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The underlying mechanisms that perpetuate liver inflammation in nonalcoholic steatohepatitis are poorly understood. We explored the hypothesis that cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in metabolic forms of fatty liver disease. Male wild-type (WT) C57BL6/N or peroxisome proliferator-activated receptor ␣ knockout (PPAR-␣ ؊/؊ ) mice were fed a lipogenic, methionine-and choline-deficient (MCD) diet or the same diet with supplementary methionine and choline (control). COX-2 was not expressed in livers of mice fed the control diet. In mice fed the MCD diet, hepatic expression of COX-2 messenger RNA and protein occurred from day 5, continued to rise, and was 10-fold higher than controls after 5 weeks, thereby paralleling the development of steatohepatitis. Upregulation of COX-2 was even more pronounced in PPAR

In mice genetically predisposed to obesity and diabetes, HF feeding leads to restriction of adipose tissue for accommodation of excess energy, causing lipid partitioning into liver, and transformation of simple steatosis to fibrosing steatohepatitis. The way in which HF feeding 'saturates' adipose stores, decreases serum adiponectin and causes hepatic inflammation in steatohepatitis may provide clues to pathogenesis of NASH in metabolic syndrome.

Methionine and choline deficiency lower IGF-1 to de-repress p53 during induction of steatohepatitis. The p53 induced by nutritional stress is biologically active in mediating mitochondrial cell death pathways, but may also be responsible for TRAIL receptor expression, thereby linking intrinsic and exogenous apoptosis pathways in NASH.

Background-Probucol remains the only conventional drug that reduces restenosis after coronary angioplasty. Apart from its weak cholesterol-lowering effect, probucol has antioxidant properties, but it remains unclear how this drug inhibits restenosis. Methods and Results-Aortic balloon-injured New Zealand White rabbits were fed 2% (wt/wt) cholesterol-enriched or normal chow, with 0.75% (wt/wt) probucol (P) or without (controls, C) for 6 weeks. Endothelial denudation of the abdominal aorta was performed at week 3 with a 3F Fogarty embolectomy catheter. The arteries were harvested after week 6 and analyzed for histology, lipids and antioxidants, and endothelial regeneration and function. Probucol significantly decreased aortic intima-to-media ratio (cholesterol-fed: C, 1.10Ϯ0.08 versus P, 0.70Ϯ0.10; normal: C, 0.89Ϯ0.02 versus P, 0.83Ϯ0.05; PϽ0.05) and the numbers of proliferating intimal smooth muscle cells and lowered serum cholesterol without altering the proportion of aortic lipids that was oxidized. Probucol promoted endothelial regeneration in the injured aorta in cholesterol-fed rabbits (25% increase in reendothelialization, PϽ0.05) and in those on normal chow (37% increase, PϽ0.01). This was associated with both improved endothelial function as assessed by enhanced aortic ring relaxation and cGMP production in response to acetylcholine and decreased intimal thickening. Conclusions-Probucol inhibits intimal thickening in balloon-damaged arteries of rabbits by promoting the regeneration of functional endothelium, without affecting the proportion of aortic lipids that was oxidized. This novel in vivo finding helps explain how probucol inhibits restenosis after coronary angioplasty and highlights potential new targets for therapeutic intervention.

Objective-To determine how hypochlorous acid (HOCl), the principal product of myeloperoxidase, modulates vascular function. Methods and Results-Rabbit arterial rings exposed to HOCl (0 to 500 mol/L) exhibited dose-and time-dependent impairment of endothelium-dependent arterial relaxation to acetylcholine and A23187, but not the NO donor, diethylamine NONOate, suggesting that HOCl targets the endothelium. This effect was not because of cytotoxicity, as HOCl treatment produced no significant change in endothelial cell morphology or lactate dehydrogenase release. We observed HOCl-mediated endothelial cell protein oxidation by immunoreactivity to HOP-1, a monoclonal antibody specific for HOCl-oxidized protein.In support of this notion, known HOCl scavengers, such as methionine and N-acetylcysteine, partially preserved endothelium-derived NO bioactivity in response to HOCl. In an unanticipated observation, HOCl-mediated impairment of NO bioactivity was prevented by manganese superoxide dismutase in a manner dependent on its enzymatic activity. Finally, we found that HOCl reduced endothelial nitric oxide synthase dimer stability, an effect that was also inhibited by superoxide dismutase. Conclusions-Taken together, these data indicate that HOCl imparts a defect in endothelial NO production due to a superoxide-dependent reduction in endothelial nitric oxide synthase dimer stability. These data provide another mechanism whereby myeloperoxidase-derived oxidants can contribute to the impairment of NO bioactivity that is characteristic of atherosclerosis. Key Words: oxidant stress Ⅲ nitric oxide Ⅲ endothelium Ⅲ superoxide A therosclerosis and its associated pathological conditions, such as hypercholesterolemia 1 and diabetes mellitus, 2 are characterized by abnormal endothelium-derived nitric oxide (EDNO) bioactivity. In atherosclerosis, vasodilatation to agonists for EDNO release, such as acetylcholine 3 or aggregating platelets, 4 is impaired or replaced by constriction. Endothelial dysfunction has been directly demonstrated in atherosclerotic human coronary arteries, 3 and there is strong evidence that it contributes to plaque activation and the pathogenesis of myocardial ischemia. 5 In fact, the presence of vascular dysfunction predicts the occurrence of clinical events in patients with atherosclerosis. 6 Available evidence suggests that vascular oxidative events play an important role in endothelial dysfunction. In animal models of atherosclerosis and hypercholesterolemia, the vascular production of superoxide anion radical is increased, 7 and this abnormality can decrease NO bioactivity through peroxynitrite formation. 8 Indeed, limiting superoxide production with oxypurinol, a xanthine oxidase inhibitor, partially restores EDNO bioactivity. 7 Similarly, in atherosclerotic rabbits, chronic treatment with polyethylene glycolatedsuperoxide dismutase (SOD) increases vascular SOD activity and partially restores NO-mediated relaxation to acetylcholine. 9 Later stages of atherosclerosis, however, are more complex. In Watan...

Increased vascular disease occurs with low albumin (human serum albumin, HSA), possibly reflecting specific inhibition of endothelial apoptosis reported for tissue culture. Despite the reported specificity for endothelial protection by HSA, the high but physiological concentrations needed appear more consistent with non-specific low-affinity interactions. We reconcile this contradiction by demonstrating protection is mediated by a partially cryptic HSA protein domain, which becomes more exposed and active following cyanogen bromide fragmentation (p < 0.001). Also, although others reported HSA radical scavenging and bound lipids as important for inhibiting apoptosis in non-endothelial cell types, we demonstrate the protective effect for endothelium is unaffected when HSA radical scavenging is blocked by alkylation, or following delipidation. Further probing the mechanism responsible, we found that the G-coupled protein inhibitors pertussis toxin and suramin reduced protection of endothelium by HSA (p < 0.005), while the tyrosine kinase inhibitor genistein had no effect. Consistent with a role for phosphoinositide 3 kinase (PI3K) was inhibition by both wortmannin and LY294002 (p < 0.05), as well as phosphorylation of Akt, while MAP kinase inhibitors had no effect. We conclude the active site in HSA inhibiting endothelial apoptosis is partially cryptic, and acts via a G-coupled protein PI3K-dependent mechanism.

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