The role of reactive oxygen species in the vascular pathology asiated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (NO) with superoxide (O2), yielding the oxidant peroynitrite (ONOO-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet Endothelium-dependent relaxation is impaired in vessels from atherosclerotic patients (1, 2) and hypercholesterolemic animal models (3-6), suggesting the functional modification of endothelium-derived relaxing factor (EDRF) in hyperlipidemia. The dynamic role ofthe endothelium in the regulation of vascular tone was established when it was observed that relaxation of isolated blood vessels by vasoactive agents, such as acetylcholine (ACh) and the calcium ionophore A23187, was dependent on an intact endothelium and a diffusible factor (EDRF) that stimulated cGMP-dependent relaxation of vascular smooth muscle cells (VSMCs; ref. 7).Nitric oxide (-NO) and EDRF share similar chemical and pharmacological properties (8) and are derived from the oxidation of a terminal guanidino group of L-arginine (9, 10).Numerous mechanisms have been suggested for the defect in vascular relaxation in atherosclerosis and hypercholesterolemic animal models. They include an increased diffusional barrier for 'NO due to intimal cell proliferation and lipid deposition (11), L-argifine depletion (3,12,13) Vessel Contraction Studies. New Zealand White rabbits (2.5-3.0 kg) were maintained on rabbit chow containing 1% cholesterol (Ralston Purina) for 6 months prior to study [cholesterol-fed (Chol-fed) group]. Age-and weight-matched controls were fed a standard diet. After exsanguination under ketamine/rompun anesthesia, vessels were isolated and changes in tension were measured in femoral artery ring segments as described (28). After maximal contraction with 70 mM KCl and recovery, phenylephrine was added to achieve 30% of maximal tone. Rings were then exposed to increasing doses of ACh; relaxation is reported as the percentage decrease in preexisting tone. After the generation of cumulative ACh dose-response curves, rings were exposed to 30 ,uM papaverine. In some experiments, rings from control and Chol-fed rabbits were incubated with 3 mM L-arginine for 30 min prior to administration of ACh. In other studies, vessels were treated with native bovine SOD (200 units/ml) before measuring ACh-induced relaxation. All studies were performed in the presence of 5 !uM indomethacin.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has recently been shown to increase cytosolic free calcium in endothelial cells. In the present study, we investigated the coronary vascular effects of recombinant human and native guinea pig VEGF/VPF in isolated canine coronary arteries in the presence and absence of intimal endothelium, indomethacin, and NG-monomethyl-L-arginine, a competitive nitric oxide synthase inhibitor. Addition of recombinant VEGF/VPF (1-660 pM) in coronary arteries that had been previously contracted with prostaglandin F2 alpha induced a slow, dose-dependent relaxation, reaching a maximum of -59.1 +/- 6.7% (mean +/- SE, n = 19). Mechanical disruption of the intimal endothelium completely abolished the observed relaxation. No direct vascular effect of recombinant VEGF/VPF on the endothelium-disrupted coronary arteries was noted. Pretreatment of endothelium-intact coronary arteries with 5 microM of indomethacin did not alter the observed relaxation (-57.3 +/- 7.0%, n = 18), whereas pretreatment with either NG-monomethyl-L-arginine or 10 microM of genistein, a known inhibitor of tyrosine kinase, significantly inhibited the relaxation. Addition of native VEGF/VPF (1-100 pM) also induced an endothelium-dependent relaxation in the isolated coronary arteries. Heating of recombinant VEGF/VPF (70 degrees C, 25 min) or prior incubation with a specific antibody raised against a VEGF/VPF peptide completely abolished the relaxation. Finally, recombinant VEGF/VPF stimulated a slow rise in cytosolic free calcium in cultured human endothelial cells that was qualitatively similar to that of native VEGF/VPF.(ABSTRACT TRUNCATED AT 250 WORDS)

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Endothelin-1 (ET-1T he endothelium plays a central role in the regulation of vascular tone both under normal circumstances and in cirrhosis by releasing endotheliumderived vasodilators and vasoconstrictors in response to a variety of biochemical and physical stimuli. 1 Nitric oxide (NO) and endothelin-1 (ET-1) are two important endothelial mediators that modulate vascular tone. Endothelial NO production is catalyzed predominately by the endothelial form of nitric oxide synthase (eNOS) and under normal circumstances is constitutively expressed and activated by calcium entry into cells. 2 ET-1 is a 21 amino acid peptide formed from a precursor, big ET-1, through the action of an endothelin-converting enzyme and is produced in a number of cell types in addition to endothelial cells, including hepatic stellate cells and biliary epithelium. 3-6 ET-1 is classically recognized as a potent paracrine vasoconstrictor, and its action is mediated by two G protein coupled receptors. 7,8 The endothelin A (ET A ) receptor mainly exists in vascular smooth muscle cells and mediates contraction and vasoconstriction. 9 Two endothelin B (ET B ) receptor types have been found: one in endothelial cells that upregulates eNOS and NO and the other in smooth muscle cells that functions similar to the ET A receptor. 10,11 Increased circulating ET-1, in part derived from increased hepatic production and

Exogenous thrombin produced a biphasic response (a potent dose-related vasodilatation followed by vasoconstriction) in nonischemic canine coronary arteries. The vasodilatation was not blocked by propranolol, atropine, or indomethacin, but was completely blocked by heparin or denudation of the intimal endothelial cells. A similar loss of vasodilating response to thrombin occurred in ischemic coronary arteries with a concomitant enhancement of vasoconstriction. This study indicates that altered responses to thrombin in coronary arteries with damaged endothelium may play an important role in the pathogenesis of coronary vasospasm.