We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L -arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L -arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L -arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS Ϫ / Ϫ mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS

Vascular endothelial growth factor (VEGF) is a heparin-binding, endothelial cell-specific mitogen. Previous studies have suggested that VEGF is a regulator ofnaturally occurring physiologic and pathologic angiogenesis. In this study we investigated the hypothesis that the angiogenic potential of VEGF is sufficient to constitute a therapeutic effect. The soluble 165-amino acid isoform ofVEGF was administered as a single intraarterial bolus to the internal iliac artery of rabbits in which the ipsilateral femoral artery was excised to induce severe, unilateral hind limb ischemia. Doses of 500-1,090 0,g of VEGF produced statistically significant augmentation of collateral vessel development by angiography as well as the number of capillaries by histology; consequent amelioration of the hemodynamic deficit in the ischemic limb was significantly greater in animals receiving VEGF than in nontreated controls (calf blood pressure ratio, 0.75±0.14 vs. 0.48±0.19, P < 0.05). Serial angiograms disclosed progressive linear extension of the collateral artery of origin (stem artery) to the distal point of parent vessel (reentry artery) reconstitution in seven of nine VEGF-treated animals. These findings establish proof of principle for the concept that the angiogenic activity of VEGF is sufficiently potent to achieve therapeutic benefit. Such a strategy might ultimately be applicable to patients with severe limb ischemia secondary to arterial occlusive disease. (J. Clin. Invest. 1994. 93:662-670.)

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Combined administration of VEGF and bFGF stimulates significantly greater and more rapid augmentation of collateral circulation, resulting in superior hemodynamic improvement compared with either VEGF or bFGF alone. This synergism of two angiogenic mitogens with different target cell specificities may have important implications for the treatment of severe arterial insufficiency in patients whose disease is not amenable to direct revascularization.