Functional Recovery Of Myocardial Infarction By Specific EBP-PR1P Peptides Bridging Injectable Cardiac Extracellular Matrix And Vascular Endothelial Growth Factor

Background and Objective Cardiovascular disease(CVD)is the leading cause of mortality worldwide,especially myocardial infarction(MI).The insufficiency of blood flow in the infarct area leads to the necrosis of cardiomyocytes,affecting cardiac function,and eventually developing into heart failure(HF).Therefore,reestablishing blood supply in the infarcted area is critical.Vascular endothelial growth factor(VEGF)is the most potent angiogenic factor.In recent years,the application of targeted peptides has developed rapidly,providing a new strategy for the sustained release of VEGF.In this study,we designed a specific EBP-PR1 P targeting peptide that specifically binds to both VEGF and extracellular matrix(c-ECM)to achieve sustained VEGF release,without affecting VEGF biological activity.Therefore,we constructed an injectable VEGF/EBP-PR1P/c-ECM scaffold and applied it in MI rats to explore the effect of VEGF/EBP-PR1P/c-ECM hydrogel on the repair of MI in rats.Methods1.PR1P/EBP-PR1 P peptides,and PR1P-biotin/EBP-PR1P-biotin were synthesized by Genscript;2.Cardiac extracellular matrix was prepared from pig left ventricle.3.The binding ability of PR1 P or EBP-PR1 P peptide with c-ECM at different concentrations and time periods was determined by ELISA.The sustained release ability of VEGF in c-ECM was determined by ELISA kit.4.MTT assay was used to detect the cellular activity of HUVEC at different concentrations of VEGF and peptides in vitro.The ability of EBP-PR1P/VEGF peptide to promote angiogenesis was detected by in vitro tube formation assay.5.The left anterior descending coronary artery of the rat was ligated with 6-0 thread.Experimental groups were: c-ECM group,VEGF/c-ECM group,VEGF/EBP-PR1P/cECM group.6.HE staining was used to observe the histomorphology of myocardial infarction.Echocardiography was used to detect cardiac function in myocardial infarction rats.Immunofluorescence staining: CD31 and α-SMA staining were used to evaluate the effect of VEGF/ EPP-PR1 P /c-ECM on angiogenesis in rats after myocardial infarction.Myocardial cell apoptosis was detected by a-MHC and TUNEL staining.Results1.Acellular c-ECM was prepared by decellularization of pig left ventricular tissue.Scanning electron microscope observation showed a porous structure with an average aperture of 100~200 μm.c-ECM collagen hydrogel was prepared by the method mentioned previously.2.It was confirmed by ELISA that EBP-PR1 P peptides could specifically bind to c-ECM through EBP domain,and more VEGF could be retained on c-ECM through the specific binding of PR1 P peptide to VEGF.In vitro sustained release experiments showed that VEGF release increased with the extension of release time.Due to the dual targeted binding effect of EBP-PR1 P,VEGF/EBP-PR1P/c-ECM group could significantly delay the release of VEGF in ECM hydrogel compared with VEGF/c-ECM group.3.In vitro proliferation experiment of HUVECs detected by MTT method,cells were grown in culture medium of VEGF,VEGF/PR1 P and VEGF/ EPP-PR1 P peptide at different concentrations(1250p M,2500 p M)for 3 days.The results showed that VEGF/EBP-PR1 P peptides could promote the proliferation of HUVECs cells in vitro.The results of in vitro tube formation experiment showed that VEGF/PR1 P group and VEGF/EBP-PR1 P group promoted in vitro tube formation of HUVECs more strongly than Control group and VEGF group,but there was no significant difference between the two groups.These results suggest that EBP-PR1 P peptides don’t affect the biological activity of VEGF.4.Echocardiography showed that VEGF/EBP-PR1P/c-ECM hydrogel delivery system can effectively promote cardiac function recovery.Statistically,significant differences were found in the ejection fraction(EF)and fractional shortening(FS)in VEGF/EBPPR1P/c-ECM group compared with the control group.In addition,there were no significant differences in other echocardiographic measures among the three groups,including left ventricular diameter during diastolic and systolic periods(LVIDd and LVIDs,respectively),and posterior left ventricular wall thickness during diastolic and systolic periods(LVPWd and IVSs,respectively).5.CD31 staining counted the number of capillaries formed,and α-SMA staining counted the number of arterioles formed.Both statistical results showed that VEGF/EPP-PR1P/cECM group generated the most blood vessels and had better angiogenesis ability than other groups.Statistical results of α-MHC and TUNEL staining showed that the numbers of cardiomyocytes apoptosis in VEGF/EBP-PR1P/c-ECM group were less than that in the other two groups.6.Western blot results showed that VEGF/EPP-Pr1p/c-ECM group had the lowest cleaved Caspase3 expression level,while BCL2 expression level was the highest,indicating the protective effects of cardiomyocytes in VEGF/EPP-PP1P/c-ECM group.Conclusions In conclusion,EBP-PR1 P peptide can specifically bind to VEGF and extracellular matrix(c-ECM),respectively,to achieve sustained release of VEGF without affecting the biological activity of VEGF.Moreover,it can promote the angiogenesis around myocardial infarction in rats,reduce the apoptosis of myocardial cells and contribute to the recovery of cardiac function.