NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis
Ramón Bataller, … , John J. Lemasters, David A. Brenner
Ramón Bataller, … , John J. Lemasters, David A. Brenner
Published November 1, 2003
Citation Information: J Clin Invest. 2003;112(9):1383-1394. https://doi.org/10.1172/JCI18212.
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Article Hepatology

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

Abstract

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II–induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox–/– mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox–/– mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47phox–/– mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Authors

Ramón Bataller, Robert F. Schwabe, Youkyung H. Choi, Liu Yang, Yong Han Paik, Jeffrey Lindquist, Ting Qian, Robert Schoonhoven, Curt H. Hagedorn, John J. Lemasters, David A. Brenner

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Figure 1

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Ang II increases ROS formation and lipid peroxidation in human HSCs via ...
Ang II increases ROS formation and lipid peroxidation in human HSCs via NADPH activity. (a) HSCs were loaded with DCFDA (10 mM) and studied with laser confocal microscopy. Ang II (10–8 M) markedly increased cell fluorescence. This effect was prevented by DPI (10–6 M). (b) Quantification of fluorescent changes in DCFDA-loaded cells using a fluorometer at excitation and emission wavelengths of 488 nm and 520 nm, respectively. Ang II increased cell fluorescence that was blocked by DPI and losartan (10–7 M). Data are representative of three independent experiments. (c) Expression of mRNA encoding p47phox, gp91phox, and Nox1 in human HSCs, as assessed by RT-PCR. Quiescent HSCs (2 days in culture after isolation from a normal human liver), human HSCs activated in culture (cells isolated from normal livers and cultured for 14 days), and HSCs activated in vivo (2 days in culture after isolation from a human cirrhotic liver) were studied. GAPDH was amplified to demonstrate equal loading. (d) Ang II phosphorylates p47phox in human HSCs. Cells were treated with Ang II for 5 minutes, and cell extracts were immunoprecipitated with anti-p47phox antibody and blotted with either anti-p47phox antibody or anti-phosphoserine (P-serine) antibody. (e) Ang II induces HNE protein adducts and upregulates heme oxygenase-1 (HO-1) protein expression in HSCs. HSCs were stimulated for 12 hours with Ang II, and cell extracts were blotted with anti-HNE and anti–heme oxygenase-1 antibodies. Cells were preincubated with buffer, DPI, or losartan before addition of Ang II. α-Tubulin was used to demonstrate equal loading.