Small Molecule Targeting of G Reduces -Adrenergic Receptor Desensitization and Normalizes Cardiac Dysfunction in an Acute Heart Failure Model

4th Annual Symposium of the American Heart Association Council on Basic Cardiovascular Sciences Cardiovascular Repair and Regeneration: Structural and Molecular Approaches in the Cellular Era July 30–August 2, 2007 Keystone Conference Center Keystone, CO Sponsored by the American Heart Association Councils on Basic Cardiovascular Sciences and Clinical Cardiology and the American Heart Association Interdisciplinary Working Group on Functional Genomics & Translational Biology. Co-sponsored by the National Heart, Lung, and Blood Institute. Conference Program Committee Conference Co-Chairs: Maurizio C. Capogrossi, Keiichi Fukuda, Sumanth D. Prabhu, and Mark Sussman This annual meeting is the fourth for this premier research conference sponsored by the American Heart Association Council on Basic Cardiovascular Sciences, the world’s leading organization of cardiovascular scientists. The conference focused on how cellular-based approaches are being manipulated to enhance the repair and regeneration capabilities of the cardiovascular system with the goal of therapeutic-based interventions. The meeting featured both invited presentations and poster abstract presentations, with participants from around the world. Abstracts for the poster presentations are provided in this special online supplement available at http://circres.ahajournals.org. by gest on Sptem er 2, 2017 http://circhajournals.org/ D ow nladed froms for the poster presentations are provided in this special online supplement available at http://circres.ahajournals.org. by gest on Sptem er 2, 2017 http://circhajournals.org/ D ow nladed from 2007 BCVS Symposium Abstracts P1 Small Molecule Targeting of G Reduces -Adrenergic Receptor Desensitization and Normalizes Cardiac Dysfunction in an Acute Heart Failure Model Liam M Casey, Frances Nwakanma, Gabriel Vorobiof, Olga Dunaevsky, Alan V Smrcka, Burns C Blaxall; Univ of Rochester Med Cntr, Rochester, NY Heart failure (HF) is a debilitating disease with poor prognosis, despite substantial therapeutic advances in the past two decades. Excess signaling through cardiac G-protein G subunits is an important component of HF pathophysiology. They recruit elevated levels of cytosolic G-protein coupled receptor kinase 2 (GRK2, a.k.a. ARK1) to agonist-stimulated -ARs in HF, leading to chronic -AR desensitization and down-regulation; these events are all hallmarks of HF. Previous data has suggested that inhibiting G signaling and its interaction with GRK2 could be of therapeutic value in HF. We recently developed a novel small molecule targeting strategy to selectively inhibit G binding interactions, and identified several G inhibitory small molecules (Bonacci et al, Science, 2006). In particular, we identified compound M119, which blocked the interaction of purified G and GRK2 in vitro. To validate this activity in a cellular setting, we isolated adult mouse cardiomyocytes, where M119 significantly reduced -AR-mediated membrane recruitment of GRK2. Furthermore, M119 significantly enhanced both adenylyl cyclase activity and cardiomyocyte contractility at baseline and in response to -AR agonist stimulation. Upon finding that the compound was biologically available following intraperitoneal injection in mice, we investigated whether the observed in vitro effects in isolated adult cardiomyocytes would translate to in vivo effects on cardiac function. Initial investigations were pursued in an acute pharmacologic HF model (30 mg/kg/day isoproterenol for seven days). Importantly, concurrent once daily injections of M119 normalized cardiac function, morphology and GRK2 expression in this acute HF model. Collectively, our study has identified a small molecule G inhibitor capable of reducing -AR desensitization, thereby enhancing -AR-mediated isolated cardiomyocyte contractility and, importantly, normalizing cardiac function and morphology in an acute HF model in vivo. Our data suggest a promising therapeutic role for small molecule inhibition of G in the