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的结果，包括原发性硬化胆管炎（PSC），原发性胆汁肝硬化（PBC），次生胆道肝硬化（SBC）。它的特征是细胞外基质（ECM）的广泛沉积，包括I型胶原蛋白I （AHSC）和门户成纤维细胞（APF）是肝脏纤维疤痕的主要来源。 APF已经由胆汁淤积性肝损伤引起的肝纤维化实施。（AIM1）在这里我们建议研究MSLN- MUC16-THY-1信号传导在MDR2 - / - 小鼠中胆固醇纤维化的发病机理中。为此，MDR2 - / - 小鼠越过MSLN - / - 小鼠，MUC16 - / - 小鼠或THY-1 - / - 小鼠。剖析MSLN-MUC16-之间的关系 thy-1，我们生成了没有MSLN和MUC16的MDR2 - / - 小鼠（三敲除MDR2 - / - MSLN - / - MUC16 - / - 小鼠）或MSLN和THY-1（MDR2 - / - MSLN - / - THY-1-/ - 小鼠）。 MDR2 - / - 和在这两个模型中，损坏的小鼠将揭示APF激活的相似性。旁分泌信号将评估APF和胆管细胞。（AIM2）我们将研究独特的机制，常见介体和介导APF激活的分子因子。我们假设MSLN诱导了非 - APF的规范TGFB/TGFBRI-SMAD2/3/4-激活。 APF中MSLN信号的其他潜在途径（例如FGFR-MSLN-AKT/ERK和JAK2/STAT3）也将进行评估。剖析机制 MSLN-THY-1途径调节APF功能，Col-GFP+THY-1+MSLN+APF将被排序，并通过 RNA-seq和质谱法。阐明新型信号通路，基因表达谱和结合将比较WT和KO APF的合作伙伴。（AIM3）将我们的发现从老鼠转化为人类人类MSLN-THY-1的体外和体内将研究人类APF。我们已经孤立了，表征了胆汁淤积患者的6种生命中的人类APF。通过表达来识别人类APF “签名基因” MSLN，CA125，THY-1，BNC1，UPK1β，Calca，GPC3。 2选定的人APF单元将使用shrna knockdown±TGFB1和RNA-Seq分析线。人类MSLN的约束伙伴将使用抗人MSLN AB通过质谱法鉴定。（AIM4）我们的中心假设是MSLN- MUC16-THY-1途径在胆汁淤积损伤的激活中起着重要作用，因此是抗纤维化疗法的靶标。我们将测试MSLN是否用抗MSLN AB-进行消融免疫毒素可以抑制“人性化” MSLN小鼠中胆固醇纤维化的发展（其中小鼠将MSLN基因替换为人MSLN基因）或肝脏异种移植Rag2 - / - GC - / - 小鼠（通过自适应生成将GFP标记的人MSLN+ APF移植到rag2 - / - gc - / - 小鼠的生活中，此技术是在我们的实验室中开发）。通过消失，将在这些小鼠中估算免疫疗法的有效性 MSLN+ APF的抑制作用。我们预计抗MSLN AB-免疫毒素将会改善胆固醇纤维化。