Frontiers in Cardiac and Vascular Regeneration

2012 International Centre for Genetic Engineering and Biotechnology THE ARTURO FALASCHI CONFERENCE SERIES ON MOLECULAR MEDICINE Frontiers in Cardiac...
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2012

International Centre for Genetic Engineering and Biotechnology THE ARTURO FALASCHI CONFERENCE SERIES ON MOLECULAR MEDICINE

Frontiers in Cardiac and Vascular Regeneration May 30 - June 2 Trieste, Italy

Programme and Book of Abstracts TOPICS: Vessel and heart formation during development - regulation of angiogenesis

in adult tissues - stem and iPS cell differentiation towards vascular cells and cardiomyocytes - stimulation of cardiac and vessel regeneration in adults - bridging the gap between vascular and cardiac research.

Under the patronage of:

comune di trieste

 

FACOLTA’ DI MEDICINA E CHIRURGIA

Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Welcome to the Trieste Conference on Frontiers in Cardiac and Vascular Regeneration, the first meeting of the ICGEB “Arturo Falaschi Conference Series on Molecular Medicine”. This meeting inaugurates a new Conference Series, dedicated to the memory of Arturo Falaschi, founder and first Director-General of the ICGEB, who passed away on 1 June 2010. It is thanks to his very passion for research and his discerning managerial talent that ICGEB exists today. Beyond the mission of education that he established, lies the concept that you cannot perform good training if you do not undertake excellent research. Twenty five years on, the ICGEB laboratories in Trieste, Cape Town and New Delhi, together with 40 Affiliated Centres worldwide, continue to provide intense training activities in the field of molecular research in medical and agricultural biotechnologies. Over 20 meetings and workshops are organised annually, counting over 1000 participants per year, and hosting international scientists of the highest standing in their fields. This conference alone brings together 30 such international speakers – world leaders in international experimental and clinical cardiology – for a rich programme juxtaposing problems related to cardiac cells with those concerning the formation of blood vessels. I am equally proud to welcome the many scientists, students and medical practitioners amongst our participants, from across Italy, Europe, Asia, the Middle East, the USA and Canada, together with those from among the ICGEB Member States, which today number over 60. I see this as a great opportunity for growth, for development, for innovation; a way to tackle the most serious, the most chronic of life threatening diseases, that causes more deaths, disability and economic strain than all other diseases. The fact that this meeting takes place in Trieste is an additional source of satisfaction for me personally. It is my great pleasure to welcome you to this beautiful city and to wish you both professional and personal gratification over the next four days.

  Mauro Giacca

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Table of Contents

General Information

5

Scientific Programme

6

Poster Session I

10

Poster Session II

12

Speakers’ Abstracts

14

Participants’ Abstracts

44

Abstract Author Index

96

List of Participants

102

About ICGEB

120

Sponsors and Acknowledgements

122

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

General Information Meeting Location Stazione Marittima Congress Centre Molo Bersaglieri 3, 34100 Trieste, Italy Tel. +39-040-304888

FVG Region “Azienda Ospedaliero-Universitaria” for a total of 14 credits. These may be assigned to registered Italian professionals, who are medical and biological staff of the local hospitals, who attend the entire meeting, and upon completion of the relevant questionnaire (distributed directly at the Registration Desk).

Hospitality arrangements Lunches and coffee breaks will be provided for the duration of the meeting at the Stazione Marittima Congress Centre. A cocktail will be held on Friday 1st June, starting 7.30 PM at the Savoia Excelsior Starhotel, directly opposite the Congress Centre. Thank you for wearing your badge at all times as this is required for admission.

External services located close to the Congress Centre Bank in the vicinity of the Congress Centre UniCredit, Piazza della Borsa 9 Tel: +39-040-6769311 Mon-Fri: 8.20-16.00

Internet Facilities Free internet access is available at the Congress Centre during the meeting.

Exchange Bureau Cambiavalute Giubbani, Piazza Ponterosso 3 Tel: +39-040-368080 Mon: 10.00-12.00; Tue-Fri: 8.00 – 17.00

Poster Sessions Poster boards are situated in the centre of the main hall of the Stazione Marittima Congress Centre. Posters should be displayed on Wednesday, 30th May upon registration and exhibited for the duration of the meeting. Specific Poster Sessions will be held in the afternoon on Thursday, 31st May and Friday, 1st June between 2.30 and 4.00 PM. A jury composed of selected scientists will award a prize for the best poster for each of the two sessions.

Parking Facilities Parking outside the Congress Centre “Park Si-Silos”, multi-storey (unattended) car park by the railway and bus stations in Piazza Libertà 9. Tel: +39-040-44924; open 24h/day at a cost of Euro 16 per day. Special rates for nearby parking facilities are offered by the local hotels. Please, check with your hotel reception. Taxi service Tel: +39-040-307730

Oral Presentations A technician is present in the conference room to provide any assistance that may be required. Speakers are requested to load and check their presentations by contacting the technician present in the conference room, either at the time of Registration or during the coffee-breaks. Oral presentations, selected from the abstracts submitted, have been allocated a 15 min time slot in the programme (12 min presentation plus discussion). Presenters are kindly asked to strictly adhere to this timing.

Pharmacies in the vicinity of the Congress Centre Farmacia al Lloyd, Via dell’Orologio 6 Tel: +39-040-300605 Mon-Fri: 8.30–13.00 and 16.00-19.30 Farmacia Campi Elisi S.N.C., Via Combi 7 Tel: +39-040 302800; Mon-Sun: 8.30-20.30 Travel Agency Cividin Viaggi, Via Imbriani 11 Tel: +39-040-3789111; Mon-Fri: 9.00–13.00 and 15.30–19.15; Sat: 9.30-12.30 Tourist Information Agenzia Turismo FVG, Via dell’Orologio 1 Tel: +39-040-3478312; E-mail: [email protected]; Mon–Fri: 9.00–19.00; Sat–Sun 9.00–13.00

Continuing Education in Medicine - ECM The conference has been accredited under the

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Programme Wednesday, 30 May 2012 11.00 - 14.30 Registration 14.30

Welcome addresses by local Authorities Welcome by Francisco E. Baralle, Director-General, ICGEB

14.40

Mauro Giacca, ICGEB Trieste and Silvano Riva, A. Buzzati Traverso Foundation, Pavia Remembrance of Arturo Falaschi

Mechanisms of vascular development Charirman: Roger Hajjar, New York 15.00

Elisabetta Dejana, Milan

Pathological development of brain microvasculature

15.30

Gera Neufeld, Haifa

Type-A plexins as versatile mediators of pro-angiogenic as well as of anti-angiogenic signal transduction

16.00

Federico Bussolino, Turin

Nervous vascular parallels: axon guidance and beyond

Coffee Break 17.00

Eli Keshet, Jerusalem

The vascular stem cell niche

17.30

Kari Alitalo, Helsinki

Therapeutic potential of vascular endothelial growth factors

18.00

Andrea Banfi, Basel

VEGF overexpression in skeletal muscle induces vascular enlargement and intussusceptive angiogenesis through reciprocal activation of Notch-1 in contiguous endothelial cells

18.15

Alessandro Carrer, Trieste

Neuropilin-1 expressing monocytes (NEMs), a novel population of bone marrow-derived myeloid cells promoting vessel maturation

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Thursday, 31 May 2012 Regulation of angiogenesis in normal and pathologic conditions Chairman: Elisabetta Dejana, Milan 9.00

Ralf Adams, Münster

Molecular regulation endothelial sprouting and angiogenic blood vessel growth

9.30

Lena Claesson-Welsh, Uppsala

How does VEGF regulate vascular permeability?

10.00

Peter Carmeliet, Leuven

Targeting endothelial metabolism: principles and strategies

10.30

Sandro De Falco, Naples

Inhibition of pathological angiogenesis targeting vessels and inflammation

Coffee Break

Gene therapy for cardiovascular disorders Chairman: Mauro Giacca, Trieste 11.30

Kenneth I. Berns, Gainesville

SPECIAL TALK SPONSORED BY THE ADRIANO BUZZATITRAVERSO FOUNDATION Evolution of AAV as a vector for gene therapy

12.00

Roger Hajjar, New York

Gene therapy for the treatment of heart failure

12.30

Andrew H. Baker, Glasgow

Modifying vein grafts by gene therapy

Lunch 14:40 - 16:00 Poster session I and coffee break

Gene therapy for cardiovascular disorders - continues Chairman: Andrew Baker, Glasgow 16.00

Mauro Giacca, Trieste

Searching for secreted factors and microRNAs promoting myocardial protection and regeneration

16.30

Christian Kupatt, Munich

AAV2.9 Thymosin beta 4 gene therapy: requirements for therapeutic neovascularization

17.00

Anna Marsano, Basel

Controlled VEGF expression in a cardiac patch improves vascularization and cardiac function in a myocardial infarction model

17.15

Sara Cioffi, Naples

Tbx1 is required in brain endothelial cells to establish vascular patterning 7

Frontiers in Cardiac and Vascular Regeneration

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Friday, 1 June 2012 From cardiac development to cardiac regeneration Chairman: Stefan Engelhardt, Munich 9.00

José Luis de la Pompa, Madrid

Notch signaling in cardiac development and disease

9.30

Juan Carlos Izpisua Belmonte, San Diego

Insights into the mechanisms underlying regeneration of the vertebrate heart

10.00

Jürgen Hescheler, Cologne

Pluripotent stem cells: basic research for later clinical application in the cardiovascular system

10.30

Piero Anversa, Boston

Cardiac stem cell and human heart failure

Coffee Break 11.30

Kenneth Chien, Boston

Driving heart progenitor cell fate and regeneration in vivo via chemically modified mRNA

12.00

Maurizio Capogrossi, Rome

Human cardiac stromal cell and heart regeneration

12.30

Ana Eulalio, Trieste

Functional screening identifies microRNAs inducing cardiac regeneration

12.45

Roberto Gimmelli, Rome

Characterization of the c-kit receptor function in cardiac regeneration by transgenic mouse models

Lunch 14:40 - 16:00 Poster session II and coffee break

Cardiac regeneration Chairman: Mark Mercola, San Diego 16.00

Robert Passier, Leiden

Human pluripotent stem cells and cardiac subtype specification

16.30

Karl-Ludwig Laugwitz, Munich

Human iPSC models of cardiac disease

17.00

Guido Tarone, Turin

Melusin and IQGAP1 are key players in ERK1/2 signaling and compensatory cardiac hypertrophy response to pressure overload

17.30

Gianfranco Matrone, Edinburgh

Role of Cyclin-Dependent Kinase-9 in cardiac injury-recovery in the zebrafish embryo