treatment of HF. P2 Encapsulation of a Small Molecule p38 Inhibitor for Cardiac Regeneration Jay C Sy, Gokulakrishnan Iyer, Milton Brown, Sergey Dikalov, Emory Univ Sch of Medicine, Atlanta, GA; Niren Murthy, Georgia Institute of Technology, Atlanta, GA; Michael E Davis; Emory Univ Sch of Medicine, Atlanta, GA Myocardial dysfunction is usually progressive and successful therapy will likely require sustained delivery. Small molecule inhibitors have great clinical potential in the treatment of cardiac disease, but their size and stability make them difficult to target to the myocardium. SB239063 has been successful in preventing cardiovascular dysfunction but treatment protocols are prolonged and may not translate to larger animals. Polyketal (PK) particles are a new class of biomaterials that hydrolyze slowly at physiological pH values and degrade to non-toxic compounds. Here we show that polyketal-encapsulated SB239063 (PKSB) timedependently inhibited TNF-induced p38 phosphorylation in RAW macrophages. In addition, PKSB, and not empty polyketals (PK), inhibited TNFstimulated extracellular superoxide production as measured by accumulation of superoxide-specific product of dihydroethidium, 2-hydroxyethidium (TNF 0.82 M; PKSB 0.37 M; p 0.05). To determine efficacy in vivo, we first established by skeletal muscle injection studies that polyketal treatment did not result in inflammation. We then performed a randomized and double-blinded study in rats subjected to myocardial infarction. Immediately following coronary artery ligation, rats were injected with PK, PKSB, or free SB239063 intramyocardially. Three days following infarction, there was a significant reduction in p38 phosphorylation within the infarct zone of PKSB rats, with no effect of PK or SB239063. In addition, only PKSB attenuated infarct-zone superoxide production (MI 27.68 M; PKSB 8.49 M; p 0.05) and TNFproduction (MI 157.73 pg/mg; PKSB 103.60 pg/mg; p 0.05). In a separate double-blinded study, we examined cardiac function by MRI at 7 and 21 days post treatment. While there was no difference at 7 days post-infarction, PKSB, and not PK or SB239063 significantly inhibited progression of dysfunction at 21 days. In summary, our data demonstrate that SB239063 retains its function following encapsulation and, in vivo following injury, performed significantly better than the free inhibitor or the control empty polymer. Thus, we conclude that polyketal encapsulation is a novel approach for delivering small, easily diffusible inhibitors for cardiac regeneration. P3 Control of Phenotypic Plasticity of Smooth Muscle Cells by BMP Signaling Through Myocardin-Related Transcription Factors Manching Ku, Brandi N Davis, Akiko Hata, Tufts Univ Sch of Medicine, Boston, MA; Peter H Nguyen, Giorgio Lagna; Tufts-New England Med Cntr, Boston, MA Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for Bone Morphogenetic Proteins (BMPs), in idiopathic pulmonary arterial hypertension (IPAH) provided a clue that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induce SMC genes in pluripotent cells, and prevents de-differentiation of pulmonary artery SMCs (PASMCs). The BMP-induced phenotype switch requires intact RhoA/ROCK signaling, but is not blocked by inhibitors of the TGF and PI-3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a SM -actin promoter was observed in response to BMP treatment in vivo. Thus, we conclude that BMP signaling modulates the phenotype of VSMCs via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with IPAH and other obliterative vascular diseases. P4 Transendocardial Autologous Bone Marrow in Chronic Myocardial Infarction Using Helical Needle Catheter: 1-Year Follow-up in an Open-Label, Nonrandomized, Single-Center Pilot Study (the TABMMI Study) Luis de la Fuente, Salvador Med Sch Buenos Aires, Buenos Aires, Argentina; Simon H Stertzer, Stanford Univ Med Cntr, Palo Alto, CA; Julio Argentieri, Eduardo Penaloza, Jorge Miano, Benjamin Koziner, Christian Bilos, Argentine Institute of Diagnosis and Treatment, Buenos Aires, Argentina; Peter A Altman; Biocardia Inc, South San Francisco, CA Aims: Cell therapy has shown benefit in preclinical and clinical studies, although debate continues on the mechanism of action and the most appropriate methods for performing such therapies. We assessed the hypothesis that helical needle transendocardial delivery of autologous bone marrow (ABM) cells around regions of hypo or akinesia in chronic post-MI patients would be safe and possibly improve ejection fraction. Methods and results: 10 stable post-MI Patients with an ejection fraction (EF) 40% were enrolled. ABM cells were aspirated from the iliac crest and delivered percutaneously with a transendocardial helical needle catheter. 86x10 cells were injected into 7.1 3.1 sites around the infarct to target the peri-infarct zones. 2D echo left ventricle EF measurements, 24 hour Holter, and exercise tolerance testing were performed at baseline, day of procedure, 1 and 12 weeks, 6 and 12 months. There were no adverse events associated with the catheter based cell transplantation procedure. At 6 and 12 months, all patients showed an improvement in left ventricle EF over baseline (35.2 4.6 to 40.8 4.5, p 0.003 at 6 months, 35.2 4.6 to 42.3 5.1, p 0.0001, at 12 months). Conclusion: ABM cells delivered with the helical needle transendocardial catheter was safe in this small uncontrolled study in patients with chronic MI. Increased ejection fraction and other positive data trends support continued development of this therapeutic strategy in larger controlled trials. P5 GSK-3 Induces Cardiomyocyte Markers in Bone Marrow–Derived Mesenchymal Stem Cells (MSCS) Jaeyeaon Cho, Shinichi Hirotani, Junichi Sadoshima; UMDNJ, Newark, NJ Although adult MSCs have cardiomyogenic properties, the underlying signaling mechanisms are not well understood. The Wnt pathway significantly affects cardiomyocyte differentiation from various stem cell populations, but its effects vary substantially depending upon experimental conditions. The goal of this study was to clarify the role of GSK-3 , a major inhibitory component of the Wnt pathway, in regulating cardiomyocyte differentiation from adult MSCs. Either human or mouse bone marrow-derived MSCs were treated with 5 M of 5azacytidine (5-Aza) without serum for 24hrs, which induced mRNA expression of cardiomyocyte markers, including Nkx2.5 and myosin heavy chain ( -MHC), within 3 days. 5-Aza treatment induced troponin I protein expression 5 days after treatment. We examined protein expression of GSK-3 and -catenin, a downstream target of GSK-3 , during 5-Aza treatments. GSK-3 was increased by 5-Aza in a time-dependent manner, reaching a peak ( 4 fold) on 3Day. This was paralleled by decreases in -catenin ( 50 %), suggesting that the activity of GSK-3 was increased, whereas the Wnt pathway was suppressed by 5-Aza. To test the effect of GSK-3 upon cardiomyocyte differentiation, MSCs were transduced with GSK-3 -adenovirus, which induced 10–15 fold expression of GSK-3 and 80–95 % downregulation of -catenin on Days 3–12. Stimulation of GSK-3 caused induction of Nkx2.5 and ANF mRNA, peaking on Days 4–5, whereas LacZ-virus did not. In order to stimulate GSK-3 by alternative methods, MSCs were isolated from conditional GSK-3 transgenic mice. Isolated MSCs were transduced with tTAor rtTA-adenovirus, to achieve GSK-3 expression regulated by the tetracycline (tet)-OFF and -ON systems, respectively. GSK-3 expression increased by the tet-OFF (4.7 fold) or tet-ON system (3 fold) induced mRNA expression of Nkx2.5, -MHC and GATA4, as well as protein expression of sarcomeric actinin and troponin I in MSCs. In contrast, MSCs treated with LiCl (10mM, an inhibitor of GSK-3 ) failed to induce cardiomyocyte markers such as troponin I. In summary, 5-Aza-induced increases in cardiomyocyte markers in MSCs were accompanied by activation of GSK-3 , and stimulation of GSK-3 induces expression of cardiomyocyte markers in adult MSCs. E54 Circulation Research Vol 101, No 5 August 31, 2007 by gest on Sptem er 2, 2017 http://circhajournals.org/ D ow nladed from P6 No Evidence of Transdifferentiation of Human Mesenchymal or Hematopoietic Stem Cells into Cardiomyocytes Following Coculture with Neonatal Rat