228. AAV Mediated Hypoxia-Inducible VEGF Expression and Neovascular Formation in Ischemic Pig Myocardium

Abstract

Rapid, efficient overexpression of angiogenic growth factors in the peri-infarct zone is a promising approach for gene therapy of acute myocardium infarction. Patients suffering from an acute myocardial infarction often require invasive treatment, and gene transfer could be performed in combination with intravascular revascularization strategies or with bypass surgery. Many angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietins, fibroblast growth factor-4 (FGF-4) and placental growth factor (PlGF) have been shown to be potent inducers of arteriogenesis. VEGF is the most potent among these factors as it is capable of increasing proliferation and migration of endothelial and vascular smooth muscle cells, and attracting stem cell homing. In our previous studies, we have induced neovascular formation in ischemic mouse hearts with adeno-associated viral vector (AAV) mediated VEGF gene transfer. To avoid the harmful effect of excessive angiogenesis, we have constructed an AAV vector (MLCVEGF) with 9 copies of the hypoxia-response element (HRE) of the human erythropoietin (EPO) gene and the promoter of the rat myosin light chain 2v (MLC-2v) controlling VEGF gene expression. Since information derived from mouse model may not be directly applicable to human and the pig heart closely resembles the human heart closely, we tested the hypotheses that MLCVEGF can also mediate hypoxia- inducible VEGF expression and induce neovascular formation in the pig heart. We injected 1|[times]|1011 genomes of MLCVEGF or CMVVEGF (AAV vector with CMV promoter driving VEGF expression) into normal and ischemic pig myocardium after 90 minutes of occlusion and reperfusion of the left anterior descending coronary artery (LAD). CMVLacZ vector (AAV vector with CMV promoter driving LacZ gene expression) was used as control. All vectors were packaged in AAV serotype 1 capsid. Myocardiums were collected 2 weeks after the vector injection. Gene expression was analyzed by real-time RT-PCR and immunohistochemical staining. Capillaries and a-actin positive vessels were quantitated on anti PECAM-1 and smooth muscle a-actin antibody stained sections. We observed robust LacZ and VEGF gene expression at CMVLacZ and CMVVEGF injection sites and found more capillaries and a-actin positive vessels at VEGF vector injected sites than CMVLacZ injection sites. VEGF expression mediated by MLCVEGF vector was low in normal myocardium and was induced about 40-fold in ischemic myocardium 2 weeks after myocardial infarction and vector injection. Conclusions: 1) AAV serotype 1 can infect pig myocardium efficiently. 2) AAV vector containing VEGF controlled by MLC-2v promoter and 9 copies of EPO HRE can mediate hypoxia-inducible gene expression and induce neovascular formation in the pig myocardium.