192. Cardiac-Specific and Hypoxia-Responsive Vascular Endothelial Growth Factor Expression in Ischemic Hearts

Abstract

A problem associated with angiogenic gene therapy is that uncontrolled gene expression of angiogenic factors may cause several side effects, including: hemangioma formation, retinopathy, occult tumor growth and atherosclerotic plaque progression. Previously, we used the hypoxia response element (HRE) isolated from the erythropoietin (Epo) gene enhancer to render vascular endothelial growth factor (VEGF) expression responsive to hypoxia induction in vitro and in ischemic myocardium in vivo. We found in our experiment that adeno-associate viral vector (AAV) can be detected in other organs in addition to the heart after intra-myocardial injection. Thus, unwanted effects may still occur even with HRE controlled VEGF expression, since hypoxia in other tissues in patients with heart failure may results in extra-cardiac VEGF gene expression. To ensure the safety of angiogenic therapy for ischemic heart disease, it is necessary that the gene expression is cardiac specific and hypoxia responsive. In this study, we explored the feasibility of using the cardiac myosin light-chain-2v (MLC-2v) promoter and Epo HRE to mediate cardiac-specific and hypoxia-responsive VEGF gene expression. We constructed an AAV vector with MLC-2v promoter and 9 copies of Epo HRE driving VEGF (MLCVEGF) expression. Gene expression mediated by this vector was studied in vitro by transfection of this vector into a rat cardiac muscle cell line (H9c2), a rat skeletal muscle cell line (L6) and a mouse fibroblast cell line (NIH3T3), and in vivo by direct injection of the vector to normal and ischemic mouse hearts. AAV vectors with CMV driving VEGF (CMVVEGF), and 9HRE plus minimum SV40 promoter driving VEGF (H9VEGF) were used as controls. RESULTS: With MLCVEGF infection, VEGF expression is higher in H9c2 than the other two non-cardiac cell lines, and the expression is hypoxia inducible. In vivo studies indicated that MLCVEGF increased VEGF expression by 74% in ischemic heart compared to normal heart. VEGF expression was detected in liver, kidney, spleen and gonad of CMVVEGF inoculated mice by RT-PCR, but no VEGF expression was detected in organs other than heart and liver of MLCVEGF inoculated mice. CONCLUSION: Our result indicates that MLC-2v promoter and 9 copies of HRE can mediate cardiac-specific and hypoxia-inducible gene expression in vitro and in vivo. Induction of angiogenesis and improvement of cardiac function by this vector, studies currently in progress, may provide a safe approach for the treatment of ischemic heart disease.