and Vascular Prosthesis Side-dependent microRNA-486-5p in Maxime Tondreau, LOEX/Laval University, Quebec, Aortic Valve Endothelium Canada; Jean-Michel Bourget,LOEX/Laval University, Casey J. Holliday-Ankeny, Georgia Institute of Quebec, Canada; Michel Fortin, LOEX/Laval University, Technology and Emory University, Atlanta, GA; Randall Quebec, Canada; Carl Bouchard, LOEX/Laval University, F. Ankeny, Georgia Institute of Technology, Atlanta, GA; Quebec, Canada; Dan Lacroix, LOEX/Laval University, Zannatul Ferdous, University of Tennessee, Knoxville, Quebec, Canada; François A. Auger, LOEX/Laval TN; Robert M. Nerem, Georgia Institute of Technology, University, Quebec, Canada Atlanta, GA; Hanjoong Jo, Georgia Institute of Purpose: There is currently a lack of clinically available Technology and Emory University, Atlanta, GA, USA small diameter vascular graft. Therefore, we created a Purpose: AV disease contributes to cardiovascular- self-assembly tissue-engineered vessel from human linked deaths; however, how it initiates/progresses is dermal fibroblasts. not well-understood. We hypothesize that different Methods: Briefly, fibroblasts were cultured with hemodynamics on the AV cusps stimulate ECs to ascorbic acid to induce the secretion of collagen and differentially regulate miRNAs, specifically miR-486-5p, the ensuing formation of cellular sheets. The sheets inducing endothelial dysfunction and disease. were rolled around a 1.6-mm mandrel and allowed Methods: This hypothesis was tested using in vitro to fuse during a maturation phase. The vessels were and in vivo approaches, high-throughput microarrays, devitalized in deionized water with no other agent in silico analyses, and functional assays. Using and the devitalized fibroblast vessels (DFV) were microarray analysis on sheared, side-specific HAVECs then conserved at 4°C. We assessed the mechanical (npatients=6), we identified/validated shear-induced properties including the burst pressure. The human changes in miRNAs. In vivo, we isolated endothelial- DFVs were implanted as infrarenal aortic interpositional enriched RNA for identification/validation of side- grafts in Sprague-Dawley rats. In vitro endothelialization dependent miRNAs in porcine AV endothelium studies were also performed using a bioreactor. (nsamples=3 pooled). We used miRWalk to determine Results: The DFVs had an estimated burst pressure potential targets of shear- (in vitro) and side-dependent of 2257 ± 325 mm Hg, a failure strain of 90 ± 12%, a (in vivo) miR-486-5p. To identify miR-486-5p’s function, Young’s modulus of 1.3 ± 0.2 MPa, and an ultimate we overexpressed miR-486-5p in HAVECs, sheared, and tensile strength of 0.7 ± 0.1 MPa. Grafted rats did not completed migration, apoptosis, and cell cycle assays. show any signs of ill health and the grafts were still We optimized miRNA inhibitor delivery to porcine and functional 4 months post-implantation. Histological mouse AVs ex vivo and in vivo, respectively. analysis revealed that DFVs supported the growth Results: We identified miR-486-5p as shear- of cells from the rat. The DFVs could easily be responsive through microarrays and qPCR in HAVECs endothelialized and supported physiological conditions (npatients=6; p<0.05). We then validated miR-486- within a bioreactor for 2 weeks. 5p as side-dependent in porcine AV endothelium by Conclusions: We believe that the simplicity of the qPCR (nsamples=9; p<0.05). Using miRWalk, filtering, graft and the low immunogenic properties of fibroblasts and preliminary overexpression studies (npatients=5), allowed human DFVs to be implanted in rats without we identified potential targets of miR-486-5p: Efna1 immunosuppression. Furthermore, the ability to easily and Prnd. Scratch assays showed miR-486-5p endothelialize the prostheses confers a major advantage overexpression leads to increased migration (npatients to DFVs when compared to synthetic grafts. =3; p<0.05). PI staining showed no cell cycle changes due to miR-486-5p (npatients=3). Annexin-V/PI staining showed a decreasing trend in early apoptosis with miR-486-5p overexpression (npatients=2). Delivery of a non-targeting miRNA inhibitor was successful in ex vivo and in vivo AVs. Conclusions: Better understanding of shear- and side-dependent miR-486-5p in AV biology/disease may provide insight into miRNA-based treatments.

ISACB’S 2012 BIENNIAL MEETING 21 ORAL PRESENTATION ABSTRACTS: SESSION 4

4.1 4.2 Platelet-derived Deoxyribose-1-Phosphate Cardioprotective Role of Myocardial Promotes Endothelial Cell Motility in vitro Ischemia-Induced Hepatic FGF21 and Angiogenesis in vivo Shu Q. Liu, Northwestern University, Evanston, IL, USA; Dina S. Vara, University of Bath, UK; Tim Woodman, Alexei Kharitonenkov, Lilly Res Labs, Indianapolis, IN, University of Bath, UK; Warwick B Dunn, University USA; Li-Qun Zhang, Northwestern University, Evanston, of Manchester, UK; Elena Garonna, Royal Veterinary IL, USA; Yu H. Wu, Northwestern University, Evanston, College London, UK; Michelangelo Campanella, IL, USA Royal Veterinary College London and University Purpose: Myocardial ischemia activates innate College London, UK; Caroline PD Wheeler-Jones, protective mechanisms, enhancing cardiomyocyte Royal Veterinary College London, UK; Giordano Pula, tolerance to ischemia. Here, we demonstrate that University of Bath, UK the liver contributes to myocardial protection by Purpose: To understand the physiological role of upregulating and releasing the secretory protein FGF21. deoxyribose-1-phosphate (dRP) release by human Methods: Myocardial ischemia was induced in mice by and mouse platelets in the homeostasis of the ligating the LAD coronary artery. Myocardial infarction cardiovascular system. was assessed by histological assays. Left ventricular Methods: The release of dRP by mouse platelets function was evaluated by echocardiography and was ablated by genetic silencing of dRP-generating hemodynamic analysis. The expression and activity of enzymes uridine phosphorylase (UP) and thymidine FGF21 and signaling molecules, including FGFR1, PI3K, phosphorylase (TP). Endothelial cell motility in vitro was Akt1, and BAD, were tested by immunoprecipitation/ tested in monolayer repair and transmigration assays. immunoblotting. The cardioprotective role of FGF21 was The angiogenesis response in vivo was tested in chick assessed in FGF21-/- and FGF21-Tg (overexpression) chorioallantoic membrane (CAM) vascularisation assays. mice. The regulatory role of FGFR1, PI3K, and Akt1 was evaluated by siRNA-mediated gene silencing. Results: Micromolar concentrations of the pro- angiogenic metabolite deoxyribose-1-phosphate Results: FGF21 was upregulated in hepatocytes and (dRP) were detected by mass spectrometry (MS) and serum from 0.5 to 3 days post myocardial ischemia. nuclear magnetic resonance (NMR) in the supernatants FGF21-/- mice exhibited increased myocardial of collagen- and thrombin-stimulated platelets. The infarction, whereas FGF21-Tg mice showed reduced genetic silencing of UP and TP in mouse platelets myocardial infarction compared to wildtype mice (TP-/-/UP-/-) significantly reduced the levels of dRP from 1 to 30 days, suggesting a cardioprotective role in platelet supernatants and impaired the ability of for FGF21. Administration of recombinant FGF21 protein-free platelet supernatants to enhance human to healthy mice or myocardial ischemia induced umbilical vein endothelial cell (HUVEC) motility. The FGFR1 phosphorylation in cardiomyocytes. FGF21- stimulation of endothelial cell migration by platelet- /- mice with myocardial ischemia exhibited reduced derived dRP correlated with the upregulation of integrin phosphorylation of FGFR1, PI3K, Akt1, and BAD β3, which was induced in a reactive oxygen species– in cardiomyocytes. siRNA-mediated FGFR1 gene dependent manner and critical for endothelial for the silencing suppressed FGFR1 expression and reduced endothelial migratory responses observed. Finally, phosphorylation of PI3K, Akt1, and BAD, resulting CAM vascularisation assays performed with protein- in an increase in myocardial infarction. PI3K or free supernatants suggested that the release of dRP by Akt1 gene silencing reduced BAD phosphorylation platelets stimulates angiogenesis in vivo. and enhanced myocardial infarction, suggesting that BAD phosphorylation is required for effective Conclusions: The pro-angiogenic metabolite dRP cardioprotection. mediates the redox-dependent upregulation of integrin β3 and enhances the cell migratory responses of Conclusions: The liver contributes to myocardial endothelial cells. The paracrine regulation of endothelial protection by upregulating and releasing FGF21, and cell motility by platelet-derived dRP is likely to play a FGF21 activates the FGFR1-PI3K-Akt1-BAD signaling role in the stimulation of postnatal angiogenesis and pathway, enhancing myocardial tolerance to ischemic the remodelling of the cardiovascular system following injury. tissue damage.

22 ISACB.ORG ORAL PRESENTATION ABSTRACTS: SESSION 4

4.3 4.4 Impact of Electrospun Scaffold Topography, Sustained Release of Rapamycin from Composition, and Biofunctionalization Electrospun Polyurethane Vascular Grafts on Intraperitoneally-Grown Vascular Jingjia Han, Drexel University, School of Biomedical Constructs Engineering, Science and Health Systems, Philadelphia, Chris A Bashur, Department of Biomedical Engineering, PA, USA; Shady Farah, School of Pharmacy, Faculty Cleveland Clinic, Cleveland, OH; Matthew J. Eagleton, of Medicine, The Hebrew University of Jerusalem, Department of Vascular Surgery, Cleveland Clinic, Jerusalem, Israel; Abraham J. Domb, School of Cleveland, OH; Anand Ramamurthi, Department of Pharmacy, Faculty of Medicine, The Hebrew University Biomedical Engineering, Cleveland Clinic, Cleveland, of Jerusalem, Jerusalem, Israel; Peter I. Lelkes, OH, USA Department of Bioengineering,Temple University, Philadelphia, PA, USA Purpose: Poor elastogenicity of adult vascular cells limits engineering of functional vascular tissues. Purpose: To incorporate rapamycin (RM) into Growing such tissues intraperitoneally using recruited polyurethane (PU) based vascular grafts via autologous peritoneal cells provides a promising electrospinning of RM-PU blends; to compare the alternative strategy. Here, we investigated how controlled release and in vitro bioavailability of RM from electrospun scaffold topography, composition, and electrospun RM-PU grafts. incorporation of elastogenic hyaluronan oligomers (HA- Methods: RM-PU fibrous scaffolds were electrospun o) impacts tissue remodeling by these cells. from RM-PU solutions at 0, 1, 5, 10, 20% RM (w/w) Methods: Randomly-oriented poly(ε-caprolactone;PCL) via three distinct blending methods. Fiber diameters conduits with different fiber diameters (0.62±0.4, and Young’s moduli of the resultant fibrous mats were 1.7±0.7, and 1.9±0.8 μm) and chemistries (PCL evaluated by SEM and Instron testing, respectively. vs. 25% collagen/PCL blend) were electrospun In vitro drug release profiles of RM-PU fibrous mats and carbodiimide-modified, with/without HA-o were determined via HPLC for 49 days. The in vitro functionalization. They were enclosed within porous, bioavailability of the released RM was analyzed be non-adhesive PTFE pouches, intra-peritoneally assessing the inhibition of cultured smooth muscle cells implanted for 2, 4, or 6wks, then either intra-aortally- using the AlamarBlue assay. The release profiles and grafted, or analyzed for cell phenotype, infiltration, gene bioavailability of RM from electrospun RM-PU vascular expression, and ECM deposition. grafts was similarly evaluated for 77 days in vitro. Results: Over 2wks, fibrous capsules formed around Results: Electrospinning of various RM-PU blended the conduits with mesothelial cells on the surface, and solutions, generated produced uniform and bead- α-SMA+ and SM22α+ cells within. Few of the CD68+ free fibers. The fiber size and the Young’s moduli of macrophages within the conduits were pro-inflammatory the RM-PU fibrous mats increased with higher RM (CD80+). The largest fiber–diameter conduits exhibited concentrations. The release profiles were dependent on greatest cell infiltration, cellularity, and collagen the RM concentration. RM was continually released over deposition. Elastin content was however limited. Greater 77 days from vascular grafts made of 20% RM - 80% collagen deposition was seen in the PCL only conduits. PU. This slowreleased RM remained bioavailable and More pronounced differences were observed after 4 was able to inhibit smooth muscle cell proliferation in and 6wks of implantation. Expression of Acta2, Myh11, vitro over the same time period. Col1a1, and Eln genes increased from 4-6wks for cells Conclusions: We were able to incorporate RM into PU within PCL conduits, but decreased for collagen/PCL. fibers via electrospinning and observed sustained RM Eln gene expression was higher with PCL conduits. release from RM-PU fibrous constructs for extended HA-o modification increased Eln, but not Col1a1, periods of time. The released RM remained bioavailable expression by recruited cells. Remodeling (α-SMA and for at least 77 days. hyaluronan distribution) occurred in the constructs 6 weeks post-autologous grafting, although occlusion occurred. Conclusions: Scaffold chemistry and fiber size impact intra-peritoneal tissue remodeling, with larger fiber diameters and incorporation of HA-o benefiting the process.

ISACB’S 2012 BIENNIAL MEETING 23 ORAL PRESENTATION ABSTRACTS: SESSION 4

5.1 Comparison of Synthetic Small-calibre Biodegradable vs. Stable ePTFE Vascular Prosthesis after Long-term Implantation in the Rat Aorta electro-spinning. After 15.5±3.0 months, in vivo ultra- Beat H. Walpoth, Department of Cardiovascular Surgery, sonography and angiography were performed to assess University Hospital of Geneva; Damiano Mugnai, patency (%) and compliance (%/100mmHg). After Department of Cardiovascular Surgery, University explantation micro-CT, immuno-histology, SEM and Hospital of Geneva; Sarra de Valence, Department of morphometry assessed (%): calcification, cellularity, Pharmaceutics & Biopharmaceutics EPGL, University intimal hyperplasia. of Geneva; Wojciech Mrowczynski, Department of Cardiovascular Surgery, University Hospital of Results: Patency was 100% for PCL and 67% for Geneva; Jean-Christophe Tille, Department of Clinical ePTFE. No aneurysms or stenoses were found. Pathology, University Hospital of Geneva; Xavier Compliance was lower for ePTFE compared to PCL Montet, Department of Radiology, University Hospital of (5.7±0.7 vs. 8.2±1.0; p<0.01), as was calcification Geneva; Robert Gurny, Department of Pharmaceutics (7.0±5.0 vs. 15.8±3.2;p<0.04). Histologically, low cellular and Biopharmaceutics EPGL, University of Geneva; ingrowth was found in ePTFE whereas PCL showed Afksendiyos Kalangos, Department of Cardiovascular significantly higher homogenous cellularity producing Surgery, University Hospital of Geneva; Michael Moeller, an autologous extra-cellular matrix, replacing the Department of Pharmaceutics & Biopharmaceutics degrading (65% MW reduction) PCL scaffold (11.3±4.0 EPGL, University of Geneva, Switzerland vs. 31.4±9.2;p<0.0001). Morphometry and SEM showed 100% neoendothelialisation for both grafts. Intimal Purpose: Shelf-ready, synthetic, small-calibre vascular hyperplasia thickness, length and area were higher in prostheses are needed in cardiovascular surgery. ePTFE compared to PCL. We assessed the long term results of synthetic, biodegradable small-calibre vascular grafts compared Conclusions: Synthetic, biodegradable small-calibre to stable ePTFE for aortic replacement in the rat. nano-fibre polycaprolactone grafts show better patency, compliance, endothelialisation, less intimal hyperplasia Methods: 14 anaesthetised Sprague Dawley rats and calcification compared to the clinically used received an infrarenal aortic graft (8-biodegradable; ePTFE grafts after long-term implantation in the rat 6-ePTFE) replacement (end-to-end; 2mm ID). aorta. Thus, such novel in situ tissue engineered grafts Biodegradable grafts (polycaprolactone=PCL) were could become a promising option for cardiovascular produced by random micro/nano-fibre (porosity 80%) revascularisation procedures.

