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: 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
• 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

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的结果，包括原发性硬化性肝硬化 胆管炎（PSC）、原发性胆汁性肝硬化（PBC）、继发性胆汁性肝硬化（SBC）。它的特点是 细胞外基质（ECM）广泛沉积，包括I型胶原。活化的肝星状细胞 肝纤维化的主要来源是肝干细胞（aHSC）和门静脉成纤维细胞（aPF）。APF已经 与胆汁淤积性肝损伤引起的肝纤维化有关。（AIM 1）在这里，我们建议研究MSLN的作用- Mdr 2-/-小鼠胆汁淤积性纤维化发病机制中的Muc 16-Thy-1信号传导为此，Mdr 2-/-小鼠 与Msln-/-小鼠、Muc 16-/-小鼠或Thy-1-/-小鼠杂交。分析Msln-Muc 16- Thy-1，我们产生了缺乏Msln和Muc 16的Mdr 2-/-小鼠（三重敲除Mdr 2-/-Msln-/-Muc 16-/-小鼠）。 或Msln和Thy-1（Mdr 2-/-Msln-/-Thy-1-/-小鼠）。Mdr 2-/-和Mdr 2-/-胆汁淤积性纤维化的时间进程比较 BDL损伤的小鼠将揭示两种模型中aPF活化的相似性。之间的旁分泌信号 将评价aPF和胆管细胞。（AIM 2）我们将研究独特的机制，共同的 介质和介导aPF活化的分子因子。我们假设MSLN诱导了一种非- 典型的TGFb/TGFbRI-Smad 2/3/4-aPF的活化。aPF中Msln信号传导的其他潜在途径 (such作为FGFR-Msln-Akt/ERK和JAK 2/STAT 3）。来剖析 Msln-Thy-1途径调节aPF功能，Col-GFP+Thy-1+Msln+ aPF将被分选纯化，并通过流式细胞术分析。 RNA-Seq和质谱。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。（AIM 3）为了将我们的发现从小鼠转化为人类， 将在体外和体内在人aPF中研究人MSLN-THY-1的表达。我们已经隔离了 表征了来自胆汁淤积患者的6个肝脏的人aPF。人aPF通过表达 MSLN、CA125、THY-1、BNC1、UPK1β、CALCA、GPC3. 2选择的人aPF细胞 使用shRNA-knockdown ± TGFb 1和RNA-Seq分析细胞系。人MSLN的结合伴侣将 使用抗人MSLN Ab通过质谱法鉴定。（AIM 4）我们的中心假设是MSLN- MUC 16-THY-1通路在应答胆汁淤积性损伤的人aPF活化中起重要作用， 因此用作抗纤维化治疗的靶点。我们将测试是否用抗MSLN抗体消融MSLN， 免疫毒素可抑制“人源化”MSLN小鼠（其中小鼠 用人MSLN基因替换msln基因）或肝异种移植Rag 2-/-gc-/-小鼠（通过过继免疫产生）。 将GFP标记的人MSLN+ aPF移植到Rag 2-/-gc-/-小鼠的肝脏中，该技术是 在我们的实验室里）。免疫治疗的有效性将通过消失来估计 和胆汁淤积性纤维化的抑制。我们预计，抗MSLN抗体免疫毒素将 改善胆汁淤积性纤维化。