Therefore, primary cholangiocytes from the double-transgenic mice are capable of adopting a myofibroblast phenotype in vitro, as has been reported for mouse and human cholangiocytes by others.7, 34, 35 To determine whether EMT occurs in vivo, we induced liver fibrosis click here in Alfp-Cre × Rosa26-YFP mice by BDL. Sirius Red staining demonstrated progressively increasing hepatic fibrosis at 2, 4, and 8 weeks post-BDL (Fig. 3; Supporting Information Fig. 2). At these timepoints (Figs. 4, 5; Supporting Information Fig. 3), there was no evidence of YFP colocalization with the mesenchymal markers S100A4, vimentin, α-SMA, or procollagen 1α2,

despite significant peribiliary staining for these markers compared with untreated controls (Supporting Information Figs. 1C, 5). Occasional YFP-negative, S100A4-positive cells appeared to infiltrate bile ducts (Fig. 5D); these may be lymphocytes or monocytes, which are known to express S100A4.36-38 Despite significant hepatic stellate cell accumulation by 8 weeks post-BDL, no YFP colocalization with the HSC marker desmin was noted (Fig. 7). These data indicate that EMT does not occur at these timepoints in the mouse BDL model. To corroborate these findings, we exposed mice to CCl4, a hepatotoxin that induces hepatic (though nonbiliary-specific) fibrosis. Although

progenitor cell proliferation has been described in this model, the phenotype depends mostly on hyperplasia of mature cholangiocytes.39, 40 After 3 weeks of Trametinib manufacturer treatment the increase in fibrosis compared to controls was modest but statistically significant (Supporting Information Fig. 2). There was no evidence of YFP costaining with mesenchymal markers noted in any of the animals despite significant peribiliary staining (Fig. 7; Supporting Information Figs. 4, 5). Neither the CCl4 model nor the BDL model produced evidence of marker colocalization in YFP-labeled hepatocytes, consistent with a recent study suggesting that hepatocyte EMT does not occur.8 BDL is the most widely used rodent model of biliary

fibrosis, although it is an imperfect model for many human diseases because the phenotype results from marked proliferation Amoxicillin of mature cholangiocytes (primarily from the large bile ducts) rather than a ductular reaction, which is proposed to involve activation of bipotential progenitor cell populations (oval cells).39, 41, 42 The ductular reaction is a dominant feature of several fibrosing biliary diseases, including biliary atresia.43, 44 To assess the possibility that EMT occurs in cholangiocyte precursors, before the expression of K19, we used the DDC dietary model, which results in an oval cell response with a marked ductular reaction and sclerosing cholangitis.45, 46 Mice fed the DDC diet for 2 weeks developed moderate fibrosis (data not shown) compared to mice fed normal chow. At 3 weeks of treatment, fibrosis was more marked and roughly equivalent by Sirius Red staining to mice 2 weeks post-BDL (Fig. 3).