Figure 1

24 ISACB.ORG ORAL PRESENTATION ABSTRACTS: SESSION 5

5.2 Vitalize Synthetic Vascular Grafts in vivo Yadong Wang, University of Pittsburgh, PA, USA; Wei Wu, University of Pittsburgh, PA, USA; Robert Allen, University of Pittsburgh, PA, USA Purpose: Current arterial tissue engineering research focuses on cell-based approaches. The purpose of this study is to investigate the potential of mammalian host to remodel synthetic polymeric grafts into viable arteries. The idea is to bypass in vitro cell seeding and culturing completely. Methods: We designed the graft to have two layers, the inner tubular core is made of the elastomeric poly(glycerol sebacate), the outer sheath is made of polycaprolactone fibers. The sterilized grafts are coated with heparin and implanted as interposition grafts in rat abdominal aorta. Results: The grafts were quickly infiltrated with cells and polymer degradation led to rapid host remodeling. The graft materials were mostly degraded within 3 months. In its place was a neo-artery that mimicked native artery mechanically, biochemically, and anatomically. The neo-arteries were well integrated with the host, remained patent and pulsed synchronously with host arteries. Conclusions: This study indicates that synthetic vascular grafts made from fast degrading elastomeric materials can be remodeled by rodents into viable arteries. It remains to be seen if this is translatable to small arteries in large animal models and humans.

ISACB’S 2012 BIENNIAL MEETING 25 POSTER PRESENTATION ABSTRACTS

P1 P2 Inflammatory Processes in Cardiovascular Investigating the Role of Perlecan in Disease and Treatment Modulating Pathological Angiogenesis in Greg A. Foster, UC Davis Biomedical Engineering, Calcific Aortic Valve Disease Robert M. Gower, UC Davis Biomedical Engineering, C. Alexander Arevalos, Rice University, Houston, TX, Chris E. Radecke, UC Davis Biomedical Engineering, Jerahme Martinez, Rice University, Dr. Mary C. Farach- Ehrin J. Armstrong, UC Davis Medical Center Internal Carson, Rice University, Dr. K. Jane Grande-Allen, Rice Medicine, Scott I. Simon, UC Davis Biomedical University Engineering. Introduction: Understudied in the pathology of CAVD Introduction: Acute myocardial infarction (MI) is the basement membrane proteoglycan perlecan associated with coronary artery disease (CAD) affects (Pln). Pln is known to regulate angiogenesis-modulated more than 2.5 million Americans annually. Studies have ossification, but it has never been studied in the context shown that 50% of patients with MI lack the traditional of modulating pathological angiogenesis in CAVD. risk factors for CAD. Therefore, there is a clinical need Methods: Freshly isolated porcine aortic valve for non-invasive assays of inflammatory cell activation endothelial cells (PAVEC) were seeded on to tissue to gauge its role in atherogenesis. culture dishes coated with full length Pln, Pln domains I, Materials and Methods: Whole blood was drawn IV, and V, and the intact basement membrane matrigel. via venipuncture and monocytes were immediately Additionally, PEGylated versions of the various domains labeled with fluorescent antibody. Data was acquired were patterned to PEGDA scaffolds to study them in on a BD FACScan cytometer. Whole blood or isolated their native conformation. The cells were imaged using monocytes were perfused though a reservoir and fluid phase contrast microscopy and probed for various was withdrawn through the flow chamber at a flow rate angiogenic markers using confocal fluorescence resulting in wall shear stress of 2 dynes/cm2. microscopy, western blotting, and qRT-PCR. Results and Discussion: We examined adhesion Results: Preliminary results have suggested differences molecule expression on monocyte subsets and found in the adhesion, morphology, and proliferation of the that CD11c was upregulated 60% on CD14++CD16+ cells exposed to the various substrates. For example, subset. Furthermore, CD11c was upregulated 300% the PAVEC seeded on to domain 1 displayed a on patients undergoing an MI compared to healthy disruption of cell to cell junctions which is an important subjects. Using vascular mimetic flow channels we step in the initiation of angiogenesis. Additionally, the detected enrichment of CD14++CD16+ monocytes cells seeded on to the intact basement membranes postprandial and for MI patients (Figure 1). Stabilizing led to the formation of early vasculogenic tubule like CD11c at low affinity diminished adhesion by 50% of structures typical of endothelial cells seeded onto control, corresponding with ~25% decrease in VLA-4 at matrigel. contact. Discussion: Fully understanding the role of perlecan Conclusions: We report on the role of CD11c as a in regulating angiogenesis-modulated ossification in biomarker of CAD and activator of VLA-4 dependent the progression of CAVD would greatly contribute to adhesion on VCAM-1. CD11c/CD18 upregulation the development of interventional treatments for the and activation is a signaling event associated with disease. This research is working to indentify a novel increased VLA-4 affinity and avidity during adhesion to target for interfering with the progression of valvular atherogenic endothelium. endothelium disruption which is seen in the pathology of CAVD. Figure 1: Enrichment of CD14++CD16+ monocytes is increased postprandial and during MI and is dependent on CD11c and VLA-4.

26 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P3 P4 Dimethylarginine Dimethylaminohydrolase Role of PI3k/Akt pathway in Cytoprotective expression in critical limb ischaemia Effect of Erythropoietin and Erythropoietin- Gareth Williams, Division of Surgery & Interventional derivatives in Ischaemic Human Myotubes Science, University College London, Royal Free Rebekah S. H. Yu Campus, London, United Kingdom; Xu Shi-wen, Purpose: To investigate the potential cytoprotective Centre for Rheumatology & Connective Tissue Disease, effects of Epo and an Epo-derivative (ARA-290) in a University College London, Royal Free Campus, London; human in vitro model of human skeletal muscle and Korsa Khan, Centre for Rheumatology & Connective determine the mechanisms by which Epo and its Tissue Disease, University College London, Royal Free derivatives inhibit apoptosis. Campus, London; Darryl Baker, Division of Surgery & Interventional Science, University College London, Royal Methods: Human myoblasts were isolated from Free Campus, London; Janice Tsui, Division of Surgery gastrocnemius muscle biopsies and differentiated into & Interventional Science, University College London, myotubes. Human myotubes were pre-treated first Royal Free Campus, London. with either Epo or ARA-290, and later with the specific pharmacological kinase inhibitors, against Jak2 (SD- 1029), or against PI3k (wortmannin) before being Purpose: The enzyme Dimethylarginine subjected to simulated ischaemia. Western blot analysis Dimethylaminohydrolase (DDAH) forms an integral part of cell lysates, using the pro-apoptotic marker cleaved of the Nitric Oxide pathway in vascular homeostasis, by caspase-3 was performed and compared with levels of breaking down inhibitory asymmetric dimethylarginine Akt and phosphorylated-Akt. (ADMA) into L-citrulline and dimethylamine. Results: Exogenous administration of Epo and ARA- Accumulation of ADMA and consequent endothelial 290 were able to ameliorate the ischaemia-induced dysfunction has been shown to result from reduced apoptosis on isolated human myotubes as shown DDAH expression in endothelial cells. However, whilst by a significant reduction in cleaved caspase-3 the majority of studies have focussed on vascular expression. Addition of either kinase inhibitor (SD- endothelial cells, our preliminary work suggests that 1029 or wortmannin), to ARA-290 or Epo pre-treated skeletal muscle itself may be a source of ADMA; we cells, abolished the reduction in apoptosis. Further, a hypothesize that raised ADMA levels in skeletal muscle reduction in apoptosis was associated with an increase homogenates from patients with critical limb ischaemia in phosphorylated-Akt on western blot analysis. (CLI) are due to changes in DDAH 1 and DDAH 2 expression in the muscle. Conclusions: The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic Methods: Skeletal muscle biopsies were collected insult suggests a potential role in tissue protection from patients with critical limb ischaemia undergoing in skeletal muscle injury. We propose that the PI3k/ lower limb amputation, and compared to non-ischaemic Akt signalling pathway is involved in mediatingthis control biopsies. Western Blotting was used to analyse cytoprotection, posing a potential future therapeutic muscle lysate, looking at DDAH 1 and DDAH 2 proteins. target. In addition, immunohistochemistry was used to obtain a qualitative measure of DDAH 1 and DDAH 2 distribution in ischaemic skeletal muscle specimens compared with non-ischaemic samples. Results: Western blot analysis showed reduced DDAH 1 and DDAH 2 expression in ischaemic skeletal muscle relative to non-ischaemic muscle. Immunohistochemistry demonstrated reduced staining for DDAH 1 and DDAH 2 of muscle fibres in ischaemic skeletal muscle when compared with non-ischaemic specimens. Conclusions: Down-regulation of DDAH 1 and 2 in critically ischaemic skeletal muscle may contribute to a rise in ADMA and subsequent Nitric Oxide pathway dysfunction, compounding ischaemic damage to the muscle.

ISACB’S 2012 BIENNIAL MEETING 27 POSTER PRESENTATION ABSTRACTS

P5 P6 IGF-1 has No Effect on the Proliferation or The Toll-like Receptor (TLR) 2 And 6 Differentiation of Myoblasts Exposed to Ligands Heat Shock Proteins (HSP) 60 And Ischaemic Conditions 70 May Play A Role In The Pathogenesis Of M Fincher, Division of Surgery & Interventional Science, Skeletal Muscle Damage In Critical Limb UCL, Royal Free Campus, UK; N Oikonomopoulos, Ischaemia (CLI) Division of Surgery & Interventional Science, UCL, Hemanshu Patel, Division of Surgery & Interventional Royal Free Campus, UK; D Abraham, Centre for Science, UCL, Royal Free Campus, UK ; Ali Navi, Rheumatology & Connective Tissue Disease, UCL, Division of Surgery & Interventional Science, UCL, Royal Free Campus, UK; D Baker, Division of Surgery & Royal Free Campus, UK; Xu Shi-wen, Centre for Interventional Science, UCL, Royal Free Campus; J Tsui, Rheumatology & Connective Tissue Disease, UCL, Division of Surgery & Interventional Science, UCL, Royal Royal Free Campus, UK; David Abraham, Centre for Free Campus Rheumatology & Connective Tissue Disease, UCL, Royal Purpose: Following revascularisation for peripheral Free Campus, UK; Daryll Baker, Division of Surgery arterial disease, patients report poor functional outcome & Interventional Science, UCL, Royal Free Campus, and no improvement in quality of life. This has been UK; Sidney Shaw Department of Clinical Research, attributed to a musculopathy. Myogenic progenitor cells University of Bern, Switzerland; Janice Tsui Division (satellite cells, SCs) provide skeletal muscle with an of Surgery & Interventional Science, UCL, Royal Free intrinsic ability to regenerate. We have demonstrated Campus, UK that ischaemia has a detrimental effect on SC function Purpose: The pathophysiology of skeletal muscle in vitro. We hypothesize that the addition of IGF-1, a damage in CLI is poorly understood. TLRs have been prominent growth factor in myogenesis that stimulates associated with ischaemia-induced tissue damage both proliferation and differentiation, will improve the and in particular TLR 2 and 6 have been implicated in function of SCs in ischaemic conditions, which may lead critically ischaemic muscle. TLR2/6 are activated by to the regeneration of ischaemic skeletal muscle. endogenous ligands such as HSP 60 and 70 which Methods: C2C12 cells (mouse myoblast cell line) were are released during tissue damage with subsequent exposed to simulated ischaemic conditions using a upregulation of pro-inflammatory cytokines such as IL- hypoxic chamber (20% CO2, 80 N2) and compared to 6. We hypothesize that the expression of TLR 2/6 and cells grown in standard normoxic conditions. Cells were their endogenous ligands HSP 60/70 is upregulated in either pretreated with IGF-1 (250ng/ml) for 24hrs or left ischaemic skeletal muscle with subsequent activation of untreated. Proliferation rates were assessed using both the signalling pathway. an MTT assay technique and cell counting. Myogenic Methods: TLR2/6 expression was studied in ischaemic differentiation was assessed by myogenin western blot. and non-ischaemic human muscle biopsies and in Results: Simulated ischaemia significantly reduced the C2C12 myotubes cultured in ischaemic conditions proliferation rate of myoblasts (P<0.001). Pretreatment using Western blot and immunofluorescence. Western with IGF-1 did not effect the proliferation rate of blot was used to measure the expression of HSP 60 myoblasts in simulated ischaemia (in both MTT assay and 70. Functional effects of TLR2/6 antagonism on and cell counting techniques) and did not significantly ischaemia-induced IL-6 release and cell death were increase the expression of myogenin in ischaemic studied by incubating myotubes with neutralizing TLR2 myoblasts. or 6 antibodies. IL-6 release was assayed by ELISA. Apoptosis was assessed using cleaved caspase-3. Conclusions: IGF-1 had no impact on the proliferation or differentiation of myoblasts when exposed to Results: TLR2/6 protein expression was significantly ischaemic conditions in vitro. Ischaemia seems to have upregulated in ischaemic muscle and in C2C12 an detrimental effect on the function of myoblasts and myotubes cultured in ischaemic conditions (p<0.05). thus the potential to stimulate their function in vivo Raised levels of HSP 60 and 70 were demonstrated in appears limited. ischaemic human muscle biopsies and in ischaemic C2C12 myotubes (p<0.05).TLR2/6 antagonism reduced ischaemia-induced IL-6 production and apoptosis in culture. Conclusions: Upregulation of TLR2/6 and HSP60 and 70 expression occurs in ischaemic muscle. Activation of TLR2/6 signalling leads to IL-6 release which contributes to inflammation and muscle damage. Inhibiting HSP 60 and 70 maybe a potential therapeutic target in reducing skeletal muscle damage in CLI.

28 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P7 P8 Functional Upregulation of Toll-like Synthetic PEG Hydrogel Extracellular Receptor 4 and 1 in Ischaemic Skeletal Matrix Mimics for the Vascularization of Muscle Engineered Tissues Ali Navi, Division of Surgery and Interventional Georgia Papavasiliou, Illinois Institute of Technology, Science, Royal Free Campus, UCL, London, UK; David Chicago, IL; Michael V. Turturro, Illinois Institute of Abraham, Centre for Rheumatology and Connective Technology, Chicago, IL; Megan Christenson, Illinois Tissue Disease, University College London, Royal Institute of Technology, Chicago, IL Free Campus, London, UK; Xu Shi-Wen, Centre Purpose: To develop synthetic PEG hydrogel for Rheumatology and Connective Tissue Disease, scaffolds with tunable gradients of cell adhesion University College London, Royal Free Campus, peptides, matrix metalloproteinase (MMP) sensitive London, UK; Sidney G. Shaw, Department of Clinical crosslinks susceptible to cell mediated proteolysis, and Experimental Research, University of Bern, 3010 Bern, compressive modulus; to quantify the spatial effects Switzerland; Daryll M. Baker, Division of Surgery and of biochemical and mechanical properties on 3D Interventional Science, Royal Free Campus, UCL, neovascularization in vitro. London, UK; Janice Tsui, Division of Surgery and Interventional Science, Royal Free Campus, UCL, Methods: Perfusion based frontal photopolymerization London, UK; (PBFP) was used to engineer PEG hydrogels with gradients in elastic modulus, immobilized YRGDS Purpose: It has been suggested that the inflammation cell adhesion ligands, and MMP-sensitive crosslinks. induced by ischaemia in peripheral arterial disease Gradients of mechanical properties, YRGDS, MMP- (PAD) has a key role in damaging lower limb skeletal sensitivity, and mechanical properties, were quantified muscle. TLR4 has been implicated in ischaemia and has by compression experiments, radiolabeling, and gel been shown to heterodimerize with TLR1. We aimed to degradation studies in collagenase, respectively. An study expression, activation and distribution of TLR4&1 in vitro model of neovascularization, composed of co- in human skeletal muscle in critical limb ischaemia (CLI). culture aggregates of endothelial and smooth muscle Methods: Human skeletal muscle biopsies were cells was used to evaluate the effect of these gradients taken from the medial head of gastrocnemius from on 3D directional vascular sprout invasion, anisotropy patients undergoing major lower limb amputation index, and sprout length by angle over 21 days. due to CLI (n=6) and from patients with no PAD Results: Gradient hydrogels exhibited an 80.4% undergoing saphenous vein harvesting for CABG decrease in modulus and a 56.2% decrease in surgery (n=6). We carried out western blot analyses immobilized YRGDS. PBFP hydrogels demonstrated and immunofluorescence to study the TLR expression, gradients in MMP sensitivity with degradation times activation and distribution in human skeletal ranging from 10-12 hours in stiffer regions to 4-6 hours muscle biopsy. in regions of decreased stiffness. Neovascularization Results: Western blot analyses on muscle homogenates in gradient hydrogels occurred bi-directionally with showed upregulation of TLR4&1 in CLI (P<0.05). vessel alignment observed in the direction parallel to the Furthermore, phosphorylated NFkB and JNK as gradient. Vessel length was found to be twice as long key known signaling pathways were found to be in the direction parallel to the gradient as compared increased in ischaemic samples (P<0.05) (Figure1). to the perpendicular direction with directionality more TLR4&1 co-immunostaining of ischaemic muscle prominent in regions of increased stiffness, MMP sections demonstrated co-localization of these sensitivity, and immobilized YRGDS. receptors, suggestive of heterodimerisation. Also, we Conclusions: We developed a novel technique that demonstrated that TLR1 is expressed on endothelium generates tunable mechanical and biochemical (CD31), neutrophils (CD43) and macrophages (CD68) gradients in scaffolds that stimulate directed 3D in CLI. neovascularization in vitro. Conclusions: We showed that TLR4&1 are upregulated and functional in CLI. Further studies are required to understand the downstream signalling, which may lead to the development of novel therapies aimed at reducing muscle damage.

