The Role of Portal Fibroblasts in Cholestatic Liver Fibrosis

门静脉成纤维细胞在胆汁淤积性肝纤维化中的作用

• 批准号: 8816891
• 负责人: Tatiana Kisseleva
• 金额: $ 34.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8816891
• 关键词: Ablation; Affect; Alcoholic Liver Diseases; Archives; Attenuated; Bile Acids; Biliary Atresia; Cell Fraction; Cells; Cicatrix; Cirrhosis; Collagen; Collagen Type I; Data; Defect; Deposition; Desmin; Development; Elastin; Etiology; Exhibits; Experimental Models; Extracellular Matrix; Fibroblast Growth Factor; Fibroblasts; Fibrosis; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Glial Fibrillary Acidic Protein; Goals; HBV Liver Disease; Healthcare; Hepatic; Hepatic Stellate Cell; Hepatitis B; Hepatitis C virus; Human; In Vitro; Injury; Interleukin-18; Ligation; Liver; Liver Fibrosis; Liver diseases; Mediating; Mesothelium; Methods; Modeling; Mus; Myofibroblast; Obstructive Liver Cirrhosis; Outcome; Pathogenesis; Pathway interactions; Patients; Phospholipids; Play; Population; Primary biliary cirrhosis; Production; Property; Proteoglycan; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Staging; Transgenic Mice; Translating; asporin; base; basonuclin; bile duct; care burden; chronic liver disease; data portal; injured; liver biopsy; liver injury; mesothelin; migration; nonalcoholic steatohepatitis; novel; novel marker; primary sclerosing cholangitis; public health relevance; receptor; response; transcriptional coactivator p75; transcriptome sequencing

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.

描述（申请人提供）：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）和中毒性肝损伤（乙型肝炎病毒）乙型肝炎病毒（HBV）、丙型肝炎病毒（HCV）、酒精性肝病和非酒精性脂肪性肝炎（NASH）是其特征。细胞外基质 (ECM) 广泛沉积，包括 I 型胶原。活化的肝脏驻留肝星状细胞 (aHSC) 和门静脉成纤维细胞 (aPF) 是肝脏纤维疤痕的主要来源，与肝纤维化有关。然而，aPFs 对胆汁淤积性肝损伤的影响。 由于细胞纯化困难和缺乏鉴定的 aP 特异性标记，损伤的特征尚不明确。本研究的目的是确定 aPF 是否在胆汁淤积性肝纤维化中发挥关键作用，并确定其激活机制。我们开发了一种基于流式细胞术的新型方法，从胶原蛋白-?1(I)-GFP 小鼠的非实质部分纯化 aPF，并鉴定了假定的 aPF 特异性标记。我们将使用小鼠胆汁淤积性肝损伤的两种模型：胆管结扎 (BDL) 和小管磷脂翻转酶缺陷 (Mdr2-/- 小鼠) 来表征 aPF 对胆汁淤积性肝纤维化的贡献，并确定对其激活至关重要的新标记物。目标1）。我们将确定我们的初步研究确定的 aP“特征基因”（包括间皮素、尿斑蛋白 1？、basonuclin 1、阿孢菌素、蛋白聚糖 4、磷脂酰肌醇蛋白聚糖 3）的表达是否在 BDL 或 Mdr2 缺陷激活的 aPF 中上调。具体而言，研究了间皮素 (Msln) 在 PF 激活中的作用 建立了 BDL-Msln-/- 和 Mdr2-/-Msln-/- aPF，以及纤维化 wt 和 Msln-/- aPF 的基因表达谱。接下来，在转基因小鼠中确定了 aPF 对 ECM 沉积的贡献，其中 Smad2 信号通路在 aPF 中被特异性删除 (AIM 2)。我们预计 aPF 中 TGF-β1/Smad2 的缺失会减轻 BDL 和 Mdr2 缺陷诱导的纤维化。我们还将确定 TGF-β1/Smad2 的消融是否会影响 aPF 中 Msln 的表达。根据我们的初步数据，与从 BDL 小鼠中分离的 wt aPF 相比，Msln-/- aPF 表现出激活缺陷。为了确定 Msln 在培养 PF 中的作用，检查了 wt 和 Msln-/- aPF 对 TGF-β、胆汁酸、IL-25 和 IL-18 的增殖、迁移和基因表达的反应 (AIM 3) 。我们在小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肝纤维化患者（包括 PSC、PBC、SBC、HCV、ALD 和 NASH）存档的肝活检中是否存在 aPF（？-SMA）来研究 PF 在胆汁淤积性肝纤维化患者中的作用。 +弹性蛋白+Thy1+间皮素+) 和aHSC (?-SMA+Desmin+GFAP+p75+) (AIM 4)。我们的目的是确定 a) PF 是否对胆汁淤积性肝纤维化患者的肌成纤维细胞群有特异性贡献； b) 不同阶段胆汁淤积性肝纤维化患者的PF数量与纤维化进展相关； c) Msln 可以作为胆汁淤积性肝纤维化患者 aPF 的新标志物。