Paracrine Mechanism Study On Bone Marrow Stem Cells And Their Mobilized Progenitor Cells-Mediated Cardiac Protection And Functional Improvement

PARTⅠHIF-1ΑINDUCED-VEGF AND INOS EXPRESSION IN BONE MARROW STEM CELL PROTECTS CARDIOMY- OCYTES AGAINST HYPOXIAObjective: HIF-1αis a proangiogenic transcription factor stabilized and activated under hypoxia. It regulates the expression of numerous target genes, including VEGF and iNOS. In this study, we hypothesized that BMSCs secrete growth factors which protect cardiomyocytes via activating HIF-1αpathway. Methods: BMSCs were obtained from transgenic mice overexpressing GFP. The protection study was carried out in vitro using co-culture of BMSCs with cardiomyocytes subjected to hypoxia or H2O2 (200μmol). LDH release, MTT uptake, DNA fragmentation and annexin-V positive cells were used as cell injury markers. The level of HIF-1αprotein as well as its activated form and VEGF were measured by ELISA. The gene expression of HIF-1α, VEGF and iNOS in BMSCs was analyzed by quantitative PCR. HIF-1αcellular localization was determined by immunohistochemistry. Results: LDH release was increased and MTT uptake was decreased after exposure of cardiomyocytes to hypoxia for 30 hours, which was prevented by co-culture cardiomyocytes with BMSCs. Cardiomyocyte apoptosis which induced by hypoxia or H2O2 was also reduced by co-culture with BMSCs. Although no significant up-regulation could be seen in HIF-1αmRNA, HIF-1αprotein and its activated form were markedly increased and translocated into nucleus or peri-nuclear area of BMSCs subjected to hypoxia, in parallel with significantly increased expression of HIF-1αtarget gene VEGF and iNOS. The increase and translocation of HIF-1αin BMSCs were completely blocked by 2-ME2, a HIF-1αinhibitor (5μmol). Moreover, VEGF and iNOS secretion as well as the protection of BMSCs on cardiomyocytes were abolished by neutralized HIF-1αantibody and 2-ME2. Additionally, the cardiac protection was duplicated by exogenous administration of VEGF in a dose dependent manner (12.5ng/ml～100ng/ml). The cardiac protection by BMSCs was also abolished by the selective iNOS inhibitor, 1400W (10mg/L) as well as a potent competitive inhibitor of all NOS, L-NAME (200μmol/L). However, SNAP (300μmol/L), a NO donor had no synergistic effect on the cardioprotective effect of BMSCs. Conclusions: Bone marrow stem cells protect cardiomyocytes against hypoxia injury by up-regulation of VEGF and iNOS via activating HIF-1α.PARTⅡEVIDENCE SUPPORTING PARACRINE HYPOTHESIS FOR BONE MARROW STEM CELLS-MEDIATED CARDIAC PROTECTION AND FUNCTIONAL IMPROVEMENTObjective: To explore the hypothesis that paracrine actions exerted by BMSCs through the release of soluble factors might be important mechanisms of hear repair and functional improvement after LAD ligation. Methods: LAD Ligation was performed in 6-wk-old Sprague-Dawley female rats. In the sham animals the ligature was left loose and no injection was performed. Before surgery, animals were randomized into five groups:①control animals that received saline (NS) injection;②control animals that received concentrated control medium (cM);③animals that received BMSCs concentrated normoxia medium (BMSCs-cNM);④animals that received BMSCs concentrated hypoxia medium (BMSCs-cHM);⑤animals that received BMSCs suspended in NS. Cardiac function was assayed by echocardiography and infarction size was measured by staining heart sections with Masson's trichome followed by quantitative assessments. Apoptosis was observed by TUNEL staining and angiogenesis was determined by immunohistochemistry. Results: Conditioned medium from hypoxic BMSCs significantly limits infarct size and improves ventricular function and markedly inhibits apoptotic cells compared to medium control. Moreover, conditioned medium from hypoxic BMSCs significantly improves the angiogenesis in infracted heart. Conclusions: Taken together, our data support BMSCs- mediated paracrine mechanisms of myocardial protection and functional improvement.PART III THE RELATIONSHIP OF GATA4 AND CARDIAC REPAIR BY MOBILIZED BONE MARROW PROGENITOR CELLSObjective: Transcription factor GATA4 regulates differentiation, growth and survival. We proposed here that MPCs are special cell types in BMSCs, which express GATA4 protein and have a better cardiac repair following transplatation into infracted myocardium. Methods: MPCs were obtained from peripheral blood of GFP transgenic mice following LAD ligation. Cell types were analyzed by FCM. Wild type mice were randomly divided into three groups for either MPCs or BMSCs or DMEM transplantation after LAD ligation. Cardiac function was assayed by echocardiography and infarction size was measured by staining heart sections with Masson's trichome followed by quantitative assessments. Gene expression was assayed by RT-PCR and/or real-time PCR or microarray. G-CSF secretion in serum was observed by ELISA. The transdifferentation of MPCs was determined by immunohisto- chemistry. Results: G-CSF was markedly up-regulated in the ischemic myocardium. A good correlation was observed between serum G-CSF and MPCs in circulation following LAD ligation. MPCs over-expressed cardiac transcription factor GATA4 besides expression of the surface markers of BMSCs. Transplantation of cultured MPCs into the ischemic border area significantly improved cardiac function by reducing infarction size. More important, compare to BMSCs, MPCs significantly protected cardiomyocytes against apoptosis and showed higher transdifferentation into cardiomyocytes when co-cultured with cardiomyocytes. The cardiac protection by MPCs was blocked by MPCs transfected with GATA4 siRNA as well as by Bcl-2 antibody. In contrast, transfection of BMSCs with GATA4 provided increased protection of cardiomyocytes against apoptosis. BMSCs transfected with GATA4 up-regulated a bunch of cardiovascular development related genes and growth factors as well as antiapoptotic gene Bcl-2.Conclusions: GATA4 is responsible for better cardiac repair of MPCs transplantation in infarcted mice.