ISACB’S 2012 BIENNIAL MEETING 29 POSTER PRESENTATION ABSTRACTS

P9 P10 Evaluation of Elastin Derived Vascular Tissue Engineering of Aortic Valve Leaflets Grafts in a Circulatory Model Using Biomimetic Composite Poly(ethylene Deon Bezuidenhout, Cardiovascular Research Unit, glycol) Diacrylate Hydrogels University of Cape Town; Tim Pennel, Cardiovascular Xing Zhang, Rice University, Houston, TX; Bin Xu, Rice Research Unit, University of Cape Town; George University, Houston, TX; Hubert Tseng, Rice University, Fercana, Department of Bioengineering, Clemson Houston, TX; Maude L. Cuchiara, Rice University, University; Peter Zilla, Cardiovascular Research Unit, Houston, TX; Jennifer L. West, Rice University, Houston, University of Cape Town; Dan Simionescu, Department TX; K. Jane Grande-Allen, Rice University, Houston, TX of Bioengineering, Clemson University Purpose: To develop anisotropic poly(ethylene Purpose: To evaluate pentagalloyl-glucose (PGG) glycol) diacrylate (PEGDA) hydrogels using two stabilized elastin-derived vascular grafts (EDVGs) in different molecular weight PEGDA molecules by the terms of their suitability as vascular substitutes. photolithographic patterning replicating mechanical Methods: EDVGs were prepared by alkaline behavior of aortic valve leaflets; To functionalize PEGDA decellularization (0.1M NaOH at 37°C for 3hrs), rinsing hydrogels with bioactive peptides mimicking the and sterilization in 0.1% peracetic acid. Batches of biological function of aortic valve leaflets. EDVG scaffolds were treated with sterile 0.1% PGG (pH Methods: PEGDA hydrogels were prepared by photo 5.5) containing 20% isopropanol for 24 hours, rinsed crosslinking. Stripe-patterned hydrogels were created and stored in sterile PBS. A subset of PGG and non- by photolithographic patterning of secondary PEGDA PGG samples were additionally covalently heparinized molecules into the network of a preformed hydrogel. via diamine coupling and reductive amination using Mechanical property was evaluated by compression, nitrous acid degraded heparin. Grafts were implanted tension and three-point bending tests. Bioactive in a rat infrarenal aortic model for 4 and 8 weeks. peptides (cell-adhesive and matrix metalloproteinase- Remodeling, patency, endothelialization and healing of sensitive peptides) were incorporated into the hydrogel the explants were determined by gross and histological network. Interaction of valvular interstitial cells with evaluation. these biomimetic PEGDA hydrogels was investigated in Results: While PGG treatment did not significantly both two-dimensional and three-dimensional cultures. affect the denaturation temperature (DT) of the tissue, Results: PEGDA hydrogels showed tunable heparinization resulted in significant increases in DT mechanical property depending on the molecular (from 52 ºC to 81-82 ºC) and heparin content (from weight of PEGDA and concentration of the prepolymer baseline noise levels of < 10mg/g to >100mg/g). Overall solution. Higher stiffness was obtained using higher the non-heparinized groups showed strong evidence concentration or lower molecular weight of PEGDA. of remodeling and recellularization, with a high patency Several photolithographic patterned hydrogels showed rate of 82%. At 8 weeks, only small fragments of the significantly different modulus when tested parallel original grafts were preserved with good neo-vessel and perpendicular to the stripe pattern under tension formation and moderate intimal hyperplasia (IH). The and bending condition (anisotropy). Bioactive peptides heparinized groups showed 100% patency, but with incorporated in the hydrogel network facilitate valvular little remodeling, presumably due to the increased cell adhesion and proliferation. crosslink density resulting from the amination of the Conclusions: PEGDA hydrogels are suitable as tissue prior to heparin attachment. Some surface scaffolds for engineering of aortic valve leaflets, endothelialization was observed in all treatment groups. considering they can be tuned mimicking the Conclusions: EDVGs are promising candidates as mechanical behavior of the leaflets, and functionalized vascular grafts, either as substrates for remodeling, or in with bioactive peptides to support valvular cell adhesion more highly crosslinked and heparinized forms. and proliferation. Moreover, PEGDA hydrogels provide a blank template for studying interaction between valvular cells and extracellular matrices, which will shed light on valvular biology and provide cues for future heart valve tissue engineering.

30 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P11 P12 Numerical Assessment of the Strain/ Heart Rate Variability With Postural Stress Fields Supported by Microstructural Changes Might Be Useful For Detection Components of Mouse Carotid Arteries Of Symptomatic Mitral Valve Prolapse Chiara Bellini, University of Calgary, Calgary, AB, Syndrome In Taiwanese Canada; Jacopo Ferruzzi, Yale University, New Haven, Ya-chu Chen, National Chiao Tung University, Hsin Chu CT, USA; Elena S. Di Martino, University of Calgary, City, Taiwan; Lung-Wen Tsai, Taipei Medical University Calgary, AB, Canada; Jay D. Humphrey, Yale University, Hospital, Taipei City, Taiwan; Ing-Fang Yang, Jen Chi New Haven, CT, USA General Hospital, Taipei City, Taiwan; Chung-Kai Tseng, Purpose: The arterial wall is a collection of China Medical University Hospital, Taichung City, independently growing elements, including cells Taiwan; Ten-Fang Yang, National Chiao Tung University, and ECM components. Residual strains allow these Hsin Chu City, Taiwan. Taipei Medical University and elements to achieve their homeostatic working point, Hospital, Taipei City, Taiwan while simultaneously ensuring material continuity of the Purpose: To evaluate if HRV parameters with postural tissue. Macroscopic measures of residual strains were changes can be used to differentiate between previously reported in terms of opening angle, i.e. the symptomatic MVPS patients and normal controls. angle between the midpoint and the tips of a radially- Methods: A total of 72 symptomatic patients (4 males cut arterial ring. Purpose of this study was to develop a and 68 females) had been echocardiographically numerical model capable of predicting the strain/stress diagnosed as having MVPS from the cardiology clinic distribution at the microstructural level under general and 101 healthy university students (51 males and configurations of the external loads. 50 females) were recruited as normal control for the Methods: Pressure-diameter tests were performed present study. A locally developed HRV system with one on carotid arteries from wildtype mice to record the modified lead II ECG was used to record the tracing. All mechanical response. Histological assays provided the records were taken during the daytime to avoid the the relative abundance of constituents. A structurally- influence of diurnal changes. The subjects were asked motivated constitutive relationship was fitted to the to rest at least 5 minutes before taking the records and mechanical data and fed to the numerical model. postural alterations (lying, sitting and standing). Opening angle measurements served as model Results: Time domain Parameters between MVPS and validation. Normal Group was statistically significantly different in Results: The model uses experimental measures of lying and sitting position (P< 0.05); Whereas, Frequency macroscopic mechanical and structural properties to domain Parameters with postural changes were shown estimate the strain/stress fields for each microstructural to have no significant differences in all postures. constituent. Results represent the first quantitative Conclusion: For time domain, HRV symptomatic MVP demonstration that loads within the physiological group is statistically significant different to normal range are mainly supported by the elastic material in control in lying and sitting positions. Lying and sitting the artery, while collagen fibers intervene at higher female MVP Time domain parameters were statistically pressures, in agreement with qualitative observations significant different to normal; whereas male only lying previously reported. RMSSD and NN50 were significantly different. For Conclusion: Understanding the contribution of frequency domain, HRV cannot significantly differentiate microstructural components in withstanding the symptomatic MVP group from normal control. external loads might clarify the mechanotransduction Table 1 Time domain HRV Parameters mechanisms through which cells sense and react to changes in the macroscopic mechanical environment. Also, it might help in estimating the intensity of perturbations that either cause the failure of ECM components or trigger specific cellular responses in disease progression.

ISACB’S 2012 BIENNIAL MEETING 31 POSTER PRESENTATION ABSTRACTS

P13 A Research of Heart Rate Variability Results: For MVPs, no difference was found in time between Symptomatic Mitral Valve Prolapse domain between male and female. For normal male and Syndrome and Normal female, frequency domain was statistically significantly different only in LF/HF ratio; Whereas, postural changes Ya-chu Chen, National Chiao Tung University, Hsin Chu were shown to have significant effects in LF/HF ratio in City, Taiwan; Lung-Wen Tsai, Taipei Medical University all postures in frequency domain. Hospital, Taipei City, Taiwan; Ing-Fang Yang, Jen Chi General Hospital, Taipei City, Taiwan; Chung-Kai Tseng, Conclusion: For time domain, HRV cannot be used China Medical University Hospital, Taichung City, to differentiate male from female in all positions, but Taiwan; Ten-Fang Yang, National Chiao Tung University, the sample size is not large enough. For frequency Hsin Chu City, Taiwan. Taipei Medical University and domain, HRV LF/HF ratio might be a useful tool in all Hospital, Taipei City, Taiwan three postures for the detection of gender differences in symptomatic MVPS and normal group. Purpose: To evaluate if HRV parameters is different between MVPS and normal. Methods: A total of 72 symptomatic echocardiographically documented MVPS patients (4 males and 68 females) and 101 healthy students (51 males and 50 females) were recruited as normals for this study. A local HRV system with one modified lead II was used. All recordings were taken during daytime to avoid diurnal ANS influences. The subjects were asked to rest 5 minutes before recording and postural alterations (lying, sitting and standing).

32 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P14 P15 Confluent Transmural Endothelialisation The Effects of Waveform on the Generation Without Hyperplasia Progression In A Loop of Flow Dependent Phospho-Smad2 and Graft Isolation Model Akt Species Tim Pennel, University of Cape Town Alex Obrejanu, University of Calgary, Calgary, AB, Purpose: We have recently described an isolation Canada, Robert D. Shepherd, University of Calgary, looped vascular graft model that clearly separates Calgary, AB, Canada, Kristina D. Rinker, University of transmural from transanastomotic endothelialisation Calgary, AB, Canada as distinctly discrete events. The present study Purpose: Endothelial cells are affected by fluid flow used the same isolation model to demonstrate that through mechanotransduction-induced signaling neointimal tissue formation accompanying transmural events. We previously identified a novel signaling endothelialisation reaches an early, stable equilibrium. pathway, involving Smad2 and Akt, in which linker Methods: High-porosity polyurethane (PU) grafts phosphorylated Smad2 is generated and translocates to (ID1.7mm; 150μm pore size) were interposed (‘welded’) the nucleus in a flow-dependent manner. The Smad co- between low-porosity ePTFE (ID1.7mm;IND 15-25μm) repressor transforming growth interacting factor (TGIF) segments and implanted in the abdominal aorta of also localizes in a similar fashion. Levels of Smad2 were Wistar rats for 6,8,12 and 24 weeks (n=8/time point). shown to follow the pattern of Akt phosphorylation Looping the graft increased their length to 8cm. Light, through inhibitor studies. Due to the nature of our immune-fluorescence (CD31) and scanning electron system, however, we were not able to determine if these microscopy were used for image analysis. events were controlled by the magnitude of fluid shear stress, or if they were dependent on flow waveform. The Results: The transanastomotic outgrowth edge never work presented here was undertaken to address this traversed the ePTFE endothelial-free zone on the question. proximal or distal segment (Proximal: 25.93±5.90mm at 6 weeks and 8.71±4.94mm at 24 weeks, Methods: HAEC were exposed to 20 hours of 2 and 10 p=0.0005), resulting in 100% isolation from the mid- dyne/cm2 shear stress from both pulsatile and steady graft endothelium. Transmural midgraft endothelial flow in a parallel-plate flow loop. Expression levels of coverage reached pre-confluence at 6 weeks (85±15%) molecular targets were determined by real-time RT-PCR and confluence between week 12(94±11%) and and western blotting. Tissue samples from a partial 24(85±28%). One of the 24-week loop grafts did carotid ligation mouse model, in which oscillatory flow not anchor in the retroperitoneum resulting in the was induced in the affected artery, were analyzed using absence midgraft endothelial coverage. There was no confocal microscopy. progression of mid-graft neo-intimal tissue hyperplasia Results: Akt phosphorylation profiles showed no at 6, 8, 12 and 24 weeks (54.99±52.02μm; 45.00±58.39; significant difference between flow patterns, whereas 45.84±46.60; 47.36±37.08μm, p=0.77). linker Smad2 phosphorylation was less robust under Conclusions: A looped interposition-graft model steady flow relative to pulsatile. Each phosphorylation provides sufficient isolation length to clearly distinguish increased at higher shear stress. Murine tissue studies transanastomotic from transmural endothelialisation were largely consistent with in vitro studies. for up to half a year. Ingrowth from surrounding Conclusions: The modulation of Smad2 and Akt tissue appears essential for transmural mid-graft signaling from fluid flow is complex, involving both endothelialisation. The lack of ongoing mid-graft shear stress magnitude and flow patterning. Further luminal narrowing confirms that sub-endothelial tissue work is necessary to completely define this pathway’s proliferation due to transmural growth is of no concern. controlling parameters.

