Antonio J. Cayatte

Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits.

To determine if endogenous local levels of nitric oxide (NO) modulate atherogenesis, we studied the effect of inhibiting NO with NG-nitro-L-arginine methyl ester (L-NAME) on early neointima formation in cholesterol-fed rabbits. Male rabbits were fed for 5 weeks with a 0.5% cholesterol diet alone or treated in addition during the last 4 weeks with L-NAME (12 mg/kg per day SC) via osmotic minipump. Endothelial cell function was assessed in isolated aortic rings by vascular reactivity and levels of cyclic GMP. In L-NAME-treated rabbits there was inhibition of endothelium-dependent relaxations to acetylcholine and the calcium ionophore A23187 as well as impaired cyclic GMP accumulation in response to acetylcholine. Neointima formation in the ascending thoracic aorta was assessed by determining media and intima cross-sectional areas with computerized image analysis. Compared with rabbits that consumed the cholesterol diet alone, L-NAME-treated rabbits had significant increases in lesion area (0.29 +/- 0.04 versus 0.15 +/- 0.03 mm2) and in lesion/media ratio (0.06 +/- 0.01 versus 0.03 +/- 0.01). Plasma levels of cholesterol and fluorescent lipid peroxide products were unchanged, suggesting no difference in cholesterol metabolism or oxidation. Because arterial blood pressure was not altered by L-NAME treatment, the increased atherogenesis could not be attributed to an increase in blood pressure. These results indicated that local inhibition of NO accelerates early neointima formation possibly because of modulating monocyte recruitment or foam cell lipid accumulation.

Superoxide Anion From the Adventitia of the Rat Thoracic Aorta Inactivates Nitric Oxide

The purpose of this study was to determine whether superoxide anion is produced endogenously in the rat aortic adventitia and whether sufficient superoxide anion is produced to interfere with the response of the rat aorta to nitric oxide. Relaxation was measured in rings of the rat thoracic aorta, which were oriented so that the adventitial or luminal surface could be preferentially exposed to nitric oxide or sodium nitroprusside. To accomplish this, the rings were mounted (1) with the adventitia facing outward, (2) with the adventitia facing inward after inverting, or (3) with the adventitia facing outward after inverting twice (to control for the inverting procedure). The relaxation to nitric oxide, but not to sodium nitroprusside, was less in rings with the adventitia facing outward compared with those in which it faced inward. In contrast, the response to nitric oxide via either surface was similar when extracellular superoxide anion was scavenged with superoxide dismutase. Incubation of rings with nitro blue tetrazolium (NBT) resulted in blue formazan staining of the adventitia, and lucigenin chemiluminescence was significantly greater when detected from the adventitial compared with the intimal aspect of the artery. The reduction of NBT in intact aortic rings was 30+/-2 pmol x min(-1) x mg(-1) and was significantly decreased by superoxide dismutase to 19+/-2 pmol x min(-1) x mg(-1) and by a synthetic superoxide dismutase mimic, Euk-8, to 11+/-2 pmol x min(-1) x mg(-1). The NADPH oxidase inhibitor, diphenyleneiodonium, decreased NBT reduction to 9+/-1 pmol x min(-1) x mg(-1), whereas inhibitors of xanthine oxidase, mitochondrial oxidases, and nitric oxide synthase were ineffective. Immunohistochemical staining indicated the localization of NADPH oxidase proteins gp91phox, p22phox, p47phox, and p67phox almost exclusively in the adventitia of the rat aorta with no substantial staining in the media. These results indicate that NADPH oxidase located in the adventitia of rat thoracic aorta generates sufficient extracellular superoxide anion to constitute a barrier capable of inactivating nitric oxide. This study suggests that adventitial superoxide anion can play a role in the pathophysiology of the arterial wall.

