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

Abstract

Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs 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 transfer restores neovascularization in a mouse model of diabetes.

Figure 1.

Left: Representative results of laser Doppler perfusion imaging recorded at serial time points after surgery in NOD and C57 mice. A color scale illustrates blood flow variations from minimal (dark blue) to maximal (red) values. As shown in upper left frame: NI, nonischemic (right) limb; T, tail; and I, ischemic (left) limb of mice. Right: Laser Doppler perfusion ratio over time after surgery in NOD and C57 mice. At day 14 after surgery, the LDPI flow ratio was significantly reduced in the NOD mice, and this difference persisted through day 35 postoperatively.

Figure 2.

CD31 staining of ischemic muscles from C57 and NOD mice at time of sacrifice (A and B) showed a significant reduction in the capillary density in diabetic mice (C).

Figure 3.

Expression of VEGF mRNA. At baseline (nonischemic hindlimbs, day 0), the level of VEGF mRNA expression was almost undetectable both in C57 and NOD mice. However, in ischemic hindlimbs, VEGF mRNA expression was significantly reduced in NOD versus C57 mice from day 3 to day 14 after surgery.

Figure 4.

Expression of VEGF protein. A: Western blot analysis of VEGF protein expression in ischemic muscles harvested at different time points after hindlimb surgery in diabetic (NOD) and normal (C57) mice. The level of VEGF protein expression was significantly reduced in NOD versus C57 mice from day 3 to day 14 after surgery. B: Positive control (human colon carcinoma, ×50 hematoxylin counter stain) for VEGF immunostaining. C: Immunostaining for VEGF in ischemic tissues of C57 and NOD mice, 7 days after surgery. Immunostaining confirmed the results of the Western blot analysis, showing a lower level of VEGF expression in the ischemic muscles of NOD versus C57 mice. Negative control slides (control) were prepared in C57 and NOD mice by substituting preimmune rabbit serum for VEGF antibody staining.

Figure 5.

VEGF supplementation in diabetic mice. (A and B) Macroscopic and histological sections of ischemic muscles stained with X-Gal solution 3 days after intramuscular injection of the adeno-nls-LacZ construct. The efficiency of transfection is indicated by the positive blue staining of the myocyte nuclei. NOD mice receiving adeno-VEGF gene transfer showed significant improvement in hindlimb perfusion as assessed by LDPI analysis of hindlimb blood flow. This improvement in hindlimb perfusion was consistent throughout the duration of the study (C).

Figure 6.

Capillary density after VEGF supplementation. NOD mice transduced with the adeno-VEGF construct (A) developed more vessels in response to ischemia than mice receiving adeno-LacZ (B). Thirty-five days after surgery, there was a significant increase in the capillary density in diabetic mice supplemented with VEGF (C).