ISACB’S 2012 BIENNIAL MEETING 33 POSTER PRESENTATION ABSTRACTS

P16 P17 Effects of Blood Volume Expansion Fluids Collagen Fiber Architecture in the on Human Aortic Endothelial Cells Longitudinal-Radial Plane of Ascending Kogan Lee, University of Calgary, Calgary, AB, Canada, Aorta is Distinctly Different among Bicuspid Andrew Walker, University of Calgary, Calgary, AB, Aortic Valve and Tricuspid Aortic Valve Canada, Robert D. Shepherd, University of Calgary, Patients with Ascending Aortic Aneurysm Calgary, AB, Canada, Clifton Johnston, Dalhousie Alkiviadis Tsamis, University of Pittsburgh, Pittsburgh, University, Halifax, Nova Scotia, Canada, Gary Dobson, PA; Julie A. Phillippi, University of Pittsburgh, Pittsburgh, Alberta Health Services, Calgary, AB, Canada, Kristina PA; Salvatore Pasta, University of Pittsburgh, Pittsburgh, D. Rinker, University of Calgary, AB, Canada PA, and Fondazione RiMED, Palermo, Italy; Antonio Purpose: Blood volume replacement is a critical issue D’Amore, University of Pittsburgh, Pittsburgh, PA, and for individuals with severe blood loss or hypovolemic Fondazione RiMED, Palermo, Italy; Simon C. Watkins, shock. However, it is unclear whether crystalloid University of Pittsburgh, Pittsburgh, PA; William Wagner, or colloid solutions are most appropriate for fluid University of Pittsburgh, Pittsburgh, PA; David A. Vorp, resuscitation. Crystalloid solutions replace volume on a University of Pittsburgh, Pittsburgh, PA; Thomas G. less than equivalent basis, and infusion of large volumes Gleason, University of Pittsburgh, Pittsburgh, PA is associated with edema and pulmonary complications. Purpose: To characterize the fiber architecture in Hydroxyethyl starch (HES) colloids resist hydrolysis, the longitudinal (LONG)-radial (RAD) plane of human allowing them to be effective osmotic agents, and ascending thoracic aortic aneurysm (ATAA) and control induce plasma volume expansion of up to 1.45 times ascending thoracic aorta (ATA). We hypothesize that the infused volume. HES have also been reported previously reported lower delamination strength of ATAA to positively influence endothelial physiology, while with bicuspid aortic valve (BAV) compared with tricuspid simultaneously exhibiting some negative clinical effects. aortic valve (TAV) is related to fiber architectural This study investigated endothelial exposure to both anomalies in the LONG-RAD plane of the tissue. crystalloid and HES under static and flow conditions to evaluate impacts on inflammatory mediators. Methods: Artificially dissected human tissue samples from BAV-ATAA, TAV-ATAA and control ATA were Methods: HAEC were treated with 25%(v/v) Pentaspan, examined under multi-photon microscopy. The medial- Voluven, and normal saline, supplemented with adventitial and medial-intimal halves were viewed in dexamethasone and/or TNF-alpha as required. the LONG-RAD plane. Images were processed using Monocyte adhesion molecules expression was an automated image-based tool to obtain the fiber determined by immunoblotting, and monocyte adhesion amplitude of angular undulation (AAU). was measured by microscopy. Viscosity dependent shear stresses were determined for flow studies. Results: For collagen fibers of the medial-intimal half, AAU was lower in BAV compared with TAV-ATAA and Results: Each of the solutions tested impacted unchanged from control ATA. AAU was higher in TAV- adhesion molecule expression in static culture, with ATAA compared with control ATA. These results suggest the HES reducing ICAM-1 and VCAM-1. This trend that collagen fibers in this region of the aorta are less was maintained upon pre-treatment with TNF-alpha, undulated about LONG axis in BAV than in TAV-ATAA, and supported by monocyte adhesion data. Under and more undulated about LONG axis in TAV-ATAA than flow, saline increased adhesion molecule expression, in control ATA. In the medial-adventitial half, the AAU of while Voluven provided mixed results. Viscosity collagen was higher in BAV and unchanged in TAV-ATAA dependent shear stress also participated in determining compared with control ATA. experimental outcomes. Conclusions: The weakened delamination strength Conclusions: HES appear to be more beneficial than previously observed for BAV-ATAA may be related to the saline to HAEC physiology based upon two factors; present findings of reduced collagen fiber undulation their chemical effect and their ability to maintain blood about the LONG axis in the medial-intimal half of BAV- viscosity at near normal values. ATAA and could be important in the progression of aortic dilatation and its propensity for dissection.

34 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P18 P19 Experimental Vascular Models for Development of Living Vascular Grafts for Evaluation of Novel Contrast Agent Peripheral Bypass Applications using a Nanoparticles Novel Transmural Perfusion Bioreactor Amber L. Doiron, Sean X.Y. Jiang, Robyn R.M. Steele, George Fercana, Clemson University, Biocompatibility Kristin L. Yaehne, Linda B. Andersen, Robert D. and Tissue Regeneration Laboratories, Clemson, SC, Shepherd, Sarah J. Childs, David T. Cramb, Richard USA; Eugene Langan, Greenville Hospital System, Frayne, Kristina D. Rinker. All author affiliations are Greenville, SC, USA; Christopher Wright, Greenville University of Calgary, Calgary, AB, Canada. Hospital System, Greenville, SC, USA ; Dan Simionescu, Purpose: Nanotechnology is a rapidly growing field Clemson University, Biocompatibility and Tissue with a wide range of applications for nanoparticles in Regeneration Laboratories, Clemson, SC, USA drug delivery and medical imaging. Understanding Purpose: To develop living, small diameter vascular nanoparticle interactions with living cells and tissues grafts of extended lengths for peripheral bypass is vital in determining their effectiveness, toxicity, and applications. biodistribution. Methods: Fresh bovine internal mammary arteries (>25 Methods: Nanoparticles of various types have been cm long) were mounted in a sterile perfusion device investigated in several vascular models to evaluate and treated with detergents and alkaline solutions adhesion, internalization, and biodistribution. Kinetics under diastolic pressures for decellularization, followed of particle uptake were assessed in human endothelial by stabilization with penta-galloyl glucose, a matrix- cells. A parallel-plate flow chamber (PPFC) was used for stabilizing polyphenol. Human aortic smooth muscle endothelial cell culture with control over shear stress. cells, aortic fibroblasts and umbilical vein endothelial In vivo techniques include zebrafish embryos, the cells were seeded onto scaffolds by injection in the chicken embryo chorioallantoic membrane (CAM) appropriate vessel tunics and maintained in culture model, and a partial carotid ligation, ApoE knockout in static conditions for 10 days followed by dynamic murine model of atherosclerosis. Fluorescence conditioning for 7 days. correlation spectroscopy was used with the CAM model Results: DAPI nuclear staining, histology and to monitor particle disappearance from blood over time. quantitative DNA analysis showed that the MR imaging and fluorescence microscopy were used to processed scaffolds were fully acellular (>90% assess particle localization. reduction in DNA content, from fresh tissue). Results: The vascular models employed provided a Desmosine and hydroxyproline assays, histology and breadth of detailed knowledge on the potential for immunohistochemistry confirmed excellent preservation nanoparticles to target tissues and attach to cells. of elastin and collagen components, as well as of As shown with the PPFC and zebrafish, nanoparticle collagen type IV and laminin. Calcein AM staining uptake varied greatly with shear stress and flow pattern. of vessels seeded with the appropriate human cells The PPFC also allowed examination of the amount of showed excellent viability and cellular infiltration in static nanoparticle contrast agent needed to cause a change and dynamic conditions. Notably, partial recellularization in magnetic resonance signal. The tortuous vessels of all three vessel tunics was confirmed with histology of the zebrafish caudal tail plexus showed the highest staining. Preliminary mechanical tests revealed burst particle uptake, localized to areas with disturbed flow. pressure values of >3000 mmHg and compliance values The CAM model highlighted the importance of particle of 0.26 % mmHg-1 x10-2. Ongoing studies include composition in determining uptake kinetics. immunohistochemical analysis to confirm identity of Conclusions: Understanding nanoparticle localization in cells present on the lumen, media and adventitia and a physiologically-relevant environment is important for stem cell seeding for potential translational applications. determining risk profiles and efficacy for drug delivery or Conclusions: Revitalized, small diameter, long vascular molecular imaging. grafts can be prepared in sterile conditions using our novel device which serves as a decellularization and functional bioreactor system for potential clinical applications.

ISACB’S 2012 BIENNIAL MEETING 35 POSTER PRESENTATION ABSTRACTS

P20 P21 Development of a Nanocomposite Based Collagen Gel Contraction Assay for Small Diameter Bypass Graft for Paediatric Evaluation of Porcine Aortic Valve Applications Interstitial Cell Glycolytic Metabolism Achala de Mel 1,2, 1Centre for Nanotechnology Peter Kamel, Rice University; Paul Qu, Rice University; & Regenerative Medicine, Division of Surgery & Deepak Nagrath, Rice University; Romain Harmancey, Interventional Science, University College London, University of Texas at Houston Medical School; Heinrich London, UK; 2 UCL Ear Institute, Royal National Taegtmeyer, University of Texas at Houston Medical Throat, Nose & Ear Hospital, 330 Gray’s Inn School; K. Jane Grande-Allen, Rice University 1 1 Road, London, UK;Giorgio Cittadella , Centre for Purpose: Despite a high incidence of calcific aortic Nanotechnology & Regenerative Medicine, Division of valve disease in metabolic syndrome, there is little Surgery & Interventional Science, University College 1 1 information about the fundamental metabolism of heart London, London UK; Hammad Lakhani , Centre for valves. Furthermore, it is appreciated for other cell types Nanotechnology & Regenerative Medicine, Division of that cell metabolism is a first responder to chemical and Surgery & Interventional Science, University College 1,3, 1 mechanical stimuli. It is unknown, however, how these London, London, UK; George Hamilton Centre for strategies -- which are often employed in the context Nanotechnology & Regenerative Medicine, Division of of valve tissue engineering -- may impact valvular Surgery & Interventional Science, University College interstitial cell (VIC) biology. London, London, UK; 3Royal Free Hampstead NHS Trust Hospital, London, UK; Alexander M Seifalian 1,3 1Centre Methods: To analyze basal metabolism, aortic VICs for Nanotechnology & Regenerative Medicine, Division were harvested from porcine hearts, seeded into fibrin of Surgery & Interventional Science, University College and collagen gels, and analyzed for gel contraction, London, London, UK; 3Royal Free Hampstead NHS Trust lactate production, and glucose consumption in Hospital, London, UK response to manipulation of concentrations of various metabolic substrates including glucose, galactose, Congenital or acquired heart disease during childhood pyruvate, and glutamine. highlights a leading cause of death within the first year, but such conditions can be treated with bypass surgery. Results: Gel contraction was a sensitive means of This need for a valid replacement for autologous assessing VIC responses to metabolic manipulation, tissues has led to the development of tissue engineered particularly in nutrient-depleted culture medium. vascular grafts (TEVGs). Currently available grafts for VIC contraction of gels was optimal at a glucose small diameter vessels do not perform adequately, but concentration of 2 g/L, slower at 1 g/L and 4.5 g/L, our group has developed an ideal heamocompatible, and extremely delayed at 0-0.5 g/L. Substitution of biostable small diameter vascular bypass graft and galactose significantly delayed contraction and reduced awaits clinical trials. Paediatric applications require lactate production. In low sugar concentrations, grafts to develop with somatic growth and thus pyruvate depletion reduced contraction. Glutamine complete tissue engineering with biodegradable depletion reduced cell metabolism and viability. nanocomposite polymer and stem cells has been looked Conclusions: Nutrient depletion and manipulation of into. Polyhedral oligomeric silsesquioxane (POSS) metabolic substrates impacts the viability, metabolism, poly(caprolactone-urea)urethane (PCL) (POSS-PCL) and contractile behavior of VICs. Nonetheless, polymer was extruded into 5mm diameter tubes with VICs readily utilize alternative substrates including small diameter pore in the lumen and larger diameter pyruvate and glutamine to compensate for low sugar. pores at the exterior using solvent exchange method. Interestingly, high concentrations of specific nutrients The grafts interior was exposed to peripheral blood reduced gel contraction, even without a substantial derived endothelial progenitor cells (EPC) and was effect on cell viability. These results begin to link VIC examined for endothelialisation. The exterior surface metabolism and macroscopic behavior such as cell- was exposed to adipose derived mesenchymal stem matrix interaction and contraction. cells (AMDSCs) and was treated with growth factors TGF-β1 and BMP-4 whilst placed in a bioreactor. EPC introduced, perfectly formed nanocomposite graft lumen became endothelialised whilst AMDSCs formed an external layer of smooth muscle cell layer with contractile and synthetic functions and was confirmed with SEM and immunohistochemistry for appropriate cell markers as well as with changes in cell morphology. In conclusion, stem cell integrated POSS-PCL based small diameter grafts are a promising option for pediatric applications.

36 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P22 Design Valvular Interstitial Cell Seeded Results: The stiffness of aligned electrospun fibers scaffolds to Mimic Heart Valve Leaflet’s with (20% w/v) PGS/PCL in a (1:1) ratio, most closely Structure and Mechanical Properties matched the native tissue’s anisotropic stiffness. Microfabricated PGS scaffolds offered elasticity and Nafiseh Masoumi, The Pennsylvania State University, appropriate porosity to the composite scaffold. Cell Bioengineering Department, University Park, PA,USA, orientation analysis showed that cell alignment was Center for Biomedical Engineering, Department of found in the direction of the long axis of the pores. Medicine, Brigham and Women’s Hospital, Harvard DNA and collagen assays showed that there was a Medical School, Cambridge MA,USA. Benjamin L. considerable amount of cellular material within all of the Larson. Harvard-MIT Division of Health Sciences and scaffolds. Technology, Cambridge, MA, USA. Jesper Hjortnaes, Department of Cardiothoracic Surgery, University Conclusions: Collectively, the results of these studies Medical Center Utrecht, Utrecht, The Netherlands, will help better define scaffolds properties and cell Center for Biomedical Engineering, Department seeding / cultivation requirements necessary for forming of Medicine, Brigham and Women’s Hospital, clinically-relevant biomimetic tissue engineered heart Harvard Medical School, Cambridge MA.USA. Ali valve leaflets. Khademhosseini , Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge MA. USA. Harvard-MIT Division of Health Science and Technology, Cambridge, MA,USA. Wyss Institute of Biologically Inspired Engineering, Harvard Medical School, Boston, MA,USA Purpose: We developed novel tri-layered scaffolds comprised of aligned elctrospun poly glycerol sebacate (PGS)/poly caprolactone (PCL) fibers and microfabricated PGS scaffolds and designed to emulate the anisotropic mechanical properties of native valvular tissues. Methods: PGS micromolding was used to create a scaffold with diamond-shaped pores. Two layers of aligned PGS/PCL electrospun scaffolds were formed and attached to the surface of a single layer PGS scaffolds to improve the mechanical stiffness of the single layer PGS microfabricated scaffold and enhance the tissue formation on the scaffold’s 3D structure Uniaxial mechanical testing was performed on native porcine tissue and one layer and composite scaffolds for comparisons. The PGS/PCL concentration and ratio were set to match the composite scaffolds mechanical property and anisotropy in accordance with native tissue mechanical characteristics. Following the aortic porcine VICs isolation, the scaffolds were seeded for 5 weeks. Scaffolds and VIC-seeded constructs were characterized for their mechanical properties, and further characterized by confocal microscopy for cell orientation and phenotypic immunofluorescence, scanning electron microscopy for observation of general tissue formation, and by DNA and collagen assays.

