Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

RESEARCH ARTICLE Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation Tian Lan1,2, Tatiana Kisseleva1, David A. Brenner1* 1 Department of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Vascular Biology Research Institute, Guangdong Pharmaceutical University, Guangzhou, China * Abstract OPEN ACCESS Citation: Lan T, Kisseleva T, Brenner DA (2015) Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation. PLoS ONE 10(7): e0129743. doi:10.1371/journal.pone.0129743 Editor: Matias A Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN Received: January 12, 2015 Accepted: May 12, 2015 Published: July 29, 2015 Copyright: © 2015 Lan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as profibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Natural Science Foundation of China (No. 81200308) and the Zhujiang Technology New Star of Guangzhou City (2013J2200025) (to Tian Lan), and by the National Institutes of Health (R01 GM041804-26, P50 AA011999-16, P42 ES010337-13, U01 AA02185602, R01 DK101737-01A1, U01 AA022614-01A1, R01 DK099205-01A1). Introduction Liver fibrosis occurs as a result of chronic liver disease and is associated with severe morbidity and mortality [1]. Chronic oxidative stress is an important etiological factor in initiating the fibrogenic process in the liver [2]. Hepatic stellate cells (HSCs) are endogenous, liver-specific mesenchymal cells that play pivotal roles in liver inflammation and fibrogenesis [1]. In the PLOS ONE | DOI:10.1371/journal.pone.0129743 July 29, 2015 1 / 19 NOX1 and NOX4 in Liver Fibrosis Competing Interests: The authors have declared that no competing interests exist. normal liver, HSCs are quiescent, desmin-positive cells, containing vitamin A lipid droplets. Upon activation by liver injury, quiescent HSCs become activated HSCs, characterized by expression of α-smooth muscle actin (α-SMA) [3], producing inflammatory cytokines, chemokines and extracellular matrix proteins [4] [5]. Reactive oxygen species (ROS) are generated by various liver injuries such as alcohol abuse, hepatitis virus infection and chronic cholestasis and contribute to hepatic fibrogenesis [6]. ROS stimulates the production of the Collagen I, acting as an intracellular signaling mediator of the fibrogenic action of TGF-β1 [7]. The multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme complexes and the mitochondrial respiratory pathway are the two major producers of endogenous ROS [8]. NOX play a central role in liver fibrogenesis. Among the seven members of the NOX family, NOX1 is structurally and functionally similar to NOX2, the classic NOX that generates the oxidative burst in neutrophil killing. Studies by us and others have shown that NOX1 and NOX2 are expressed in HSCs and deficiencies of NOX1 or NOX2 decrease liver inflammation and fibrosis in the carbon tetrachloride (CCL4) and bile duct ligation (BDL) models [5, 9]. Angiotensin II (Ang II) also induces NOX1 to promote HSCs proliferation and aggravate liver fibrosis [5, 9]. In contrast, NOX4, a nonphagocytic NOX homolog is expressed in the liver, and is different from the other NOX isoforms because it does not require the recruitment of cytosolic structural subunits to form the active enzyme to produce ROS [10, 11]. NOX4 is critical in lung and kidney fibrosis by activating and transforming of myofibroblasts [12, 13]. In the liver, NOX4 is expressed in hepatocytes, stellate cells, and endothelial cells [14]. NOX4 has been found to be upregulated in hepatitis virus C, and to contribute to the formation of ROS, most likely via TGF-β1 induction [10]. The role of NOX4 in liver injury and fibrosis has only been assessed in the BDL model using NOX4 deficient mice [15]. A concern about these previous studies is that they were performed by breeding homozygous knock-out mice compared to wild type strain matched control mice, which could result in artifact genetic drift in the two groups. Recently, small molecule NOX1/4 dual inhibitors such as GKT137831 have been developed that show good orally bioavailability and tolerability when administered orally in animal model of pulmonary fibrosis [16] and liver fibrosis [15]. Thus, we hypothesize that deficiency of either NOX1 or NOX4 attenuates HSCs activation and liver fibrosis. The overall goal of this study was to determine the roles of NOX1 and NOX4 on the proliferative and fibrogenic phenotypes of HSCs and its contribution to liver fibrosis. We report for the first time a direct comparison of the long-term effects of NOX1 and NOX4 deficiency in the development and progression of liver fibrosis, by comparing liver fibrosis in CCl4-induced NOX1KO and NOX4KO mice and their respective wild-type (WT) littermates. Our results demonstrate that both NOX1 a (...truncated)