17.45

Gabriella Minchiotti, Naples

The G-protein coupled receptor APJ keeps the balance between proliferation and cardiovascular differentiation of multipotent mesodermal progenitors in embryonic stem cells

18.00

Aftab Ahmad Chatta, Lahore

Inhibition of CSC specific miRNAs results in differentiation of CSC into cardiomyocytes

18.15

Giuseppe Ristagno, Milan

Cardiac effect of modified embryonic mesoangioblasts expressing PlGF, MMP9 or both after myocardial infarction in the mouse

19.30 Cocktail and cultural event at the Savoia Excelsior Palace Starhotel 8

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Saturday, 2 June 2012 Cardiac regeneration continues Chairman: Thierry Pedrazzini, Lausanne 9.00

Mark Mercola, San Diego

Development of a pharmacological approach to myocardial regeneration

9.30

Fabio Recchia, Philadelphia and Pisa

Cardiac imaging in regenerative medicine

10.00

Ivonne Schulman, Miami

Cell therapy for chronic ischemic heart disease: from concept to clinic

Coffee Break

Cardiac regulatory RNAs Chairman: Jürgen Hescheler, Cologne 11.00

Thierry Pedrazzini, Lausanne

Novel non-coding RNAs in cardiac regeneration

11.30

Stefan Engelhardt, Munich

MicroRNA control of tissue fibrosis

12.00

Gianluigi Condorelli, Rome and San Diego

Translational control of myocardial function

12.30 Closing remarks and Meeting closure

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POSTER SESSION I

Thursday, 31 May - 14.30-16.00 1. A proteomic evaluation of smooth muscle cells in thoracic aortic aneurisms

Ceyda Acilan, Ph.D., Betul Baykal, Muge Serhatlı, MSc., Omer Kacar, Adiguzel, MSc., Altug Tuncer, M.D., Kemal Baysal, M.D., Ph.D., Ahmet T. Baykal, Ph.D.

MSc.,

Zelal

2. Nkx2-3 homeodomain transcription factor as a master determinant for the vascular patterning of spleen and intestinal lymphoid tissues Péter Balogh, Zoltán Kellermayer, Árpád Lábadi, Tamás Czömpöly, Hans-Henning Arnold

3. VEGF

over-expression in skeletal muscle induces vascular enlargement and intussusceptive angiogenesis through reciprocal activation of Notch-1 in contiguous endothelial cells Roberto Gianni-Barrera, Marianna Trani, Silvia Reginato, Ruslan Hlushchuk, Michael Heberer, Valentin Djonov and Andrea Banfi

4. Neuropilin-1 expressing monocytes (NEMs), a novel population of bone marrow-derived

myeloid cells promoting vessel maturation Carrer A., Moimas S., Zacchigna S., Maione F., Mano M., Zentilin L., Kazemi, M., Bussolino F., Giraudo E., Giacca M.

5. Transcriptional analysis reveals new candidate modulators of IFN-alpha response in endothelial cells Francesco Ciccarese, Angela Grassi, Barbara Di Camillo, Gianna Toffolo and Stefano Indraccolo

6. Tbx1 is required in brain endothelial cells to establish vascular patterning Sara Cioffi, Marchesa Bilio, Stefania Martucciello, Elizabeth Illingworth

7. Nitric oxide modulation of endothelial progenitor cells: increased contribution to skeletal muscle remodeling? Valentina Conti, Emanuele Azzoni, Silvia Brunelli

8. Therapeutic potential of Askina Gel in regenerative medicine Delle Monache Simona, Giuliani Gianfranco and Angelucci Adriano

Antonio,

Evtoski

Zoran,

Sanità

Patrizia,

Amicucci

9. Engraftment responses of human bone marrow stromal cells are enhanced by Notch ligands in vitro Giulia Detela, Owen Bain, Prof. Anthony Mathur, Dr. Ivan Wall.

10. Soluble VEGFR-1 interaction with α5β1 integrin triggers an endothelial cell dynamic and

angiogenic phenotype Angela Orecchia, Amel Mettouchi, Paolo Uva, Glenn Simon, Diego Arcelli, Simona Avitabile, Gianluca Ragone, Guerrino Meneguzzi, Karl Pfenninger, Giovanna Zambruno and Cristina Maria Failla

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11. Effect of sterilization by gamma-irradiation on biocompatibility of starch-based polymers and composites Abdel Wahab El Naggar, Magdy M Senna, Soheir Korraa and Sanaa Refaay

12. Controlled VEGF expression ensures safe angiogenesis and functional improvement in a model of myocardial infarction Melly L, Marsano A, Helmrich U, Heberer M, Eckstein F, Carrel T, Cook S, Giraud MN, Tevaearai H, Banfi A

13. VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC

Milivojevic Milena, Petrovic Isidora, Nikcevic Gordana, Zaric Jelena, Ruegg Curzio, Stevanovic Milena

14. The role fo transcription factors ZBP-89, SP3, NF-Y and EGR1 in the regulation of the SOX18 promoter activity Petrovic Isidora, Kovacevic-Grujicic Natasa, Milivojevic Milena, Stevanovic Milena

15. Isolation and propagation of mesenchymal stem cells from human cord tissue and blood in Iraq Farooq I Mohammad, Majeed Irsheed Sabah, Ali Hayder, Shaimaa Yusif Abdualfatah, Ban Sabah and Baraa Abdualhadi

16. Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery F. Papadopulos, B. Weber, M. Buzzi, M. Gargiulo, G. Pasquinelli

17. Defining the role of Neuropilin 1 in the cardiac neural crest Alice Plein, Dr. Alex Fantin, Christiana Ruhrberg

18. Human umbilical cord blood progenitors coordinate a regenerative angiogenic microenvironment in ischemic tissues David M. Putman, Kevin Y. Liu, Heather C. Broughton, Gillian I. Bell, David A. Hess

19. Gene expression profile of the early chick hemangioblast Ana Teresa Tavares, Vera Teixeira, Antonio Duarte

20. Dose- and time-dependent angiogenesis by controlled delivery of matrix-bound growth factors Sacchi Veronica, Martino Mikael, Morton Tatjana, Hofmann Gianni-Barrera Roberto, Hubbell Jeffrey, Redl Heinz and Banfi Andrea

Anna,

Groppa

Elena,

21. Ultrastructural characteristics of myogenic cells in failing myocardium of a patient on chemotherapy Ivan Zahradník, Jozef Bartunek, Marc Vanderheyden, Marta Novotová.

22. Post-translational modification by acetylation regulates VEGFR2 activity

Annalisa Zecchin, Lucia Pattarini, Maria Ines Gutierrez, Miguel Mano, Mike P. Myers, S. Pantano and Mauro Giacca

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POSTER SESSION II

Friday, 1 June - 14.30-16.00 23. ACE polymorphism and obesity: cardiovascular risk factors among Sudanese Fatima E. Abukunna, Abdulhadi N. H

Nagwa

M.

Mohamed,

Dina

A.

Hassan,

Hussein

A.

M,

24. Potential treatment modality OF ischemic myocardial rat heart with mesenchymal stem cell transplantation Esin Akbay, Handan Sevim, Özer Aylin Gürpinar, Serdar Günaydin, Mehmet Ali Onur

25. Pharmacological attenuation of Cardiac Stem Cell senescence in vitro increases their reparative ability in vivo Elisa Avolio, Angela Caragnano, Carlo Vascotto, Giuseppe Gianfranceschi, Ugolino Livi, Rajesh Katare, Paolo Madeddu, Daniela Cesselli, Carlo Alberto Beltrami and Antonio Paolo Beltrami

26. Engraftment responses of human bone marrow stromal cells and unselected mononuclear cells are dependent on the physical extracellular matrix but not the chemotactic factor SDF-1 Owen Bain, Giulia Detela,Christopher Mason, Anthony Mathur, Ivan Wall

27. iPS technology as a tool to investigate atrial fibrillation

Benzoni P., Bisleri G., De Luca A., Crescini E., Barbuti A., Baruscotti M., Muneretto C., Richaud Y., Raya A., and Dell’Era P.

28. Gap junctional coupling with cardiomyocytes is necessary but not sufficient for

cardiomyogenic differentiation of cocultured human mesenchymal stem cells Arti A. Ramkisoensing, Daniël A. Pijnappels, Jim Swildens, Marie José Goumans, Willem E. Fibbe, Martin J. Schalij, Douwe E. Atsma, Antoine A.F. de Vries

29. Effects of SOX2 overexpression on relative cell cycle distribution and cell migration of NT2/D1 cell clones Danijela Drakulic, Aleksandar Krstic, Andrijana Klajn, Milena Stevanovic

30. Effects of SOX2 over-expression on pluripotency, proliferation and differentiation of embryonal carcinoma stem cells NT2/D1 Milena Stevanovic, Danijela Drakulic, Aleksandar Krstic and Andrijana Klajn

31. Open chromatin conformation at Notch1 target genes is essential to sustain neonatal cardiomyocyte proliferation Giulia Felician, Chiara Collesi, and Mauro Giacca

Marina

Lusic,

Matteo

Dal

Ferro,

Lorena

Zentilin

32. The role of Ccbe1, a novel gene coding for an EGF-like domain protein, in the process of organogenesis of the heart João Furtado, Margaret Bento, and José A. Belo

33. Analysis of molecular profiling data to understand the whole-genome effects

of HGF stimulus in the heart Stefano Gatti, Christian Leo, Simona Gallo, Valentina Sala, Enrico Bucci, Alice Poli, Daniela Cantarella, Enzo Medico, Tiziana Crepaldi

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34. Characterization

of the c-Kit receptor function in cardiac regeneration by transgenic mouse models Gimmelli R., Di Siena S., Barbagallo F., Campolo F., Dolci S., Nori S., Isidori A., Lenzi A., Naro F., Pellegrini M.