ISACB’S 2012 BIENNIAL MEETING 37 POSTER PRESENTATION ABSTRACTS

P23 P24 Modulation, Mechanism and Angiogenic Targeting Extracellular DNA to Deliver IGF-1 Potential of Macrophage Polarization (M1/ to the Injured Heart M2) on Electrospun Bioresorbable Vascular Raffay S. Khan, Emory University, Atlanta, GA, Jay C. Sy, Grafts Emory University, Atlanta, GA, Milton E. Brown, Emory Koyal Garg, Virginia Commonwealth University, VA; University, Atlanta, GA, Mario D. Martinez, Nicholas A. Pullen, Virginia Commonwealth University, Emory University, Atlanta, GA, Niren Murthy, Georgia VA; Carole A. Oskeritzian, Virginia Commonwealth Tech, Atlanta, GA, Michael E. Davis, Emory University, University, VA; John J. Ryan, Virginia Commonwealth Atlanta, GA. University, VA; Gary L. Bowlin, Virginia Commonwealth Purpose: During myocardial infarction (MI), necrosis University, VA of myocardial cells releases large amounts of DNA, Purpose: (a) Modulate the macrophage phenotype representing a potential target for drug delivery. (M1/M2) by varying the pore-size of electrospun Hoechst, a DNA binding molecule used to stain nuclei, polydioxanone (PDO). (b) Assess the angiogenic can be functionalized to contain reactive groups such potential of macrophages on PDO in vitro and in vivo as biotin. Insulin-like growth factor-1 (IGF-1), a small (Directed in vivo angiogenesis assay (DIVAA)). (c) molecule with a short half-life is cardio-protective. We Study the involvement of MyD88, TLR-4 and NFkB in hypothesized that conjugating IGF-1 to Hoechst would macrophage polarization. increase targeting of IGF-1 to injured myocardium. Methods: Bone marrow derived murine macrophages Methods: Hoechst-IGF1 (H-IGF1) was synthesized (BMMΦs, 106 cells) were seeded on TCP (24 well plate) by binding Hoechst-biotin to biotinylated IGF- and PDO scaffolds (15 mm discs) of varying pore- 1 via a fluorescent streptavidin linker. H-IGF1 cell sizes (1μm, 10μm and 14μm). After 24 hours, the cell impermeability and activity were assessed in vitro using lysates were analyzed for arginase (Arg1), and iNOS cultured macrophages and cardiac progenitor cells expression (Western blot) and cell culture supernatants (CPCs) respectively. To determine in vivo targeting of were analyzed for nitric oxide (NO2), TNF-α, IL-6, VEGF, H-IGF1 to MI, mice underwent coronary artery ischemia- TGF-β1 (ELISA). A 3D bead assay was used to quantify reperfusion. Six hours following MI, mice were injected the endothelial cell sprouting induced by conditioned intravenously with 70ng of H-IGF1, S-IGF1 (streptavidin media from BMMΦ:PDO interaction. BMMΦs were also bound IGF-1 only) or PBS followed by in vivo imaging. prepared from MyD88-/- and TLR-4-/- knockout mice Results: Intact macrophages did not show nuclear and seeded. staining with H-IGF1, while permeabilized cells had Results: Arg1, TGF-β1, VEGF (M2 markers) expression an increase in fluorescent Hoechst staining, indicating was statistically higher on the 14μm pore-size. The HIGF1 was cell impermeable but could still bind DNA. expression of iNOS, NO2-, TNF-α, IL-6 (M1 markers) H-IGF1 activity, demonstrated by Akt phosphorylation was higher on the 1μm pore-size. The 3D bead assay in CPCs was similar to native IGF-1. At 30 minutes showed larger average length of sprouts and higher post-injection, 3.2% of injected H-IGF1 accumulated percentage density of sprouts from the conditioned in infarcted hearts compared with 1.8% of S-IGF1, with media of BMMΦ:PDO (14μm) interaction. BMMΦ from levels unchanged at 2 hours post-injection as confirmed both MyD88-/- and TLR-4-/- mice showed impaired by IGF-1 ELISA. Intravenous administration of H-IGF1 iNOS and Arg1 expression on both PDO and TCP pre-bound to exogenous DNA, resulted in reduced indicating that they are both involved in BMMΦ:PDO IGF-1 targeting suggesting that H-IGF1 localization interaction. The results from the ongoing studies on depended on binding DNA. NFkB inhibition and the DIVAA assay should provide Conclusion: Our data demonstrate that targeted, greater insight into the mechanism of phenotype intravenous delivery of IGF-1 can be achieved by modulation and the angiogenic response of PDO conjugation to Hoechst-derivatives. in vivo, respectively.

38 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P25 Creation of a Mouse Aortic Valve Interstitial Cell-Line Paul W. Riem Vis, Dept. of Cardiothoracic Surgery, University Medical Center Utrecht, The Netherlands; Koen Braat, Dept. of Cell Biology, University Medical Center Utrecht, The Netherlands; Hanna Talacua, Dept. of Cardiothoracic Surgery, University Medical Center Utrecht, The Netherlands, Lex van Herwerden, Dept. of Cardiothoracic Surgery, University Medical Center Utrecht, The Netherlands, Jolanda Kluin, Dept. of Cardiothoracic Surgery, University Medical Center Utrecht, The Netherlands. Purpose: Calcific aortic valve disease (CAVD) is life- threatening, but the only curative therapy is valve replacement. To understand mechanisms behind onset and progression of the disease, mouse KO-models are used to induce vessel- and valve disease. However, cellular mechanisms are difficult to study in these models, because the ability to expand murine aortic valve interstaitial cells (VICs) in vitro is limited. We describe a method to produce immortal cell-lines from mouse aortic VICs. Methods: An aortic valve was obtained from one mouse and fresh serum from 10 others. Following collagenase digestion, cells were cultured in media supplemented with fresh serum. Immortalization was performed using a SV40LT construct. Subsequently, cells were characterized with immunocytochemistry and differentiation. After several passages, these characterizations were repeated and a growth curve was constructed. Results: At p6, enough cells were obtained for retroviral transduction with SV40LT. SV40LT-presence was verified in selected colonies. Cells stained positively for interstitial cell markers α-SMA, SMemb and Vimentin, but also weakly for smooth muscle marker desmin. These results were similar at p12. The growth curve indicated rapid proliferation of cells, while differentiation with BMP2 showed increased activity of alkaline phosphatase and alizarin red, showing more mineralization. Conclusion: Our results show that the described method can be used to expand and immortalize mouse aortic VICs, which can subsequently be used for in vitro or in vivo experiments to study CAVD in mouse models. This allows better molecular / mechanistic understanding of the underlying process.

ISACB’S 2012 BIENNIAL MEETING 39 POSTER PRESENTATION ABSTRACTS

P26 Interleukin-10 Controls The Protective Methods: TEVMs were produced from human Effects Of Circulationg Microparticles arterial smooth muscle cells and were incubated From Septic Shock Patients On Tissue- with microparticles isolated from 16 septic patients. Microparticles were extracted from whole blood of Engineered Vascular Media septic subjects. TEVMs were incubated for 24 h with Hadj Ahmed Mostefai, INSERM, U694, Angers, France; microparticles and used for the study of vascular Université d’Angers, Angers, France; Jean-Michel contraction, direct measurements of nitric oxide and Bourget, Centre LOEX de l’Université Laval, Génie superoxide anion production by electron paramagnetic tissulaire et régénération : LOEX - Centre de recherche resonance and quantification of mRNA cytokine FRSQ du Centre hospitalier affilié universitaire de expressions. Québec, and Département de Chirurgie, Faculté de Results: Septic MPs increased contraction induced Médecine, Université Laval, Québec, QC, Canada; by histamine in TEVM. This effect was not associated Ferhat Meziani, Laboratoire HIFIH, UPRES EA 3859, IFR with inflammation, neither linked to the activation of 132, Université d’Angers, PRES UNAM, Département NF-kB pathway, nor to the increase of inducible nitric de Réanimation Médicale et de Médecine Hyperbare, oxide synthase and cyclooxygenase-2 expressions. Centre Hospitalo-Universitaire, Angers, France; Maria In contrast, mRNA expression of interleukin-10 was Carmen Martinez, INSERM, U694, Angers, France; enhanced. In addition, we investigated the effect of Université d’Angers, Angers, France ; Alain Mercat, interleukin-10 on vascular hyporeactivity induced Laboratoire HIFIH, UPRES EA 3859, IFR 132, Université by lipopolysaccharide. Although interleukin-10 d’Angers, PRES UNAM, Département de Réanimation treatment did not modify the contractile response in Médicale et de Médecine Hyperbare, Centre Hospitalo- TEVM by itself, this interleukin restored contraction Universitaire, Angers, France; Pierre Asfar, Laboratoire in lipopolysaccharide-treated TEVM towards control HIFIH, UPRES EA 3859, IFR 132, Université d’Angers, vessels. PRES UNAM, Département de Réanimation Médicale et de Médecine Hyperbare, Centre Hospitalo-Universitaire, Conclusions: These data demonstrate an association Angers, France; Lucie Germain, Centre LOEX de between the capability of septic MPs to restore vascular l’Université Laval, Génie tissulaire et régénération : LOEX hyporeactivity induced by lipopolysaccharide and their - Centre de recherche FRSQ du Centre hospitalier affilié ability to increase interleukin-10 in the tissue-engineered universitaire de Québec, and Département de Chirurgie, blood vessel model. Faculté de Médecine, Université Laval, Québec, QC, Canada; Ramaroson Andriantsitohaina, INSERM, U694, Angers, France; Université d’Angers, Angers, France. Purpose: During sepsis, the links between inflammation and coagulation can be orchestrated by the interaction between circulating and vascular cells, that under activation, release microparticles. Recently, we reported that increased circulating microparticles in septic patients play a pivotal role in ex vivo vascular function suggesting that they are protective against vascular hyporeactivity. The present study was designed to investigate the effects of septic microparticles on the contractile response of tissue-engineered vascular media (TEVM).

40 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P27 P28 ECM-related Gene Expression and Hydrogel Composition Influences Interstitial Thrombogenicity of Endothelial Cells Grown Cell Fate in a 3D in vitro Heart Valve Model on Electrospun Hybrid Fibrous Scaffolds for Hjortnaes J., Brigham and Women’s Hospital, Harvard Vascular Grafts Medical School, Boston, USA; University Medical Jingjia Han, Drexel University, School of Biomedical Center Utrecht, Utrecht, The Netherlands; Camci-Unal Engineering, Science and Health Systems, Philadelphia, G., Massachusetts Institute of Technology, Cambridge, PA; Jonathan Gerstenhaber, Drexel University, School of USA; Kluin J., University Medical Center Utrecht, Biomedical Engineering, Science and Health Systems, Utrecht, The Netherlands; Schoen F.J., Brigham and Philadelphia, PA; Philip Lazarovici, School of Biomedical Women’s Hospital, Harvard Medical School, Boston, Engineering, Science and Health Systems, Philadelphia, USA;, Khademhosseini A., Massachusetts Institute PA; Peter I. Lelkes, Department of Bioengineering, of Technology, Cambridge, USA; Wyss Institute for Temple University, Philadelphia, PA Biologically Inspired Engineering, Harvard University, Boston, USA; Aikawa E., Brigham and Women’s Hospital, Harvard Medical School, Boston, USA; Purpose: To investigate the ECM-related gene Purpose: Valvular interstitial cells (VICs) maintain expression and thrombogenicity of endothelial cells (EC) the health and structural integrity of heart valves grown on electrospun fibrous scaffolds; to elucidate and contribute to valvular disease. Collagen and the role of surface topology versus matrix composition glycosaminoglycans are important components of the on the modulation of ECMrelated differential gene valve extracellular matrix. By creating a composite expression and EC thrombogenicity. biomaterial of these natural polymers, we aim to Methods: Human aortic endothelial cells (HAECs) engineer a 3D tissue model that can simulate the in situ were cultured for 24 h on glass cover slips coated physiological environment of VICs and be used to study with electrospun (PLGA-Gelatin-Elastin – PGE) mats calcific aortic valve disease. (PGE, 3D), PGE solution (PGE film, 2D) and various Methods: Porcine aortic VICs were encapsulated in defined ECM protein solutions. ECM-related gene hydrogels comprised of hyaluronic acid methacrylate expression of HAECs was evaluated for cells cultured (HAMA) and gelatin methacrylate (GelMA). Different on PGE, PGE film, gelatin and collagen IV via a focused compositions of biomaterials varying in adhesion sites, PCR microarray, which surveyed 84 genes encoding stiffness and porosity were used. Cell-laden constructs ECM and adhesion molecules. Both basal and TNF-α were cultured in normal media for 21 days and VIC induced tissue factor (TF) mRNA expression and protein behavior was analyzed in relation to the biomaterial activity levels were assessed for HAECs cultured on all properties. Additionally, the effect of culturing heart substrates. valve-like constructs in osteogenic medium was Results: HAECs cultured for 24 h on 3D electrospun evaluated. PGE fibrous scaffolds were able to form confluent Results: Compression testing and scanning electron monolayers, expressing similar level of VE-cadherin, as microscopy demonstrated an increase in stiffness they do on their 2D counterparts (PGE film) and other (compressive modulus: 3.07±0.72 to 112±40.94 kPa) protein substrates. A differential expression “profile” and decrease in porosity when concentrations of of HAECs cultured on PGE scaffolds was observed as HAMA and GelMA in the hydrogels were increased. compared to cells on other substrates examined; cells Cellular viability remained above 80.2±7.3%. Calcific on PGE scaffolds were more quiescent and passivated nodule formation was observed in hydrogels after 21 than those on gelatin. In addition, cells cultured on PGE days of culture in osteogenic media. Alpha-smooth scaffolds expressed TF mRNA levels similar to those on muscle actin, a marker for activated VICs (with minimal gelatin and exhibited similar TF activity levels as HAECS expression in native valves), was induced only in cultured on collagen IV. composite hydrogels when cultured in osteogenic Conclusions: HAECs grown on electrospun PGE media, indicating VIC activation. scaffolds essentially exhibited “healthy” phenotype and Conclusions: Our construct simulates in situ VIC were more quiescent and less thrombogenic than those behavior where VICs remain quiescent until stimulated on gelatin. by biomechanical or pathological cues. This composite hydrogel platform can be used as a model system to elucidate mechanisms of calcific aortic valve disease.

ISACB’S 2012 BIENNIAL MEETING 41 POSTER PRESENTATION ABSTRACTS

P29 P30 Regulation of thrombotic and Vitronectin Coating Increases Cell Seeding inflammatory responses in baboon on Fibrin Microthread Sutures for Cell endothelial outgrowth cells under Delivery to the Heart various hemodynamic conditions Mark C. Kowaleski, Worcester Polytechnic Institute, Randall F. Ankeny, Georgia Institute of Technology, Worcester, MA; Kaitlyn Marengo, Worcester Polytechnic Parker H. Petit Institute for Bioengineering and Institute, Worcester, MA; George D. Pins, Worcester Bioscience, Atlanta, GA, USA; Robert M. Nerem, Polytechnic Institute, Worcester, MA; Glenn R. Gaudette, Georgia Institute of Technology, Parker H. Petit Institute Worcester Polytechnic Institute, Worcester, MA for Bioengineering and Bioscience, Atlanta, GA, USA Purpose: Exogenous cellular therapies have shown Purpose: Due to isolation ease and proliferative ability, potential to regenerate the myocardium post infarct. endothelial outgrowth cells (EOCs) are a prominent However, effectiveness has been limited by the ability candidate as a tissue engineering cell source. to efficiently and locally deliver cells to a region of Interestingly, studies highlighted phenotype differences interest. Cell-seeded biological sutures, formed from between EOCs and mature vascular endothelial cells fibrin microthreads, overcome these issues and have under steady laminar flow. This study investigates demonstrated increased cell engraftment. To increase thrombotic and inflammatory pathways under differing the number of cells that can be seeded on these hemodynamic conditions in baboon EOCs. biological sutures, we investigated coating them with ECM proteins. Methods: Baboon late EOCs were isolated from circulating EPCs and exposed to four hemodynamic Methods: Biological sutures were produced by bundling conditions using the cone-and-plate viscometer: twelve fibrin microthreads. Surface modified sutures steady, laminar (LS, 10 dynes/cm2), oscillatory (OS, were created by coating with poly-l-lysine or vitronectin. 0±10 dynes/cm2), pulsatile (10±10 dynes/cm2), or Sutures were then seeded with 100,000 human reversing (RS, 2±10 dynes/cm2) shear stress. Following mesenchymal stem cells for 24hrs. Seeded sutures shear, RNA was collected and analyzed, using RT- were spun in an in vitro shear stress model to assess PCR, to determine inflammatory and thrombotic cell adhesion. To evaluate the ability of cells to align pathway-associated genes. Further, to determine how along the length of the suture, nuclear alignment was these hemodynamic conditions affected functional determined. inflammation, monocyte adhesion assays were Results: Vitronectin coating significantly increased the performed. number of cells seeded on fibrin sutures (19,604±1,829 Results: To determine EOCs’ response to shear, eNOS, cells/cm) compared to unmodified sutures (6,821±739 an endothelial marker upregulated by LS, was analyzed. cells/cm; p<0.05) and sutures modified with poly-l- LS and PS upregulated eNOS compared to OS. We lysine (4,226±1,003 cells/cm; p<0.05). Spinning resulted next analyzed the inflammatory genes, ICAM-1 and in similar decreases in cell number in all groups. VCAM-1, showing no significant difference between Unmodified sutures had more aligned nuclei between the four hemodynamic conditions. Interestingly, we suture surfaces (p<0.05 vs. poly-l-lysine and vitronectin). found that LS significantly decreased monocytes All suture types had increased alignment compared to bound to the endothelium when compared to OS cells grown on plates (p<0.001). and RS while PS significantly decreased monocytes Conclusions: Vitronectin coating increases the number bound to the endothelium when compared to only OS. of cells seeded on fibrin microthread based biological When investigating the thrombotic pathway, we found sutures. Cell adhesion strength was not altered with that anti-thrombogenic markers CD39, EPCR and vitronectin coating, suggesting that delivery of stem thrombomodulin were increased by LS compared to OS. cells to the heart may be similar to the 65% delivery We found no significant differences in tissue factor. efficiency seen with uncoated fibrin microthread sutures. Conclusions: These results show that shear stress plays an important role in inflammatory and thrombotic responses in EOCs and is an important consideration when using EOCs.

