The Role of Portal Fibroblasts in Cholestatic Liver Fibrosis

摘要：胆汁淤积性纤维化是包括原发性硬化在内的慢性肝病的结果。 胆管炎(PSC)、原发性胆汁性肝硬变(PBC)、继发性胆汁性肝硬变(SBC)。它的特点是 细胞外基质(ECM)广泛沉积，包括I型胶原，激活的肝星状细胞 AHSCs和门脉成纤维细胞(APF)是肝脏纤维性瘢痕的主要来源。APF已经被 与胆汁淤积性肝损伤引起的肝纤维化有关。(AIM1)在这里，我们建议研究MSLN- Muc16-Thy-1信号在mdr2-/-小鼠胆汁淤积性纤维化发病机制中的作用为此，mdr2-/-小鼠 与Msln-/-小鼠、Muc16-/-小鼠或Thy-1-/-小鼠杂交。为了剖析Msln-Muc16-之间的关系 THY-1，我们产生了同时缺乏Msln和Muc16的mdr2-/-小鼠(三重敲除mdr2-/-msln-/-Muc16-/-小鼠) 或Msln和Thy-1(mdr2-/-Msln-/-Thy-1-/-小鼠)。Mdr2-/-和mdr2-/-患者胆汁淤积性纤维化的时程比较 BDL损伤的小鼠将在两种模型中显示APF激活的相似之处。旁分泌信号在 将对APFS和胆管细胞进行评估。(AIM2)我们将研究独特的机制，共同的 介体和介导APF活化的分子因子。我们假设MSLN诱导了一个非- 规范的TGFb/TGFbRI-Smad2/3/4-APF的激活。APFS中Msln信号的其他潜在途径 (如FGFR-MSLN-AKT/ERK和JAK2/STAT3)也将得到评估。剖析其作用机制 Msln-Thy-1途径调节APF功能，对Col-GFP+Thy-1+Msln+APFS进行分类纯化，并进行分析 RNA-Seq和质谱学。阐明新的信号通路、基因表达谱和结合 WT和KO APFS的合作伙伴将进行比较。(AIM3)为了将我们的发现从小鼠转化为人类， 人MSLN-THY-1的体外和体内研究将在人的APF中进行。我们已经隔离了 从6例胆汁淤积症患者的肝脏中鉴定人类APFS。人类APF是通过表达识别的 MsLn、CA125、THY-1、Bnc1、UPK1、β、Calca、GPC3。2个精选人APF细胞 将使用shRNA-Knokdown±TGFb1和RNA-Seq来分析品系。人MSLN的结合伙伴将 用抗人MSLN单抗进行质谱学鉴定。(AIM4)我们的中心假设是MSLN- MUC16-THY-1通路在胆汁淤积性损伤中激活人APF中起重要作用 因此成为抗纤维化治疗的靶点。我们将测试用抗MSLN抗体消融MSLN是否- 免疫毒素可抑制“人源化”MSLN小鼠的胆汁淤积性纤维化的发展(其中小鼠 Msln基因被人MSLN基因替换)或异种肝移植Rag2-/-GC-/-小鼠(过继产生 将GFP标记的人MSLN+APFS移植到Rag2-/-GC-/-小鼠的肝脏中，这项技术是 在我们实验室开发的)。免疫治疗的有效性将通过这些小鼠的消失来评估。 MSLN+APFS，抑制胆汁淤积纤维化。我们预计抗MSLN抗体免疫毒素将 改善淤胆性纤维化。

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.