35. AAV2.9 Thymosin beta 4 gene therapy: requirements fo therapeutic neovascularization

R. Hinkel, T. Trenkwalder, F. Gesenhues, A. Pfosser, G. Stachel, F. Götz, C. Lebherz, I. Bock-Marquette, E. Hannappel, C. Kupatt

36. Expression profile of striated muscle signaling protein Ankrd2 in neonatal cardiomoyocytes and its putative role in heart function and development Snezana Kojic, Valentina Martinelli, Jovana Jasnic-Savovic, Ljiljana Aleksandra Nestorovic, Dragica Radojkovic and Georgine Faulkner

rat

Rakicevic,

37. Hyperpolarized 13C-pyruvate to study carbohydrate metabolism in beating heart by

magnetic resonance imaging V. Lionetti, G. D. Aquaro, F. Frijia, L. Menchetti, F. Santarelli, V. Positano, S. L. Romano, G. Giovannetti, L. Landini, M. Lombardi, F.A. Recchia

38. Identification of novel genes regulating AAV transduction of cardiomyocytes and other post-mitotic cells by high-throughput, genome-wide siRNA screening Miguel Mano, Jasmina Lovric, Rudy Ippodrino, Lorena Zentilin and Mauro Giacca

39. Functional characterization of stem cell-derived cardiomoyocytes Irene C. Marcu, Pernilla Hoffmann, Marisa Jaconi, Nina D. Ullrich

40. Carbon nanotubes as a synthetic substrate for in vitro physiological heart tissue engineering

Valentina Martinelli, Giada Cellot, Francesca Maria Toma, Carlin S. Long, John H. Caldwell, Lorena Zentilin, Mauro Giacca, Antonio Turco, Maurizio Prato, Laura Ballerini, and Luisa Mestroni

41. Role of Cyclin-Dependent Kinase 9 in cardiac injury-recovery in the zebrafish embryo G. Matrone, K.S. Wilson, J.J. Mullins, C.S. Tucker & M.A. Denvir

42. Ultrastructural characteristics of myogenic cells in hypertrophying myocardium of rodents Marta Novotová and Ivan Zahradník

43. Nuclear architecture dynamics during human myoblast in vitro differentiation analyzed by 2D and 3D FISH Natalia Rozwadowska, Tomasz Kolanowski, Ewa Wiland, Marta Olszewska, Maciej Kurpisz

Piotr

Pawlak,

Marcin

Siatkowski,

44. Identification of novel cardioprotective factors by in vivo gene selection using AAV vectors

Giulia Ruozi, Serena Zacchigna, Matteo Dal Ferro, Francesca Bortolotti, Antonella Falcione, Lorena Zentilin and Mauro Giacca

45. Induction of cardiomyogenic markers in murine and human adult stem cells isolated from different sources upon electric stimulation Zamperone A., Pietronave S., Pavesi A., Oltolina F., Consolo F., Novelli E., Diena M., Fiore G.B., Soncini M., Radaelli A. and Prat M.

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Speakers’ Abstracts (in order of presentation)

Note: The Abstracts published in this meeting book should be treated as personal communications and be cited only with the consent of the author

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Pathological development of brain microvasculature Dejana E. IFOM-IEO-Campus and Milan University, Milan, Italy Brain microvasculature constitute a highly specialized and selective vascular barrier between blood and the central nervous system, called blood brain barrier (BBB). In these vessels endothelial cells present a highly developed system of tight junctions (TJs), absence of fenestration and low pinocytotic activity. Cells of the brain parenchyma, the astrocytes, contribute to the BBB-coverage with their foot processes, which constitute about the eighty percent of the basal aspect of the vessels. As a consequence, circulating solutes do not readily enter the brain parenchyma unless through specific endothelial “transporters”. Thus, BBB also limits the passage of anti-cancer drugs from the blood to the brain. Therefore, it would be therapeutically useful to develop systems to modulate BBB permeability. To this aim it is important to define the molecular mechanisms that regulate the establishment and maintenance of BBB properties. Data from our laboratory suggest a key role of the Wnt/β-catenin signaling pathways in the induction, regulation and maintenance of the BBB characteristics during embryonic and post-natal development. In endothelial cells, Wnt signaling induces barrier differentiation by increasing the stabilization and the transcriptional activity of β-catenin. On the contrary, inactivation of β-catenin causes significant downregulation of junctional proteins, and consequent BBB breakdown. Besides β-catenin, other three proteins, CCM1, CCM2 and CCM3, expressed by brain endothelial cells, are emerging as key modulators of the organization and function of the BBB. Indeed, mutations occurring in any of the genes encoding these proteins, leads to Cerebral Cavernous Malformation (CCM), a pathology characterized by brain vascular malformations. The endothelium in the lesions presents very few tight junctions and gaps are observed between endothelial cells. In addition, the vascular basal lamina is disorganized and the astrocytes do not take contact with the endothelial wall. The structural alterations of the BBB observed in CCM lesions are associated with severe clinical manifestations, such as cerebral haemorrhages and stroke. Additional data point to a possible link between the β-catenin and TGF beta pathways and the functions of CCM proteins in the regulation of BBB stability. These data open new therapeutic opportunities for this so far incurable disease

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Type-A plexins as versatile mediators of pro-angiogenic as well as of anti-angiogenic signal transduction Adi Sabag, Tanya Smolkin, Ofra Kessler and Gera Neufeld Cancer research and Vascular Biology Center, Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel. The seven members of the class-3 semaphorin subfamily are the only secreted factors of their class. In addition to their role as axon guidance factors have been found to function as inhibitors of angiogenesis and tumor progression. To transduce their inhibitory signals, most class-3 semaphorins, with the exception of sema3E, bind to one of the two neuropilin receptors. The neuropilins in turn cannot transduce semaphorin signals on their own and associate with one of the five plexin receptors capable of associating with neuropilins, and these serve as the signal transducing elements of the semaphorin receptor complex. We have recently observed that class-3 semaphorin receptor complexes contain more than one plexin. Thus, to transduce signals of sema3A in endothelial cells and glioblastoma cells three receptors, neuropilin-1, plexin-A1 and plexin-A4 are absolutely required and the lack of even one of them inhibits completely signal transduction. Likewise, we found that in these same cells signal transduction of sema3B requires the presence of plexin-A2 as well as plexin-A4 in addition to a neuropilin, and that none of the other plexins can substitutes for either of these plexins. These experiments demonstrate that receptor complexes for different semaphorins can contain shared components like the plexin-A4 receptor as well as unique components which determine specificity like plexin-A2. Furthermore both plexins and neuropilins can associate with other receptors that convey completely different signals and modulate their activities. For example, neuropilin-1 as well as plexin-A4 can associate with the VEGF receptor VEGFR-2 and potentiate VEGF165 induced signal transduction. Our experiments suggest that the characterization of receptor complexes for both pro- and anti-angiogenic factors is of crucial importance for the understanding of the mechanisms that fine-tune angiogenesis.

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Nervous vascular parallels: axon guidance and beyond Marco Arese, Enrico Giraudo, Guido Serini, Federico Bussolino Department of Oncological Sciences. University of Torino The vascular and nervous systems are organized with well defined and accurate networks, which represent the anatomical structure enabling their functions. In recent years, it has been clearly demonstrated that these two systems share in common several mechanisms and specificities. For instance, the networking properties of the nervous and vascular systems are governed by common cues that in the brain regulate axon connections and in the vasculature remodel the primitive plexus towards the vascular tree. As paradigmatic example of axon guidance molecule, we discuss the role of semaphorins in physiological and pathological angiogenesis. Finally, we discuss the presence in blood vessels of neurexin and neuroligin, two proteins that finely modulate synaptic activity in the brain. This observation is suggestive of an intriguing new class of molecular and functional parallels between neurons and vascular cells.

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The vascular stem cell niche Tamar Licht, Myriam Grunewald and Eli Keshet Dept. of Dev. Biol. & Cancer Res., The Hebrew University-Hadassah Med. Sch., Jerusalem 91120, Israel It is thought that the organ vasculature in a number of adult organs is an integral component of the respective stem cell niche. However, this proposition is mostly based on the intimate proximity of tissue stem cells to blood vessels and evidence that nearby vessels actually govern SC function is rudimentary. To address this issue, we use conditional VEGF-based vascular manipulations for expanding or, conversely, reducing the vasculature in selected adult organs. Neuronal stem cells (NSCs) residing in the hippocampus can promote adult neurogenesis associated with enhanced cognitive function. Previous studies by us and others have shown that hippocampus-specific induction VEGF augments adult neurogenesis and improves learning and memory. Via switching-off transgenic VEGF after it has induced durable vessels at the site where NSCs reside, we show that mere expansion of the vasculature is sufficient to maintain a 4-fold elevated neurogenic rate. Intriguingly, the sustained increase in neurogenesis rate does not come on the expanse of accelerated depletion of the exhaustible reservoir of hippocampal NSCs overriding the natural, rapid age-dependent decay of hippocampal neurogenesis. The hematopoietic stem cell (HSC) niche is thought to be composed of both osteoblasts and endothelial cells but the relative contribution of each is unknown. We show that switching-on VEGF in adult mice is sufficient for retaining mobilized BM HSCs in the liver and spleen and for generating a vessel-expanded niche conducive for full blown extra-medullary hematopoiesis associated with overall increase in HSC number. These studies thus provide a further support for a vascular stem cell niche.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Therapeutic Potential of Vascular Endothelial Growth Factors Kari Alitalo and collaborators Molecular/Cancer Biology Program and Finnish Institute for Molecular Medicine My laboratory studies vascular growth factors to facilitate therapeutics development for cardiovascular diseases and cancer. – Because of the importance of the growth of new blood vessels, or angiogenesis, in tumor progression, the first anti-angiogenic agents have been approved for clinical use. However, most patients are either refractory or eventually acquire resistance to anti-angiogenic therapeutics. A combination of angiogenesis and lymphangiogenesis inhibitors based on based on solid knowledge of the major interacting angiogenesis signaling pathways could be used to significantly advance the efficacy of tumor therapy. – The idea of proangiogenic therapy is to grow new functional blood vessels and thus restore blood flow to ischemic tissue. Several attempts have been made to stimulate angiogenesis and arteriogenesis in tissue ischemia, with limited success. VEGF-B, a coronary vascular growth factor, has recently stimulated renewed interest in such therapy in cardiac ischemia. – The growth of lymphatic vessels, lymphangiogenesis, is actively involved in a number of pathological processes including tissue inflammation and tumor dissemination but is insufficient in patients suffering from lymphedema, a debilitating condition characterized by chronic tissue edema and impaired immunity. Lymphangiogenic growth factors, such as VEGF-C provide possibilities to treat lymphedema. – Thus, there is considerable potential for the development of therapeutics based the biological functions of vascular endothelial growth factors. The angiopoietin (Ang) - Tie signalling system has important functions in cardiovascular development and regulation of vessel stability. Ang1 is dispensable in quiescent vessels but modulates the vascular response after injury, while Ang2 functions as a context-dependent antagonist of Ang1. Inhibition of Ang2 attenuates tumor angiogenesis and improves the anti-angiogenic effect of VEGF inhibition. Recent results suggest that Ang1 and Ang2 inhibitors have applications also in cardiovascular medicine. Bry M et al., Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation.Circulation 122:1725-33, 2010. Norrmén C et al., Biological basis of therapeutic lymphangiogenesis. Circulation 123: 1335-1351, 2011. Saharinen P, Alitalo K. The yin, the yang, and the angiopoietin-1. J Clin. Invest. 121: 21572159, 2011.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Molecular regulation endothelial sprouting and angiogenic blood vessel growth Ralf H. Adams1 and colleagues Max Planck Institute for Molecular Biomedicine, Department Tissue Morphogenesis, and University of Muenster, Faculty of Medicine, Muenster, Germany