Cardiomyocytes Remco Koninckx, Jr, Karen Hensen, Virga Jesse Hosp, Hasselt, Belgium; Olga Marinkina, Ivo Lambrichts, Univ of Hasselt, Diepenbeek, Belgium; Hanne Jongen, Urbain Mees, Virga Jesse Hosp, Hasselt, Belgium; Paul Steels, Univ of Hasselt, Diepenbeek, Belgium; Marc Hendrikx, Jean-Luc Rummens, Virga Jesse Hosp, Hasselt, Belgium Background: Several clinical trials have shown that stem cell based therapy may improve heart function. Both bone marrow derived mesenchymal (MSC) as well as hematopoietic stem cells (HSC) are reported to be multipotent. This study investigates whether neonatal rat cardiomyocytes (NRCM), when co-cultured, can induce transdifferentiation of either MSC or HSC into cardiomyocytes (CM). Methods and results: Ex vivo expanded human bone marrow derived MSC showed expression of CD49c, CD73, CD90, CD105 but not of CD34, CD45, CD106 and CD184. The expanded MSC kept their multipotent characteristics. In contrast, HSC were freshly isolated by flow-sorting based on their expression of CD133 CD34 . Co-cultures, using cell tracker green (5-chloromethylfluorescein diacetate) labelled MSC or HSC and cell tracker red (5-(and6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamin) labelled NRCM, were performed at different ratios (1:6, 1:4, 1:3 and 1:1). Since several reports indicate that dimethylsulfoxide (DMSO) and 5-azacytidin (5-aza) induce myocardial differentiation, 1% DMSO for 48h or 3 M 5-aza for 24h was added to the co-cultures and compared to conditions without additives. After three weeks green and red labelled cells were separated by flow-sorting and cardiac gene expression was analyzed by RT-PCR. Co-culturing MSC induced the expression of troponin T (TnT) and GATA-4. However, no expression of -actinin, myosin heavy chain (MHC) or troponin I (TnI) could be detected. Furthermore, co-culturing HSC, could only induce the expression of TnT, but no expression of -actinin, MHC, TnI or GATA-4 was observed. Transmission electron microscopy confirmed the absence of sarcomeric organization both in co-cultured MSC and HSC. Conclusions: This in vitro co-culture study obtained no convincing evidence of transdifferentiation of either MSC or HSC into CM. Despite the induction of TnT and GATA-4, several cardiac specific genes are not expressed after 3 weeks of co-culture. No influence of DMSO or 5-aza on the transdifferentiation of MSC or HSC could be detected. Therefore, the reported functional improvement following cell based therapy for myocardial infarction may be due to other mechanisms than transdifferentiation of MSC or HSC. P7 Spontaneously Recruited Bone Marrow Progenitor Cells Are Cardiogenic and Vasculogenic in Chronically Ischemic Myocardium: Role of Hypoxia and Oxidants in Their Spatial Distribution Nicanor I Moldovan, Omer I Butt, Davis Heart and Lung Rsch Institute, Ohio State Univ, Columbus, OH; Brad W Blaser, Comprehensive Cancer Cntr, Ohio State Univ, Columbus, OH; Leni Moldovan, Guanglong He, Jay L Zweier; Davis Heart and Lung Rsch Institute, Ohio State Univ, Columbus, OH We tested the hypothesis that ischemic hearts have a natural healing capacity based on the contribution of bone marrow derived progenitor cells, and inquired into the reasons of its limited efficiency. Methods. In mice transplanted with bone marrow from LacZ expressing donors, we performed coronary ligation lasting for 7 days. Cellular composition of the hearts was determined after whole-organ incubation in the beta-galactosidase substrate, followed by immunostaining of paraffin sections. We assessed actual oxygen distribution in the ischemic myocardium in vivo using implanted oxygen-sensitive probes detected by electron paramagnetic resonance (EPR). We also determined the pattern of reactive oxygen species (ROS) formation by staining with fluorescent probes DCF-DA (for H2O2 and/or secondary oxidants) and hydroethidine (for superoxide). Results. LacZ positive cells (i.e. of bone marrow origin) accumulating mostly in the border zone co-expressed in a mosaic pattern: a) progenitor cells markers c-Kit and ABC-G2 (‘side population’ marker, also found in endothelial cells); b) troponin