The Role of Portal Fibroblasts in Cholestatic Liver Fibrosis

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ABSTRACT: Cholestatic fibrosis is the outcome of chronic liver diseases, including primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), secondary biliary cirrhosis (SBC). It is characterized by extensive deposition of extracellular matrix (ECM), including collagen Type I. Activated hepatic stellate cells (aHSCs) and portal fibroblasts (aPFs) are the major source of the fibrous scar in the liver. aPFs have been implicated in liver fibrosis caused by cholestatic liver injury. (AIM1) Here we propose to study the role of Msln- Muc16-Thy-1 signaling in the pathogenesis of cholestatic fibrosis in Mdr2-/- mice. For this purpose, Mdr2-/- mice are crossed to Msln-/- mice, Muc16-/- mice, or Thy-1-/- mice. To dissect the relationship between Msln-Muc16- Thy-1, we generated Mdr2-/- mice devoid of both Msln and Muc16 (triple knockout Mdr2-/-Msln-/-Muc16-/- mice) or Msln and Thy-1 (Mdr2-/-Msln-/-Thy-1-/- mice). The time course comparison of cholestatic fibrosis in Mdr2-/- and BDL-injured mice will reveal similarities of aPF activation in both models. The paracrine signaling between aPFs and cholangiocytes will be evaluated. (AIM2) We will investigate the unique mechanisms, common mediators, and molecular factors that mediate activation of aPFs. We hypothesize that Msln induces a non- canonical TGFb/TGFbRI-Smad2/3/4-activation of aPFs. Additional potential pathways of Msln signaling in aPFs (such as FGFR-Msln-Akt/ERK and JAK2/STAT3) will also be evaluated. To dissect the mechanism by which Msln-Thy-1 pathway regulates aPF functions, Col-GFP+Thy-1+Msln+ aPFs will be sort purified, and analyzed by RNA-Seq and mass spectrometry. To elucidate novel signaling pathways, gene expression profiles and binding partners of WT and KO aPFs will be compared. (AIM3) To translate our findings from mice to humans, the role of human MSLN-THY-1 will be studied in human aPFs in vitro and in vivo. We have already isolated and characterized human aPFs from 6 livers of patients with cholestasis. Human aPFs are identified by expression of the “signature genes” MSLN, CA125, THY-1, BNC1, UPK1β, CALCA, GPC3. 2 selected human aPF cell lines will be analyzed using shRNA-knockdown ± TGFb1, and RNA-Seq. Binding partners of human MSLN will be identified by mass spectrometry using anti-human MSLN Ab. (AIM4) Our central hypothesis is that MSLN- MUC16-THY-1 pathway plays an important role in activation of human aPFs in response to cholestatic injury, and therefore serves as a target for anti-fibrotic therapy. We will test if ablation of MSLN with anti-MSLN Ab- immunotoxins can suppress development of cholestatic fibrosis in “humanized” MSLN mice (in which mouse msln gene was replaced with human MSLN gene) or liver xenograft Rag2-/-gc-/- mice (generated by adoptive transplantation of GFP-labeled human MSLN+ aPFs into the livers of Rag2-/-gc-/- mice, this technique is developed in our laboratory). Effectiveness of immunotherapy will be estimated in these mice by disappearance of MSLN+ aPFs, and suppression of cholestatic fibrosis. We anticipate that anti-MSLN Ab-immunotoxins will improve cholestatic fibrosis.