To Investigate The Efficacy Of Direct Gene Transfer Of Nacked DNA Encoding Vascular Endothelial Growth Factor Into Ischemic Canine Myocardium

BackgroundCurrent treatment options for patients with advanced ischemic heart disease include medical therapy or coronary revascularization by percutaneous coronary angioplasty(PTCA) or coronary bypass surgery(CABG). However, a significant number of these patients are not candidates for standard revascularization procedures or have incomplete revascularization with these procedures. For example, in patients with 2- and 3-vessel or long and diffuse disease. Furthmore, full functional recovery of the ishemic myocardium may not be achieved if revascularization is delayed. For these patients, gene therapy is thought to be a exciting new strategy. This strategy is designed to promote the development of supplemental collateral blood vessels that will constitute endogenous bypass conduits around occluded native arteries, a strategy termed "therapeutic angiogenesis" or "molecular bypass". Vascular endothelial growth factor(VEGF) is one of the most important genes in the stratefy because of its traits in angiogenesis.AIM and MethodsPART I : AIM: To construct a eukaryotic expression vector of human VEGF-165 gene. METHODS: pcDNA3.1 (-) /VEGF-165 eukaryotic expressionplasmid was constructed by inserting the VEGF-165cDNA into pcDNAS.l (-) ,and new plasmid were confirmed by digested with gene enzyme and DNA sequence analysis.PART II: AIM: To investigate the transfection and expression of pcDNAS.l (-) /VEGF-165 eukaryotic expression plasmid in myocardial cells.METHODS : Rat primary cultured myocardial cells were transiently transfected with LipofectAMINE2000. RT-PCR, ELISA, Western blot and imnumohistochemical method were used to detect the expression of VEGF gene, and MTT to detect the biological activity of the conditioned medium after transfection.PART III: AIM.- To investigate the efficacy of direct gene transfer of nacked DNA encoding VEGF-165 into ischemic canine myocardium. METHODS : 24 canines were underwent left thoracotomy followed by the ligation of left anterior descending coronary artery. Constructed pcDNA3.1(-)/VEGF165 eukaryotic expression plasmid was directly injected into the canine myocardium. Coronary angiography(CAG) and immunohistocheistry of factor VIII related antigen were used to detect it's biological effect and evaluate collateral circulation of the occluded artery.Results1. The coding sequence of VEGF-165 in the new plasmid was confirmed by DNAsequence analysis.and the new plasmid was found to contain the VEGF-165 cDNA sequence by agarose gel electrophoresis analysis.2. There were signifected increases of VEGF mRNA and protein in the myocardial cells transfected with pcDNA3.1 ( - ) /VEGF-165. The conditioned medium after transfection showed the biological activity to stimulate the proliferation of endothelial cells.3. In 3 month after operation, CAG showed better collateral circulation in VEGF group, and there was a significant increase in the number of vessels when compared with control group.Conclusion1. The pcDNA3.1 (-) /VEGF-165, a eukaryotic expression plasmid forhVEGF-165 gene is constructed.2. High levels of VEGF mRNA and protein expression can be obtained in the myocardial cells transfected with pcDNA3.1 (-) /VEGF-165 eukaryotic expression plamid.The expressed protein has the biological activity.3. Direct gene transfer of nacked DNA encoding vascular endothelial growth factor into ischemic canine myocardium can increase capillary number and enhance collateral circulation of coronary, therefore it may be an novel therapeutic approach for the treatment of myocardial ischemia.