Engineered zinc finger-activating vascular endothelial growth factor transcription factor plasmid DNA induces therapeutic angiogenesis in rabbits with hindlimb ischemia

Abstract

Background - Therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) exists in multiple isoforms. We investigated an engineered zinc finger-containing transcription factor plasmid designed to activate the endogenous VEGF gene (ZFP-VEGF). Methods and Results - New Zealand White rabbits (n=56) underwent unilateral femoral artery ligation and excision. At day 10 postoperatively, the ischemic muscle received ZFP treatment (500 μg ZFP-VEGF plasmid) or no ZFP treatment (β-galactosidase, empty, or no plasmid). Group 1 (n=13) was harvested 3 days after injection to examine VEGF mRNA by real-time polymerase chain reaction and protein by ELISA. Groups 2 (n=13) and 3 (n=10) were harvested 11 days after injection. Group 2 was studied by histology and group 3, by histology and changes in blood flow. Groups 4 and 5 (n=10 each) were harvested 22 and 32 days after injection, respectively, and studied for changes in blood flow. In group 1, VEGF mRNA copy numbers were significantly higher for VEGF 121, VEGF165, VEGF189, and protein in the ZFP-VEGF-treatment versus no-ZFP-treatment arms. In groups 2 and 3, capillary density and proliferating cells were significantly greater and apoptosis significantly lower in the treatment versus no-treatment arms. Changes in the blood flow ratio of the ischemic to the nonischemic limb were significantly greater in the treatment versus no-ZFP-treatment groups (6.57±1.52% versus 3.38±0.87%, P<0.005; 13.15±1.77% versus 6.13±1.55%, P<0.001; and 20.16±2.84% versus 13.88±3.14%, P<0.01, for groups 3, 4, and 5, respectively). Conclusions - This engineered ZFP-VEGF-activating transcription factor may provide a novel approach to treat peripheral arterial disease.