Background-Oxidative stress plays important role in the pathogenesis of atherosclerosis and coronary artery disease (CAD). We aimed to determine the sources and selected molecular mechanisms of oxidative stress in CAD. Methods and Results-We examined basal and NAD(P)H oxidase-mediated superoxide (O 2 · Ϫ ) production using lucigenin chemiluminescence, ferricytochrome c and dihydroethidium fluorescence in human coronary arteries from 19 CAD and 17 non-CAD patients undergoing heart transplantation. NAD(P)H oxidase subunits and xanthine oxidase expression were measured. Superoxide production was greater in coronary arteries from patients with CAD, even in vessels without overt atherosclerotic plaques, and was doubled within branching points of coronary arteries. Studies using pharmacological inhibitors and specific substrates showed that NAD(P)H oxidases (60%) and xanthine oxidase (25%) are primary sources of O 2 · Ϫ in CAD. Losartan significantly inhibited superoxide production in coronary arteries. NAD(P)H oxidase activity and protein levels of the NADPH oxidase subunits p22phox, p67phox, and p47phox were significantly increased in CAD, as were mRNA levels for p22phox and nox2, and no NAD(P)H oxidase subunit mRNA levels correlated with NAD(P)H oxidase activity in vessels from individual patients. Activity and protein expression of xanthine oxidase were increased in CAD, whereas xanthine dehydrogenase levels were not changed. Key Words: endothelium Ⅲ NAD(P)H oxidase Ⅲ nitric oxide Ⅲ oxidant stress Ⅲ reactive oxygen species I ncreased vascular production of reactive oxygen species is a characteristic feature of vascular disease states, including coronary artery disease (CAD). In particular, superoxide (O 2 · Ϫ ) and products of O 2 · Ϫ promote atherosclerosis by quenching nitric oxide (NO) and activating redox-sensitive signaling pathways 1 that modulate vessel remodeling and plaque stability. 2 Accordingly, endothelial dysfunction associated with overproduction of O 2 · Ϫ has been shown to provide prognostic information in patients with coronary artery disease. 3,4 Potential sources of vascular O 2 · Ϫ include the NAD(P)H oxidases, xanthine oxidase, cyclooxygenases, nitric oxide synthases, or mitochondrial oxidases. 5 Recent studies have shown that the NAD(P)H oxidases and xanthine oxidase have important roles in human vessels. 6 Azumi et al first showed that NAD(P)H oxidase is present in human coronary arteries. 7 Several molecular homologues of the NAD(P)H oxidase large membrane subunit, (gp91phox; nox2) are present in vascular cells, 5 and could contribute to ROS production during development of CAD. More recent studies have shown that NAD(P)H oxidase subunit expression is correlated with both severity of atherosclerosis 5 and with features of plaque stability in human coronary arteries. 8 Despite these important findings, a systematic analysis of the sources of O 2 · Ϫ production in human coronary arteries is lacking. Furthermore, it is not clear how coronary O 2 · Ϫ production is regulated in the presence of ...

BACKGROUND AND PURPOSEInflammation plays a key role in atherosclerosis. The protective role of angiotensin 1-7 (Ang-(1-7)) in vascular pathologies suggested the therapeutic use of low MW, non-peptide Ang-(1-7) mimetics, such as AVE0991. The mechanisms underlying the vasoprotective effects of AVE0991, a Mas receptor agonist, remain to be explored. EXPERIMENTAL APPROACHWe investigated the effects of AVE0991 on the spontaneous atherosclerosis in apolipoprotein E (ApoE)À/À mice, in the context of vascular inflammation and plaque stability. KEY RESULTSAVE0991 has significant anti-atherosclerotic properties in ApoEÀ/À mice and increases plaque stability, by reducing plaque macrophage content, without effects on collagen. Using the descending aorta of chow-fed ApoEÀ/À mice, before significant atherosclerotic plaque develops, we gained insight to early events in atherosclerosis. Interestingly, perivascular adipose tissue (PVAT) and adventitial infiltration with macrophages and T-cells precedes atherosclerotic plaque or the impairment of endothelium-dependent NO bioavailability (a measure of endothelial function). AVE0991 inhibited perivascular inflammation, by reducing chemokine expression in PVAT and through direct actions on monocytes/macrophages inhibiting their activation, characterized by production of IL-1β, TNF-α, CCL2 and CXCL10, and differentiation to M1 phenotype. Pretreatment with AVE0991 inhibited migration of THP-1 monocytes towards supernatants of activated adipocytes (SW872). Mas receptors were expressed in PVAT and in THP-1 cells in vitro, and the anti-inflammatory effects of AVE0991 were partly Mas dependent. CONCLUSIONS AND IMPLICATIONSThe selective Mas receptor agonist AVE0991 exhibited anti-atherosclerotic and anti-inflammatory actions, affecting monocyte/macrophage differentiation and recruitment to the perivascular space during early stages of atherosclerosis in ApoEÀ/À mice.

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2•−) production in human AAA.Methods and resultsAAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2•− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size.ConclusionsIncreased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients.

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Our results show for the first time that inhibition of FLAP by MK-886 reduces development of atherosclerosis in gene-targeted apoE/LDLR-DKO mice.

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BackgroundMitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice.Methods and ResultsAlda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids.ConclusionsCollectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation.