Contribution of Cyclooxygenase End Products and Oxidative Stress to Intrahepatic Endothelial Dysfunction in Early Non-Alcoholic Fatty Liver Disease.

INTRODUCTION:Metabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD. MATERIALS AND METHODS:Sprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels. RESULTS:HFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response. CONCLUSIONS:Our study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease

Metabolic syndrome assessment.

<p>(A) Rat body weight. Rats fed with HFD (n = 21) increased rat body weight compared with CD rats (n = 19). (B) Glucose intolerance. HFD (n = 5) rats exhibited higher fasting hyperglycemia and at every time point after glucose administration compared with CD (n = 3) rats. (C) Arterial hypertension. Higher mean arterial pressure (MAP), diastolic arterial pressure (DAP) and systolic arterial pressure (SAP) were observed in HFD (n = 4) rats as compared with CD rats (n = 6). Values are mean ± SEM.</p

Characteristics of rats receiving control diet (CD) and high-fat diet (HFD).

<p>Characteristics of rats receiving control diet (CD) and high-fat diet (HFD).</p

In vivo assessment of liver circulation.

<p>Portal pressure was increased in HFD rats (n = 7) compared with CD rats (n = 9). This increase was associated with a significant change in portal blood flow reflecting an increase in hepatic vascular resistance. Values are mean ± SEM.</p

Oxidative stress and nitric oxide (NO).

<p>(A) <i>Left panel</i>: <i>Top</i>. Representative western blot of nitrotyrosinated proteins (3-NT) of livers from HFD and CD rats (n = 4, in each group). <i>Bottom</i>. Densitometry analysis of 3-NT to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio. HFD rats exhibited a higher level of oxidative stress. <i>Right panel</i>: Malondialdehyde (MDA) content in HFD and CD rats (n = 5, in each group). (B) <i>Top</i>. Representative western blot of phosphorylated endothelial NO synthase (p-eNOS) of livers from HFD and CD rats (n = 4, in each group). <i>Bottom</i>. Densitometry analysis of p-eNOS to GAPDH ratio. eNOS activity was decreased in HFD rats. (C) NO bioavailability. GMPc intrahepatic levels were significantly different between CD and HFD rats (n = 4, in each group). Values are mean ± SEM.</p

Isolated perfused liver studies.

<p>(A) Endothelium-dependent relaxation to acetylcholine (ACh) after preincubation with N^W-nitro-L-arginine (L-NNA) or vehicle in CD and HFD rats. Vasodilation in CD rats (n = 5) was significantly reduced by endothelial NO synthase inhibition compared with vehicle (n = 4). Conversely, in HFD rats relaxation to ACh was unaffected by L-NNA (n = 4) compared with vehicle (n = 5). (B) Cyclooxygenase blockade and antioxidant preincubation improved vasodilator response in NAFLD rats. HFD preincubated with indomethacin (n = 5) or tempol (n = 4), improved endothelial dysfunction compared with vehicle (n = 4). (C) Effect of SQ29548 on the concentration-response curve to ACh in rats with NAFLD. Tromboxane A2 receptor selective antagonist (n = 4) did not improve vasodilation compared with vehicle (n = 4).</p