S17834, a new inhibitor of cell adhesion and atherosclerosis that targets nadph oxidase.

microdant stress is involved in the events that accompany endothelial cell expression of adhesion molecules and leukocyte adherence in many disease states, including atherosclerosis. A recently discovered benzo(b)pyran-4-one derivative, S17834 (10 to 50 micromol/L), reduced tumor necrosis factor-stimulated vascular cell adhesion molecule-1 (VCAM) mRNA accumulation and protein expression in human umbilical vein endothelial cells. Intercellular cell adhesion molecule-1 and E-selectin were also inhibited by S17834, but platelet endothelial cell adhesion molecule-1 was not. Adherence of U937 monocytic cells to the endothelial cells as well as to plastic plates coated with soluble VCAM, intercellular cell adhesion molecule-1, P-selectin, and E-selectin was also decreased. Consistent with an antioxidant mechanism of action, S17834 (10 to 50 micromol/L) inhibited tumor necrosis factor-stimulated release of superoxide from endothelial cells measured by cytochrome c reduction. S17834 had no effect on superoxide produced by xanthine oxidase, indicating that rather than by acting as a scavenger of superoxide anion, the drug acts by inhibiting the production of free radicals. Indeed, S17834 inhibited NADPH oxidase activity of endothelial cell membranes. The ability to inhibit superoxide anion production appears to be key in the effect of S17834 on superoxide anion production and VCAM expression, because these actions were mimicked by adenovirus-mediated overexpression of superoxide dismutase. Furthermore, these actions may be relevant in vivo, because S17834 reduced aortic superoxide anion levels by 40% and aortic atherosclerotic lesions by 60% in apolipoprotein E-deficient mice. These results indicate that S17834 inhibits adhesion molecule expression and adherence of leukocytes to endothelial cells as well as aortic atherogenesis and that perhaps these effects can be explained by its ability to inhibit endogenous superoxide anion production.

Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice.

Oxygen-derived free radicals are involved in the vascular response to angiotensin II (Ang II), but the role of NADPH oxidase, its subunit proteins, and their vascular localization remain controversial. Our purpose was to address the role of NADPH oxidase in the blood pressure (BP), aortic hypertrophic, and oxidant responses to Ang II by taking advantage of knockout (KO) mice that are genetically deficient in gp91(phox), an NADPH oxidase subunit protein. The baseline BP was significantly lower in KO mice than in wild-type (WT) (92+/-2 [KO] versus 101+/-1 [WT] mm Hg, P<0.01), but infusion of Ang II for 6 days caused similar increases in BP in the 2 strains (33+/-4 [KO] versus 38+/-2 [WT] mm Hg, P>0.4). Ang II increased aortic superoxide anion production 2-fold in the aorta of WT mice but did not do so in KO mice. Aortic medial area increased in WT (0.12+/-0.02 to 0.17+/-0.02 mm(2), P<0.05), but did not do so in KO mice (0.10+/-0.01 to 0.11+/-0.01 mm(2), P>0.05). Histochemistry and polymerase chain reaction demonstrated gp91(phox) localized in endothelium and adventitia of WT mice. Levels of reactive oxidant species as indicated by 3-nitrotyrosine immunoreactivity increased in these regions in WT but not in KO mouse aorta in response to Ang II. These results indicate an essential role in vivo of gp91(phox) and NADPH oxidase-derived superoxide anion in the regulation of basal BP and a pressure-independent vascular hypertrophic and oxidant stress response to Ang II.

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.

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).

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.

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.

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.

Atherosclerosis. Potential targets for stabilization and regression.

Reviewed are various aspects of atherosclerotic plaque stabilization and regression in humans and experimental animals. Plaque regression is a function of the dynamic balance among initiation, progression, stabilization, and removal of plaque constituents. Pseudoregression, the result of the triad thrombolysis, age- or lesion-dependent arterial dilatation, and relaxation of vasospasm, may readily give rise to angiographic misinterpretation. Although lowering of plasma cholesterol and low density lipoprotein-cholesterol has demonstrated significant clinical benefits in a number of clinical trials, the magnitude of angiographic regressive changes is relatively small despite aggressive lipid-lowering regimens. The emerging need for alternative or complementary therapeutic interventions has been emphasized. In particular, they should be targeted to pivotal cellular or molecular mechanisms in initiation, progression, or stabilization. Potentially important therapeutic targets include the use of antioxidants or free radical scavengers such as Probucol or its analogues, butylated hydroxytoluene, tocopherols, and possibly the tocotrienols. Other therapeutic targets include intimal monocyte-macrophage recruitment, macrophage cholesterol acyltransferase inhibition, stimulation of the high density lipoprotein-mediated reverse cholesterol transport system, smooth muscle cell migration to and proliferation in the arterial intima, and intimal connective tissue synthesis. Whether the isoprenylated proteins associated with the cholesterol biosynthetic pathway will give rise to compounds regulating smooth muscle cell growth has yet to be determined. Because of the importance of thrombosis in the pathogenesis and progression of lesions, the need to develop interventional strategies targeted at endothelial cell thromboresistance and thromboregulation must assume a high priority in future research and development. Other areas of therapeutic promise include the calcium channel blockers and angiotensin converting enzyme inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin.