42 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P31 P32 A New Generation of Transcatheter Phenotypic And Functional Effects of Heart Valves Elevated Glucose Concentration on Human Benyamin Rahmani, Centre for Nanotechnology Valve Interstitial Cells & Regenerative Medicine, Division of Surgery & Alba Singla, Facultat de Biologia, University of Interventional Sciences, University College London, Barcelona, Barcelona, Spain; Najma Latif, Harefield UK; Spyridon Tzamtzis, Cardiovascular Engineering Heart Science Centre, Imperial College, Harefield, & Medical Devices Group, Department of Mechanical Middx, UK; Magdi H. Yacou, Harefield Heart Science Engineering, University College London, UK; Michael Centre, Imperial College, Harefield, Middx, UK; Adrian Mullen, The Heart Hospital, University College London H. Chester, Harefield Heart Science Centre, Imperial Hospitals, UK; John Yap, The Heart Hospital, University College, Harefield, Middx, UK; College London Hospitals, UK; Alexander M. Seifalian, Purpose: Diabetes is an established risk factor for Centre for Nanotechnology & Regenerative Medicine, many types of cardiovascular disease, including aortic Division of Surgery & Interventional Sciences, University stenosis. However, the effect of glucose concentration College London, UK; Gaetano Burriesci, Cardiovascular on the function of valve interstitial cells (VICs) has not Engineering & Medical Devices Group, Department of been investigated. Mechanical Engineering, University College London, UK. Methods: Human VICs were isolated from all four Purpose: Transcatheter aortic valve implantation (TAVI) valves, obtained from unused donor hearts, and is becoming the procedure of choice for high risk maintained under standard culture conditions. Cells patients with severe aortic stenosis. Despite remarkable were maintained in media containing 0, 0.4, 1, 5 or 10% benefits of current TAVI procedures, they are challenged serum with either low, 5mM glucose (LG) or high, 25mM complications such as lack of repositionability, glucose (HG). The effect of PDGF (0, 1, 5, 10, 30 ng/mL) paravalvular leakage and atrio-ventricular block. was also assessed in LG and HG media. Proliferation of Methods: A new generation of TAVI devices has been cells was analysed with MTS assay and apoptosis was developed in the form of a self-expandable Nitinol assessed via TUNEL staining. stent encompassing leaflets synthesised from novel Results: VICs demonstrated a concentration-dependent nanocomposite polymers. The polymers are composed increase in proliferation in response to serum. There was of a polycarbonate-ureaurethane soft segment (PCU) no significant difference in response to serum between and hard segment derived from polyhedral oligomeric media containing LG (401±25%) and HG (366±25%) silsesquioxane (POSS) nanoparticles. POSS-PCU after 7 days incubation (P>0.05, n=4). Treatment with nanocomposites have been found to possess enhanced PDGF in LG (119±6.8) and HG (112±6.3) media also physiochemical properties, resistance to calcification showed no significant difference in proliferation after 7 and thrombosis with superior in-vivo biostability. days (p>0.05, n=8). Cells retained a similar spindle–like POSS-PCU has been successfully implanted in human morphology after 7 days incubation in both LG and HG in the form of a bypass graft and the world’s first media. In addition, there was no evidence of increased synthetic trachea. The valve design allows for controlled levels of apoptosis in either glucose concentrations. deployment of the prosthesis, and is fully retrievable in Conclusion: VIC viability and proliferation is unaffected the case of misplacement. by acute incubation with pathophysiological levels Results: The leaflet and frame design was optimised of glucose. Further studies are required to assess by numerical simulation, and a new manufacturing the effects of chronic exposure to high glucose process was developed to produce initial prototypes. concentration and the propensity of VICs to differentiate No structural damage was observed after testing the into pathological cell phenotypes that may participate in valves for collapse and reexpansion over several cycles. the calcification process. Owing to its polymeric leaflets membrane, the valve can be crimped in to a catheter equal to or less than 18 Fr to provide easier vascular access, and the design has improved anchoring and sealing with no need for excessive radial force. Conclusions: The proposed valve is a novel solution, which can overcome the main limitations of current TAVI devices and is currently under pre-clinical evaluation.

ISACB’S 2012 BIENNIAL MEETING 43 POSTER PRESENTATION ABSTRACTS

P33 P34 Porcine Model of Prosthetic Limb Bypass Cell Specific Invasion of Hydrogels Graft Modified by Adhesive Protein Regulated Through Polymerisation with Assemblies and Seeded with Autologous Different MMP Cleavage Sites Endothelium Neil Davies, Cardiovascular Research Unit, University of Jaroslav Chlupac, Institute of Physiology, Academy of Cape Town, South Africa; Mona Bracher, Cardiovascular Sciences of the Czech Republic v.v.i. and Transplant Research Unit, University of Cape Town, South Africa; Surgery Department, Institute for Clinical and Deon Bezuidenhout, Cardiovascular Research Unit, Experimental Medicine, Prague, Czech Republic; Tomas University of Cape Town, South Africa; Peter Zilla, Riedel, Eduard Brynda, Institute of Macromolecular Cardiovascular Research Unit, University of Cape Town, Chemistry, Academy of Science of the Czech Republic South Africa v.v.i., Prague, Czech Republic; Rudolf Poledne, Purpose: We have begun to explore the utility of Laboratory for Atherosclerosis Research, Institute for hydrogels crosslinked with specific MMP cleavage Clinical and Experimental Medicine, Prague, Czech sites in modifying the behaviour of different primary cell Republic; Elena Filova, Lucie Bacakova, Institute types. of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Methods: 3-D cell spheroids (smooth muscle cells (SMC) or fibroblasts) were cultured in polyethylene Purpose: To develop a porcine model of lower limb based hydrogels that were crosslinked with either a prosthetic bypass graft, to assess two animal species pan-MMP or with MMP-14 or MMP-9 specific cleavage and to perform double-stage seeding of autologous peptides. All hydrogels were also modified with the cell endothelial cells onto the lumen of vascular grafts. binding substrate RGD. Cellular invasion was quantified Methods: Five domestic and six minipigs were by image analysis of phalloiden stained spheroids. used. Different anatomical settings of supra- and Results: Hydrogels formed with the different MMP infrainguinal bypass were evaluated. Clinically used PET substrates had very similar physical properties and (polyethylene-terephtalate) vascular prostheses (VUP enzyme specificity was preserved after incorporation Joint-Stock Comp., Brno, Czech Republic) of 6 mm into hydrogels. Cellular invasion was completely inner diameter were modified with fibrin and fibronectin inhibited by the MMP inhibitor GM6001. Both cell assemblies deposited by layer-by-layer technique. types sprouted similarly into pan-MMP gels. For both Endothelial cell were harvested from inner jugular vein, cell types invasion was comparably reduced into the cultured in vitro and seeded onto the lumen before MMP-9 gels but the cells behaved entirely differently in implantation. Unseeded control graft was implanted in MMP-14 specific hydrogels. In these hydrogels SMC contralateral limb. Bypass graft explants were observed had a significant doubling of sprout length above that in in confocal microscope. the pan-MMP hydrogel whilst fibroblasts sprout length Results: Five animals were implanted for evaluation halved. of bypass setting without cell seeding. Domestic pigs showed undesirable weight gain while performing cell culture. Five seeding procedures succeeded and one cell culture failure occurred. The seeding density achieved on vascular prostheses was 60±40 cells per cm2. Mean period of double-stage seeding procedure was 50±16 days. Both seeded and control grafts occluded in two of the five seeding procedures, one graft thrombosed due to poor outflow tract and one seeded graft remained patent 22 days while control graft got occluded. Conclusions: The most feasible model seems to be bilateral aorto-internal iliac bypass graft with external iliac ligature performed in a minipig. Pre-coating of fibrin / fibronectin does help with autologous cell seeding and culturing in the lumen of PET vascular prosthesis which Conclusions: This novel finding opens up a new avenue may also inhance the bypass graft patency. for the cell type specific manipulation of cell behaviour. The approach of using specific cleavage sites may be an additional tool for the optimisation of tissue engineering matrices. It could also have general utility in exploring cellular mechanisms of invasion.

44 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P35 P36 Beware of interspecies differences in Development of Nitric Oxide-eluting vascular tissue engineering: in vitro and in Nanocomposite Materials Using vivo discrepancies Nanoparticles for Cardiovascular Murielle Rémy, INSERM U1026, BIOTIS, University Applications Bordeaux Segalen, Bordeaux F-33076, France; Marlène Noora Naghavi, UCL Centre for Nanotechnology Durand, CHU de Bordeaux, CIC-IT Biomatériaux, F-33000 and Regenerative Medicine, Division of Surgery and Bordeaux, France; Patrick Menu, Groupe Mécanique et Interventional Science, University College London (UCL), Ingénierie Cellulaire et Tissulaire, CNRS 7563, Faculté de London; Achala de Mel, UCL Centre for Nanotechnology Médecine, Nancy Université-UHP, Vandoeuvre-lès-Nancy and Regenerative Medicine, Division of Surgery and 54505, France; JC Voegel, INSERM U977, Université de Interventional Science, University College London (UCL), Strasbourg, Faculté de Chirurgie Dentaire, Strasbourg London; Brian G Cousins, UCL Centre for Nanotechnology 67085, France; JF Ponsot, Bioprotec; P Dos Santos, and Regenerative Medicine, Division of Surgery and PTIB, Bordeaux F-33000, France; MF Harmand, LEMI, Interventional Science, University College London (UCL), Technopole Montesquieu, Martillac F-33650, France; London; George Hamilton, Royal Free London NHS Laurence Bordenave, INSERM U1026, BIOTIS, University Foundation Trust Hospital, London; Alexander M. Seifalian, Bordeaux Segalen, Bordeaux F-33076, University Hospital UCL Centre for Nanotechnology and Regenerative of Bordeaux, Bordeaux F-33000, France ; CHU de Medicine, Division of Surgery and Interventional Science, Bordeaux, CIC-IT Biomatériaux, F-33000 Bordeaux, France. University College London (UCL), and Royal Free London Purpose: Polyelectrolyte multilayers (PEMs) proposed NHS Foundation Trust Hospital, London to functionalize biomaterials are efficient in rabbits Purpose: To develop nitric oxide (NO)-eluting towards in vitro endothelialization and in vivo patency. We nanocomposite biomaterials using nanoparticles explored whether results obtained in small animals can be conjugated with NO donors, which induce anti- extended to large ones. thrombogenic properties in the development of Methods: PEMs were fabricated from alternate deposition cardiovascular devices. of anionic PSS (poly[styrene sulfonate]) and cationic PAH Methods: NO-releasing fumed silica nanoparticles (poly[allylamine]). Sheep peripheral blood mononuclear (FSNPs) were synthesised via the reaction of cells were isolated and cultured to obtain progenitor S-nitrosothiol NO donors on to the surface of FSNPs. derived endothelial cells (PDECs) on PEM-, fibronectin- FTIR, NMR, acid titration, and Ellman’s tests confirmed and collagen-coated substrates before characterization the attachment of NO donors on to FSNPs. NO- (CD31, VE-cadh, von Willebrand factor…). Cell FSNPs were dispersed in to polyhedral oligomeric attachment and proliferation were quantitatively assessed silsesquioxane-poly(carbonate-urea)urethane (POSS- on all substrates. PDEC-seeded PEMs and PEM-coated PCU) nanocomposite polymer at 2, 4, and 8 wt% and veins were submitted to shear stress (SS) (parallel flow bypass grafts were fabricated using extrusion phase chamber and BioDynamic test instrument), respectively. inversion techniques. NO release from grafts was PEM-coated vessels were implanted in sheep. measured in a physiological pulsatile flow circuit using Results: We obtained well characterized sheep PDECs. the amperometric technique, and their biocompatibility Unlike previous studies (rabbit) performed with PEMs was evaluated in whole blood as a measure of terminated by PAH, no noticeable differences appeared thrombogenicity. between the PEM-, fibronectin- and collagen-coated Results: The results confirmed the functionalisation of surfaces in terms of :PDEC differentiation time, confluent FSNPs with NO donors and release profiles that could monolayer constitution, cell spreading, proliferation, be controlled by changing the amount of FSNPs in the specific marker expression. After 6h exposure to 1.2 Pa polymer matrix. All FSNPs dispersed in to POSS-PCU sheep PDECs lining PEMs withstood SS and markers were capable of NO elution and the optimal release profile were present. Fluorescent PEMs coating veins were still was evident with 2 wt% NO-FSNP at physiologically present and not altered after 48h exposure to 0.5 Pa. Only relevant concentrations of ~ 8×10-10 mol cm-2min-1 a reduced number of animals were implanted (coronary over a 7 h period. NO-eluting grafts enhanced anti- and carotid position) because of PEM thrombogenicity in thrombogenic properties when compared with controls sheep. To address the discrepancy between rabbit and to confirm the protective roles of NO and attenuation of sheep, we compared PEM hemocompatibility in vitro with thrombosis. blood from both species and again found differences. Conclusions: This study used POSS-PCU Conclusions: our findings indicate that precautions must nanocomposite, already in used in human as tracheal be taken in extrapolation between species. We thank and lacrimal duct conduits. NO-releasing FSNPs were program ANR-07-TecSan-022-01 “Subvacel”. incorporated in to POSS-PCU, and controlled release was confirmed in vitro. NO-eluting bypass grafts with anti- thrombogenic properties represent a new generation of materials in the development of cardiovascular devices.

ISACB’S 2012 BIENNIAL MEETING 45 POSTER PRESENTATION ABSTRACTS

P37 P38 Human Endometrial Stem Cells as a Useful Cellular Content and Distribution in Source for cardiovascular Regeneration Intraluminal Thrombus (ILT) from Abdominal Aortic Aneurysm (AAA) Farzaneh Khademi1, Department of Tissue Engineering and Cell Therapy, Faculty of Advanced Medical David A. Vorp, PhD, Departments of Bioengineering, Technologies, Tehran University of Medical Sciences, Cardiothoracic Surgery and Surgery, University of Tehran, Iran1, Jafar Ai1,2,3, Stem Cell and Transgenic Pittsburgh, Jayashree Rao, MS, University of Pittsburgh, Technology Research Center, Department of Pathology, Matthew P. Scanlon, BS, Penn State University, Shiraz University of Medical Sciences, Shiraz, Iran2, Madhusudanan Nair, MD, University of Pittsburgh, Research Center for Science and Technology in Steven A. Leers, MD, University of Pittsburgh,Michel Medicine, Imam Hospital, Tehran University of Medical S. Makaroun, MD, University of Pittsburgh, Jay D. Sciences, Tehran, Iran3, Alexander M. Seifalian4 , UCL Humphrey, PhD, Department of Biomedical Engineering, Centre for Nanotechnology & Regenerative Medicine, Yale University. Division of Surgery & InterventionalScience, University Purpose: ILT is present in the majority of abdominal College London, London, United Kingdom . 4 aortic aneurysms, but little is known about the cellular Purpose: The ease of isolation, expand rapidly from content or its role in AAA pathobiology. Our purpose patients without leading to major ethical and technical was to assess the cellular content and distribution problems, higher abilities for proliferation, differentiation within ILT. and fast angiogenesis and no immunological response, Methods: ILT was freshly harvested from patients aim of this study was introduction of human endometrial undergoing elective AAA repair surgery. Sections stem cells (hEnSCs) as a valuable source of adult stem were cut and stained for specific immunohistological cells for cardiovascular application. . examination via fluorescent microscopy; cells were also Methods: The hEnSC were isolated of tissue’s biopsy extracted, treated with antibodies and analyzed using from 20 women at the age of 18-40 years of old. Cells flow cytometry. See Table for all markers. are exposed to vessel induction medium that endothelial Results: and myogenic differentiation was assess via quantitative reverse transcription polymerase chain reaction (Real- time PCR) , immunofluresent staining for endothelial and smooth muscle -specific genes and proteins, including fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), CD31, myosin, sarcomeric actin and desmin as well as functional tests in a three-dimensional cultures for angiogenesis were performed. Results: Cultured cells on appropriate differentiation medium were differentiated to endothelial and smooth muscle-like cells, shown by Real-time PCR and immunostaning after differentiation period. Conclusions: In the presence study, the differentiated Our data suggest that there is a preponderance of cells features were closely resembled human adult cells positive for CD68, CD105, and HLA-DR epitopes endothelial and smooth muscle cells by in vitro testing. – all markers for activated macrophages. All present These cells will be provided possible sources of cells for cell types are located within a nucleated stratus near cardiovascular repopulation. the luminal edge of the thrombus reducing in number exponentially with depth from the lumen. Conclusions: Our findings suggest a local inflammatory response within the luminal region of ILT. It is possible that these cells might contribute to the further degradation of the aortic wall and/or contribute to the remodeling of either the ILT or AAA wall.

