Experimental Studies On The Treatment Of Acute Myocardial Infarction Through RAd-mediated Vascular Endothelial Growth Factor121 Gene Transfer In A Rat Model

Background:Acute myocardial infarction is an ischemic cardiovascular disease that has a severe clinical manifestation and a high mortality rate.Coronary artery bypass grafting(CABG)and percutaneous coronary intervention(PCI)in the treatment of coronary heart disease is more effective than in the diffuse coronary artery disease and old myocardial infarction with poor heart function.Vascular endothelial growth factor(VEGF)gene therapy is a new progress for treatment of ischemic heart disease.The principal cellular target of VEGF is the vascular endothelial cell.It has a powerful promoting endothelial cell proliferation and promoting angiogenesis effect through binding the specific receptor of the vascular endothelial cell.Studies found that the role of other angiogenesis factors is through wholly or partly upregulate the expression of VEGF.Promoting the expression of VEGF or giving exogenous VEGF gene can increase angiogenesis in ischemia myocardial that provides the new treatment of ischemia myocardial disease.Objectives:To establish acute myocardial infarction rat model and to study the effect of adenovirus vector mediated vascular endothelial growth factor121 (Ad-VEGF₁₂₁)gene transfer direct injection in ischemic myocardial,ventricular structure and function,and myocardial apoptosis and its possible mechanism.Methods:1.Established the adenovirus vector expressing h-VEGF:the amount of Ad-VEGF was amplified with 293T cell.PCR assay was used to identify the augmented of Ad-VEGF.2.78 male SD rats(250～300g)were randomly divided into 4 groups:AMI+ Ad-VEGF(VEGF group),AMI+ normal saline(MI/NS group),AMI group,and Sham group.All of three groups(VEGF group,A MI/NS group,and AMI group) were underwent the ligation of left anterior descending branch(LAD)to perform AMI rat model.However,the LAD of the sham group was not ligated.3.60μl Ad-VEGF was injected into the area of myocardial infarction in VEGF group and 60μl(×10¹⁰vp)normal saline was injected into the area of myocardial infarction in MI/NS group using micro syringe after 10～15 minutes ligation of LAD. On the other hand,after ligation of LAD,thorax was closed in the AMI group.After 14 days,all of the groups underwent echocardiography examination to determine left ventricular structure and function;then rats were anaesthetized and sacrificed to remove the heart and take out the left ventricle.Upper half of left ventricle was frozen in -80℃,then RNA extraction and semi-quantitative RT-PCR were performed to examine mRNA expression of bax,bcl-2,caspase-3,and hVEGF₁₂₁.Lower half of left ventricular was fixed with formalin to perform HE stain and the MI area was determined by Image Pro Plus 6.0;capillary density which was counted within infarct and peri-infarct area was examined by immunohistochemistry vWF stain;apoptotic index of cardiomyocytes were assessed by TUNEL.Results:1.The titre of Ad-VEGF was 1.155×10¹²vp/ml post augmentation.PCR assay showed a 476bp fragments in agarose gel electrophoresis which presented pAd-hVEGF₁₂₁.2.Myocardial infarction of rats was successfully constructed.ECG showed that ST segment and QRS complex were elevated.14 day after MI,gross examination show that left ventricle local myocardial pale,ventricular wall thining.HE stain of heart samples displayed local transmural infarction in anterior wall of left ventricle. MI model success rate was 67.9%.3.At the end of the test,the weight of rat is less after MI.Echocardiography demonstrated that LVDd and LVDs got larger after MI.Results of statistics analysis were as follows:the left ventricular EF of rats transferred with Ad-VEGF group was better than that of saline and AMI group,but there was no statistic difference.4.HE stain show that infarcted areas in transferred Ad-VEGF group has no various difference compared with AMI and saline groups.Capillary density in VEGF group was much more than that of AMI group and MI/NS group(p＜0.05).There is no obvious new capillary in sham group.5.RT-PCR result showed that the expression of bcl-2,bax and caspase-3 had significant increase after MI(p＜0.01).Compared with AMI and MI/NS groups,the expression of bcl-2 and bax had no significant change in VEGF group.TUNEL result showed that the distribution of apoptosis was mainly distributed at the area surroundings in the infarction.The apoptotic index of apoptotic cardiomyocytes in VEGF group was dropped.There was obviously difference between the VEGF group and the AMI or MI/NS group(p＜0.05).Conclusion:1.Ligation of LAD can constructed myocardial infarction model of rats successfully.2.Direct intramyocardial injection of Ad-hVEGF₁₂₁(1×10¹⁰vp)can improve left ventricular function.This maybe related to the increase of density of capillary and decrease of the apoptotic index.3.Treatment with Ad-hVEGF₁₂₁could decrease the apoptotic index through improving the bcl-2/bax ratio and downregulating the expression of caspase-3 rather than upregulating the expression of bcl-2.4.Ad-hVEGF₁₂₁is safe,no side-effects were found.