633. Synergistic Effects of Naked DNA Vectors Encoding VEGF and bFGF on Angiogenesis and Blood Flow Recovery in Ischemic Hindlimb

Abstract

Therapeutic angiogenesis to enhance collateral vessel growth is a promising strategy for the treatment of vascular occlusive diseases. Although local delivery of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) has shown favorable results, few studies have examined in detail the combined effects of VEGF and bFGF gene delivery on collateral development. Here, we evaluated potential synergism of the naked DNA vectors encoding VEGF and bFGF by using a skeletal muscle-based ex vivo angiogenesis assay and by comparing blood flow recovery and limb loss score in a murine model of hindlimb ischemia (C57BL/6) where femoral artery was excised. In the ex vivo angiogenesis assay, VEGF+bFGF combination group displayed larger sprouting area than LacZ, VEGF, or bFGF group. Reflecting this capillary sprouting data, the combination group had the highest amount of secreted VEGF in the conditioned medium. Consistent with these ex vivo results, regional blood flow recovery at Day 14 as measured by Laser Doppler Imaging was also highest in the VEGF+bFGF combination group followed by bFGF, VEGF, and LacZ control group. The limb loss score was lowest (no necrosis) in the combination group, whereas there were 1|[sim]|4 cases of limb or toe necrosis in other groups. Currently, histological and genetic analyses of the ischemic muscles in the combination group are underway to find the evidence of increased angiogenesis and/or arteriogenesis and upregulation of the genes that may be associated with arteriogenesis, such as cardiac ankyrin repeat protein (CARP), TGF-|[beta]|1, and early growth response factor-1 (Egr-1). Our study has an implication for the development of combined arteriogenic gene therapy that uses the two best characterized angiogenic growth factors, VEGF and bFGF.