NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.

Sinusoidal Endothelial Dysfunction Precedes Inflammation and Fibrosis in a Model of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Most morbidity associated with the metabolic syndrome is related to vascular complications, in which endothelial dysfunction is a major pathogenic factor. However, whether NAFLD is associated with endothelial dysfunction within the hepatic vasculature is unknown. The aims of this study were to explore, in a model of diet-induced overweight that expresses most features of the metabolic syndrome, whether early NAFLD is associated with liver endothelial dysfunction. Wistar Kyoto rats were fed a cafeteria diet (CafD; 65% of fat, mostly saturated) or a control diet (CD) for 1 month. CafD rats developed features of the metabolic syndrome (overweight, arterial hypertension, hypertryglyceridemia, hyperglucemia and insulin resistance) and liver steatosis without inflammation or fibrosis. CafD rats had a significantly higher in vivo hepatic vascular resistance than CD. In liver perfusion livers from CafD rats had an increased portal perfusion pressure and decreased endothelium-dependent vasodilation. This was associated with a decreased Akt-dependent eNOS phosphorylation and NOS activity. In summary, we demonstrate in a rat model of the metabolic syndrome that shows features of NAFLD, that liver endothelial dysfunction occurs before the development of fibrosis or inflammation.