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: 10441586
• 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/10441586
• 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; Injury; 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; 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（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）。其特点是 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。 aPF 已 与胆汁淤积性肝损伤引起的肝纤维化有关。 （AIM1）在这里我们建议研究Msln-的作用 Muc16-Thy-1 信号传导在 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。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-/-与Mdr2-/-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。之间的旁分泌信号传导 将评估 aPF 和胆管细胞。 （AIM2）我们将研究独特的机制、常见的 介导 aPF 激活的介质和分子因子。我们假设 Msln 诱导非 典型的 TGFb/TGFbRI-Smad2/3/4-aPF 激活。 aPF 中 Msln 信号传导的其他潜在途径 （例如 FGFR-Msln-Akt/ERK 和 JAK2/STAT3）也将进行评估。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 测序和质谱分析。阐明新的信号传导途径、基因表达谱和结合 WT 和 KO aPF 的合作伙伴将进行比较。 （AIM3）为了将我们的发现从小鼠身上转化为人类，作用 将在体外和体内研究人类 aPF 中人类 MSLN-THY-1 的作用。我们已经隔离并 对来自胆汁淤积患者的 6 个肝脏的人类 aPF 进行了表征。人类 aPF 通过表达来识别 “特征基因”MSLN、CA125、THY-1、BNC1、UPK1β、CALCA、GPC3。 2 选定的人aPF细胞 将使用shRNA敲低±TGFb1和RNA-Seq来分析品系。人类 MSLN 的结合伴侣将 使用抗人 MSLN Ab 通过质谱分析进行鉴定。 (AIM4) 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， 因此可作为抗纤维化治疗的靶点。我们将测试是否使用抗 MSLN 抗体消融 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 我们实验室开发的）。将通过消失来评估这些小鼠中免疫疗法的有效性 MSLN+ aPF，并抑制胆汁淤积性纤维化。我们预计抗 MSLN 抗体免疫毒素将 改善胆汁淤积性纤维化。