The Thromboxane Receptor Antagonist S18886 but Not Aspirin Inhibits Atherogenesis in Apo E–Deficient Mice: Evidence That Eicosanoids Other Than Thromboxane Contribute to Atherosclerosis

Atherosclerosis involves a complex array of factors, including leukocyte adhesion and platelet vasoactive factors. Aspirin, which is used to prevent secondary complications of atherosclerosis, inhibits platelet production of thromboxane (Tx) A(2). The actions of TxA(2) as well as of other arachidonic acid products, such as prostaglandin (PG) H(2), PGF(2alpha), hydroxyeicosatetraenoic acids, and isoprostanes, can be effectively antagonized by blocking thromboxane (TP) receptors. The purpose of this study was to determine the role of platelet-derived TxA(2) in atherosclerotic lesion development by comparing the effects of aspirin and the TP receptor antagonist S18886. The effect of 11 weeks of treatment with aspirin (30 mg. kg(-1). d(-1)) or S18886 (5 mg. kg(-1). d(-1)) on aortic root atherosclerotic lesions, serum levels of intercellular adhesion molecule-1 (ICAM-1), and the TxA(2) metabolite TxB(2) was determined in apolipoprotein E-deficient mice at 21 weeks of age. Both treatments did not affect body or heart weight or serum cholesterol levels. Aspirin, to a greater extent than S18886, significantly decreased serum TxB(2) levels, indicating the greater efficacy of aspirin in preventing platelet synthesis of TxA(2). S18886, but not aspirin, significantly decreased aortic root lesions as well as serum ICAM-1 levels. S18886 also prevented the increased expression of ICAM-1 in cultured human endothelial cells stimulated by the TP receptor agonist U46619. These results indicate that inhibition of platelet TxA(2) synthesis with aspirin has no significant effect on atherogenesis or adhesion molecule levels. The effects of S18886 suggest that blockade of TP receptors inhibits atherosclerosis by a mechanism independent of platelet-derived TxA(2), perhaps by preventing the expression of adhesion molecules whose expression is stimulated by eicosanoids other than TxA(2).

Pathogenesis of the Atherosclerotic Lesion: Implications for diabetes mellitus

In this review, we have highlighted pivotal cellular and molecular events in the initiation and progression of atherosclerosis. Key components of lesion initiation are an enhanced focal intimal influx and accumulation of lipoproteins, including LDL in hemodynamically determined lesion-prone areas, focal monocyte-macrophage recruitment, intimal generation of ROS, and oxidative modification of lipoproteins (including LDL [Ox-LDL]). Modified lipoproteins are taken up by the non-downregulating macrophage scavenger receptor, with foam cell formation and the development of the so-called fatty streak. One transitional event in lesion progression is foam cell necrosis, likely attributable to the cytotoxicity of both intimal free radicals and Ox-LDL, with development of an extracellular metabolically inert lipid core. Another is the migration to and proliferation within the intima of medial SMCs, leading to the synthesis of plaque collagens, elastin, and proteoglycans. Mural thrombosis plays a significant role in the late-stage progression of lesions. Regression of lesions is considered a function of the dynamic balance among components of initiation, progression, plaque stabilization, and removal of plaque constituents—the so-called regression quartet. Here, we critically examine how components of diabetes mellitus might impact not only lesion development, but also lesion regression. It is concluded that some components of diabetes mellitus augment key mechanisms in lesion initiation and progression and will likely retard the processes of plaque regression. Specifically, we focus on the various influences of diabetes mellitus on lipoprotein influx and accumulation, free radical generation and Ox-LDL, monocyte-macrophage recruitment, thrombosis and impaired fibrinolysis, and the reverse cholesterol transport system. The importance of nonenzymatic protein glycosylation in modifying a number of these processes is emphasized.

Paracrine Role of Adventitial Superoxide Anion in Mediating Spontaneous Tone of the Isolated Rat Aorta in Angiotensin II-Induced Hypertension