1

Angiogenesis is the main process mediating the expansion of the blood vessel network during development, tissue regeneration or in pathological conditions such as cancer. The formation of new endothelial sprouts, a key step in the angiogenic growth program, involves the selection of endothelial tip cells, which lack a lumen, are highly motile, extend numerous filopodia, and lead new sprouts. Angiogenic sprouting is induced by tissue–derived, pro–angiogenic signals such as vascular endothelial growth factor (VEGF), which activates and triggers signaling by cognate receptor tyrosine kinases in the endothelium. However, this response is strongly modulated by intrinsic signaling interactions between endothelial cells (ECs). For example, expression of the ligand Delta-like 4 (Dll4) in tip cells activates Notch receptors in adjacent (stalk) ECs and is thought to downregulate VEGF receptor expression in these cells. Thus, the tip cell phenotype is suppressed in stalk cells and a balance between sprouting and the necessary preservation of existing endothelial tubes is established. Our work is providing further insight into the regulation of sprouting angiogenesis. The Notch ligand Jagged1 is a potent pro-angiogenic regulator with the opposite role as Dll4. In contrast to current models, we found that Notch controls VEGFR2 only moderately whereas VEGFR3 is strongly regulated. Moreover, blocking of Notch enables angiogenic growth even in mutant animals lacking endothelial VEGFR2 expression. We also found that endothelial sprouting and proliferation extension depend on VEGF receptor endocytosis. Ephrin-B2, a ligand for Eph family receptor tyrosine kinases, is required for endothelial cell motility, VEGF receptor endocytosis and the activation of downstream signal transduction cascades. More recently, we have identified Disabled 2, a clathrin-associated sorting protein, and the cell polarity protein PAR-3 as novel interaction partners of ephrin-B2 and VEGF receptors. These results establish that regional VEGF receptor endocytosis, which is controlled by a complex containing Dab2, PAR-3 and ephrin-B2, play a key role in the spatial organization of angiogenic growth. Acknowledgement: This study has been supported by the Max Planck Society, the University of Muenster and the German Research Foundation.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

How does VEGF regulate vascular permeability? Lena Claesson-Welsh Uppsala University Regulation of vascular endothelial growth factor (VEGF)-induced permeability is critical in physiological and pathological processes. Cardiovascular diseases as well as cancer growth and metastatic spread are progagated by excess vascular permeability. VEGF was originally identiifed as vascular permeability factor (VPF), but the molecular regulation downstream of VEGF has not been clarified We have identified a VEGF/VEGFR2-induced signaling chain that regulates vascular permeability. Autophosphorylation at Y951 in the kinase insert of VEGFR2 presents a binding site for the Src Homology 2-domain of T cell specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, through increased phosphorylation at pY418 and reduced phosphorylation at pY527 in c-Src. Tsad silencing blocked VEGF-induced c-Src activation but did not affect other VEGF-induced pathways involving phospholipase Cgamma, extracellular regulated kinase and endothelial nitric oxide. VEGF-induced rearrangement of vascular endothelial (VE)-cadherin positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression and TSAd-mediated translocation of active c-Src to junctions. In accordance, TSAd formed a complex with vascular endothelial (VE)-cadherin, VEGFR2 and c-Src at endothelial junctions. Vessels in tsad-/- mice showed undisturbed flow and pressure but failed to respond to VEGF with extravasation of Evans blue, dextran and microspheres in the skin and the trachea. Moreover, macrophage inflammatory protein-2, but not VEGF, induced extravasation of inflammatory cells in the tsad-/- cremaster muscle. We conclude that TSAd is required for VEGF-induced c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Targeting endothelial metabolism: principles and strategies Peter Carmeliet Center for Transgene Technology & Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven Angiogenesis, the growth of new blood vessels, plays a crucial role in numerous diseases, including cancer. Anti-angiogenesis therapies have been developed to deprive the tumor of nutrients. Clinically approved anti-angiogenic drugs offered prolonged survival to numerous cancer patients. However, the success of anti-angiogenic VEGF-targeted therapy is limited in certain cases by intrinsic refractoriness and acquired resistance. New strategies are needed to block tumor angiogenesis via alternative mechanisms. We are therefore exploring whether targeting endothelial metabolism can be a possible alternative therapeutic strategy for anti-angiogenic therapy.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Inhibition of pathological angiogenesis targeting vessels and inflammation. Laura Tudisco1, Ivana Apicella1, Menotti Ruvo2, Yoshio Hirano3, Jayakrishna Ambati3, Sandro De Falco1 1 Angiogenesis Lab, Institute of Genetics and Biophysics and and Bioimaging, CNR, Napoli Italy

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Institute of Biostructures

In the last years it has been definitively demonstrated that the pro-angiogenic members of vascular endothelial growth factor family, VEGF-A, VEGF-B and PlGF, which accomplish their action interacting with the two receptors VEGFR-1 (recognized by all three growth factors) and VEGFR-2 (specifically recognized by VEGF-A), play a central role in the modulation of pathological angiogenesis. These molecular players have become the preferential targets for anti-angiogenic therapy. Indeed, the first anti-angiogenic drugs approved for cancer and age-related macular degeneration treatments, have as target VEGF-A (Avastin and Lucentis) or the VEGF receptors (Sunitinib or Sorafenib, that are multi-target tyrosine kinase inhibitors). Currently a monoclonal antibody anti-PlGF is in phase 2 of clinical trials for tumor treatment. Recently, a great attention has been devoted on PlGF/VEGFR-1 axis because it is important not only for the stimulation of endothelial cells but mainly for the modulation of inflammatory response associated to the pathological angiogenesis, and for vessel stabilization. Indeed, it has been reported that overexpression of PlGF in tumor cells induced an impressive recruitment of inflammatory cells (F4/80 positive cells) and increased significantly the number of vessels surrounded by smooth muscle cells. It is important also consider that due to the strict biochemical and functional relationship between VEGFs and related receptors it appear evident how the ability to interfere with more than one of these factors may represent an advantage in term of therapeutic outcome. Indeed the upregulation of PlGF in tumors represents one of tumor escape strategy consequent to VEGF inhibition. We have recently reported the identification of a new synthetic tetrameric tripeptide named 4.23.5 that specifically binds VEGFR-1 preventing its activation by VEGF-A, VEGF-B and PlGF, at low micromolar range. Data on its anti-angiogenic properties in syngenic and xenograft cancer models as well as in choroid neovascularization model will be presented.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Evolution of AAV as a Vector for Gene Therapy Kenneth I. Berns University of Florida Genetics Institute, Gainesville, FL, USA Adeno-associated virus (AAV) is one of the most frequently used vectors for gene therapy. A successful vector must fulfill several criteria: it must not be toxic; it must be able to infect the target tissue; it must be able to be expressed in the nucleus; it must sccessfully contend with the host immune system; and, in most instances, expression should be long lasting. AAV vectors have been developed which meet all these criteria. A primary advantage is that AAV has not been implicated as the cause of any disease. Another potential advantage is that the only viral DNA that AAV vectors contain is the terminal repeat sequence of 145 bases at each end. The remainder of the vector genome contains the inserted transgene and associated regulatory sequences. The successful development of AAV as a vector has required detailed knowledge of the basic biology of AAV and its application. Among the issues which had to be resolved are differential targeting by different serotypes, differential interactions with the host immune system of different serotypes, and detailed knowledge of the 3 dimensional structure of the viral capsid and its various biological properties. Finally, knowledge of the factors affecting intracellular trafficking of the viral particle has proven to be of great importance. As a consequence of the basic knowledge which has been obtained, it has been possible to develop vectors which have been clinically efficacious in the treatment of one form of congenital blindness and hemophilia B.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Gene Therapy for the Treatment of Heart Failure Roger J. Hajjar Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY, USA Congestive heart failure remains a progressive disease with a desperate need for innovative therapies to reverse the course of ventricular dysfunction. Recent advances in understanding the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology have placed heart failure within reach of gene-based therapies. One of the key abnormalities in both human and experimental HF is a defect in sarcoplasmic reticulum (SR) function, which is responsible for abnormal intracellular Ca2+ handling. Deficient SR Ca2+ uptake during relaxation has been identified in failing hearts from both humans and animal models and has been associated with a decrease in the activity of the SR Ca2+-ATPase (SERCA2a). Over the last ten years we have undertaken a program of targeting important calcium cycling proteins in experimental models of heart by somatic gene transfer. This has led to the completion of a first-in-man phase 1 clinical trial of gene therapy for heart failure using adeno-associated vector (AAV) type 1 carrying SERCA2a. In this Phase I trial, there was evidence of clinically meaningful improvements in functional status and/or cardiac function which were observed in the majority of patients at various time points. The safety profile of AAV gene therapy along with the positive biological signals obtained from this phase 1 trial has led to the initiation and recent completion of a phase 2 trial of AAV1.SERCA2a in NYHA class III/IV patients. In the phase 2 trial, gene transfer of SERCA2a was found to be safe and associated with benefit in clinical outcomes, symptoms, functional status, NT-proBNP and cardiac structure.Furthermore, the recent successful and safe completion of the CUPID trial along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Modifying Vein Grafts by Gene Therapy Andrew H. Baker Institute of Cardiovascular and Medical Sciences University of Glasgow Patency rates for coronary artery bypass grafting (CABG) procedures using autologous saphenous vein (SV) remain poor. The vast majority of failures are due to neointima formation and superimposed atherosclerosis. There remains a requirement to develop a novel therapy through which to improve patency rates. Since many of the molecular and cellular mechanisms that lead to neointima formation have been identified, a number of strategies have emerged. These include modulation of smooth muscle cell migration, proliferation and/or apoptosis, acceleration of endothelial regeneration and improvement in endothelial function. Vein grafting is highly suited for human gene therapy since it allows ex vivo manipulation of the vein prior to grafting into the coronary circulation of the patient. This has clear safety advantages over in vivo gene therapeutic applications but, due to the short clinical window through which gene transfer can be administered, efficient vector systems are required. Adenoviral vectors have proven efficient for gene delivery in this context although expression of transgenes using first-generation vectors is transient in nature and associated with inflammation. However, this may be advantageous since a number of candidate therapeutic transgenes have been targeted to either block vascular smooth muscle cell migration and/or proliferation or to promote apoptosis. Our research has shown that overexpression of certain genes, including TIMP-3, p53 and NogoB has a beneficial effect by reducing neointima formation. This represents a valid therapeutic strategy to prevent vein graft failure in patients. We have also developed novel adenovirus vectors with improved capacity to transduce vascular cells.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Searching for secreted factors and microRNAs promoting myocardial protection and regeneration Mauro Giacca Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy The identification of novel genes and pathways controlling prenatal cardiomyocyte proliferation or regulating cardiomyocyte survival during the adult life holds paramount interest in view of developing new therapeutic approaches for patients with ischemic cardiomyopathy and heart failure. We are undertaking two complementary approaches in this respect. The first one exploits the capacity of viral vectors based on the Adeno-Associated Virus (AAV) to transduce myocardial cells in vivo with high efficiency and to promote the expression of their transgenes for prolonged periods of time. The exquisite tropism of these vectors for post-mitotic tissues such as cardiomyocytes and skeletal muscle fibers is explained by the lack of expression of DNA damage response proteins in these cells, in particular the members of the Mre11-Rad50-Nbs1 complex, which in replicating cells restrict vector transduction. Using AAV vectors, we have undertaken an exhaustive approach to select factors exerting beneficial effect upon myocardial damage by the construction of a library of AAV vectors delivering the entire mouse secretome (1600+ mouse secreted proteins). Preliminary screening of a subset of 100 vectors from this library has led to the identification of a few novel factors exerting a powerful cardioprotective action in vitro and in vivo. Among these factors is the neuropeptide hormone ghrelin, which we found to markedly ameliorate myocardial function upon AAV delivery after infarction in vivo. A second approach entails high throughput screening of microRNAs promoting primary neonatal cardiomyocyte proliferation in vitro. Out of a library consisting of ~1000 human microRNAs, we identified 40 human microRNAs with outstanding activity in promoting expansion of cardiomyocytes in cell culture, massive cardiac hyperplasty in the neonatal heart and remarkable improvement of cardiac function after infarction in adult animals upon cardiac delivery using an AAV9 vector. The identified factors and microRNAs hold great promise for the development of innovative therapeutic strategies for myocardial infarction and heart failure. This work was supported by Advanced Grant 250124 “FunSel” from the European Research Council (ERC)