I in proliferating clusters of mononuclear cells, as well as in well-developed, small cells with a single nucleus morphologically integrated with resident cardiomyocytes at the border zone. c) F4/80 (monocyte/macrophage marker); d) smooth muscle alpha actin (in myofibroblasts). Many mature cardiomyocytes in the infarct penumbra also expressed LacZ at lower intensity, indicating cell fusion. At the core of the infarct we detected an abrupt decrease in oxygenation, concordant with a paucity of organized microvessels, and increased ROS formation detectable by fluorescence. Paradoxically at the border zone (site of bone marrow cells engraftment, differentiation and neovascularization), we found a slight hyperoxia. Conclusion. At one week post-infarction, we found substantial accumulation and proliferation of bone marrow derived progenitor cells, showing cardiogenic and vasculogenic differentiation, and possibly cell fusion. Spatial distribution of this activity, excluded from hypoxic zones (also marked by increased ROS), indicates the role of tissue oxygenation as a limiting factor in the myocardial repair after infarction. P8 Evidence for Paracrine Stimulation of Neovascularization by Bone Marrow–Derived Progenitor Cells: Assessment by in Vivo EPR Oximetry Omer I Butt, Robert W Carruth, Periannan Kuppusamy, Jay L Zweier, Nicanor I Moldovan; Davis Heart and Lung Rsch Institute, Ohio State Univ, Columbus, OH Background: The activity of bone marrow progenitor cells during neovascularization is highly debated, because contradictory data in favor of direct incorporation, cell fusion or distant paracrine action are available. Therefore, a reliable assay to sort out these possibilities is highly desirable. Methods: We purified c-Kit and sca-1 positive bone marrow cells by Magnetic Activated Cell Sorting (MACS), and demonstrated that they have cardiovascular (endothelial and smooth muscle cells) differentiation potential, by cultivation for 3 weeks in EGM-2 medium (Clonetics). For in vivo quantification of angiogenic activity, we developed a novel oxygen biosensor, containing an oxygen-sensitive spin probe detectable by Electron Paramagnetic Resonance (EPR), placed inside of nanoporous filter-limited biocompatible capsules. These devices were implanted subcutaneously in lots of six C57/Bl6 mice, as it follows: (i) alone; (ii) adding 200 l Matrigel in front of the biosensor; and (iii) supplementing the Matrigel plug with freshly isolated c-kit /sca-1 bone marrow cells. Implant-reported pO2 was recorded non-invasively for up to 10 weeks. Tissues surrounding the implants were directly analyzed by microphotography to document the extent of neovascularization, and collected for immunohistochemistry. Results: Around control biosensors and in the cell-free plugs, we detected only collagen deposition, along with F4/80 macrophages and giant cells. In presence of progenitor cells, outside of gels there was an explosive development of host-derived neovasculature. The gels themselves showed progressive colonization by adipose tissue containing capillaries, apparently of host origin as well. After four weeks, the sensors near progenitor cellssupplemented plugs detected almost twice the oxygen concentrations (16.2 /0.2 mm Hg) as compared to controls (9.1 /0.7 mm Hg). This difference remained significantly larger (p 0.05, n 4) for six more weeks. Conclusion: Bone marrow progenitor cells powerfully stimulate host neovascularization, with little evidence of direct incorporation. Our approach could be used to control vascularization in ischemic hearts, as well as in the vicinity of biomedical implants affected by fibrous encapsulation. P9 Stem Cell Therapy Combined with Transmyocardial Laser Revascularization: A Novel Therapeutic Strategy for Patients with Refractory Angina Luis Henrique W Gowdak, Isolmar T Schettert, Marcos Rienzo, Carlos Eduardo Rochitte, Luiz Antonio M Cesar, Luiz Augusto F Lisboa, Luis Alberto O Dallan, Luiz Augusto F Lisboa, Jose Eduardo Krieger, Jose Antonio F Ramires, Sergio A de Oliveira; Heart Institute (InCor), Univ of Sao Paulo Med Sch, Sao Paulo, Brazil Background: