Characteristics of gene transfer and VEGF expression using naked plasmid vectors in the rat heart / 대한내과학회지

ABSTRACT

BACKGROUND: The purpose of this study was to compare gene expression among newly designed eukaryotic expression vectors, and to characterize the pattern of vascular endothelial growth factor(VEGF) expression using the most potent plasmids DNA vector. METHODS: After exposure of a beating rat heart (Sprague-Dawley, 250-300g), 5 different types of plasmid DNA was injected directly into the myocardium. Reporter protein was analyzed by ELISA in the extracted heart. RESULTS: The vector harboring cytomegalovirus (CMV) promoter and enhancer induced the strongest expression of reporter gene (chloramphenicol acetyl transferase; CAT) compared to those of pC3.1, pEF1a, RSV, pActin in the rat heart via direct injection of plasmid DNA into the apex (p<0.001). Using pCN-CAT, gene expression showed a dose-dependent response over a range of 0.3-10 (mu)g. CAT expression could be detected up to 30 days after 10 (mu)g of pCN-CAT injection with the maximal expression on day 5. In X-gal staining of injected pCN-lacZ gene, beta-galactosidase was found only around the needle track in the apex. The expressed hVEGF121 had biologic activity with vascular permeability assay (Miles assay) in guinea pigs. After injection of pCN-hVEGF121 into the apex of the rat heart, the expression of VEGF protein was dose-dependent over the range of 25 and 500 (mu)g. VEGF expression was detected up to 14 days with its peak on day 2 after injection of 250 (mu)g of pCN-hVEGF121. When plasmid was injected into the apex of the rat heart, the expression of VEGF in the heart showed concentration gradient from the apex to the base. However, the expression of CAT was detected only in the apex. CONCLUSION: Plasmids vector with hCMV IE promoter/enhancer will provide clear advantages over other previously developed plasmids and the information regarding the behaviors of VEGF expression may be useful in angiogenic gene therapy of the heart.