Vascular Smooth Muscle Cells Attenuate Myocardial Ischemia/Reperfusion-induced Apoptosis And Antophagy By Secreting BFGF

Background:Myocardial ischemia reperfusion injury contributes to adverse cardiovascular outcomes after myocardial ischemia,cardiac surgery or circulatory arrest。Vascular smooth muscle cells(VSMCs)has been demonstrated that VSMCs block the progression of cardiac remodeling and thus promoting cardiac function in a rat myocardial infarction model.However,the detailed molecular mechanism of how VSMCs contributes to recovery in myocardial ischemia/reperfusion remains not fully understood。The aim of this study was to investigate the mechanism by which vascular smooth muscle cells reduce myocardial ischemia-reperfusion injury.Methods:We have isolated,identified and cultured VSMCs from rats to co-culture with rat cardiomyocyte H9C2.To culture H9C2 cells under hypoxia,we utilized CoC12-containing medium to culture for 8 h and then was replaced with normal media for additional 16 h.Cell viability was examined by MTT assay and apoptosis was determined by flow cytometry.Infarcted area of myocardial tissue was measured by TTC staining.Results:1.cell viability of H9C2 was decreased in myocardial I/R which was induced by CoC12 600μmol/L8/16h compared to normal control cell,in addition the apoptosis detection by flow cytometry assay showed significantly increased apoptotic ratio in myocardial I/R.VSMCs was shown to promote cell viability and inhibit apoptosis of H9C2 cells under hypoxia,which exhibited upregulated anti-apoptotic protein Bcl-2 and autophagy-related protein p62,whereas pro-apoptotic protein cleaved caspase-3 and the level of LC3II/LC3I were downregulatedo 2.Then,we conducted ELISA and found that the level of bFGF in media of VSMCs was markedly higher than that in media without VSMCs.Flow cytometry analysis also demonstrated FGFR1 inhibitor blocked the protection of H9C2 from VSMCs;however,bFGF greatly decreased apoptosis rate of H9C2 in myocardial I/R relative to treatment without bFGF.So we confirmed VSMCs played the contributory role in cell viability of H9C2 under hypoxia by secreting bFGF。3.Westbolt analysis found that PI3K/Akt pathway was inhibited by myocardial I/R,which was rescued by co-culturing with VSMCs.Noticeably,phosphorylated of FGFR1 and Akt were markedly decreased by treatment of FGFR1 inhibitor and PI3K inhibitor LY294002,while bFGF increased the expression level of p-FGFRl and p-AKT.Furthermore,phosphorylated of the AKT downstream genes mTOR and Fox03a were also significantly reduced while bFGF increased the expression level of p-Fox03a and p-mTOR.These results indicated VSMCs-secreted bFGF played a suppressive role in apoptosis and autophagy of cardiomyocyte,in large part,via activation of PI3K/Akt pathway which exerted its function through PI3K/Akt pathway.4.Finally,in vivo,the results demonstrated that VSMCs transplantation contributed to recovery of myocardial ischemia.Conclusion:We determine that VSMCs promote recovery of infarcted cardiomyocyte through secretion of bFGF,which then activating PI3K/Akt pathway to inhibit apoptosis and autophagy.These findings provide more insights into the molecular mechanism underlying VSMCs contributing to recovery of myocardial I/R and facilitate developing therapeutical strategies for treating heart diseases.