46 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P39 The Influence of Cannulation on Methods: Tissue specimen of AAV and NAAV Metalloproteinase Expression in Venous from both groups were compared by histology, Aneurysm Complicating Vascular Access immunohistochemistry (IHC) and zymography analysis of Metalloproteinases (MMP) 2 and 9. Histologic Arteriovenous Fistula sections were stained with hematoxylin-eosin-safran Xavier Bérard, Department of Vascular surgery, and orcein, and IHC specimens with antibodies to Bordeaux University, CHU de Bordeaux, CIC-IT CD34, CD68 and MMP-9. Further analyses comprised Biomatériaux, F-33000 Bordeaux, France ; Sébastien RT-PCR of MMP-2 and western blotting, ELISA and RT- Déglise, Department of Physiology, Laboratory of PCR of the inhibitor of matrix metalloproteinase (TIMP) Experimental Medicine, University Hospital, 1005 TIMP-1 and TIMP-2. Lausanne, Switzerland; Richard Daculsi, Univ. Bordeaux, Results: In both groups, histological analysis found Bioingénierie tissulaire, INSERM U1026 BIOTIS, an extensive fibrous remodeling in the media. CD68 F-33000 Bordeaux, France ; Samantha Delmond, CHU immunostaining was more pronounced in the AAV from de Bordeaux, CIC-IT Biomatériaux, F-33000 Bordeaux, group 1 than in group 2. The activity of proactive MMP- France; Chantal Bourget, Univ. Bordeaux, Bioingénierie 9 was significantly increased in group 1 as compared to tissulaire, INSERM U1026 BIOTIS, F-33000 Bordeaux, group 2 but remained identical regarding MMP-2. In a France; François Saucy, Department of Physiology, selection of patients from both groups, MMP-2, TIMP-1 Laboratory of Experimental Medicine, University and TIMP-2 were increased in the AAV as compared to Hospital, 1005 Lausanne, Switzerland ; Jean-Marc NAAV, but only mRNA expression of MMP-2 reached Corpataux, Department of Physiology, Laboratory significance. of Experimental Medicine, University Hospital, 1005 Lausanne, Switzerland; Jacques-Antoine Haefliger, Conclusions: Metalloproteinases MMP-9 and MMP- Department of Physiology, Laboratory of Experimental 2 seem to play a role in the pathobiology of venous Medicine, University Hospital, 1005 Lausanne, aneurysm complicating AVF. Repeated cannulation is Switzerland; Dominique Midy, Department of Vascular associated with more inflammation and intense activity surgery, Bordeaux University, France; Laurence of proactive MMP-9. Bordenave, CHU de Bordeaux, CIC-IT Biomatériaux, F-33000 Bordeaux, Univ. Bordeaux, Bioingénierie tissulaire, INSERM U1026 BIOTIS, F-33000 Bordeaux, France. Purpose: To report the histological findings and the gene expression in aneurysm complicating vascular access arteriovenous fistula (AVF). In 2 vascular surgery departments, tissue samples of aneurysmal arterialized vein (AAV) and non-aneurysmal non-arterialized vein (NAAV) were collected from patient operated for the maintenance of their vascular access. The AAV samples were obtained from cannulated AVFs in group 1 and from non-cannulated AVF in group 2.

ISACB’S 2012 BIENNIAL MEETING 47 POSTER PRESENTATION ABSTRACTS

P40 High-Frequency Pulsatile Shear Stress Increases Mitochondrial DNA Damage By JNK Activation In Endothelial Cells Nelson Jen, University of Southern California, CA; Tzung Hsiai, University of Southern California Purpose: To study the effects of hemodynamic effects associated with increased heart rates in atrial fibrillation (AF) on the endothelium. Methods: A dynamic flow system was used to implement pulsatile blood flow in response to tachycardia at arterial carotid bifurcations. Human Aortic Endothelial Cells (HAOEC) were exposed pulsatile shear stress at 23±5 dyne·cm-2 and CL=429 (PSS140, 140 beats per minute) and compared to static condition. Following one hour of flow, we measured JNK activation by immunoblot and measured MitoSOX Red (a mitochondrial superoxide indicator) fluorescence intensity by FACS analysis. Following four hours of flow, mitochondrial DNA damage was quantified by PCR.

Results: In HAEC, PSS140 induced peak JNK activation after one hour and increased fluorescent MitoSOX intensity for the same time-point. Furthermore, PSS140 significantly increased mitochondrial DNA damage after four hours. JNK knockdown by siRNA attenuated increased MitoSOX intensity and mitochondrial DNA damage.

Conclusions: Our findings suggest that PSS140 induces mitochondrial dysfunction by JNK activation mediated mitochondrial DNA damage.

48 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P41 Biocompatibility Evaluation of Methods: aTPU grafts were fabricated by Bioresorbable Vascular Substitutes electrospinning [void fraction: 80%, pore size: 4 μm, fiber diameter: 1.32 μm]. The fabricated grafts Nargiz Seyidova, Ludwig Boltzmann Cluster for and ePTFE controls were evaluated in-vitro for cell Cardiovascular Research, Division of Biomedical attachment and proliferation. Prostheses, aTPU (n=28) Research, Medical University Vienna; Christian Grasl, and ePTFE (n=28), were implanted into the abdominal Ludwig Boltzmann Cluster for Cardiovascular Research, aorta in rats for 1 week, 1, 6 and 12 months. The Center for Medical Physics and Biomedical Engineering, retrieved grafts were evaluated for patency, thrombosis, Medical University Vienna; Martin Stoiber, Ludwig aneurysmal dilation and material degradation. The Boltzmann Cluster for Cardiovascular Research, Center biomechanical analysis was performed as well. Cell for Medical Physics and Biomedical Engineering, migration was calculated by computer assisted Medical University Vienna; Catharina Schreiber, Ludwig morphometry. Boltzmann Cluster for Cardiovascular Research, Division of Biomedical Research, Medical University Vienna; Results: Cell attachment and proliferation in-vitro Ingrid Walter, Department of Pathobiology, Veterinary was significantly lower in ePTFE grafts (p<0.001) than University; Konstanze Seidler, Institute for Applied in aTPU grafts. All vascular prostheses were patent Synthetic Chemistry, Technical University Vienna; S. and showed no evidence of thrombus formation or Clark Ligon, Institute for Applied Synthetic Chemistry, aneurismal dilatation. Within 1 week of implantation Technical University Vienna; Stefan Baudis, Institute aTPU graft walls showed significantly increased growth for Applied Synthetic Chemistry, Technical University factors (PDGF, VEGF, p<0.001). At all time points, Vienna; Robert Liska, Institute for Applied Synthetic aTPU conduits revealed significantly higher host cell Chemistry, Technical University Vienna; David Bernhard, populations as controls (p<0.001). The remodelling of Cardiac Research Laboratory, Department of Surgery, elastomeric conduits was already observable within Medical University Vienna; Heinrich Schima, Ludwig 1 month. Biomechanical analysis revealed sufficient Boltzmann Cluster for Cardiovascular Research, Center tensile strength of aTPU grafts. for Medical Physics and Biomedical Engineering, Conclusion: aTPU grafts demonstrate good in-vitro and Medical University Vienna; Helga Bergmeister, Ludwig in-vivo biocompatibility. Porosity and pore size facilitate Boltzmann Cluster for Cardiovascular Research, Division host tissue ingrowth and remodelling. Biomechanical of Biomedical Research, Medical University Vienna. properties are sufficient even in long-term applications. Purpose: Due to innate surface thrombogenicity and biomechanical mismatch with the host vessels, currently used synthetic conduits, such as ePTFE, are prone to thrombosis and intimal hyperplasia formation. To overcome these limitations, biodegradable scaffolds, with the potential to induce regenerative remodelling, are considered. We evaluated the application of new biodegradable, thermoplastic polyurethane (aTPU) and ePTFE vascular prostheses.

ISACB’S 2012 BIENNIAL MEETING 49 POSTER PRESENTATION ABSTRACTS

P42 Reactive Oxygen Species and Zinc Mediate Results: ROS accumulation was observed in SMCs BAV Aortopathy from BAV-TAA patients but not in TAV-TAA or normal patients. N-acetylcysteine, a zincchelator and ROS- Julie A. Phillippi, Department of Cardiothoracic Surgery, scavenger decreased MMP activity and abrogated University of Pittsburgh; Benjamin R. Green, Department ROS-induced down-regulation of elastin. Collagen of Cardiothoracic Surgery, University of Pittsburgh and elastin fiber architecture was altered in the aortas Medical Center; Michael A. Eskay, Department of of BAV patients when compared with aortas from TAV Cardiothoracic Surgery, University of Pittsburgh; Mary patients. P. Kotlarczyk, Department of Cardiothoracic Surgery, University of Pittsburgh; Michael R. Hill, Department Conclusions: These results suggest a substantial role of Bioengineering, University of Pittsburgh; Anne M. for ROS and zinc in mediating the BAV aortopathy. ROS Robertson, Department of Mechanical Engineering and accumulation coupled with abnormal zinc regulation Materials Science, University of Pittsburgh; Gregory may cause aortic ECM degeneration, leading to the A. Gibson, Center for Biologic Imaging, University of changes seen in matrix architecture. ROS detection Pittsburgh; Yi Hong, Department of Surgery, University and amelioration could offer novel diagnostic and of Pittsburgh; William R. Wagner, Department of therapeutic interventions for patients with BAV who are Bioengineering, University of Pittsburgh; Claudette at risk for aortic catastrophe. M. St. Croix, Department of Environmental and Occupational Health, University of Pittsburgh; Simon C. Watkins, Department of Cell Biology and Physiology, University of Pittsburgh; David A. Vorp, Department of Bioengineering, University of Pittsburgh; and Thomas G. Gleason, Department of Cardiothoracic Surgery, University of Pittsburgh Purpose: The goal is to identify the mechanism that mediates extracellular matrix (ECM) degeneration associated with bicuspid aortic valve (BAV) aortopathy. Prior reports from this group demonstrated a weakened oxidative stress response in smooth muscle cells (SMCs) and altered biomechanical strength in aneurysmal BAV patients compared with normal patients. These observations led to the hypothesis that ROS accumulation causes abnormal matrix architecture specific to the BAV aortopathy. Methods: Human SMCs were isolated from aortic specimens harvested during ascending aortic replacement due to thoracic aortic aneurysm (TAA) in patients with BAV or tricuspid aortic valve (TAV) and from normal donors. SMCs were cultured in 2-D or within 3-D poly (ester urethane) urea scaffolds. ROS and intracellular free zinc were monitored using fluorescent probes. MMP activity was quantified following ROS manipulation. ECM architecture was assessed via multi-photon microscopy and quantification of fiber alignment.

50 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P43 P44 Pre-implantation Collapse In The Sorin Decellularized Aortic Allografts Implanted In Perceval S Sutureless Prosthesis Does Not Sheep: A Pathological Analysis Affect Pericardial Graft Structure Mila Della Barbera, Department of Cardiological, Thoracic Mila Della Barbera, Department of Cardiological, Thoracic and Vascular Sciences, University of Padua, Italy; Igor and Vascular Sciences, University of Padua, Italy; Cristina Tudorache, Klinik für Herz-, Thorax-, Transplantations-, Basso, Department of Cardiological, Thoracic and und Gefäßchirurghie Medizinische Hochschule Vascular Sciences, University of Padua, Italy; Marialuisa Hannover, Germany; Marialuisa Valente, Department of Valente, Department of Cardiological, Thoracic and Cardiological, Thoracic and Vascular Sciences, University Vascular Sciences, University of Padua, Italy; Gaetano of Padua, Italy; Tanja Meyer, Klinik für Herz-, Thorax-, Thiene, Department of Cardiological, Thoracic and Transplantations-, und Gefäßchirurghie Medizinische Vascular Sciences, University of Padua, Italy Hochschule Hannover, Germany; Karolina Theodoridis, Klinik für Herz-, Thorax-, Transplantations-, und Purpose: : Sutureless valves have been conceived Gefäßchirurghie Medizinische Hochschule Hannover, to perform aortic valve replacement by simplifing the Germany; Cristina Basso, Department of Cardiological, procedure and shortening the cross-clamping time as Thoracic and Vascular Sciences, University of Padua, to limit myocardial damage due to cardiac arrest. At Italy; Serghei Cerbotari, Klinik für Herz-, Thorax-, difference from transcatheter aortic valve implantation Transplantations-, und Gefäßchirurghie Medizinische (TAVI), sutureless procedure is associated to native Hochschule Hannover, Germany; Axel Haverich, Klinik für cusps resection, and to reduced valve collapse time Herz-, Thorax-, Transplantations-, und Gefäßchirurghie before implantation. The Sorin Perceval S provides short Medizinische Hochschule Hannover, Germany; Gaetano collapse time, with limited involvement of the pericardial Thiene, Department of Cardiological, Thoracic and cusps, and does not undergo, after deployment, to Vascular Sciences, University of Padua, Italy prosthetic cusp ballooning except only to anchoring stent level. Purpose: Aim of the study was to characterize cell phenotype repopulation in decellularized aortic allograft Methods: Eleven Sorin Perceval S Sutureless prostheses roots implanted in sheep. underwent to different collapse times (four 15 and 60 min each and three 180 min) followed by deployment and Methods: 4 female sheep were studied: 1 control not ballooning. Two unimplanted pericardial sheets and four decellularized, unimplanted; 1 control decellularized, uncollapsed valves served as control. Gross, histological unimplanted; 2 decellularized, implanted for 14 and staining, including Picrosirius red, scanning electron 18.5 months. Gross, x-ray and light microscopy analysis microscopy (SEM) and morphometry were carried out to was carried out and consisted histological staining study collagen fibers waveness periodicity. and immunohistochemical staining (mouse anti-human -SMC actin, vimentin, eNOS, CD57, rabbit anti-human Results: Gross examination revealed optimal pericardial α VWF and CD31 antibodies). cusp coaptation. No tears, perforations or folding were observed. Prosthetic frame showed a preserved Results: The decellularization appeared complete with shape. Histology and SEM exhibited neither breaks no smooth muscle cells in the lamellar units of the aortic nor differences in collagen periodicity (16.55±2.89 μm tunica media and no interstitial cells with the cusps in in 15 min, 17.01±3.11 μm in 60 min, 16.45±2.13 μm in the treated, unimplanted allografts and no evidence of 180 min collapsed valves) when compared to controls endothelial lining. (17.47±2.50 μm in unmounted pericardium, 16.51±2.65 Both explanted decellularized aortic allografts appeared μm in uncollapsed valves) (p = NS). normally functioning with soft and pliable cusps, free Conclusions: At difference with TAVI, pre-implantation from calcium and tears. Repopulation occurred in both collapse, deployment and ballooning do not affect implanted decellularized allografts. The phenomenon the structural integrity of the collagen network of the was observed in the outer part of aortic wall with α-SMC pericardial cusp tissue in Sorin Perceval S Sutureless actin positive (most probably smooth muscle cells) prosthesis. infiltrating the elastic lamellar units. Repopulation was seen sporadically also in the intima with the formation of a neo-myointimal layer. Recellularization in the cusps was limited to ventricularis and spongiosa with α-SMC actin positive neo-interstitial cells and a few CD57 positive neural crest cells. Endothelium was regularly restored. Conclusions: Repopulation in decellularized aortic allograft occurs after implantation at both aortic wall and cusp level. It consists of α-SMC actin positive cells. The source is the adventitia for the outer part of the aortic wall tunica media and the blood itself for cusps and myointimal aortic wall layer.

