Background —The effect of aging on angiogenesis in ischemic vascular disease has not been studied. Accordingly, we investigated the hypothesis that angiogenesis is impaired as a function of age. Methods and Results —Forty days after the resection of 1 femoral artery, collateral vessel development was significantly impaired in old (aged 4 to 5 years; n=7) versus young (aged 6 to 8 months; n=6) New Zealand White (NZW) rabbits on the basis of reduced hindlimb perfusion (ischemic: normal blood pressure ratio=0.58±0.05 versus 0.77±0.06; P <0.005), reduced number of angiographically visible vessels (angiographic score=0.48±0.05 versus 0.70±0.05; P <0.01), and lower capillary density in the ischemic limb (130.3±5.8/mm 2 versus 171.4±9.5/mm 2 ; P <0.001). Angiogenesis was also impaired in old (aged 2 years) versus young (aged 12 weeks) mice as shown by reduced hindlimb perfusion (measured by laser Doppler imaging) and lower capillary density (353.0±14.3/mm 2 versus 713.3±63.4/mm 2 ; P <0.01). Impaired angiogenesis in old animals was the result of impaired endothelial function (lower basal NO release and decreased vasodilation in response to acetylcholine) and a lower expression of vascular endothelial growth factor (VEGF) in ischemic tissues (by Northern blot, Western blot, and immunohistochemistry). When recombinant VEGF protein was administered to young and old rabbits, both groups exhibited a significant and similar increase in blood pressure ratio, angiographic score, and capillary density. Conclusions —Angiogenesis responsible for collateral development in limb ischemia is impaired with aging; responsible mechanisms include age-related endothelial dysfunction and reduced VEGF expression. Advanced age, however, does not preclude augmentation of collateral vessel development in response to exogenous angiogenic cytokines.

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.

Background —Vascular endothelial growth factor (VEGF), an endothelial cell mitogen that promotes angiogenesis, was initially identified as a vascular permeability factor (VPF). Abundant evidence suggests that angiogenesis is preceded and/or accompanied by enhanced microvascular permeability. The mechanism by which VEGF/VPF increases vascular permeability (VP), however, has remained enigmatic. Accordingly, we used an in vivo assay of VP (Miles assay) to study the putative mediators of VEGF/VPF-induced permeability. Methods and Results —VEGF/VPF and positive controls (platelet-activating factor [PAF], histamine, and bradykinin) all increased vascular permeability. Prior administration of the tyrosine kinase inhibitors genistein or herbimycin A prevented VEGF/VPF-induced permeability. Placenta growth factor, which binds to Flt -1/VEGF-R1 but not Flk -1/KDR/VEGF-R2 receptor tyrosine kinase, failed to increase permeability. Other growth factors such as basic fibroblast growth factor (FGF), acidic FGF, platelet-derived growth factor-BB, transforming growth factor-β, scatter factor, and granulocyte macrophage-colony stimulating factor (8 to 128 ng) failed to increase permeability. VEGF/VPF-induced permeability was significantly attenuated by the nitric oxide (NO) synthase inhibitors N ω -nitro- l -arginine (10 mg/kg) or N ω -nitro- l -arginine methyl ester (20 mg/kg) and the cyclooxygenase inhibitor indomethacin (5 mg/kg). The inactive enantiomer N ω -nitro- d -arginine methyl ester (20 mg/kg) did not inhibit VEGF/VPF-induced permeability. In vitro studies confirmed that VEGF/VPF stimulates synthesis of NO and prostaglandin metabolites in microvascular endothelial cells. Finally, NO donors and the prostacyclin analogue taprostene administered together but not alone reproduced the increase in permeability observed with VEGF/VPF. Conclusions —These results implicate NO and prostacyclin produced by the interaction of VEGF/VPF with its Flk -1/KDR/VEGF-R2 receptor as mediators of VEGF/VPF-induced vascular permeability. Moreover, this property appears unique to VEGF/VPF among angiogenic cytokines.