The relationship between vascular generation of superoxide anion and spontaneous tone observed in the isolated aorta was studied in hypertensive rats infused with angiotensin II. Aortic rings from hypertensive, but not from sham-operated rats, demonstrated oscillatory spontaneous tone that represented 52+/-5.6% of the maximal contraction to KCl. Spontaneous tone was prevented by calcium-free buffer or by blocking calcium influx through L-type calcium channels with nifedipine. The production of superoxide anion measured by lucigenin chemiluminescence was up to 15-fold higher than in sham-operated rat aorta. The adventitial site of production of superoxide anion was suggested by the fact that lucigenin chemiluminescence was 5.5-fold higher from the adventitia than from the intima. This was confirmed histochemically by demonstrating that the adventitia was the site of reduction of nitroblue tetrazolium as well as immunohistochemical staining of NAD(P)H oxidase subunit proteins. A causal link between superoxide anion production by NAD(P)H oxidase and the spontaneous tone is suggested by the fact that superoxide dismutase or the inhibitor of NAD(P)H oxidase, diphenylene iodonium, decreased both superoxide anion production and spontaneous tone. L-NAME or removal of the endothelium from the aorta had no significant effect on superoxide anion levels or spontaneous tone. However, although superoxide dismutase decreased superoxide anion levels in the presence of L-NAME or in endothelium-denuded rings, it no longer inhibited the tone. This suggests that the effect on tone of superoxide anion originating in the adventitia is mediated by inactivating endothelium-derived nitric oxide, which promotes smooth muscle calcium influx and spontaneous tone. The adventitia is not a passive bystander during the development of hypertension, but rather it may have an important role in the regulation of smooth muscle tone.

The Thromboxane A2 Receptor Antagonist S18886 Prevents Enhanced Atherogenesis Caused by Diabetes Mellitus

Background— S18886 is an orally active thromboxane A2 (TXA2) receptor (TP) antagonist in clinical development for use in secondary prevention of thrombotic events in cardiovascular disease. We previously showed that S18886 inhibits atherosclerosis in apolipoprotein E–deficient (apoE−/−) mice by a mechanism independent of platelet-derived TXA2. Atherosclerosis is accelerated by diabetes and is associated with increased TXA2 and other eicosanoids that stimulate TP. The purpose of this study was to determine whether S18886 lessens the enhanced atherogenesis in diabetic apoE−/− mice. Methods and Results— Diabetes mellitus was induced in apoE−/− mice with streptozotocin and was treated or not with S18886 (5 mg · kg−1 · d−1). After 6 weeks, aortic lesion area was increased >4-fold by diabetes in apoE−/− mice, associated with similar increases in serum glucose and cholesterol. S18886 largely prevented the diabetes-related increase in lesion area without affecting the hyperglycemia or hypercholesterolemia. S18886 prevented deterioration of endothelial function and endothelial nitric oxide synthase expression, as well as increases in intimal markers of inflammation associated with diabetes. In human aortic endothelial cells in culture, S18886 also prevented the induction of vascular cell adhesion molecule-1 and prevented the decrease in endothelial nitric oxide synthase expression caused by high glucose. Conclusions— The TP antagonist inhibits inflammation and accelerated atherogenesis caused by diabetes, most likely by counteracting effects on endothelial function and adhesion molecule expression of eicosanoids stimulated by the diabetic milieu.

Decreased sensitivity to nitric oxide in the aorta of severely hypercholesterolemic apolipoprotein E-deficient mice.

A normal response to nitric oxide donors has been cited as evidence that impaired endothelium-dependent vasodilation during hypercholesterolemia is due to decreased synthesis of nitric oxide. This tenet was examined by determining responses to nitric oxide gas as well as to acetylcholine and sodium nitroprusside in the isolated aorta of apolipoprotein E-deficient mice fed normal or Western-type cholesterol-rich diet until 21 or 35 weeks of age. In mice fed normal chow, relaxation to all agents remained comparable to that obtained in wild-type mice. In mice fed Western diet, the relaxation to acetylcholine as well as to nitric oxide was decreased at 35 weeks of age. At 21 weeks of age, decreased sensitivity to nitric oxide was observed despite a normal response to acetylcholine. The response to sodium nitroprusside was normal in all groups. A decrease in aortic superoxide dismutase activity as well as an increase in aortic superoxide anion generated in the presence of NADH as measured by lucigenin chemiluminescence was observed in the group fed Western diet at 35 weeks. This provides evidence that altered superoxide anion could contribute to the deterioration in nitric oxide sensitivity that underlies the impaired endothelium-dependent relaxation. These data indicate that decreased sensitivity to nitric oxide may contribute to the development of impaired endothelium-dependent relaxation in hypercholesterolemia. The response to sodium nitroprusside appears not to reflect the decreased sensitivity of vascular smooth muscle to authentic nitric oxide.