Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase–generated superoxide
Adam C. Straub, Katherine A. Clark, Mark A. Ross, Ashwin G. Chandra, Song Li, Xiang Gao, Patrick J. Pagano, Donna B. Stolz, Aaron Barchowsky
Adam C. Straub, Katherine A. Clark, Mark A. Ross, Ashwin G. Chandra, Song Li, Xiang Gao, Patrick J. Pagano, Donna B. Stolz, Aaron Barchowsky
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Research Article Hepatology

Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase–generated superoxide

Abstract

Environmental arsenic exposure, through drinking contaminated water, is a significant risk factor for developing vascular diseases and is associated with liver portal hypertension, vascular shunting, and portal fibrosis through unknown mechanisms. We found that the addition of low doses of arsenite to the drinking water of mice resulted in marked pathologic remodeling in liver sinusoidal endothelial cells (SECs), including SEC defenestration, capillarization, increased junctional PECAM-1 expression, protein nitration, and decreased liver clearance of modified albumin. Furthermore, the pathologic changes observed after in vivo exposure were recapitulated in isolated mouse SECs exposed to arsenic in culture. To investigate the role of NADPH oxidase–generated ROS in this remodeling, we examined the effect of arsenite in the drinking water of mice deficient for the p47 subunit of the NADPH oxidase and found that knockout mice were protected from arsenite-induced capillarization and protein nitration. Furthermore, ex vivo arsenic exposure increased SEC superoxide generation, and this effect was inhibited by addition of a Nox2 inhibitor and quenched by the cell-permeant superoxide scavenger. In addition, inhibiting either oxidant generation or Rac1-GTPase blocked ex vivo arsenic-stimulated SEC differentiation and dysfunction. Our data indicate that a Nox2-based oxidase is required for SEC capillarization and that it may play a central role in vessel remodeling following environmentally relevant arsenic exposures.

Authors

Adam C. Straub, Katherine A. Clark, Mark A. Ross, Ashwin G. Chandra, Song Li, Xiang Gao, Patrick J. Pagano, Donna B. Stolz, Aaron Barchowsky

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Figure 6

Arsenic-stimulated superoxide generation is inhibited by tempol and gp91ds-tat peptide.

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Arsenic-stimulated superoxide generation is inhibited by tempol and gp91...
(A) Primary SECs isolated from mice were pre-loaded with 5 μM dihydroethidium for 10 min prior to a 30-min arsenite exposure. Cells were fixed and imaged for hydroethidium (HE; red) fluorescence and DAPI (blue) stained nuclei. Scale bar: 10 μm. (B) Percent (mean ± SD) positive hydroethidium staining, normalized to the percentage of positive nuclei staining (n = 4 cultures from 2 livers). (C) SECs were preloaded with 5 μM dihydroethidium with and without 1 mM tempol 10 min prior to a 30-min, 2.5-μM arsenite exposure and then imaged and analyzed as in B (n = 4 cultures). (D) Cells were preincubated with dihydroethidium in the presence or absence of 10 μM gp91ds-tat (gp-tat) or scrambled-tat (scr-tat) peptide for 30 min prior to a 2.5-μM arsenite exposure (n = 6 cultures from 3 livers). In C and D, significance of differences was determined by ANOVA followed by Newman-Keuls post test. **P < 0.01, ***P < 0.001 versus control; ###P < 0.001 versus arsenite.