Reactive oxygen species (ROS) play a key role in chronic liver injury and fibrosis. Homologues of NADPH oxidases (NOXs) are major sources of ROS, but the exact role of the individual homologues in liver disease is unknown. Our goal was to determine the role of NOX4 in liver fibrosis induced by bile duct ligation (BDL) with the aid of the pharmacological inhibitor GKT137831, and genetic deletion of NOX4 in mice. GKT136731 was either applied for the full term of BDL (preventive arm), or starting at 10 days post-operatively (therapeutic arm). Primary hepatic stellate cells (HSC) from control mice with and without BDL were analyzed and the effect of NOX4 inhibition on HSC activation was also studied. FasL or TNFα/actinomycin D induced apoptosis was studied in wild type and NOX4−/− hepatocytes. Results: NOX4 was upregulated by a TGF-β/Smad3-dependent mechanism in HSC. Downregulation of NOX4 decreased ROS production and the activation of NOX4−/− HSC was attenuated. NOX4−/− hepatocytes were more resistant to FasL or TNFα/actinomycin D induced apoptosis. Similarly, after pharmacological NOX4 inhibition, ROS production, the expression of fibrogenic markers and hepatocyte apoptosis were reduced. NOX4 was expressed in human livers with stage 2–3 autoimmune hepatitis. Fibrosis was attenuated by the genetic deletion of NOX4. BDL mice gavaged with GKT137831 both in the preventive or the therapeutic arm displayed less ROS production; significantly attenuated fibrosis and decreased hepatocyte apoptosis. In conclusion, NOX4 plays a key role in liver fibrosis. GKT137831 is a potent inhibitor of fibrosis and hepatocyte apoptosis; therefore it is a promising therapeutic agent for future translational studies.

BACKGROUND & AIMS-Hepatocyte apoptosis and activation of hepatic stellate cells (HSC) are critical events in fibrogenesis. We previously demonstrated that phagocytosis of apoptotic hepatocytes by HSC is profibrogenic. Based on this, as well as the observation that NADPH oxidase induction is central to fibrogenesis, our aim was to study the phagocytic NADPH oxidase, NOX2.

Hepatic stellate cells (HSC), the key fibrogenic cells of the liver, transdifferentiate into myofibroblasts upon phagocytosis of apoptotic hepatocytes. Galectin-3, a β-galactoside-binding lectin, is a regulator of the phagocytic process. In this study, our aim was to study the mechanism by which extracellular galectin-3 modulates HSC phagocytosis and activation. The role of galectin-3 in engulfment was evaluated by phagocytosis and integrin binding assays in primary HSC. Galectin-3 expression was studied by real-time PCR and enzyme-linked immunosorbent assay, and in vivo studies were done in wild-type and galectin-3(-/-) mice. We found that HSC from galectin-3(-/-) mice displayed decreased phagocytic activity, expression of transforming growth factor-β1, and procollagen α1(I). Recombinant galectin-3 reversed this defect, suggesting that extracellular galectin-3 is required for HSC activation. Galectin-3 facilitated the α(v)β(3) heterodimer-dependent binding, indicating that galectin-3 modulates HSC phagocytosis via cross-linking this integrin and enhancing the tethering of apoptotic cells. Blocking integrin α(v)β(3) resulted in decreased phagocytosis. Galectin-3 expression and release were induced in active HSC engulfing apoptotic cells, and this was mediated by the nuclear factor-κB signaling. The upregulation of galectin-3 in active HSC was further confirmed in vivo in bile duct-ligated (BDL) rats. Galectin-3(-/-) mice displayed significantly decreased fibrosis, with reduced expression of α-smooth muscle actin and procollagen α1(I) following BDL. In summary, extracellular galectin-3 plays a key role in liver fibrosis by mediating HSC phagocytosis, activation, and subsequent autocrine and paracrine signaling by a feedforward mechanism.

Advanced glycation end products (AGEs) accumulate in patients with diabetes, yet the link between AGEs and the inflammatory and fibrogenic activity in non-alcoholic steatohepatitis (NASH) has not been explored. TNFα converting enzyme (TACE) is at the center of inflammatory processes. As the main natural regulator of TACE activity is the tissue inhibitor of metalloproteinase 3 (Timp3), we hypothesized that AGEs induce TACE through NADPH oxidase 2 (NOX2); and the downregulation of Sirtuin 1 (Sirt1)/Timp3 pathways mediate fibrogenic activity in NASH. The role of NOX2, Sirt1, Timp3 and TACE were evaluated in the choline deficient L-amino acid defined (CDAA) or western diet-fed wild type (wt) and NOX2−/− mice. To restore Timp3, the mice were injected with Ad-Timp3. Sirt1 and Timp3 expressions were studied in livers from NASH patients, and we found that their levels were significantly lower than in healthy controls. In the wt mice on the CDAA or western diet Sirt1 and Timp3 expressions were lower whereas production of reactive oxidative species and TACE activity significantly increased with an increase in active TNFα production, and the induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen α1 (I), αSMA and TGFβ expression. The NOX2−/− mice on the CDAA or western diet had no significant change in Sirt1, Timp3, TACE activity or the fibrosis markers assessed. In vitro, AGEs exposure decreased Sirt1 and Timp3 in hepatic stellate cells by a NOX2-dependent pathway, and TACE was induced after exposure to AGEs. Conclusion TACE activation is central to the pathogenesis of NASH, and is mediated by AGEs via NOX2 induction and the downregulation of Sirt1/Timp3 pathways.

Hepatocellular carcinoma (HCC) carries a poor prognosis with no effective treatment available other than liver transplantation for selected patients. Vascular invasion of HCC is one of the most important negative predictor of survival. As the regulation of invasion of HCC cells is not well understood, our aim was to study the mechanisms by which galectin 3, a β-galactosidase binding lectin mediates HCC cell migration. HCC was induced by N-diethylnitrosamine (DEN) in wild type and galectin 3−/− mice, and tumor formation, histology, and tumor cell invasion were assessed. The galectin 3−/− mice developed significantly smaller tumor burden with a less invasive phenotype than the wild type animals. Galectin 3 was upregulated in the wild type HCC tumor tissue, but not in the surrounding parenchyma. Galectin 3 expression in HCC was induced by NF-κB transactivation as determined by chromatin immunoprecipitation assays. In vitro studies assessed the pro-migratory effects of galectin 3. The migration of hepatoma cells was significantly decreased after transfection by the galectin 3 siRNA and also after using the Rho kinase (ROCK) inhibitor Y-27632. The reorganization of the actin cytoskeleton, RhoA GTP ase activity and the phosphorylation of MLC2 were decreased in the galectin 3 siRNA-transfected cells. In addition, in vitro and in vivo evidence showed that galectin 3 deficiency reduced hepatoma cell proliferation and increased their apoptosis rate. In conclusion, galectin 3 is an important lectin that is induced in HCC cells, and promotes hepatoma cell motility and invasion by an autocrine pathway. Targeting galectin 3 therefore could be an important novel treatment strategy to halt disease progression.