Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver - PubMed (original) (raw)

Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver

Adam C Straub et al. Hepatology. 2007 Jan.

Abstract

Trivalent arsenic [As(III)] is a well-known environmental toxicant that causes a wide range of organ-specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)-induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)-exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM-1 and laminin-1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin-1 expression and SEC caveolae, as well as increased membrane-bound Rac1-GTPase.

Conclusion: These data demonstrate that exposure to As(III) causes functional changes in SEC signaling for sinusoidal capillarization that may be initial events in pathogenic changes in the liver.

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Conflict of interest statement

Potential conflict of interest: Dr. Stoltz and Dr. Barchowsky received grants from NIH. Dr. Straub received grants from University of Pittsburgh. Dr. Savay received grants from Dartmouth Medical School.

Figures

Fig. 1

Fig. 1

Arsenic-stimulated capillarization of the liver sinusoidal endothelium. SEM images of sinusoidal vessels were obtained from thick sections of livers excised from control mice or mice exposed to 250 ppb As(III) for 5 weeks. In the graph, data are presented as mean ± SD porosity (open bars = control, closed bars = As(III)-exposed, n = 3), as determined in experimental procedures. Two-way ANOVA and Bonferroni’s post-test demonstrated no significant differences between zones, a highly significant effect of arsenic exposure relative to control (***P < 0.001).

Fig. 2

Fig. 2

Arsenic-stimulated capillarization, basement membrane formation, and increased hepatocyte microvilli. (A) TEM images of sinusoidal vessels were captured from ultrathin sections of livers. Representative images are presented with portions magnified (B) to illustrate changes in the space of Disse and to show increased caveolae (arrows) in As(III)-exposed mice. (Bars = 500 nm) Abbreviations: SD, space of Disse; L, sinusoid lumen.

Fig. 3

Fig. 3

Arsenic(III) induced expression of sinusoidal PECAM-1 and laminin protein. (A) Cryosections were immunostained for PECAM-1 (green) or laminin-1 (red). Merged images show DRAQ 5 stained nuclei (blue) (bar = 50 μm). (B) Images of portal vein and periportal PECAM-1 (green) and nuclear staining demonstrate PECAM changes in sinusoids, but not in the portal vein endothelium (L = lumen). (C) Quantitative morphometric analysis of midlobular PECAM-1 and laminin-1 protein staining is presented as the mean ± SD percentage of total positive-staining pixels for the respective protein per 400× microscopic field (**P < 0.01 and *P < 0.05, n = 5 mice).

Fig. 4

Fig. 4

As(III) stimulates vascularization of the PBVP and hepatic artery contraction. Serial sections were prepared from livers from control or As(III)-exposed mice. Sections were stained with either hematoxylin & eosin [(A) and (C)] or immunostained for PECAM-1 (green) and α-SMC actin (red) [(B) and (D)]. Final image magnification was 400× (BD = biliary ducts, HA = hepatic arteries). (E) The mean ± SD of PECAM-positive structures [* in images (B) and (D)] surrounding at least 2 biliary ducts per section from 5 mice is presented (***P < 0.001). (F) The luminal areas of hepatic arterioles were calculated. The data are the mean ± SD of the average luminal areas of at least 2 hepatic arterioles per section from 5 mice in each group. (**P < 0.01).

Fig. 5

Fig. 5

Co-localization of As(III)-stimulated caveolin-1 and PECAM-1 protein expression. Sections of livers were immunostained for PECAM-1 (green) or caveolin-1 (red). Nuclei were stained with DRAQ 5 (blue). The white bar = 25 μm. The graph presents mean ± SD percentage of caveolin-1 positive pixels per 400× microscopic field (**P < 0.01; n = 5).

Fig. 6

Fig. 6

Chronic As(III)-stimulated mobilization of Rac1 to SEC luminal membranes. Luminal SEC membranes of control and As(III) exposed mice were separated from total liver cell membranes (light fraction), as described in “Materials and Methods”. Rac1 and actin (loading control) abundance was measured by Western blotting. Each lane presents protein abundance in liver and SEC membrane fractions from individual mice.

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