| Objective: Reestablishment of blood circulation is an effective treatment for myocardial infarction(MI).Vascular endothelial growth factor(VEGF)effectively promotes the formation of new blood vessels during the initial stages of angiogenesis.However,VEGF-induced neovascularization lacks vascular basement membranes and perivascular perivascular cells,increasing vascular permeability.Angiopoietin 1(Ang-1)plays an important role in promoting vascular maturation and enhancing the integrity of the endothelial cell barrier during the later stages of angiogenesis.The Ang-1 domain peptide chain is reported to be composed of seven amino acids QHREDGS(AMP),which has similar angiogenic activity to Ang-1.After myocardial infarction,the expression of matrix metalloproteinase 2(MMP-2)increased gradually.We used this feature to connect PLGLAG(TIMP),a shear peptide chain of MMP-2,to AMP to form QHREDGSPLGLAG(TIMP-AMP),and achieve the purpose of sequential release.Therefore,the objective of our study was to observe the therapeutic effect of this hydrogel system in MI by giving exogenous VEGF and AMP in sync with endogenous neovascularization via acellular cardiac extracellular matrix(c-ECM)sequence.Methods and Contents:(1)Injectable c-ECM hydrogels were prepared by physical and chemical methods.(2)Western Blot was used to detect the expression of MMP-2trend after myocardial infarction.(3)Binding assay and release assay were used to detect the binding ability of TIMP-AMP to c-ECM and the targeted cleavage of TIMP by MMP-2 to release AMP.(4)MTT assay and in vitro tubular assay were used to detect the biological activities of AMP and TIMP-AMP;(5)Preparation of rat myocardial infarction model and hydrogel therapy;(6)TTC staining method was used to verify whether the preparation of the model was successful;(7)Ultrasound detection of cardiac function in small animals;(8)The number of angiogenesis,vessel wall thickness,cardiomyocyte apoptosis and intercellular connections were detected by immunofluorescence staining.Results:(1)Porcine left ventricular tissue was decellularized to prepare acellular cECM,which was gelated by physical method.(2)Western Blot showed that the expression of MMP-2 was upregulated gradually after myocardial infarction.Binding assay showed that both TIMP-AMP and AMP could be tightly attached to c-ECM through chemical crosslinking.The results of molecular scissors MMP-2 release assay showed that under the action of MMP-2 in vitro,the release amount of AMP increased with the extension of time.(3)The biological activities of AMP and TIMPAMP were similar by MTT method.The results showed that there was no significant difference between them at the same concentration.In addition,AMP and TIMPAMP showed similar biological activity at various concentrations,namely promoting the proliferation of HUVEC.In vitro tube formation showed that VEGF group,VEGF+ ANG-1,VEGF+AMP and VEGF+TIMP-AMP could promote the formation of tube like structure in vitro.(4)After TTC staining,red represents the non-infarcted area and white represents the infarcted area,indicating the successful preparation of the rat myocardial infarction model;(5)Echocardiography showed that the ejection fraction(EF)and fractional shortening(FS)of the CBD-VEGF/c-ECM/TIMP-AMP hydrogel group were significantly different from those of the control group.In addition,there was no significant statistical difference in other echocardiographic indicators.(6)v WF,α-SMA and SM22 a staining showed that the number of regenerated blood vessels in the CBD-VEGF/c-ECM/TIMP-AMP hydrogel group increased significantly;TUNEL staining showed that the number of TUNEL positive nuclei was significantly reduced in the CBD-VEGF/c-ECM/TIMP-AMP hydrogel group;ZO1 staining showed that the ZO1 positive area ratio of CBD-VEGF/cECM/TIMP-AMP hydrogel group was significantly increased.Conclusions: CBD-VEGF/c-ECM/TIMP-AMP hydrogel can effectively increase angiogenesis,reduce cardiomyocyte apoptosis,and promote the connection between cells,so as to promote the recovery of cardiac function in rats with myocardial infarction. |
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