| Background:Coronary artery bypass grafting has been well recognized to ameliorate angina pectoris, prevent myocardial infarction, and improve long-term survival in patients with atherosclerotic coronary disease. However, many of these patients are not candidates for further direct revascularization because of diffuse myocardial lesion with poor runoff vessels on angiography, lack of available conduits, unacceptably high operative risk, or often, a combination of these factors. It is well known that coronary collateral vessels protect myocardial tissues from acute or chronic ischemia. In addition to physiologic factors such as coronary perfusion pressure and flow, some growth factors have become of importance because they can induce angiogenesis. Induced angiogenesis consequently has drawn attention as a therapeutic modality of ischemic myocardium. Hepatocyte growth factor (HGF) is a recently characterized growth factor that has a disulfide-linked heterodimer structure and an apparent molecular weight of SOkDa.Its receptor has been identified as c-Met, a transmembrane tyrosine kinase proto-oncogene. HGF has been shown to have numerous functions including mitogenesis, motogenesis, morphogenesis, and angiogenesis. Furthermore, HGF and c-Met have been implicated in capillary endothelial cell regeneration in the ischemically injured heart. Replication-deficient adenoviruses (Ad) have been used to transfer various genes coding angiogenic factors.Ad vectors present several advantagesincluding : ( 1 ) low pathogenicity in individuals without immunosuppression; (2) high capacity to carry foreign DNA; (3) high viral liter-, (4) efficient gene expression in quiescent cells such as skeletal muscle fibers or cardiomyocytes; (5) transient expression in dividing cells, which is particularly adapted to the time course of vessel neofomation. To investigate the possibility of adenovirus-mediated HGF gene transfer in the treatment of ischemic heart disease, we constructed a replication-deficient recombinant adenovirus vector carrying human HGF and examined its effect on the proliferation of endothelial cells in vitro. To evaluate the ability of Ad-HGF in vivo, we employed an acute myocardial ischemia model to direct intramyocardial injection Ad vectors or infusion into rat left ventricular cavity. While many cardiac studies have been carried out in small animal models that may be informative in term of cardiac function and ventricular performance under various inotropic and loading conditions, it is important to use large animal models with physiology similar to that of humans for implication of potential applications to human gene therapy protocols. For this reason, we employed a dog acute myocardial ischemia model to study the effect of direct intramyocardial injection of replication-deficient recombinant Ad vectors carrying the HGF gene. Methods;1. Construction of the replication-deficient recombinant adenovirus carrying HGF.Recombinant plasmids PXCGFP (containing green fluorescence protein GFP gene), PXCHGF (containing hepatocyte growth factor gene) wereconstructed by molecular cloning. Recombinant adenovirus Ad-GFP,Ad-HGF were produced by homologous recombination in 293 cells, amplified also in 293 cells on a large scale, and purified by ultracentnfugation in CSC1 step gradient solutions. At OD260nm the amount of viral particles was determined, the purity was evaluated by the ratio of OD260nm/OD280nm, and the viral titers was determined with plaque assay.2. Ad-HGF transfection efficiency and proliferate vascular endothelial cells in vitro.Fetal cardiomyocytes (FCM) were isolated and cultured. FCM infected with Ad-GFP were examined for transfection efficiency through observing green fluorescence protein by fluorescence microscopy on days 1, 2, 3, 5, 7, 14, 21, 28 after infection. The HGF gene expression was examined by ELISA and immunohistochemstry . The effects of Ad-HGF on proliferation of endothelial cell line EVC304 were examined by MTT assay.3. Effect of Ad-HGF gene on induc...
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