The Role of Portal Fibroblasts in Cholestatic Liver Fibrosis

描述(申请人提供)：肝纤维化是慢性肝脏疾病的结果，包括淤胆性肝病(原发性硬化性胆管炎(PSC)、原发性胆汁性肝硬化症(PBC)、继发性胆汁性肝硬化症(SBC)和中毒性肝损伤(乙肝病毒、丙型肝炎病毒)、酒精性肝病和非酒精性脂肪性肝炎(NASH)。它的特点是广泛沉积细胞外基质(ECM)，包括I型胶原。活化的驻肝肝星状细胞(AHSCs)和门脉成纤维细胞(APF)是肝内纤维瘢痕的主要来源。APFS与胆汁淤积性肝损伤引起的肝纤维化有关。然而，APFS在胆汁淤积性肝脏中的作用 由于细胞纯化困难和缺乏识别的AP特异性标志物，损伤的特征尚未得到很好的描述。本研究的目的是确定APF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。我们开发了一种基于流式细胞术的新方法，从胶原-绿色荧光蛋白1(I)-α小鼠的非实质部分中分离纯化APF，并鉴定了可能的APF特异性标志物。我们将使用两种胆汁淤积性肝损伤的小鼠模型：胆管结扎(BDL)和胆管磷脂翻转酶缺乏(MDR2-/-小鼠)，以表征APFS在胆汁淤积性肝纤维化中的作用，并寻找对其激活至关重要的新标记物(AIM 1)。我们将确定我们的初步研究确定的AP“标志性基因”(包括间硫蛋白、Uroplakin 1β、basonuclin 1、asporin、蛋白多糖4、胶质原蛋白3)在Bd1或MDR2缺乏激活的APF中是否表达上调。具体地说，研究了间充质蛋白(MSLN)在PF激活中的作用 Bd1-msln-/-和mdr2-/-msln-/-APFS，建立了肝纤维化wt和msln-/-APFS的基因表达谱。其次，在APFS(AIM-2)特异缺失Smad2信号通路的转基因小鼠中，确定APFS对ECM沉积的贡献。我们预计，APFS中的转化生长因子1/Smad2基因缺失可减轻BDL和MDR2缺乏症所致的纤维化。我们还将确定消融转化生长因子1/Smad2是否会影响APFS中MSLN的表达。根据我们的初步数据，与从BDL-小鼠分离的wt APF相比，msln-/-APF在激活方面存在缺陷。为了确定MSLN在培养的PFS中的作用，检测了WT和MSLN-/-APF对转化生长因子、胆汁酸、IL-25和IL-18的增殖、迁移和基因表达的反应(目的3)。我们在老鼠身上的发现必须转化为患者。我们建议通过分析不同病因的肝纤维化患者的肝活检档案来研究PFS在胆汁淤积性肝纤维化患者中的作用，包括PSC、PBC、SBC、丙型肝炎病毒、ALD和NASH是否存在APF(α-SMA+弹性蛋白+Thy1+间充质蛋白+)和AHSCs(α-SMA+结蛋白+GFAP+p75+)(AIM4)。我们的目标是确定a)PFS是否对淤胆性肝纤维化患者的肌成纤维细胞数量有显著贡献；b)不同阶段的淤胆性肝纤维化患者PFS的数量与纤维化进展相关；c)MSLN可作为淤胆性肝纤维化患者APFS的新标记物。

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-α1(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 1β, 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-ß1/Smad2 in aPFs attenuates fibrosis induced by BDL and Mdr2-deficiency. We will also determine if ablation of TGF-ß1/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-ß, 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 (α-SMA+Elastin+Thy1+Mesothelin+) and aHSCs (α-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.