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

AAV2.9 Thymosin beta 4 gene therapy: requirements for therapeutic neovascularization R. Hinkel1, T. Trenkwalder1, F. Gesenhues1, A. Pfosser1, G. Stachel1, F. Götz1, C. Lebherz1, I. Bock-Marquette2, E. Hannappel3, C. Kupatt1 1 Internal Medicine I, Klinikum Großhadern - LMU, München; Germany 2UT Southwestern Medical Center, Dallas, USA; 3Institut für Biochemie, Universität Nürnberg-Erlangen, Erlangen, Germany

Thymosin beta4 (TB4), a vasoactive peptide, has been shown to promote angiogenesis in the developing heart. For therapeutic use, however, conductance vessel growth is viewed as inevitable, which is not provided by all vessel growth factors at the same rate. For this process, microvessel maturation and smooth muscle cell proliferation of collaterals appear necessary. We investigated the potential of TB4 to induce therapeutic neovascularization. Methods: In vivo, rabbits (n=5) underwent femoral artery excision on day0 and 5x1012 AAV2/9 expressing Tb4, Ang-2 or Lac-Z were injected intramuscular. Application of Tb4 was performed over the whole limb or in the upper and lower limb separately. Injection of AAV2/9-Ang-2 was performed selectively into the lower limb. In an additional group L-NAME, an unselective NO inhibitor was applied. Quantification of collaterals was measured via angiography on day7 and day35 (%d7). Regional perfusion was assessed by application of fluorescent microspheres (%d7). HPLC-Anaylsis of Tb4 protein levels and Immunohistochemistry for PECAM-1 positive cells (capillaries/muscle fiber = c/mf) and vessel maturation (NG-2 positive pericytes/capillary = p/c) were quantified. Results: Overexpression of Tb4 in chronic ischemia increased capillary density (1.96±0.1 vs. control 1.37±0.1c/mf), collateral growth (175±15% vs. 95±6 %d7 in controls) and blood perfusion (187±5%d7 vs. control 110±6%d7). Furthermore, pericyte coverage was stimulated in the Tb4-treated group (0.69±0.1p/c) compared to control (0.46±0.1 p/c). Transduction of Tb4 only into the lower limb was sufficient to induce collateral growth (177±4%d7) and blood perfusion in the upper limb (207±8%d7), whereas selective injection into the region of collateral formation in the upper limb resulted only in a slight improvement of arteriogenesis (150±3%d7) and blooflow (142±6%d7). Furthermore, nitric oxide mediated increases in flow and shear stress were necessary for collateral growth, since the unselective NO-inhibitor L-NAME prevented arteriogenesis (118±8%) and perfusion gain (123±13%) after LL-TB4 transduction. Conclusion: TB4 induces arteriogenesis of conductance vessels in the upper limb, even when applied remotely in the lower limb. The mechanisms include microcirculatory maturation, i.e.pericyte coverage, and backward signaling via shear stress and nitric oxide formation.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Notch signaling in cardiac development and disease José Luis de la Pompa CNIC, Madrid Spain Notch is an evolutionarily conserved signaling pathway that regulates cell-fate specification, differentiation, and patterning. Mutations of Notch ligands and receptors are implicated in numerous congenital and acquired human diseases. Notch acts locally, specifying individual fates among a group of equivalent neighboring cells or directing a field of cells towards a given developmental fate. Notch activity is crucial in organs with complex architecture, such as the heart, that requires the coordinated development of multiple parts. In this talk I will discuss our recent advances in the field of Notch signaling in cardiac development and its impact in disease. I will describe how Notch signaling patterns the embryonic endocardium, enabling region-specific differentiation and critical interactions of the endocardium (or its derived mesenchyme) with other cardiac tissues (cardiac neural crest, myocardium), so that specialized structures (cardiac valves and chambers) are generated. I will also discuss extensively the implication of Notch in cardiac pathologies, including aortic valve disease, and cardiomyopathies.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Insights into the mechanisms underlying regeneration of the vertebrate heart Chris Jopling1, Guillermo Sune1, Eduard Sleep1, Marina Raya1, Merce Marti1, Carme Fabregat1, Angel Raya1, Juan Carlos Izpisua Belmonte1,2 1 2

Center of Regenerative Medicine in Barcelona, Barcelona, Spain Salk Institute for Biological Studies, La Jolla, USA

