EPCS Modified By 6hre-vegf₁₆₅ Gene Improved Neovascularization In Rats Ischemic Myocardium After Ami

AimsThe current study found that exogenous VEGF165 gene can promote angiogenesis in ischemic tissue, and the multiple copies of Hypoxia Response Element (HRE) has been reported to regulate the expression of VEGF165 gene both in vivo and in vitro to reduce its side effects。Although many forms of stem / progenitor cells and even genetically modified stem cells have been reported to promote angiogenesis in ischemic tissue ,and improve the function of ischemic tissue, the therapeutic angiogenesis function of multiple copies of HRE regulated VEGF165 gene modified Endothelial Progenitor Cells (EPCs) in ischemic myocardium of Acute Myocardial infarction (AMI) rats is still unclear. The purpose of this study is to study the therapeutic function of the EPCs modified by 6HRE regulated VEGF165 gene in angiogenesis and cardiac function after myocardial infarction. Main methodsFirst of all, r6HRE-CMV-VEGF165-EGFP and rCMV-VEGF165-EGFP recombinant plasmids were successfully constructed by using molecular biology techniques, and EPCs were isolated from SD rat bone marrow,cultured in vitro as described in literature. The expression of CD133,CD34,VEGFR-2 and Dil-acLDL was conducted to identify EPCs, through immunofluorescent staining. Second, the recombinant plasmids were transfected into EPCs by Lipofectamine, and then hypoxia model was constructed in vitro to identify the regulate gene expression function of the hypoxia promoter. Finally, the gene-modified EPCs transplanted by tail vein injection into the successful establishment of AMI model of SD rats, then VEGF165 mRNA and protein expression levels in ischemic myocardium at 1 week post-AMI were measured by reverse transcription-polymerase chain reaction (RT-PCR) and by Western blot, the capillary density in ischemic myocardium at 4 week post-AMI was measured by immunohistochemistry and the cardiac function of rat at 4 week post-AMI was detected by echocardiography. Results1) LipofectAMINE 2000 is a safe and efficient way of EPCs transfection;2) 6HRE-CMV hypoxia promoter induced VEGF165 gene dramatic overexpression in hypoxia environment ,but showing only low expression in normal environment both in vivo and in vitro.3) VEGF165 mRNA and protein expression levels in ischemic myocardium at 1 week post-AMI and the capillary density in ischemic myocardium at 4 week post-AMI were significantly higher in r6HRE-CMV-VEGF165-EGFP recombinant plasmids transfected EPCs transplanted group than that in rCMV-VEGF165-EGFP recombinant plasmids transfected EPCs transplanted group and in none plasmid transfected EPCs transplanted group.4) Rat left ventricular end diastolic pressure (LVEDP) was significantly lower in r6HRE-CMV-VEGF165-EGFP recombinant plasmids transfected EPCs transplanted group than in the other experimental groups (P<0.05), while the left ventricular ejection fraction (EF) was significantly higher in r6HRE-CMV-VEGF165-EGFP recombinant plasmids transfected EPCs transplanted group than in other experimental groups (P<0.05). ConclusionOur results suggest that VEGF165 gene mediated by 6HRE-CMV promoter modify EPCs transplantation was more safety and efficient to improve neovascularization and heart function after AMI than EPCs and EPCs modified by VEGF165 transplantation .