Background-We hypothesized that ischemia-induced expression of vascular endothelial growth factor (VEGF) and the production of NO stimulate coronary collateral growth. Methods and Results-To test this hypothesis, we measured coronary collateral blood flow and VEGF expression in myocardial interstitial fluid in a canine model of repetitive myocardial ischemia under control conditions and during antagonism of NO synthase. Collateralization was induced by multiple (1/h; 8/d), brief (2 minutes) occlusions of the left anterior descending coronary artery for 21 days. In controls, collateral blood flow (microspheres) progressively increased to 89Ϯ9 mL ⅐ min Ϫ1 ⅐ 100 g Ϫ1 on day 21, which was equivalent to perfusion in the normal zone. Reactive hyperemic responses (a measure of the severity of ischemia) decreased as collateral blood flow increased. In N G -nitro-L-arginine methyl ester (L-NAME)-and L-NAMEϩnifedipine-treated dogs, to block the production of NO and control hypertension, respectively, collateral blood flow did not increase and reactive hyperemia was robust throughout the occlusion protocol (PϽ0.01 versus control). VEGF expression (Western analyses of VEGF 164 in myocardial interstitial fluid) in controls peaked at day 3 of the repetitive occlusions but waned thereafter. In sham-operated dogs (instrumentation but no occlusions), expression of VEGF was low during the entire protocol. In contrast, VEGF expression was elevated throughout the 21 days of repetitive occlusions after L-NAME. Reverse transcriptase-polymerase chain reaction analyses revealed that the predominant splice variant expressed was VEGF 164 . Conclusions-NO is an important regulator of coronary collateral growth, and the expression of VEGF is induced by ischemia. Furthermore, the induction of coronary collateralization by VEGF appears to require the production of NO.

Background-The in vivo mechanism by which inhibition of NO synthase impairs ischemia-induced coronary vascular growth is unknown. We hypothesized that production of the growth inhibitor angiostatin increases during decreased NO production, blunting angiogenesis and collateral growth. Methods and Results-Measurements were made in myocardial tissue or interstitial fluid (MIF) from dogs undergoing repetitive coronary occlusions under control conditions or during antagonism of NO synthase (N G -nitro-L-arginine methyl ester [L-NAME]) for 7, 14, or 21 days. A sham group was instrumented identically but received no occlusions. In controls, capillary density in the ischemic zone increased initially but returned to baseline at the later times. In the L-NAME group, capillary density was lower at 7 days compared with that of controls. MIF from control dogs induced in vitro endothelial tube formation and cell proliferation, significantly greater than that from the L-NAME group. MIF from shams did not stimulate tube formation. In controls or shams, tube formation or cell proliferation did not change after administration of antiangiostatin, but this antibody restored the responses to control levels in the L-NAME group. Angiostatin expression in MIF was increased in the L-NAME group compared with controls and shams. The activities of tissue matrix metalloproteinases (MMPs) MMP-2 and MMP-9, which generate angiostatin, were increased in the L-NAME group. Conclusions-Inhibition of NO synthase increased expression of angiostatin and activities of MMP-2 and MMP-9. Our findings indicate that angiostatin inhibits coronary angiogenesis during compromised NO production and may underscore the impairment of coronary angiogenesis during endothelial dysfunction.

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