Although mammalian hearts show almost no ability to regenerate, there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response. In contrast to mammals, several non-mammalian vertebrate species are able to regenerate their hearts, including the zebrafish (zf) which can fully regenerate its heart after amputation. To address the source of newly formed cardiomyocytes during zf heart regeneration, we first established a genetic strategy to trace the lineage of cardiomyocytes in the adult fish, based on the Cre/lox system widely used in the mouse. Utilizing this system, we will show that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes. Furthermore, we will show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure, detachment from one another and the expression of cell-cycle progression regulators. Specifically, we will show that the gene product of polo-like kinase 1 is an essential component of cardiomyocyte proliferation during heart regeneration. Our data provide the first direct evidence for the source of proliferating cardiomyocytes during zf heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process. Hypoxia plays an important role in many biological/pathological processes. In particular, hypoxia is associated with cardiac ischemia which, while initially inducing a protective response, will ultimately lead to the death of cardiomyocytes and loss of tissue, severely impacting cardiac functionality. We surmised that ventricular amputation would lead to hypoxia induction in the myocardium of zf and that this may play a role in regulating the regeneration of the missing cardiac tissue. Using a combination of O2 perturbation, conditional transgenics and in vitro cell culture, we will show how hypoxia induces cardiomyocytes to dedifferentiate and proliferate in zf. These results indicate that hypoxia plays a positive role during heart regeneration, which should be taken into account in future strategies aimed at inducing heart regeneration in humans.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Pluripotent stem cells: basic research for later clinical application in the cardiovascular system Juergen Hescheler Institute for Neurophysiology, University of Cologne It is our aim to provide a fundamental basis to the development of new medical treatments. This presentation will give an overview on our recent research work on human embryonic in comparison with induced pluripotent stem cells. Starting from basic investigations on the physiological properties of cardiomyocytes developed from pluripotent stem cells we have established in vitro and in vivo transplantation models enabling us to systematically investigate and optimize the physiological integration and regeneration of the diseased tissue. Our main focus is the cardiac infarction model. Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as the most promising cell type for cardiac cell replacement therapy. iPS cells are functionally highly similar to embryonic stem (ES) cells, but in addition have the advantage of being ethically uncontroversial and obtainable from readily accessible autologous sources. Moreover, iPSCMs also provide an interesting new tool to study the pathophysiology of monogenetic diseases of the cardiovascular system. A functional integration of iPSCMs is crucial for efficiency and safety, but has not been demonstrated, yet. Thus, we investigated the electrical integration of transplanted CMs into host tissue. Genetically modified murine iPSCM, expressing eGFP and a puromycin resistance under control of the alpha-MHC promoter, were purified by antibiotic selection. Purified iPSCM were injected into adult mouse hearts. At different times after transplantation recipients were sacrificed and viable ventricular tissue slices were prepared. Slices were focally stimulated by a u iPSCMs iPSCMs nipolar electrode placed in host tissue. Recordings of action potentials were performed by glass microelectrodes in transplanted iPSCM, which could be identified by their green fluorescence, and in host cardiomyocytes within the tissue slices. Translation from the laboratory into the clinic is one of the key problems of stem cell research. Although proof of principle for the therapeutic use of iPS cells in cardiac diseases has been shown both at the laboratory scale and in animal models, the methods used today for generation, cultivation, differentiation and selection are not yet suitable for the clinic.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Cardiac stem cell and human heart failure Piero Anversa Departments of Anaesthesia and Medicine, Center for Regenerative Medicine Brigham & Women's Hospital, Harvard Medical School, Boston, MA The identification of cardiac progenitor cells in small and large mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. We established the conditions for the isolation and expansion of c-kit-positive hCSCs from small fragments of myocardium discarded at surgery. Additionally, we tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunodeficient and immunosuppressed animals independently of cell fusion. We have documented that c-kit-positive human cardiac cells possess the fundamental properties of stem cells: they are self-renewing, clonogenic and multipotent. hCSCs differentiate predominantly into cardiomyocytes and to a lesser extent into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles and capillary profiles. Importantly, the differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. Although the human myocardium shows an immature phenotype, it contracts regionally and contributes to the improvement in the hemodynamic performance of the infarcted left ventricle. These experimental findings constituted the basis for pre-clinical studies in large animal models and for the recent phase 1 trial SCIPIO (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy). One million autologous hCSCs were administered by intracoronary infusion ~4 months after multi-by-pass surgery in patients with chronic ischemic cardiomyopathy. No hCSC-related adverse effects were reported. The initial results of the SCIPIO trial are very encouraging; intracoronary infusion of autologous CSCs improved left ventricular systolic function and reduced infarct size in patients with severe heart failure after myocardial infarction.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Driving heart progenitor cell fate and regeneration in vivo via chemically modified mRNA Kenneth R. Chien Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Fairchild Lab, Harvard University, Cambridge, MA 02138, USA and MGH Cardiovascular Research Center A family of multipotent heart progenitors is responsible for the diversification and expansion of cardiac myocyte, smooth muscle, and endothelial cell lineages during cardiogenesis1-3. These progenitors persist after birth4-10 and may represent an opportunity for cell-based regenerative therapeutics. However, an effort to unlocking their potential has met with limited success11-14. The localized, transient and efficient delivery to the heart of paracrine factors which control the expansion and differentiation of resident heart progenitors might represent a viable alternative therapeutic strategy, akin to the known clinical utility of cytokines to selectively augment specific blood cell lineages. Herein, we establish chemically modified mRNA (modRNA) as a platform for localized, transient and highly efficient expression of paracrine factors in the murine heart. We show that VEGF-A modRNA administered in the setting of myocardial infarction (MI) markedly expanded the pool of Wilm’s tumor 1 (Wt1)expressing epicardial heart progenitors. Moreover, VEGF-A acted as a fate switch, driving rare pre-existing epicardial progenitors away from a previously described interstitial fibroblast-like state7 and towards a cardiac and vascular fate following MI. As a result, VEGF-A modRNA markedly increased epicardial progenitors and their vascular cell lineages, increase capillary density, reduced cardiac fibrosis, and improved cardiac function. These results show that VEGF-A modRNA represents a novel heart progenitor cell fate switch following injury and provide a new cell-free therapeutic paradigm to achieve in vivo recruitment and subsequent differentiation of endogenous heart progenitors for cardiovascular regeneration.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Human cardiac stromal cell and heart regeneration Maurizio Capogrossi Istituto Dermopatico dell’Imamcolata-IRCCS, Via dei Monti di Creta 104, 00167 Rome Stromal cells can be isolated from a variety of adult tissues and organs, the most extensively characterized being those of bone marrow origin [bone marrow mesenchymal stromal cells (BMStC)]. Recently, stromal cells have been also isolated from the cardiac tissue (CStC). The objective of the present work was to compare adult human CStC to syngeneic BMStC in order to establish their ability to differentiate into cardiovascular cells in vitro and repair the infarcted rat heart. Although CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC. In conclusion, although CStC and BMStC share a common phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Human pluripotent stem cells and cardiac subtype specification Robert Passier LUMC, Dept of Anatomy & Embryology, 2300 RC Leiden, the Netherlands Pluripotent human embryonic stem cells (hESC) have the potential to differentiate to any cell type of the human body. This characteristic has sparked researchers to study the use of hESC for regenerative medicine, drug screenings and embryonic development. We have recently optimized differentiation of hESC to cardiomyocytes, including growth factor directed differentiations as monolayers or as three-dimensional aggregates (embryoid bodies or EBs). Previously, we have demonstrated that hESC-derived cardiomyocytes (hESC-CM) faithfully recapitulate the early molecular events during embryonic development. Recently, we have generated a cardiac reporter line by introducing Green Fluorescent Protein (GFP), in the genomic locus of the early cardiac transcription factor NKX2-5, which enables us to visualize the derivation of NKX2-5+ cardiomyocytes during in vitro differentiation and purify these cells by Fluorescent Activated Cell Sorting (FACS). The combination of different transcription factor-coupled fluorescent reporters in this so-called “rainbow” hESC cell line, covering sequential stages of the cardiac lineage, will allow us to identify and characterize pathways for specific subtypes of the cardiac lineage at early and later stages during differentiation. Furthermore, a better understanding of these developmentally related processes will be further important for progress in fields of tissue engineering, disease modelling, drug toxicity and discovery, which most likely will lead to improved tailor-made therapies and better and safer medicines on the market.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Human iPSC models of cardiac disease Laugwitz K-L Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München Much of what is known about the molecular pathways that lead to human cardiovascular disorders has come from studying animal models, particularly genetically modified mice. In some cases it is possible to translate genetic discoveries from humans to mice (e.g. non-sense mutations), but in most circumstances there are no direct correlates for human genetic variants such as single nucleotide polymorphisms (SNPs) or copy number variants. Therefore, it is imperative to replicate relevant features of human cardiovascular physiology in the context of the human genome. Recent advances in stem cell biology now raise the possibility of generating human models of cardiovascular physiology and disease. Generating patient-specific cells and tissues has recently emerged with the demonstration that exogenous expression of four proteins in human skin fibroblasts (e.g. c-MYC, KLF4, OCT4, and SOX2) is sufficient to induce pluripotency in the cells. Although so-called induced pluripotent stem cells (iPSCs) are not perfectly equivalent to human ES cells, they retain important properties of ES cells such as the capacity for long-term propagation and the ability to differentiate into all human somatic cell types. Factor-based reprogramming enables us of the long-standing ambitions of stem cell biology: the ability to generate pluripotent cells from specific patients and figuratively, move a patient`s disease into the Petri dish. This will be discussed for human monogenetic cardiovascular diseases, e.g. LQT syndromes, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVC). Recent advances describing the derivation of human iPSCs from peripheral, frozen blood brings the stem cell field an important step closer to bio-banked blood samples and eventual clinical use. Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Gödel A, Höhnke C, Hofmann F, Seyfarth M, Sinnecker D, Schömig A & Laugwitz K-L (2010). Patient-specific induced pluripotent stem cell models for long-QT syndrome. N Engl J Med., Epub Jul 21. Jung B, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Pfeiffer S, Bellin M, Dorn T, Goedel A, Dirschinger R, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL. (2011). Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol. Med., Epub Dec 15.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Melusin and IQGAP1 are key players in ERK1/2 Signaling and compensatory cardiac hypertrophy response to pressure overload Mauro Sbroggiò, Alessandro Bertero, Daniela Carnevale, Giuseppe Lembo, Mara Brancaccio and Guido Tarone Molecular Biotechnology Center University of Torino, Angiocardioneurology, I.R.C.C.S. “Neuromed”, Pozzilli, ITALY

