| Objective Reconstruction of blood circulation is an effective method to treat myocardial infarction(MI).According to reports,vascular endothelial growth factor(VEGF)-derived specific angiogenic peptides(VEGF mimic peptides,VMP)can activate their receptors and have angiogenesis-like activity similar to VEGF.However,it is not clear whether it can play an effective role in the treatment of myocardial infarction.In this study,in order to increase the effective therapeutic concentration in the ischemic area,we constructed a targeting peptide CBD-VMP peptide that can specifically bind to collagen.This targeting peptide(CBD-VMP)can specifically bind to collagen without affecting the angiogenic effect of VMP.Subsequently,we combined it with the decellularized cardioc extracellular matrix(c-ECM)collagen hydrogel and co-injected it into the rat myocardial infarction site.To study the therapeutic effect of CBD-VMP/c-ECM collagen hydrogel in myocardial infarction.Methods and Contents1.An injectable c-ECM hydrogel was obtained through a series of decellularized operations,the c-ECM gel and the collagen gel are mixed in a ratio of 1: 1 to obtain a c-ECM collagen hydrogel.2.MTT assays were used to detect the biological activity of CBD-VMP peptide and VMP peptide.3.ELISA assays were used to detect the binding ability of peptides to decellularized matrix collagen hydrogel.4.CBD-VMP/collagen scaffolds and VMP/collagen scaffolds were implanted subcutaneously into rat to detect whether the CBD-VMP peptide can effectively promote angiogenesis in vivo.5.Preparation of animal models with MI.6.TTC staining was used to verify whether the myocardial infarction model is successful.7.Perform an echocardiogram for overall cardiac function testing.8.Histological and immunohistochemistry staining for angiogenesis and cardiomyocyte apoptosis detection.Results1.The porcine left ventricular tissue was decellularized and showed a porous structure by SEM scanning.The average pore size was 100-200 μm,which could promote the penetration of cells and nutrients.A c-ECM collagen hydrogel was prepared as described.2.The biological activity of CBD-VMP and VMP peptides obtained by MTT method is similar to that of VEGF.The results showed no significant difference between the three at the same concentration,which is consistent with other reports.In addition,CBD-VMP and VMP have similar abilities at various concentrations,that is,to promote the proliferation of HUVEC.3.ELISA test found that the collagen-binding peptide(CBD)was able to bind to the decellularized matrix collagen hydrogel better.The results showed that as CBD-VMP and VMP concentrations were increased,more CBD-VMP remained on the decellularized matrix collagen hydrogel compared to VMP.4.Animal subcutaneous implantation experiments found that CBD-VMP peptide can promote angiogenesis in vivo.Various peptides are loaded onto collagen scaffolds and implanted subcutaneously in rats.Two weeks after implantation,more cell infiltration and blood vessel formation were observed in the CBD-VMP/collagen group,and HE staining confirmed these results.In addition,α-SMA is a specific marker of blood vessels,and both VMP and CBD-VMP peptides can induce more angiogenesis.However,the CBD-VMP peptide can specifically bind to the collagen scaffold and promote angiogenesis more significantly.5.The model rats were sacrificed,their hearts were taken out and stained with TTC solution.The red part was the non-infarcted area and the white part was the infarct area.The results showed that the rat model of myocardial infarction was successfully prepared.6.Echocardiography shows that promoting angiogenesis can effectively promote the recovery of cardiac function.Statistics found that in the CBD-VMP/c-ECM collagen hydrogel group,the ejection fraction(EF)and the left ventricular fractional shortening(FS)were significantly different from the control group.In addition,there were no significant differences in other echocardiographic indicators in these three groups,including diastolic and systolic left ventricular diameters(LVIDd and LVIDs,respectively),and diastolic and systolic left ventricular posterior wall thickness(LVPWd and LVPWs).7.CBD-VMP/c-ECM collagen hydrogel not only promotes angiogenesis but also protects cardiomyocytes in MI rats.Three months after the operation,the infracted cardiac tissue was taken and sliced for immunostaining.α-SMA staining found that the number of regenerated blood vessels in CBD-VMP/c-ECM collagen hydrogel group and VMP/c-ECM collagen hydrogel group increased significantly;TUNEL staining showed the number of TUNEL positive cells in CBD-VMP/c-ECM collagen hydrogel group.The significant decrease indicates that CBD-VMP/c-ECM collagen hydrogel can reduce apoptosis after MI and protect cardiomyocytes.Conclusions In summary,the combination of CBD-VMP peptide and injectable c-ECM collagen hydrogel can effectively increase angiogenesis and reduce cardiomyocyte apoptosis,thereby promoting the recovery of cardiac function myocardial infarction in rats. |
