Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF

Diabetes is a major risk factor for coronary and peripheral artery diseases . Although diabetic patients often present with advanced forms of these dis eases, it is not known whether the compensatory mechanisms to vascular isch emia are affected in this condition. Accordingly, we sought to determine wh ether diabetes could: 1) impair the development of new collateral vessel fo rmation in response to tissue ischemia and 2) inhibit cytokine-induced ther apeutic neovascularization, Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 m ice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler stud ies after the surgery. In NOD mice, measurement of the Doppler Bow ratio be tween the ischemic and the normal limb indicated that restoration of perfus ion in the ischemic hindlimb was significantly impaired. At day 14 after su rgery, Doppler flow ratio in the NOD mice was 0.49 +/- 0.04 versus 0.73 +/- 0.06 for the C57 mice (P less than or equal to 0.005), This impairment in blood Bow recovery persisted throughout the duration of the study with Dopp ler Bow ratio values at day 35 of 0.50 +/- 0.05 versus 0.90 +/- 0.07 in the NOD and C57 mice, respectively (P less than or equal to 0.001), CD31 immun ostaining confirmed the laser Doppler data by showing a significant reducti on in capillary density in the NOD mice at 35 days after surgery (302 +/- 4 capillaries/mm(2) versus 782 +/- 78 in C57 mice (P less than or equal to 0 .005). The reduction in neovascularization in the NOD mice was the result o f a lower level of vascular endothelial growth factor (VEGF) in the ischemi c tissues, as assessed by Northern blot, Western blot and immunohistochemis try. The central role of VEGF was confirmed by showing that normal levels o f neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection o f an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impa irs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfe r restores neovascularization in a mouse model of diabetes.