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Department

of

Melusin is a muscle-specific protein involved in triggering compensatory cardiac hypertrophy in response to pressure overload via activation of AKT and ERK1/2 MAPKs signaling pathways. To define the mechanism of melusin action, we searched for molecular partners involved in the melusin-dependent signal transduction. We demonstrated that melusin forms a supramolecular complex with c-Raf, MEK1/2 and ERK1/2 and that melusin-bound MAPKs are activated by pressure overload. Both the cytosolic tyrosine kinase FAK (Focal Adhesion Kinase) and the scaffold protein IQGAP1 are part of the melusin supramolecular complex and are required for ERK1/2 activation in response to pressure overload. To asses the in vivo role of this molecular complex we investigate the heart remodeling in mice lacking IQGAP1 expression. IQGAP1-null mice have unaltered basal heart function and develop normal hypertrophy in response to acute aortic banding. When subjected to chronic pressure overload, however, they develop thinning of left ventricular walls, chamber dilation, and a decrease in contractility, in an accelerated fashion compared to wild type mice. This unfavourable cardiac remodeling is characterized by blunted reactivation of the fetal gene program, impaired cardiomyocyte hypertrophy and increased cardiomyocyte apoptosis. IQGAP1-null mice show strongly impaired phosphorylation of MEK1/2-ERK1/2 and AKT following 4 days of pressure overload, but normal activation these kinases after 10 min, indicating that IQGAP1 is required for a specific, temporally regulated wave of both ERK1/2 and AKT signaling in response to pressure overload. These findings point to melusin and IQGAP1 as a key players in organizing a specific ERK1/2 signalosome in cardiomyocytes and in regulating adaptive long-term left ventricle remodeling in response to pressure overload.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Development of a pharmacological approach to myocardial regeneration Erik Willems, Wenqing Cai, Marion Lanier, Karl Okolotowicz, Marcia Dawson, John Cashman, and Mark Mercola Sanford-Burnham Medical Research Institute, La Jolla, CA 92037 USA; Human Biomolecular Research Institute, San Diego, CA 92121 USA Since current therapies for heart failure do not specifically create new myocytes, there has been tremendous interest in defining natural and synthetic molecules to regenerate myocardium from endogenous stem or progenitor cells. Towards this end, we have developed automated assays using progenitors from mouse and human embryonic stem cells expected to resemble functionally adult cardiac stem cells. Using these assays in high throughput screens of chemical and microRNA libraries, we have discovered chemical compounds and miRs that promote cardiomyocyte differentiation through the production of committed cardiac progenitors and differentiation of cardiomyocytes. An example of such a compound is a novel inhibitor of TGFbeta signaling. This compound selectively clears the type II TGFbeta receptor (TGFBR2) from the cell surface and targets it for proteasomal degradation in a ubiquitination-independent manner. Unlike previous inhibitors of TGFbeta signaling that target the receptor kinase activity, this compound is selective for TGFbeta over the closely related Activin receptor mediated signaling. Probing cardiogenesis, the compound aided in revealing a bimodal role for Nodal and TGFbeta signaling. During development, Nodal and TGFbeta both induce cardiogenic mesoderm, but subsequently Nodal expression declines and TGFbeta becomes uniquely responsible for inhibiting cardiomyocyte differentiation while simultaneously promoting vascular endothelial and smooth muscle differentiation. Mechanistically, Nodal/TGFbeta signaling represses cardiomyogenic differentiation, functioning by blocking an epigenetic switch involving Baf60c, Gata4 and Tbx5 that open chromatin of cardiac enhancers enhancing accessibility to transcriptional machinery. Since TGFbeta is involved in fibrosis and inflammation after myocardial injury, our results indicate that it might also antagonize creation of new myocytes and, therefore, suggest that transient inhibition of might facilitate regeneration.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Cardiac imaging in regenerative medicine Fabio A. Recchia Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy; Department of Physiology, Temple University School of Medicine, Philadelphia, USA Cardiac stem cell therapy typically targets selected regions of the heart, therefore classical functional evaluations of global function, such as ejection fraction or dP/dtmax, may largely underestimate its efficacy. This is particularly important during the first few months following cell delivery, when the cardioreparative effects may not rapidly translate into an overall improvement of contractile function. Unfortunately, most of the published studies on stem cell therapy, both in experimental models and in humans, have assessed the functional outcome based on ejection fraction. While this approach can be justified by practical aspects that render ejection fraction a standard and widely used index, its limitation might explain the inconsistent findings relative to the therapeutic efficacy of stem cell delivery. Modern technology offers better tools for non-invasive measurements of cardiac function and metabolism at regional level. In our laboratory, we have induced small size myocardial infarctions in pigs, which did not affect ejection fraction both in untreated and stem celltreated hearts. However, standard and “tagged” cardiac magnetic resonance imaging (MRI), combined with positron emission tomography (PET), revealed marked differences in infarct size, contractile function, blood perfusion and glucose uptake between treated and untreated hearts. The effectiveness of stem cell delivery, documented by our study, would have been completely ignored without the “magnifying lens” of MRI and PET imaging, which was focused on pre-defined segments of the ventricular wall in order to obtain rigorous comparisons of equivalent regions. A more recently developed technology, named 13C hyperpolarization, will likely evolve into a new precious tool for regional assessment of ventricular metabolism with MRI, thus replacing PET and the associated use of radioisotopes. Our Center is currently testing 13C-labeled myocardial energy substrates such as pyruvate and acetate. They are first subjected to a strong magnetic field and then injected intravenously during MRI performed with a clinical standard scanner. This imaging allows a global and segmental evaluation of substrate uptake and generation of the catabolites lactic acid and CO2 and even of Krebs cycle intermediate metabolites. Finally, at least in pre-clinical studies, stem cells can be transduced with genes encoding for proteins that render them detectable with MRI. In infarcted rats, we have successfully tested ferritin as a reporter protein detectable with a clinical MRI scanner, showing that stem cells can be tracked in vivo also after their differentiation into mature lineages. Based on these premises, it is conceivable that, in a near future, MRI, widely available at many research and clinical centers, might become the gold standard for simultaneous assessment of cardiac morphological, functional and metabolic changes occurring after stem cell delivery, with the additional possibility to track cell homing and differentiation in pre-clinical studies.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Cell therapy for chronic ischemic heart disease: from concept to clinic Ivonne H. Schulman, MD University of Miami Miller School of Medicine, Miami, USA. Heart failure is an increasingly frequent cardiovascular disorder that affects 15-20 million people worldwide. After myocardial infarction, the heart has insufficient regenerative capacity and undergoes progressive remodeling events that lead to left ventricular dysfunction and ultimately heart failure. Although pharmacological and interventional advances have reduced the morbidity and mortality of heart failure, there is an ongoing need for novel therapeutic strategies that prevent or reverse progressive ventricular remodeling. The development of cell-based therapy as a strategy to repair or regenerate injured cardiac tissue offers extraordinary promise for a powerful anti-remodeling therapy. Clinical trials are currently underway in our laboratory testing autologous and allogeneic bone marrow-derived mesenchymal stem cells (MSCs) for myocardial regeneration and reverse remodeling in heart failure patients with ischemic cardiomyopathy. In addition, the effectiveness of various methods of cell delivery and accuracy of diverse imaging modalities to assess therapeutic efficacy is under investigation. Optimization of stem cell therapy has the potential to provide long-term improvements for those suffering from heart failure. MSCs contribute to cardiac repair after myocardial injury via mechanisms that include trilineage differentiation, anti-inflammatory effects, immunomodulation, neovascularization, and modulation of the cardiac stem cell niche. These findings have in turn guided rationally designed translational clinical investigations. Collectively, there is a growing understanding of the parameters that underlie successful cell-based approaches for improving heart structure and function in ischemic cardiomyopathy.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Novel non-coding RNAs in cardiac regeneration Samir Ounzain1, Mark Ibberson2, Stefania Crippa1, Mohammed Nemir1, Alexandre Sarre3, Gaelle Boisset4, Keith Harshman5, Ioannis Xenarios2, Dario Diviani6, Daniel Schorderet4, Thierry Pedrazzini1 Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, Lausanne; Switzerland; 2Swiss Institute of Bioinformatics, Lausanne, Switzerland; 3Cardiovascular Assessment Facility, University of Lausanne, Lausanne, Switzerland; 4Institute for Research in Ophthalmology, Sion, Switzerland; 5Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland; 6Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland 1

Recently, the notion of promoting cardiac regeneration has engendered considerable research interest. Unlike the mammalian heart, which has a poor regenerative capacity, certain fish retain a robust capacity for regeneration into adult life. This includes the zebrafish, which can fully regenerate its heart after amputation of up to 20% of the ventricle. This capacity is a consequence of the differential utilization of regenerative genetic circuits, many of which are absent in the adult mammalian heart. These circuits are enacted by the integrated execution of specific transcriptional programs, which themselves are modulated by coding and noncoding regulatory factors, including small noncoding microRNAs (miRNAs). Recent studies have revealed central roles for miRNAs as core regulators of gene expression during cardiac development and disease, with the integration of miRNAs into the regulatory circuitry of the heart providing regulatory interactions to control cardiac gene expression. To systematically characterize these regulatory circuits we have generated global gene and miRNA expression profiles in the poorly regenerating mouse (ligation of the left descending coronary artery) and regenerating zebrafish (20% ventricular apex resection) models of cardiac injury and regeneration. We have developed and employed a novel integrated bioinformatic approach to identify differentially regulated miRNA dependant genetic programs in the mouse and zebrafish injury models. Many well characterized miRNA networks implicated in cardiac fibrosis (miR-133, -29, -30, -21, -208, -499) and hypertrophy (miR-1, -133, -208) were differentially modulated in the two species, highlighting the fundamentally different response of the mouse and zebrafish to cardiac injury at the miRNA level (miRome). Additionally, this global miRome characterization has allowed us to identify novel miRNA regulatory circuits potentially involved in critical biological pathways. These pathways have been implicated in the regenerative response including ECM deposition/ fibrosis, cell cycle control, cytokinesis and sarcomeric assembly/disassembly. Through the direct manipulation of these novel candidate miRNA circuits in the mouse, we hope to reactivate the regenerative potential of the adult mammalian heart in response to cardiac injury.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

MicroRNA control of tissue fibrosis Stefan Engelhardt Institut für Pharmakologie und Toxikologie, Technische Universitaet Muenchen (TUM) Munich Heart Alliance, Munich, Germany Muscle responds to a wide variety of stressors by hypertrophic growth of myocytes and interstitial fibrosis. In various muscular disorders, the hypertrophic response may temporarily serve a compensatory role but becomes detrimental when prohypertrophic stimulation persists. As such, cardiac hypertrophy has been identified as a risk factor for impaired cardiac function, future heart failure and life threatening arrhythmias. In contrast, muscle fibrosis has long been regarded as a secondary phenomenon thought to primarily result from myocyte pathology and cell death. Our recent work and that of others on small RNAs expressed in muscle suggests that fibroblasts may play an early and important role in muscular disease. MiRNAs interact specifically with mRNAs by repressing their translation or inducing their degradation. They are thus, by their impact on gene expression, key factors in the development and maintenance of tissue, both in health and disease states. The majority of work in the cardiovascular field has focussed on miRNAs whose cellular levels change under disease-causing conditions, based on the assumption that disease relevance and deregulation are tightly linked. In this context, we have identified miR-21 as one of the strongest upregulated microRNAs in myocardial disease and to be serve a key role in tissue fibrosis. We found that miR-21 regulates ERK-MAPkinase signalling in cardiac fibroblasts, which impacts on cardiomyocyte hypertrophy and cardiac function. Experimental therapeutic studies in animal models demonstrated inhibition of miR-21 as an effective therapeutic strategy against cardiac, pulmonary, kindney and skeletal muscle fibrosis. This talk will summarize our current state of knowledge on the role of miRNAs in tissue fibrosis and discuss prospects for RNA-based therapeutic approaches to target tissue fibrosis.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Translational control of myocardial function Deng Hong Zhang1, Riccardo Contu1, Juliana Smetana1, Gianluigi Condorelli1-2 University of California San Diego, La Jolla; and Istituto Clinico Humanitas IRCCS, Milan

1

2

National Research Council of Italy

mRNA translation is fundamentally involved in the regulation of cardiac hypertrophy. One of the critical molecules controlling mRNA translation and thus cell growth and in the response to energy state changes is a gene product called Mechanistic target of rapamycin (MTOR), a serine-threonine kinase. mTOR can be found in multiprotein complexes containing, among other partners, either raptor, forming TORC1, or raptor, forming TORC2. TORC1 and TORC2 selectively phosphorylate substrates with specific activities. Myocardial mTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. We previously demonstrated that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. We found that deletion of 4E-BP1 determined an increase of 4E-BP2, another member of the 4E-BP family. Deleting 4E-BP2 in the context of mTOR/4E-BP1 double knockout, further ameliorated cardiac funciton and survival, strenghthening the concept that 4E-BP is a critical regulator of the effects of mTOR in the heart. Our results demonstrated a critical role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Participants’ Abstracts

(in alphabetical order according to presenter)

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

ACE polymorphism and obesity a cardiovascular risk factors among Sudanese Fatima E. Abukunna, Nagwa M. Mohamed, Dina A. Hassan, Hussein A. M, Abdulhadi N. H Central Laboratory-Ministry of Sciences& Technology- Khartoum- Sudan In this study, the association of angiotensin converting enzyme (ACE) insertion (I)/deletion (D) polymorphism and obesity a cardiovascular disease factors in Sudanese adults was investigated.The subjects were divided according to the BMI, into two groups (Obese& over weight/ Normal weight). Body composition, lipid profile and C reactive protein were compared among groups. Obesity related dyslipidemia seems to affect overweight/obese males more than females. While obesity related hypertension is a phenomenon among females probably due to release of CRP. * The finding of ACE polymorphism will be included later.

