Background & Aims Biliary-committed progenitor cells (small cholangiocytes, SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, FoxA2 is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Methods mRNA expression in SMCCs and LGCCs was assessed by PCR array analysis. Liver tissues and hepatic stellate cells from PSC and PBC patients were tested by real-time PCR for methylation, senescence and fibrosis markers. Bile duct ligation (BDL) and MDR2 knockout mice (MDR2−/−) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. Results We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs, and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes DNMT1 and DNMT3B. Serum ALT and AST levels in NOD/SCID mice engrafted with SMCCs after BDL showed significant changes compared with vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2−/− mouse liver as well as in isolated hepatic stellate cells by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. Conclusions The definitive endoderm marker and the positive regulator of biliary development, FoxA2, mediates the therapeutic effect of biliary-committed progenitor cells during cholestatic liver injury.

Secretin receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2 mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR, Mdr2, and SR/Mdr2 mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2 mice was reduced in SR/Mdr2 mice. Enhanced senescence levels in cholangiocytes from Mdr2 mice were reversed to normal in SR/Mdr2 mice. However, senescence was decreased in HSCs from Mdr2 mice but returned to normal values in SR/Mdr2 mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.

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Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to play a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR21−/− mice underwent sham or bile duct ligation (BDL) for 1 wk, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and Smad-7 expression. In vitro, immortalized murine biliary cell lines (IMCL) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. Also, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers TGF-β1 and α-SMA. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared to control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.