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

基本信息

批准号：
10312314
负责人：
Tatiana Kisseleva
金额：
$ 48.66万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10312314
关键词：
Abbreviations Ablation Area Binding Biological Assay CA-125 Antigen Calcitonin Cell Line Cell Nucleus Cells Cholestasis Chromatin Cicatrix Cirrhosis Collagen Collagen Type I Complex Consensus Deposition Development Down-Regulation Effectiveness Extracellular Matrix Fibroblast Growth Factor Fibroblast Growth Factor Receptors Fibroblasts Fibrosis GPC3 gene Gene Expression Gene Expression Profile Genes Genetic Transcription Goals Healthcare Hepatic Hepatic Stellate Cell Hepatotoxicity Human Immunoprecipitation Immunotherapy Immunotoxins In Vitro JAK2 gene Kidney Knock-out Label Laboratories Liver Liver Fibrosis Lung MSLN gene Mass Spectrum Analysis Mediating Mediator of activation protein Mesenchymal Modeling Molecular Mucins Mus Myofibroblast Obstructive Liver Cirrhosis Organ Outcome Paracrine Communication Pathogenesis Pathway interactions Patients Phenotype Play Primary biliary cirrhosis Property Proteins RNA analysis Recording of previous events Regulation Role STAT3 gene Series Signal Pathway Signal Transduction Signaling Protein Small Nuclear RNA Source Stimulus Techniques Testing Time Transcriptional Regulation Translating Translational Research Transplantation Transposase antifibrotic treatment asporin basonuclin care burden cholangiocyte cholestatic injury chronic liver disease design experimental study humanized mouse improved in vivo injured insight knock-down liver injury liver xenograft mesothelin novel novel strategies primary sclerosing cholangitis response small hairpin RNA transcriptome sequencing

项目摘要

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.

摘要：胆汁淤积性纤维化是慢性肝病（包括原发性硬化症）的结果。胆管炎（PSC）、原发性胆汁性肝硬化（PBC）、继发性胆汁性肝硬化（SBC）。细胞外基质 (ECM) 广泛沉积，包括 I 型胶原蛋白。激活的肝星状细胞 (aHSC) 和门静脉成纤维细胞 (aPF) 是肝脏纤维疤痕的主要来源。与胆汁淤积性肝损伤（AIM1）引起的肝纤维化有关，我们建议研究 Msln- 的作用。 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-/- 小鼠杂交以剖析 Msln-Muc16- 之间的关系。 Thy-1，我们生成了缺乏 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-/- 中胆汁淤积性纤维化的时程比较。 BDL 损伤的小鼠将揭示两种模型中 aPF 激活的相似性。将评估 aPF 和胆管细胞 (AIM2) 我们将研究独特的机制和常见的机制。我们发现 Msln 会诱导非-介导 aPF 的激活。 aPF 中 Msln 信号传导的典型 TGFb/TGFbRI-Smad2/3/4 激活。（例如 FGFR-Msln-Akt/ERK 和 JAK2/STAT3）也将进行评估，以剖析其机制。 Msln-Thy-1途径调节aPF功能，Col-GFP+Thy-1+Msln+ aPF将被分选纯化，并通过以下方法进行分析： RNA-Seq 和质谱分析阐明新的信号通路、基因表达谱和结合。将比较 WT 和 KO aPF 的伴侣 (AIM3) 为了将我们的发现从小鼠转移到人类，其作用。我们已经在体外和体内研究了人类 MSLN-THY-1 的作用。通过表达鉴定了来自 6 个胆汁淤积患者肝脏的人类 aPF。 “特征基因”MSLN、CA125、THY-1、BNC1、UPK1β、CALCA、GPC3 2 选择的人 aPF 细胞。将使用 shRNA 敲低 ± TGFb1 和人类 MSLN 的 RNA-Seq 分析细胞系。使用抗人 MSLN 抗体 (AIM4) 通过质谱分析进行鉴定我们的中心假设是 MSLN- MUC16-THY-1 通路在人类 aPF 响应胆汁淤积损伤的激活中发挥重要作用，因此，我们将测试是否用抗 MSLN Ab 消融 MSLN。免疫毒素可以抑制“人源化”MSLN 小鼠（其中小鼠 msln 基因替换为人 MSLN 基因）或肝异种移植 Rag2-/-gc-/- 小鼠（由过继产生）将 GFP 标记的人 MSLN+ aPF 移植到 Rag2-/-gc-/- 小鼠的肝脏中，该技术是将通过消失来评估这些小鼠中免疫疗法的有效性我们预计抗 MSLN Ab 免疫毒素将发挥作用。改善胆汁淤积性纤维化。