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

A Proteomic Evaluation of Smooth Muscle Cells in Thoracic Aortic Aneurysms Ceyda Acilan1, Ph.D., Betul Baykal1,2, Muge Serhatlı1, MSc., Omer Kacar1, MSc., Zelal Adiguzel1, MSc., Altug Tuncer3, M.D., Kemal Baysal1,4, M.D., Ph.D., Ahmet T. Baykal1*, Ph.D. TUBITAK, MRC, Genetic Engineering & Biotechnology Institute, 2International Centre for Genetic Engineering and Biotechnology, 3Kartal Kosuyolu Advanced Training and Research Hospital, 4Dokuz Eylul University, Medical Faculty, Biochemistry Department

1

Aortic aneurysms (AA), a common disease affecting up to ~9% of individuals above the age 65, are characterized as localized degeneration of the aorta leading to weakening of the wall and widening of the vessel. While the exact mechanisms are yet to be determined, current studies indicate that the degradation of extracellular matrix proteins and apoptosis of vascular smooth muscle cells (SMCs) within the aorta may result in extendibility, dilatation and rupture of the vessel. Among the cells that form the aortic wall, SMCs are implicated as key components involved in disease development as numerous molecular changes have been reported to occur in these cells. Most current studies involve either investigation of proteins comprising a group or pathway in SMCs or global analyses in the whole aortic tissue. In order to determine which proteins are important in the development of thoracic aortic aneurysms, we performed comparative proteomic analyses using isolated, cultured SMCs from thoracic aortic aneurysms (TAA) versus controls. We hypothesized that cultured SMCs represent tissue characteristics and investigation of molecular changes in this sub-fraction of aorta should be able to better reveal the underlying reasons for this disease. Label-free LC-MS/MS analysis of cell extracts resulted in the identification of 256 proteins from which 26 significant differentially regulated proteins were identified and characterized. Both previously described and new proteins were identified that were involved in oxidative stress, extracellular matrix formation, protein folding, energy metabolism, and 14-3-3 pathway. Here, we attempted to build a multi protein model to shed light on the cellular mechanism of TAA. Keywords: Label-free proteomics, thoracic aortic aneurysm, smooth muscle cell, SerpinH1/ HSP47, oxidative stress, protein expression

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Inhibition of CSC specific miRNAs result in differentiation of CSC into cardiomyocytes 1

Aftab Ahmad, 2Edilamar M. de Oliveira, 3Dafina Ibrani, 4Yao Liang Tang, 5M.Ian Phillips

School of Biological Sciences, University of the Punjab, Lahore. Pakistan University of Sao Paulo, Sao Paulo, Brazil. 3University of California. Los Angeles, CA, USA 4,5 KGI, Claremont. CA. USA 1 2

Cardiomyocytes can be derived from hESC and human cardiomyocyte progenitor cells by treatment with activin A (Acv), bone morphogenic protein (BMP) or transforming growth factor, beta receptor III (TGF-βIII). We hypothesized that inhibition of specific miRNA in mouse cardiac stem cells (CSC) and CSC with GATA-4 (CSCG) could induce differentiation in cardiomyocytes by activation of their target genes. We studied 569 unique miRNAs probes in mouse heart cells (MHtC), CSC and CSCG and identified high expression of miR762, 21 and 31 in CSC and CSCG compared to MHtC. CSC and CSCG were cultured in matrigel and anti-mRNAs were transfected with oligofectamine reagent twice (3 and 15 days) after plating cells. We inhibited miR-762; 762+21; 762+31 and 762+21+31, which present as target genes activin A, BMP and TGF-β receptors. The target genes as TGF-βIII, activin A, BMP as well as Smad-4 and Dicer were analyzed by RT-PCR. Beating cells were recorded on Confocal microscopy.  As a positive control the cells were treated with activin A and BMP-4 cytokines and also compared to MHtC exrpession. The anti-sense was tested at 1, 5, 10, 50 and 100nM concentration. After 72hs of the treatment the target genes were analyzed by RT-PCR. In CSC and CSCG the TGF-βIII, activin AR, BMPR and Smad4 expression increased up 50nM and beginning inhibition with 100nM concentration. The maximum miRNA expression inhibition was observed 6hs after the treatment, and 24hs the miRNA expression was similar of the control cells, however the target genes expression was increased.  As expected cytokines treatment increased the target genes and signaling pathway increasing Smad-4. In CSC the miR-762, 762+21 and 762+21+31 anti-sense increased the target genes TGFβIII, AcvR, but not BMPR. Furthermore, increase the transcription factor Smad-4 and MLC-2a. α-MHC expression increased with 762+21 anti-sense association. In CSCG the 762+31 and 762+21+31 inhibition induced the genes, including TGFβIII, AcvR, but not BMPR. Also, increase Smad4 and MLC-2a and α-MHC. The results show that CSC and CSCG differentiate in cardiomyocytes by activation of TGFβIII and AcvR signaling pathway, but not BMPR. 

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Potential treatment modality for ischemic myocardial rat heart with mesenchymal stem cell transplantation Esin Akbay a, Handan Sevim a , Özer Aylin Gürpinar a, Serdar Günaydin b,Mehmet Ali Onur a Hacettepe University, Faculty of Science, Department of Biology, Beytepe,06800, Ankara, Turkey b Kirikkale University, Faculty of Medicine, Department of Thoracic and Cardiovascular Surgery, Kirikkale, Turkey a

Stem cells are investigated for their potential use in regenerative medicine (1). Several human organs are not capable of functional regeneration following a tissue defect and react with scar formation (2). For example, after ischemic cardiomyopathy and myocardial infarction (MI), the damaged myocardium is replaced by fibrotic noncontractile cells. These diseases are typified by the irreversible loss of cardiac muscle cells, which are essential for maintaining cardiac integrity and function. This ischemic heart disease accounts for 50% of all cardiovascular deaths. (3). For stem cell transplantation, undifferentiated precursor cells are applied to defective tissue for therapeutic regeneration (2). In this study, mesenchymal stem cells collecting from rat bone marrow were transplanted into the ischemic rat myocardium and healing of damaged tissue was investigated by using electrocardiographic and histological methods. In our study, the experiments were performed in 20 male Wistar rats with an initial body weight of   ~ 300 gram. Bone marrow was collected from tibias and femurs of five rats. After the ether inhalation, both tibias and femurs were removed and proximal ends were cut. Samples were collected in a centrifuge tube with normal cell culture medium (DMEM/F12) containing 1% penicillin/streptomicin. Bone marrow were homogenized by pipetting and centrifuged at 800 rpm for 5 minutes. The marrow pellet were resuspended in special medium and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 ºC. After three days of incubation, unattached cells were removed by changing medium. The attached cells were incubated in same medium under standard culture conditions. When the cells become confluent, they were trypsinized. Mesenchymal stem cells were passaged three times. Mesenchymal stem cells were characterized for CD13 and CD29 by using immunoflourescence staining. To evaluate the effects of trypsinization, cells were characterized by using same antibodies after 1st, 2nd and 3rd passages. Ischemic rat heart model was performed with a modified method of Pfeffer et al. (4). The rats were anesthetized by ketalar injection (0.2 mg/kg). After thoracotomy was performed, the left coronary artery was heat-cauterized between the pulmonary artery outlow tract. Cell transplantation was performed within 5 minute after induction MI. The thorax and the skin then immediately closed and rats were left to heal.  

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Pharmacological attenuation of Cardiac Stem Cell senescence in vitro increases their reparative ability in vivo Elisa Avolio, Angela Caragnano, Carlo Vascotto, Giuseppe Gianfranceschi, Ugolino Livi, Rajesh Katare, Paolo Madeddu, Daniela Cesselli, Carlo Alberto Beltrami and Antonio Paolo Beltrami Department of Medical and Biological Sciences, University of Udine Recently, we demonstrated that both age and pathology exert detrimental effects on human Cardiac Stem Cells (CSC) by attenuating their telomerase activity, reducing telomeric length, determining telomere erosion and the occurrence of telomere induced dysfunction foci, eventually leading to an impairment of CSC function in vitro. Aims:  to investigate whether CSC senescence is associated with a reduction in their reparative ability in vivo, to identify the molecular determinants possibly responsible for CSC senescence, to screen drugs able to interfere with CSC senescence and to verify if CSC drug treatment in vitro is effective in restoring the reparative potential of senescent CSC in vivo. Methods and Results: Western blot analysis of CSC obtained from either controls (C) or failing hearts (F) was carried out to identify pathways possibly associated with CSC senescence. Drug screening assays were performed on growing cultures of F-CSCs, exposing them to Rapamycin, DETA/NO or Resveratrol for three days. At the end of the treatment, cells were analyzed to quantify cellular senescence, cell proliferation, and cell death. The reparative capacity of CSC was evaluated in a mouse model of acute myocardial infarction (AMI). Cardiac function and histology were studied 2 weeks post-AMI. F-CSC are characterized by a significant activation of TORC1 complex and reduced in vivo reparative potential. A short term pharmacological treatment of F-CSCs resulted in a significant: a. reduction of p16+ cells, and increase of apoptosis in rapamycin treated cells, b. reduction of p21+ and γH2A.X+Ki67- cells, together with an increase in CSC proliferation in resveratrol treated cells, and c. decrease of γH2A.X+Ki67- cells in DETA/NO treated cells. In vitro treatment of CSC obtained from pathologic hearts with 10nM Rapamycin and 0.5µM Resveratrol prior to their in vivo administration to infarcted mice restored their reparative ability in vivo.    

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Frontiers in Cardiac and Vascular Regeneration

May 30 - June 2 Trieste, Italy

Engraftment responses of human bone marrow stromal cells and unselected mononuclear cells are dependent on the physical extracellular matrix but not the chemotactic factor SDF1 Owen Bain, Giulia Detela,Christopher Mason, Anthony Mathur, Ivan Wall University College London, Barts and the London NHS Trust Recently, a growing number of clinical trials have assessed bone marrow-derived cells as candidate therapies for ischemic injury to the myocardium. However, only mild improvements in cardiac function are reported. Furthermore, cell engraftment and survival are very low as many cells either undergo anoikis or migrate away. Several pre-clinical studies have reported that preconditioning bone marrow-derived stromal cells (MSCs) with factors such as SDF1 increases their engraftment in ischemic myocardial tissue, resulting in improved cardiac output. Therefore, we hypothesised that pre-stimulating MSCs with SDF1 would enhance cellular responses associated with cell engraftment and provide clinically relevant data about how to improve engraftment of cells in the heart. We tested the effect of SDF1 on early attachment and subsequent migration of human MSCs on fibronectin versus BSA-blocked tissue culture plastic. Additionally, we sought to address whether conditions that support angiogenesis induce formation of vessel-like structures by MSCs in vitro. We also addressed the effect of SDF1 on mediating these same cellular functions in whole bone marrow mononuclear cells (BMCs) isolated from patients with ischemic heart disease. To mimic the ischemic environment that prevails in injured myocardial tissue, assays were conducted under hypoxic conditions (2% oxygen tension), with normoxia (20%) used as a control. We found that early attachment to fibronectin was significantly greater than BSA control, both in normoxia and hypoxia (p