Hepatic stellate cell specific endoglin deletion modulates TGF-β signaling and aggravates liver fibrosis in two experimental models of murine liver injury

M Alsamman; V Sterzer; S Meurer; H Sahin; R Weiskirchen; C Trautwein; D Scholten

doi:10.1055/s-0033-1360866

Zeitschrift für Gastroenterologie, Inhaltsverzeichnis

Hepatic stellate cell specific endoglin deletion modulates TGF-β signaling and aggravates liver fibrosis in two experimental models of murine liver injury

M Alsamman ¹, V Sterzer ¹, S Meurer ², H Sahin ¹, R Weiskirchen ², C Trautwein ¹, D Scholten ¹

¹University Hospital RWTH Aachen, Department of Medicine III, Aachen, Germany
²University Hospital RWTH Aachen, Institute of Clinical Chemistry and Pathobiochemistry, Aachen, Germany

Abstract

Background: Hepatic stellate cells (HSC) are the major source for extracellular matrix (ECM) production in liver fibrosis. Endoglin (ENG) is a type III auxiliary receptor for TGF-β that is expressed on HSCs. Due to different splicing there are two ENG isoforms, L- and S-Endoglin, which modulate TGF-β signaling differentially. TGF-β is the a profibrotic cytokine expressed in response to liver injury. This study analyzes the role of ENG and TGF-β signaling in two models of experimental liver fibrosis by cell line specific endoglin deletion in HSCs.

Methods: Using the Cre-LoxP genetic recombination system we created HSC specific ENG(-/-) mice by crossing GFAP-Cre to ENG(flox/flox) mice. Mice were subjected to liver injury by CCl4 treatment (8 weeks) or bile duct ligation (BDL) (21 days). Liver fibrosis was analyzed by hydroxyproline measurement and Sirius Red. For in vitro analysis HSCs from ENG(flox/flox) mice were isolated and treated with adenoviral CMV-Cre-RSV-GFP expressing vector. TGF-β signaling was analyzed by western blots and qPCR. The expression of L- and S-Endoglin was analyzed in wildtype mice by qPCR in response to liver injury.

Results: In response to liver injury the balance of L- and S-Endoglin expression changes in HSCs of wildtype mice. Whereas L-Endoglin is the predominantly expressed isoform, the expression of S-Endoglin is increased 3-fold after CCl4 treatment. Analyzing fibrotic effects of ENG deficiency, livers of GFAP-Cre(+)-ENG(d/d) mice (n = 12) showed 43% (p < 0.01) higher hydroxyproline content compared to GFAP-Cre(-)-Eng(f/f) litter mates reflecting increased liver fibrosis. Sirius red stainings underlined these findings, showing 48% (p < 0.05) more collagen deposition in livers of Cre(+) mice with endoglin deficient stellate cells. Similar results could be obtained for mice subjected to bile duct ligation. Compared to GFAP-Cre(-)-ENG(f/f) mice GFAP-Cre(+)-ENG(d/d) litter mates (n = 8) showed 100% (p = 0.02) more Collagen deposition in Sirius Red stained liver sections. Hydroxyproline levels were increased by 46% (p < 0.01). Western blot analysis of TGF-β signaling in vitro using adenoviral Cre treated ENG(flox/flox) primary HSCs demonstrated a differential effect of ENG on TGF-β signaling most likely due to opposing effects of ENG isoforms. In cell culture experiments using a novel murine HSC cell line overexpression of ENG resulted in a downregulation of Collagen and CTGF expression.

Conclusion: Of both ENG isoforms L-Endoglin is expressed predominantly in the liver. However, in response to injury S-Endoglin expression is 3-fold increased. Endoglin deficiency in hepatic stellate cells significantly aggravates liver fibrosis in response to injury. L- and S Endoglin promote TGF-β signaling through different Smad proteins resulting in activating (L-ENG) or quiescing (S-ENG) effects. This study suggests that S-Endogin modulated TGF-β signaling can have a protective effect against liver fibrosis.