TEMPORALLY DELIVERED COMBINATION PRO-ANGIOGENIC GENE THERAPY FOR NEOVASCULARIZATION OF CHRONIC HINDLIMB ISCHEMIA IN DIABETES

Abstract

BACKGROUND: Diabetes mellitus increases the risk of developing cardiovascular diseases and is associated with macro and microvascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue have been investigated, including delivery of pro-angiogenic growth factors and the genes that encode them, but results have been disappointing. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We sought to examine the revascularization effect of multiple pro-angiogenic genes on chronic hindlimb ischemia in both non-diabetic and diabetic mouse models. METHODS: Spontaneously type II diabetic male mice (db/db; with C57/Bl6 genetic Background) and their control (db/m) between 8and 10-week-old were studied. Unilateral hindlimb ischemia was induced in all animals by ligation and stripping of the left femoral artery. For gene delivery, plasmids containing pro-angiogenic genes were administrated intramuscularly directly into the ischemic distal hindlimb muscle, below the ligation site at 4 weeks (for early delivery) and at 6 weeks (for late delivery) after ligation procedure. Gene combinations included vascular endothelial growth factor (VEGF) and angiopoietin 1, 2 (Ang-1, 2). Relative blood flow to the foot was measured under standardized conditions by laser Doppler perfusion imaging (LDPI). Perfusion measurements were performed on postoperative days 1, 3, 7, week 2, 3, 4, 5, 6, 7 and 8. The perfusion ratio (occludedto-non-occluded leg) was calculated for each animal. Animals from all groups were sacrificed at various times after gene delivery for tissue sampling (PCR, westerns, immunohistochemistry). RESULTS: Diabetic mice showed reduced ability to induce angiogenesis following ischemia compared to non-diabetic mice. In response to VEGF gene therapy, non-diabetic mice responded with greater neovascularization compared to diabetic mice. Ang-1 mono-therapy promoted an angiogenic response in diabetic mice, but not in non-diabetic mice. Ang2 mono-therapy did not promote angiogenesis in either model. Temporally separated delivery of VEGF (early) and Ang-1 (late) promoted more neovascularization than any single gene therapy. Delivery of VEGF and Ang-2 (early) and Ang-1 (late) showed the greatest angiogenic response in both diabetic and non-diabetic mice. CONCLUSION: Diabetic mice show a reduced angiogenic response to VEGF and increased angiogenic response to Ang1 in compared to non-diabetic mice. Temporally delivery of VEGF, Ang-2 and Ang-1 gene combination resulted a greater perfusion recovery and a greater angiogenic response in ischemic hindlimb skeletal muscle, compared to single gene or multiple gene combinations without temporal separation in both diabetic and non-diabetic settings.