Fetuin belongs to a group of fetal glycoproteins whose specific function is not known. In this study we investigated the effect of bovine fetuin on exogenous fatty acid incorporation into lipid classes by fetal rabbit aortic smooth muscle cells (SMC) and human fetal skin fibroblasts. When compared with albumin, the addition of fetuin to the culture medium caused a dramatic increase in labeled fatty acid incorporation (nanomoles/mg of protein) by SMC into triglycerides (albumin (control) 2.8 +/- 0.3 + fetuin 178.3 +/- 13.7). This effect was noted at a wide range of fetuin concentrations (0.2-5%) at oleate:fetuin molar ratios of 3.3-0.13, respectively. Similar effects were noted using human fetal skin fibroblasts with both labeled oleic and arachidonic acids (0.1 mM) as substrates (arachidonic acid incorporation into triglycerides, albumin (control) 76.9 +/- 16.2 + fetuin 684.6 +/- 64.1). Stimulation of fatty acid incorporation into di- and monoglycerides was also noted. Although the amount of unbound fatty acid in the presence of fetuin was greater than with albumin, experiments done under conditions that create identical unbound oleate levels (by varying fatty acid concentration) still showed increased fatty acid incorporation into triglycerides by SMC when exposed to fetuin. This marked effect of fetuin on triglyceride accumulation in cells was confirmed by lipid analysis, strong positive staining with oil red O, and transmission of electron microscopy. Furthermore, the potential physiological role of fetuin in terms of fatty acid and transport was attested by (a) the presence of significant amounts of free fatty acids associated with fetuin; and (b) by the stimulatory effect of fetuin, even when added to culture media containing other fatty acid carriers. These results show that (a) fetuin is far more efficient than albumin in incorporating fatty acids into cells; and (b) this might represent a novel function for fetuin during development.

Association of a lipoprotein-like particle with bovine fetuin.

Fetuin is a major protein of fetal bovine serum that exhibits heterogeneity and has been found to be associated with some biological active growth factors. Preliminary studies indicated that commercial fetuin preparations contain lipids. We investigated in detail the nature of lipids associated with fetuin by using ultracentrifugation and agarose gel chromatography followed by lipid analysis. Fetuin was associated with a variety of lipids, predominantly cholesterol, cholesteryl ester with smaller amounts of phospholipids, triglycerides, and free fatty acids. Adjustment of fetuin preparation for various densities followed by ultracentrifugation resulted in a fraction with a density 1.063-1.21 g/ml (1-2% of total protein) that contained the bulk of the lipids. This fraction eluted as a single peak upon high pressure liquid chromatography and agarose gel chromatography. Delipidation of the lipoprotein-like particle followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a major band in the range of fetuin itself. These studies suggest that a fraction of fetuin (or isoform) binds lipids forming a particle with floating characteristics similar to high density lipoproteins.

Morphological and smooth muscle cell phenotypic changes in fetal rabbit aorta during early development.

Previous studies from our laboratory noted a) high aortic cholesterol esterification activity in the fetal rabbit, and b) increased susceptibility of fetal aortic explants to smooth muscle cell proliferation in culture, two features commonly associated with atherogenesis. This prompted us to examine in detail morphological development of fetal aorta and its relationship to fetal plasma cholesterol levels. Our studies made three important observations: 1) plasma cholesterol levels were high in early fetus which decreased at term; 2) in early fetus aortic endothelial cells appear to protrude into the lumen, whereas at birth the cells become flat, forming a continuous endothelium sheet; 3) in early fetus, smooth muscle cells exist predominantly in synthetic phenotype, while at birth the cells appear contractile. Despite the presence of synthetic smooth muscle cells and hypercholesterolemia in early fetal life, no accumulation of lipids was evident under transmission electron microscopy.

Fetal aortic cholesterol concentration and metabolism: relationship to plasma cholesterol and potential role of placental factors.

The relationship of fetal plasma cholesterol levels to aortic free and esterified cholesterol concentration was determined in the rabbit during gestation. Plasma cholesterol levels (mg/dl) in early fetuses were markedly high and decreased at term. While aortic free cholesterol content increased with fetal age, cholesteryl ester concentration did not change significantly and no pathological accumulation was evident. This occurred despite high fetal aortic cholesterol esterification activity noted in earlier studies. We evaluated the potential effect of rabbit placental extracts on lipid metabolism and cellular proliferation in fetal aortic explants to explore the role of placental factors in affecting lipid metabolism. Labeled [14C]oleate and [3H]thymidine were used to investigate the rates of incorporation of (a) oleate into lipids, and (b) thymidine into DNA, respectively. Placental extracts at term (but not from 22 days gestation) significantly decreased oleate incorporation (P less than 0.05) into cholesteryl esters, phospholipids and diglycerides. This effect of placental extracts was noted both in absence or presence of serum in the culture medium, and was predominantly found in fraction of Mr greater than 100,000. [3H]Thymidine incorporation (dpm/g protein) into DNA was significantly decreased (P less than 0.01) by placental extracts. These studies suggest that placental extracts contain factor(s) influencing fetal aortic lipid metabolism in culture.

Isolation and characterization of a novel lipoprotein particle from human placental extracts.

While it is believed that placental tissue is very active in lipid metabolism, the nature of lipid containing particles secreted (if any) by this tissue is not known. Lipoprotein profile of human placental tissue was analysed by gel filtration, gel electrophoresis and electron microscopy. Our studies demonstrated the presence of lipoproteins with unusual compositions. A novel major lipoprotein (which eluted in the same position on plasma VLDL) was characterized. While this lipoprotein floated at density greater than 1.006 gr/ml and contained apo B (same as plasma VLDL) it differed from plasma VLDL in a) size, b) contining a significant amount of apo Al, and c) carried bulk of the cholesterol (80% in free form) and phospholipids. This study suggests that placental tissue might contain unique lipoproteins perhaps serving specific metabolic needs.