Optimization of adenovirus-mediated endothelial nitric oxide synthase delivery in rat hindlimb ischemia

Adenovirus-mediated overexpression of endothelial nitric oxide synthase (eNOS) induces collateral artery development and substantially increases blood flow after induction of experimental acute hindlimb ischemia. However, the optimal technique of gene delivery for this or any other form of gene therapy in limb ischemia is still unknown. The purpose of this study was to determine the effect of the two most commonly used techniques, intra-arterial and intramuscular injection, on blood flow recovery, collateral artery development, and preservation of muscle mass. We compared intra-arterial injection under vascular isolation, intra-arterial injection under transient vascular occlusion, and intramuscular injection of phosphate buffered saline (PBS) or adenovirus encoding either the eNOS (AdeNOS) or LacZ (AdlacZ) gene after induction of acute hindlimb ischemia. Delivery of AdeNOS by both intra-arterial injection techniques increased eNOS activity (22.30 versus 10.56, P<0.01), blood flow (0.90+/-0.02 versus 0.69+/-0.07, P<0.001) and collateral artery development (17.56484 versus 13.74259, P<0.05) more than by intramuscular delivery. Intra-arterial injection under transient vascular occlusion led to better preservation of muscle mass, muscle architecture, and clinical ischemic index, but led to greater transgene expression in distant organs and contralateral limb muscles. Intra-arterial injection of AdeNOS under transient vascular occlusion is the optimal technique to reverse severe hindlimb ischemia in the rat. This is the first systematic comparison of different delivery techniques used in gene therapy of experimental hindlimb ischemia.