ISACB’S 2012 BIENNIAL MEETING 51 POSTER PRESENTATION ABSTRACTS

P45 P46 Optimising vascular graft micro- Early Aberrant Angiogenesis due to Elastic architecture to improve surgical Fiber Fragmentation in Aortic Valve Disease applicability and tissue regeneration Amy Opoka, Division of Cardiology, Cincinnati Children’s Sarra de Valence, Dept. of Pharmaceutics & Hospital Medical Center; Amy L. Juraszek, Division of Biopharmaceutics EPGL, University of Geneva, Jean- Cardiology, University of Texas Southwestern; Hanna Christophe Tille, Dept. of Clinical Pathology, University Osinska, Division of Cardiology, Cincinnati Children’s Hospital of Geneva, Jean-Pierre Giliberto, Dept. of Hospital Medical Center; J. Michael Smith, Division Cardiovascular Surgery, University Hospital of Geneva, of Cardiothoracic Surgery, University of Cincinnati; Wojciech Mrowczynski, Dept. of Cardiovascular Surgery, Walter H. Merrill, Division of Cardiothoracic Surgery, University Hospital of Geneva, Robert Gurny, Dept. of University of Cincinnati; Pirooz Eghtesady, Division of Pharmaceutics & Biopharmaceutics EPGL, University Cardiothoracic Surgery, Cincinnati Children’s Hospital of Geneva, Michael Möller, Dept. of Pharmaceutics & Medical Center; Robert P. Mecham, Department of Cell Biopharmaceutics EPGL, University of Geneva, Beat H. Biology, Washington University; Kevin E. Bove, Division Walpoth, Dept. of Cardiovascular Surgery, University of Pathology, Cincinnati Children’s Hospital Medical Hospital of Geneva, Switzerland Center; Robert B. Hinton, Division of Cardiology, Cincinnati Children’s Hospital Medical Center Purpose: Shelf-ready synthetic biodegradable vascular grafts are promising alternatives to autologous material Purpose: Elastic fiber fragmentation (EFF) is a for clinical small diameter vascular. The production of universal hallmark of aortic valve disease (AVD), and electro-spun micro and nano-fiber based prostheses is neovascularization has been identified as a late finding simple and cost-effective. An important parameter for due to inflammation. EFF stimulates angiogenesis, tissue regeneration in such scaffolds is pore size(too and genetic syndromes due to mutant elastic fiber small impedes cell infiltration; too large creates blood components are associated with valve disease. We leakage). In this study, bilayered vascular grafts with hypothesized that angiogenesis due to EFF would be an two distinct micro-architectures were prepared and early AVD finding, preceding inflammation. evaluated. Methods: To examine disease progression, regional Methods: The bilayered grafts were made by anatomy and pathology of valve tissue was examined electrospinning polycaprolactone into a high using histochemistry, immunohistochemistry and porosity(82%) graft and adding a low porosity(65%) electron microscopy from early (<40yo) and late barrier layer either on the luminal or the adventitial (≥40yo) non-syndromic AVD specimens. To assess side. Grafts were characterized in vitro for fiber size, the effects of elastic fiber dysregulation, Williams and pore size, total porosity, water and blood leakage, Marfan syndrome aortic valves also were analyzed. mechanical strength, burst pressure, and suture Angiogenesis (VEGF-A, CD-34 and chondromodulin), retention. The two types of grafts were then evaluated EFF (elastin, fibrillin-1, emilin-1, fibulin-4 and -5), and in vivo in the rat abdominal aorta replacement model for inflammation/atherosclerosis (CD-68, LRP-5) 3 and 12 weeks. were assessed. Results: The in vitro blood leakage through these Results: Bicuspid aortic valve was more common in barrier grafts was significantly reduced compared to early AVD (n=21), and cardiovascular comorbidities were the single layer high porosity graft(0.6 vs. 1.5 ml/cm2/ more common in late AVD (n=11). Early AVD specimens min). In vivo, all the grafts were patent and cell invasion demonstrated angiogenesis without atherosclerosis. A at 3 and 12 weeks(6.4±2.3% and 12.5±0.7% for the distinct pattern of elastic fiber components surrounded outside barrier grafts vs. 23.5±5.5% and 35.3±5.3% early AVD neovessels, including increased emilin and for the inside barrier grafts) and neovascularization at 3 decreased fibulin-5. Different types of EFF were present weeks(3.2±1.1 vs. 14.7±3.1 capillaries/field of view) was in WS (n=6) and MFS (n=4) valves. WS but not MFS significantly reduced in outside barrier grafts, but there aortic valves demonstrated angiogenesis. Distinct was no significant difference between the grafts for matrix differences were observed by anatomic region endothelialization rate or intimal hyperplasia. between groups. Conclusions: By tailoring the micro architecture Conclusions: Aberrant angiogenesis is an early of multi-layered grafts an optimization of tissue mechanism in AVD pathogenesis preceding regeneration and surgical applicability was achieved in inflammation, implicating elastic fiber dysregulation as synthetic biodegradable vascular grafts. an inciting factor. Elucidation of underlying mechanisms may inform the development of new pharmacologic therapeutics and durable bioprostheses.

52 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P47 P48 VEGF-bound Stent: Arterial Shear Stress IL1-β and TNF-α-Induced Perturbations Augments The Differentiation in Cross-Talk Between Canonical Wnts Yoshinori Suzuki, Genome Biotechnology Laboratories, and TGF-β 1: A Mechanistic Target for Kanazawa Institute of Technology, Ishikawa, Japan; Abdominal Aortic Aneurysm (AAA) Therapy? Kimiko Yamamoto, Department of Biomedical Shyam M Manisastry, Department of Biomedical Engineering, Graduate School of Medicine, University Engineering, Cleveland Clinic, Cleveland, OH, and of Tokyo, Tokyo, Japan; Joji Ando, Department Anand Ramamurthi, Department of Biomedical of Biomedical Engineering, Graduate School of Engineering, Cleveland Clinic, Cleveland, OH Medicine, University of Tokyo, Tokyo, Japan; Kunio Matsumoto, Cancer Research Institute, Kanazawa Purpose: AAAs involve slow disruption of elastic matrix University, Ishikawa, Japan; Takehisa Matsuda, Genome in the aortal wall, by inflammatory cell-generated MMPs Biotechnology Laboratories, Kanazawa Institute of upon cytokine stimulation. Separate studies have shown Technology, Ishikawa, Japan TGF-β over-expression to stabilize experimental AAAs, and stimulate elastic matrix regenerative repair by AAA Purpose: Endothelializatioin of intravascular artificial SMCs. We investigated temporal changes in expression devices may promise to express nonthrombogenic of genes involved in signaling pathways triggered by potential. To this end, we are developing the surface cytokine stimulation of SMCs that culminates in elastin technology of in situ capturing of endothelial progenitor disruption, and how these correlate with endogenous cells (EPCs) on VEGF-bound device surface under TGF-β signaling. arterial flow, followed by rapid differentiation into endothelial cells (ECs). In fact, our extensive stent Methods: Rat aortic SMCs were cultured with IL1β or implantation study in porcine model showed capture of TNF-α alone (0&100 ng/ml) and together (50 ng/ml each) EPCs and subsequent endothelialization. In this study, over 4-120h and over 22d. Based on an initial WNT we investigated how differentiation potential of EPCs PCR array, we further screened for Wnts 1, 3, 4, 5A, cultured on VEGF-bound surface is influenced by a 7A, MMPs 2,7, 9 and 24, TIMPS 1-4, TGF-β, and matrix simulated arterial shear stress. genes elastin, collagen, LOX, & fibrillin. TCF activation was studied through Topflash/Fopflash ratio analysis. Methods: EPCs, obtained from human peripheral Western blots were performed for select proteins and blood, were cultured on VEGF-bound surface under TEM used to gauge de novo elastic matrix deposition a simulated arterial flow (shear stress 15 dynes/cm2; and quality at 22d. corresponds to arteries) for 24 h using the parallel plate- type device. Results: Bootstrapping analysis showed that IL1β+ TNF-α suppress canonical Wnts (1,3A,4) greatly Results: EPCs adhered well and proliferated on VEGF- enhance non-canonical Wnts (5A,7A), gradually bound surface. The loading of shear stress suppressed suppress TGF-β. These temporally coincided with the expression of mRNAs (CD34 and CD133), both of exponential increases in MMPs 2 and 9 mRNA and which are markers for EPCs, promoted the expression protein and elastin gene down-regulation, reflected in both mRNAs {CD31 and von Willebrand factor (vWF)} sporadic, non fibrillar elastic matrix. and protein (vWF) , all of which are markers for ECs. The loading of shear stress promoted the expression of Conclusions: Our results suggest that elastin and cell ephrinB2 mRNA which is a marker for arterial ECs, and homeostasis initially maintained by the canonical Wnts did not changed the expression of EphB4 mRNA which & TGF−β is perturbed by cytokine-activation of Wnts 5A is a marker for venous ECs. and 7A resulting in enhanced disruption and suppressed regeneration of elastin matrix. The results suggest a Conclusions: The arterial shear stress accelerated potential mechanistic target for therapy to concurrently differentiation of EPCs adhered on VEGF-bound surface reverse both these effects to stabilize AAAs. in vitro, strongly suggesting that the hydrodynamic shear stress is one key factor for rapid differentiation in vivo. This supports evidence of rapid endothelializatoin in porcine model.

ISACB’S 2012 BIENNIAL MEETING 53 POSTER PRESENTATION ABSTRACTS

P49 Genetically Engineered Vasculature for Methods: Human vascular networks can be Production of Protein Therapeutics in vivo bioengineered in vivo by the self-assembly of endothelial colony-forming cells (ECFCs) from peripheral Ruei-Zeng Lin, Department of Cardiac Surgery, blood and mesenchymal stem cells (MSCs) from Children’s Hospital Boston, and Harvard Medical bone marrow. Because we have good control over the School, Boston, MA; Shou-Ching S. Jaminet, Center engraftment of these cells, we now propose to use for Vascular Biology, Department of Pathology, this approach for gene therapy. To this aim, we chose Beth Israel Deaconess Medical Center, and Harvard erythropoietin (EPO) as a proof-of-concept model; we Medical School, Boston, MA; Juan M. Melero-Martin, genetically engineered human ECFCs to express EPO Department of Cardiac Surgery, Children’s Hospital under the control of a tetracycline-regulated system, Boston, and Harvard Medical School, Boston, MA and generated subcutaneous vascular networks Purpose: The use of recombinant proteins is a standard capable of systemic EPO release in immunodeficient practice in medicine. However, therapeutic proteins mice. are very expensive to produce, which can hamper the Results: Implants contained ECFC-lined blood vessels development of new products from basic research to that formed functional anastomoses with the mouse clinical trials. In addition, protein-based treatments vasculature, allowing direct delivery of EPO into the are expensive, involve parenteral administration, and bloodstream. After activation of EPO expression, require sustained patient compliance, drawbacks that erythropoiesis was induced in both normal and anemic have stimulated our search for alternative solutions. For mice, a process that was completely reversible. decades, ex vivo gene therapy has been postulated as a potential alternative to deliver recombinant proteins. Conclusions: We propose this ECFC-based gene- However, returning transfected cells back into patients delivery strategy as a novel platform technology for the remains a challenge. delivery of a diverse range of therapeutic proteins in clinical and research applications.

54 ISACB.ORG POSTER PRESENTATION ABSTRACTS

P50 Increased 18F-FDG Uptake in the Aortic Results: Mean 6-day aortic diameter increases for Wall of β-aminopropionitrile Exposed Rats control AAA and BAPN exposed rats that ultimately Is Predictive of Rupture in a Novel AAA ruptured were not significantly different at 258.3% and 276.2% (p=0.683), respectively. Areas of significantly Rupture Model greater focal 18F-FDG uptake, correlated with sites of Sean J English MD1, Moran R Piert MD2, Jose A Diaz rupture, along the left anterolateral aspects of AAAs that MD1, David Gordon MD3, Abhijit Ghosh PhD1, Phillip S ultimately ruptured were observed compared to areas Sherman BS2, Carole A Quesada BS2, Angela Pechota of greatest 18F-FDG uptake by control AAAs (p=0.004). BS1, Cathy E Luke1, Megan A Elfline MS1, Brendan S No aortic dissection was observed in any case of AAA McEvoy BS1, Jonathan L Eliason MD1, Peter K Henke rupture, and no significant differences in mean thrombus MD1, and Gilbert R Upchurch, Jr MD4. 1.) Conrad Jobst area to total AAA wall area ratios were determined for Vascular Research Laboratory, Dept of Surgery, Univ control and ruptured AAAs. Neutrophil populations of Michigan Health System, Ann Arbor, MI, USA 2.) were significantly decreased (p=0.008) in ruptured AAA Div of Nuclear Medicine, Dept of Radiology, Univ of tissues compared to control AAA tissues; however Michigan Health System, Ann Arbor, MI, USA 3.) Dept macrophage populations (p=0.013), as well as MMP2 of Pathology, Univ of Michigan Health System, Ann (0.002) and MMP9 (p=0.031) activities were significantly Arbor, MI, USA 4.) Div of Vascular and Endovascular increased in ruptured AAA tissues compared to control Surgery, Dept of Surgery, Univ of Virginia Health System, AAA tissues. Charlottesville, VA, USA Conclusions: BAPN can be used to generate aortic Purpose: A reproducible model of abdominal aortic aneurysm rupture in rats, in the presence of mural aneurysm (AAA) rupture is needed, and clinical decision thrombus and absence of aortic dissection. Decreased making would greatly benefit from non-invasive neutrophil and increased macrophage populations prediction of AAA rupture. Increased metabolic activity indirectly correlated with increased MMP9 activity and in the porcine pancreatic elastase (PPE) perfusion increased 18F-FDG uptake in the AAA wall of animals model in Sprague-Dawley rats stimulated to rupture that ruptured supports the role of macrophages in with β-aminopropionitrile (BAPN) was evaluated with aortic rupture. In this model, focally increased 18F-FDG 18F-fluorodeoxyglucose (18F-FDG) micro-positron uptake on micro-PET imaging was predictive of rat emission tomography (micro-PET) as a surrogate marker AAA rupture. of tissue inflammation. Methods: BAPN (N=25, 300mg/kg SC) was administered daily starting three days prior to PPE exposure and thereafter until rupture or harvest on postoperative day fourteen (12U/mL), and control rats underwent PPE and normal saline (3mL/kg SC) exposure until harvest on postoperative day six (N=7) or fourteen (N=7). Aortic intraluminal diameters were measured using ultrasound (US, 12MHz), and associated size increases were determined. Both groups underwent dynamic micro-PET imaging with 18F-FDG (18F-FDG 1mCi IV, 90 min). Maximum aortic to mean psoas muscle ratios (AMR) for 18F-FDG uptake were determined for areas of greatest focal 18F-FDG uptake over the left anterolateral AAA wall. Stereologic estimates of AAA mural thrombus area to total AAA wall area ratios were determined for control AAA and ruptured AAA tissue. Utilizing control AAA and ruptured AAA sections, stereologic estimates of neutrophil and macrophage populations per unit area of AAA wall were determined using H&E and CD68 immunohistochemistry, respectively. Matrix metalloproteinase (MMP) 2 and 9 activities in integrated optical density (IOD) were assessed for control and ruptured AAA tissue.

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