Abstract 403: E-selectin Transfer by Recombinant Adeno-associated Virus Improves Angiogenesis in a Murine Model of Hindlimb Ischemia

Abstract

Novel cell-based therapies, including optimization of the wound microenvironment via chemical or genetic means, hold promise to revolutionize the treatment of chronic limb ischemia and ischemic wounds. We examined the hypothesis that increasing expression in ischemic tissue of the endothelial adhesion molecule E-selectin by adeno-associated viral transduction would promote angiogenesis in a murine model of hindlimb ischemia. 12-week-old female C57BL/6 mice (N=10) underwent unilateral femoral artery ligation. After ligation, each mouse was injected in the ipsilateral semimebranosus muscle with 1x108 viral particles of either adeno-associated virus recombinant with human E-selectin gene (AAV/E-selectin) or adeno-associated virus recombinant with green fluorescent protein gene as control (AAV/GFP) suspended in phosphate-buffered saline (N=5/group). Ischemia was confirmed by laser Doppler perfusion imaging on post-ligation day one. Mice were sacrificed on post-ligation day seven and semimembranosus muscle harvested from the ligated limb. Muscle was fixed, embedded, and sectioned in 5 μm intervals. To assess the efficacy of viral transduction, we performed immunohistochemistry (IHC) of each section from the AAV/GFP group with anti-GFP antibody and from the AAV/E-selectin group with anti-human E-selectin antibody. Angiogenesis was evaluated by capillary density with IHC of all slides for endothelial cell marker VEGFR2 with anti-VEGFR2 antibody. Relative fluorescence index per high-powered field (RFI) was recorded for each slide. E-selectin RFI was 4 times greater, VEGFR2 RFI was 2 times greater, and E-selectin/VEGFR2 RFI ratio was 2 times greater in AAV/E-selectin treated tissue than in control. In conclusion, this study demonstrates the AAV/E-selectin vector as an effective means to upregulate E-selectin expression in ischemic muscle and supports E-selectin upregulation as a possible means to improve ischemia-induced angiogenesis.