Liver fibrosis is driven by protease-activated receptor-1 expressed by hepatic stellate cells in experimental chronic liver injury

Background: Blood coagulation protease activity is proposed to drive hepatic fibrosis through activation of protease-activated receptors (PARs). Whole-body PAR-1 deficiency reduces experimental hepatic fibrosis, and in vitro studies suggest a potential contribution by PAR-1 expressed by hepatic stellate cells. However, owing to a lack of specific tools, the cell-specific role of PAR-1 in experimental hepatic fibrosis has never been formally investigated. Using a novel mouse expressing a conditional PAR-1 allele, we tested the hypothesis that PAR-1 expressed by hepatic stellate cells contributes to hepatic fibrosis.

Methods: PAR-1^flox/flox mice were crossed with mice expressing Cre recombinase controlled by the lecithin retinol acyltransferase (LRAT) promoter, which induces recombination in hepatic stellate cells. Male PAR-1^flox/flox/LRATCre and PAR-1^flox/flox mice were challenged twice weekly with carbon tetrachloride (CCl₄, 1 mL/kg i.p.) for 6 weeks to induce liver fibrosis.

Results: PAR-1 mRNA levels were reduced (>95%) in hepatic stellate cells isolated from PAR-1^flox/flox/LRATCre mice. Hepatic stellate cell activation was evident in CCl₄-challenged PAR-1^flox/flox mice, indicated by increased α-smooth muscle actin labeling and induction of several profibrogenic genes. CCl₄-challenged PAR-1^flox/flox mice displayed robust hepatic collagen deposition, indicated by picrosirius red staining and type I collagen immunolabeling. Notably, stellate cell activation and collagen deposition were significantly reduced (>30%) in PAR-1^flox/flox/LRATCre mice. Importantly, the reduction in liver fibrosis was not a consequence of reduced acute CCl₄ hepatotoxicity in PAR-1^flox/flox/LRATCre mice.

Conclusions: The results constitute the first direct experimental evidence that PAR-1 expressed by stellate cells directly promotes their profibrogenic phenotype and hepatic fibrosis in vivo.