The Role of Portal Fibroblasts in Cholestatic Liver Fibrosis

描述（由申请方提供）：肝纤维化是慢性肝病的结果，包括胆汁淤积性肝病（原发性硬化性胆管炎（PSC）、原发性胆汁性肝硬化（PBC）、继发性胆汁性肝硬化（SBC）和毒性肝损伤（B型肝炎维鲁（HBV）、丙型肝炎病毒（HCV）、酒精性肝病和非酒精性脂肪性肝炎（NASH））。其特征在于细胞外基质（ECM）的广泛沉积，包括I型胶原。激活的肝脏驻留肝星状细胞（aHSC）和门静脉成纤维细胞（aPF）是肝脏中纤维性瘢痕的主要来源。aPF与胆汁淤积性肝损伤引起的肝纤维化有关。然而，aPF对胆汁淤积性肝脏的贡献 由于细胞纯化的困难和缺乏鉴定的aP特异性标志物，损伤没有被很好地表征。本研究的目的是确定aPFs是否在胆汁淤积性肝纤维化中起关键作用，并确定其激活机制。我们已经开发了一种新的流式细胞术为基础的方法，从胶原蛋白的非实质部分的aPF纯化？1（I）-GFP小鼠，并且已经鉴定了推定的aPF特异性标志物。我们将使用两种小鼠胆汁淤积性肝损伤模型：胆管结扎（BDL）和小管磷脂翻转酶缺乏（Mdr 2-/-小鼠），以表征aPF对胆汁淤积性肝纤维化的贡献，并鉴定其活化的新标志物（AIM 1）。我们将确定通过我们的初步研究鉴定的aP“标签基因”（包括间皮素，尿斑蛋白1？，基底核蛋白1、asporin、蛋白聚糖4、glipican 3）在由BDL或Mdr 2缺陷激活的aPF中上调。具体地，研究了间皮素（Msln）在PF活化中的作用。 BDL-Msln-/-和Mdr 2-/-Msln-/-aPF，并建立了纤维化wt和Msln-/-aPF的基因表达谱。接下来，在转基因小鼠中确定aPF对ECM沉积的贡献，其中Smad 2信号传导途径在aPF（AIM 2）中特异性缺失。我们预计，删除TGF-？aPF中的1/Smad 2减弱由BDL和Mdr 2缺乏诱导的纤维化。我们还将确定是否消融TGF-？1/Smad 2影响aPF中Msln的表达。基于我们的初步数据，当与从BDL-小鼠分离的wt aPF相比时，Msln-/-aPF表现出活化缺陷。为了确定Msln在培养的PF中的作用，wt和Msln-/-aPF对TGF-β的反应，检查胆汁酸、IL-25和IL-18的增殖、迁移和基因表达（AIM 3）。我们在老鼠身上的发现必须转化到病人身上。我们建议通过分析来自不同病因肝纤维化患者的存档肝活检来研究PFs在胆汁淤积性肝纤维化患者中的作用，包括PSC、PBC、SBC、HCV、ALD和NASH，以确定是否存在aPFs（？SMA+弹性蛋白+Thy 1+间皮素+）和aHSC（？SMA+结蛋白+GFAP+p75+）（AIM 4）。我们的目的是确定a）PF是否对患有胆汁淤积性肝纤维化的患者中的肌成纤维细胞群体有特异性贡献; B）患有胆汁淤积性肝纤维化的不同阶段的患者中的PF的数量与纤维化进展相关;以及c）Msln可以作为患有胆汁淤积性肝纤维化的患者中的aPF的新标志物。

DESCRIPTION (provided by applicant): Hepatic fibrosis is the outcome of chronic liver diseases, including cholestatic liver disease (primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), secondary biliary cirrhosis (SBC) and toxic liver injury (hepatitis B viru (HBV), hepatitis C virus (HCV), alcoholic liver disease and non-alcoholic steatohepatitis (NASH). It is characterized by extensive deposition of extracellular matrix (ECM), including collagen Type I. Activated liver resident 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. However, the contribution of aPFs to cholestatic liver injury is not well characterized due to difficulties in cell purification and lack of identified aP specific markers. The goal of this study is to determine if aPFs play a critical role in cholestati liver fibrosis and identify the mechanisms of their activation. We have developed a novel flow cytometry-based method of aPF purification from the non-parenchymal fraction of Collagen-?1(I)-GFP mice and have identified putative aPF specific markers. We will use two models of cholestatic liver injury in mice: bile duct ligation (BDL) and deficiency of canalicular phospholipd flippase (Mdr2-/- mice) to characterize the contribution of aPFs to cholestatic liver fibrosis and identify novel markers critical for their activation (AIM 1). We will determine if expression of aP "signature genes" identified by our preliminary study (including mesothelin, uroplakin 1?, basonuclin 1, asporin, proteoglycan 4, glipican 3) is upregulated in aPFs activated by either BDL or Mdr2-deficiency. Specifically, the role of mesothelin (Msln) in PF activation is investigated in BDL-Msln-/- and Mdr2-/-Msln-/- aPFs, and the gene expression profile of fibrogenic wt and Msln-/- aPFs is established. Next, the contribution of aPFs to ECM deposition is determined in transgenic mice, in which Smad2 signaling pathway is deleted specifically in aPFs (AIM 2). We anticipate that deletion of TGF-?1/Smad2 in aPFs attenuates fibrosis induced by BDL and Mdr2-deficiency. We will also determine if ablation of TGF- ?1/Smad2 affects expression of Msln in aPFs. Based on our preliminary data, Msln-/- aPFs exhibit a defect in activation when compared with wt aPFs isolated from BDL-mice. To determine the role of Msln in cultured PFs, responses of wt and Msln-/- aPFs to TGF-?, bile acids, IL-25 and IL-18 are examined with respect to proliferation, migration, and gene expression (AIM 3). Our findings in mice must be translated into patients. We propose to study the role of PFs in patients with cholestatic liver fibrosis by analyzing archived liver biopsies from patients with liver fibrosis of different etiologies, including PSC, PBC, SBC, HCV, ALD, and NASH for the presence of aPFs (?-SMA+Elastin+Thy1+Mesothelin+) and aHSCs (?-SMA+Desmin+GFAP+p75+) (AIM 4). We aim to determine if a) PFs contribute speifically to the myofibroblast population in patients with cholestatic liver fibrosis; b) the number of PFs in patients with different stages of cholestatic lver fibrosis correlates with the fibrosis progression; and c) Msln can serve as a new marker of aPFs in patients with cholestatic liver fibrosis.