Transmyocardial laser revascularization (TMLR) is a therapeutic option for patients (pt) with severe ischemic heart disease (IHD) not amenable to conventional myocardial revascularization, either by CABG or PCI. Recently, cell therapy with autologous bone marrow cells (BMC) has been tested in clinical trials for severe IHD. Objective: To test the hypothesis that TMLR combined with intramyocardial injection of BMC is safe, and may help increase the functional capacity of pt with refractory angina, despite maximally tolerated medical therapy. Methods: 11 pt (10 men), 67 5 years old, with refractory angina for multivessel disease and previous myocardial revascularization procedures (CABG/PCI), not candidates for another procedure due to the extension of the disease were enrolled. TMLR (11 3 laser drills) was performed via a limited thoracotomy using a commercially available CO2 Heart Laser System. BMC were obtained immediately prior to surgery, and the lymphomonocytic fraction separated by density gradient centrifugation. During surgery, 5mL containing approximately 2.4 0.2x10 BMC (CD34 2.1 0.5%) were delivered by multiple injections in the ischemic myocardium. Before (B) and 6 months (6M) after the procedure, pt underwent clinical evaluation, and myocardial perfusion assessment by cardiac magnetic resonance imaging (MRI) during pharmacological stress with adenosine. Results: Injected segments (n 12) with BMC included the anterior (n 5), lateral (n 4), and inferior (n 3) walls. No major complications or deaths occurred during the procedure. There was a reduction in the ischemic score as assessed by MRI from 1.61 0.11 (B) to 0.94 0.10 (6M) (P .01). Clinically, there was a reduction in functional class of angina from 3.5 0.2 (B) to 1.3 0.2 (6M) (P .0001). Conclusions: In this initial experience, the combined strategy of TMLR plus cell therapy appeared to be safe, and may have synergistically acted to reduce myocardial ischemia, with clinically relevant improvement in functional capacity. Provided these data are confirmed in a larger, randomized, controlled trial with longer follow-up, this strategy could be used as a novel therapeutic option for treating pt with refractory angina. P10 Phenotypic Characterization of CD133 Cells Suggests a Potent Hemangioblast Population Jason C Kovacic, Victor Chang Cardiac Rsch Institute, Sydney, NSW, Australia; David Ma, St Vincent’s Hosp, Sydney, NSW, Australia; Helen Tao, Andrea Herbert, Victor Chang Cardiac Rsch Institute, Sydney, NSW, Australia; John Moore, St Vincent’s Hosp, Sydney, NSW, Australia; Robert M Graham; Victor Chang Cardiac Rsch Institute, Sydney, NSW, Australia Background: Although recently described, CD133 cells may represent a potent hemangioblast population. As they may be obtained from adult humans, CD133 cells are therefore an attractive autologous option for therapeutic angiogenesis. However, scant data exists regarding their precise phenotype, particularly when derived from patients with ischemic heart disease (IHD). Methods: Peripheral blood CD133 cells from 19 patients with stable IHD, obtained before and after GCSF mobilization, were characterized by fluorescent-activated cell sorting (FACS). At peak GCSF mobilization, concentrated CD133 cells were obtained using anti-human CD133 conjugated magnetic beads and a CliniMACS system (mean purity 59.5%). Total RNA from CD133 -enriched cells was reverse transcribed and quantitative real time-polymerase chain reaction (qRT-PCR) performed using primers for: -actin, VEGF, HGF, PDGF-B, MCP-1, TNF , PlGF. Results: FACS demonstrated expression of both hematopoietic and endothelial antigens, with high-level CD45 (100%), CD31 ( 90%) and vWF ( 80%); intermediate-level ICAM-1 ( 30%) and E-selectin (13–29%); and low but detectable VEGFR-2 (2–4%), VE-Cadherin (1–4%) and VCAM-1 (1–4%). CD133 cells also expressed a range of stem cell markers: CD34 ( 90%), c-kit (80–95%) and MDR-1 ( 10%). GCSF administration was associated with reduced CD38 (75% vs 60%, p 0.05) and increased c-kit (80% vs 92%, p 0.05) expression by CD133 cells; suggestive that GCSF-mobilized CD133 cells were less differentiated (more primitive) than those obtained in the basal state. qRT-PCR indicated that the RNA expression of angiogenic cytokines by CD133 cells was similar to (VEGF, HGF, 2007 BCVS Symposium Abstracts E55 by gest on Sptem er 2, 2017 http://circhajournals.org/ D ow nladed from TNF ) or less than (PDGF-B, MCP-1, PlGF, all p 0.05) that of other relevant comparator cell populations; suggesting that CD133 cells are unlikely to make a major contribution to angiogenesis via paracrine mechanisms. Conclusions: While expressing hematopoietic and endothelial markers, CD133 cells from patients with stable IHD exhibit the antigenic and functional characteristics of potent progenitor cells. This phenotype supports the contention that CD133 cells are a hemangioblast population, and thus warrant further study as a therapeutic agent. P11 Epigenetic Reprogramming During SS-Dependent Differentiation of Mouse Embryonic Stem Cells: Role of Nitric Oxide and Histone Deacetylases Barbara Illi, Francesco Spallotta, Cntr Cardiologico Monzino, Milan, Italy; Jessica Rosati, Stefania Mattiussi, Istituto Dermopatico dell’Immacolata, Rome, Italy; Maurizio Capogrossi, Carlo Gaetano; Istituto Dermopatico dell’ Immacolata, Rome, Italy Introduction: Shear stress (SS) reprograms mouse embryonic stem cells (ES) to express cardiovascular markers at least in part through the occurrence of epigenetic modifications. Nitric oxide (NO) is involved in SS signalling in vascular cells and facilitates cardiomyogenesis in ES. Here we show the results of experiments performed to assess the molecular mechanism activated by NO in the SS-dependent cardiovascular differentiation of ES. Methods and Results: Exposure of ES to the NO synthase inhibitor S-methyl-thiosourea (SMT), inhibited the SS-dependent expression of cardiovascular markers in ES, while nitric oxide donors anticipated cardiovascular differentiation in ES deprived from leukaemia inhibitory factor (LIF). This result paralleled with a marked decrease in Oct4 expression, indicating a role for NO in the cardiovascular commitment of ES cells. The expression of developmentally regulated genes requires the fine tuning of the chromatin condensation/decondensation process in a temporally and spatially-regulated manner, a phenomenon strictly dependent on chromatin modifying enzymes’ activity such as Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs). We found that after 1 hour of LIF deprivation, class II HDACs shuttled from the cytoplasm to the nucleus of ES cells, confirming a role of these chromatin modifiers in the epigenetic reprogramming of ES cells’ fate. This phenomenon was transient as these molecules returned to the cytoplasm in 6 hours. Intriguingly, the direct exposure of ES to NO donors allowed the nuclear retention of these enzymes beyond the 6 hours timepoint and reduced significantly histone H3 acetylation, indicating the presence of a prolonged histone deacetylase activity in the ES nuclei upon NO treatment. The HDAC inhibitor Trichosatin A (TSA) blocked ES differentiation towards the endothelial lineage, while class I and II HDACs specific inhibitors induced the expression of endothelial and neuronal markers respectively. Conclusions: These data suggest a direct role of NO in the regulation of class II HDACs function and in the chromatin remodelling of ES cells and may envisage new epigenetic strategies to reprogram stem cells’ fate which may be useful for cardiovascular cell-based therapies. P12 Correlation Analysis of Endothelial Colony Forming Units and Endothelial Microparticles in Patients with Cerebrovascular Risks Soon-tae Lee, Kon Chu, Keun-Hwa Jung, Hee-Kwon Park, Vit Nary Choe, Dong-Hyun Kim, Woo-Seok Im, Jeong-Eun Park, Jin-Hee Kim, Jae-Joon Bahn, Sang Kun Lee, Manho Kim, Jae-Kyu Roh; Clinical Rsch Institute, Seoul National Univ Hosp, Seoul, Republic of Korea Endothelial progenitor cell (EPC) is a surrogate biologic marker for vascular regeneration that is inversely correlated with cardiovascular risk, and endothelial microparticle (MP) is a possible indicator of vascular endothelial damage. We isolated EPC colony-forming units and endothelial microparticles concomitantly and investigated their correlations. We enrolled patients (n 72) with variable cerebrovascular risks, which included patients with headache, chronic stroke, or traditional cerebrovascular risk factors. Heparinized venous blood samples (25mL) taken from each subjects were used for the isolation of EPC. Mononuclear cells were fractionated by centrifugation on Histopaque 1077 gradients at 400g for 40min, resuspended using the EGM-2 BulletKit system, seeded on gelatin-coated 12-well plates, and incubated in a 5% CO2 incubator at 37°C. This method allowed colony formations of EPCs or circulating angiogenic cells (CACs), defined as a central core of rounded cells surrounded by elongated spindle-shaped cells at 7 days in culture. MP-rich plasma was prepared from citrated venous blood samples (5mL) using serial ultracentrifugation at 1500g for 15min and 13000g for 2min. Samples were frozen at -70’C till analyses. After thawing at room temperature, we counted MPs ( 1 micrometer) using FACScalibur with anti-CD31, anti-CD42, anti-Annexin V, and anti-CD62E antibodies. This method allowed the fractionation of endothelial, platelet, and total microparticles. Further correlation analysis showed that EPC colony counts were correlated with endothelial MPs (p 0.039 for CD31 CD42MPs, p 0.040 for CD31 AnnexinV MPs), but not with platelet microparticles (p 0.129 for CD42 MPs). In particular, EPC colony count was more significantly correlated with logarithmic values of CD31 CD42(p 0.009), CD31 AnnexinV (p 0.007), and CD62E endothelial MPs (p 0.036). In this study, we show that there exists an inverse correlation between EPC colony counts and circulating endothelial MPs, which suggests the reduced circulating endothelial regeneration pool in patients with active endothelial damage. P13 Int6: New Target for Angiogenesis (1) eIF3e/int6 Specifically Targets HIF-2 for Degradation by Hypoxiaand pVHL-Independent Regulation Li Chen, Kazuyo Uchida, Tokyo Metropolitan Institute of Med Science, Bunkyo-ku, Tokyo, Japan; Alexander Endler, Sch of Basic Medicine, Tongji Univ, Shanghai, China; Osamu Iijima, Alphagen Co, Ltd, Chiyoda-ku, Tokyo, Japan; Futoshi Shibasaki; Tokyo Metropolitan Institute of Med Science, Bunkyo-ku, Tokyo, Japan Abstract: Hypoxia-inducible factors (HIFs) -1 and -2 are structurally similar as regards their DNA-binding and dimerization domains, but differ in their activation targets. In order to address this discrepancy, we performed yeast two-hybrid analysis and identified Int6/eIF3e/p48 as a Hypoxia-inducible factors (HIFs) -1 and -2 are structurally similar as regards their DNA-binding and dimerization domains, but differ in their activation targets. In order to address this discrepancy, we performed yeast two-hybrid analysis and identified Int6/eIF3e/p48 as a novel target gene product involved in HIF-2 regulation. The int6 gene was previously identified as a frequent integration site of the mouse mammary tumor virus (MMTV). Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells and HRE-reporter assays, we report the specific interaction of HIF-2 (but not HIF-1 or HIF-3 ) with Int6. The results indicate that the direct interaction of Int6 induces HIF-2 degradation. This degradation was found to be both hypoxiaand pVHL-independent. Furthermore, int6-siRNA increased endogenous HIF-2 expression, and followed by inducing sets of critical angiogenic factors comprising VEGF and bFGF mRNA in HeLa cells. Moreover, HIF-2 and the related factors are also expressed concomitantly in human endothelial cells. We proofed that Int6-siRNA induced mRNA of HIF-2 , bFGF, IL6, and IL8 in HUVEC cells, and these induction triggered to mediate the cord of formation on matrigels. These results indicate that Int6 is a novel and critical determinant of HIF-2 -dependent angiogenesis not only in HeLa cells, but also in human endothelial cells. Thus, int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, and obstructive vessel diseases. P14 AMP-Kinase Activates Ubiquitin Ligases in Cardiomyocytes Kedryn K Baskin, Peter Razeghi, Heinrich Taegtmeyer; Univ of Texas Health Science Cntr,