Beta-catenin-driven hepatobiliary reprogramming as a therapeutic modality for cholangiopathies

β-连环蛋白驱动的肝胆重编程作为胆管病的治疗方式

基本信息

批准号：
10618307
负责人：
Kari N Nejak-Bowen
金额：
$ 40.09万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10618307
关键词：
Acceleration Agonist Alkaline Phosphatase Animal Model Bile fluid Biliary Cell Nucleus Cells ChIP-seq Cholestasis Chronic Cirrhosis Cre lox recombination system Defect Development Diet Disease Epithelium Etiology Exposure to Fibrosis Gene Expression Genetic Genotype Hepatic Hepatobiliary Hepatocyte Heterozygote Immune Impairment In Vitro Inflammation Injury Intrahepatic Cholestasis Knock-out Knockout Mice Label LacZ Genes Life Ligation Liver Liver Failure Location LoxP-flanked allele Mediating Medical Methods Modality Modeling Mus Mutate Organoids Pathologic Pathway interactions Patients Phenotype Physiology Population Process Prognostic Marker Progressive Disease Proliferating Regulation Reporter Reporter Genes Resolution Role Serum Severities Signal Pathway Signal Transduction Source Structure System Testing Therapeutic Time Toxic effect Transgenic Mice Transgenic Organisms Transplantation Up-Regulation WNT Signaling Pathway Work beta catenin bile duct biliary tract cholangiocyte disease diagnosis functional restoration improved in vivo in vivo Model insight intrahepatic liver development liver transplantation mouse model mutant novel overexpression pressure repaired therapeutic development transcription factor transcriptome sequencing transcriptomics transdifferentiation tumorigenesis

项目摘要

Cholangiopathies are chronic, progressive diseases of the biliary tree, and can be either acquired or genetic. Regardless of etiology, cholangiopathies share common pathologic mechanisms, including inflammation, aberrant ductular proliferation, fibrosis, ductopenia, and cholestasis, which can over time result in tumorigenesis, cirrhosis, or liver failure. Despite recent advances in our understanding and diagnosis of these diseases, there are no proven therapeutic treatments for the majority of cholangiopathies. Thus, mechanistic-based studies that emphasize therapeutic development are desperately needed. Previous work has identified the Wnt/β-catenin signaling pathway as a modulatable target in mouse models of biliary injury such as 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Overexpression of a mutated non-degradable form of β-catenin in transgenic (TG) mice subjected to long-term DDC results in a significant reduction in serum alkaline phosphatase, a common prognostic marker for biliary injury, and a concurrent increase in bile flow. Notably, this improvement was associated with widespread expression of biliary marker A6 in the hepatocytes (HC) of these TG mice. Further analysis revealed that TG had increased expression of biliary markers in HC as early as 1 month after DDC. During biliary injury, HC are known to alter their phenotype and acquire cholangiocyte (CC)-like features, a process known as cellular reprogramming. HC reprogramming may contribute to biliary repair by modifying bile to reduce toxicity, serving as a source of de novo CC to repair the biliary epithelium, or creating new channels to facilitate bile flow. Thus, the overarching hypothesis of the proposal is that activation of Wnt/β-catenin signaling in HC during cholestasis will induce reprogramming to a CC-like phenotype, and that this process will aid in restoring bile flow and reducing the severity of cholestatic liver disease. In aim 1, we will unambiguously determine if β-catenin-overexpressing HC fully differentiate into functional CC or maintain an intermediate phenotype during cholestasis by isolating permanently labeled HC and their progeny and analyzing them through phenotypic characterization, functional tests, and transcriptomic analysis. In aim 2, we will characterize the mechanism by which Wnt/β-catenin activates a biliary phenotype in HC by using unbiased methods to identify the transcription factors downstream of β-catenin in cholestasis, as well as evaluating the contribution of Yap signaling as a potential downstream effector of β-catenin using an in vivo two-gene reporter system. In aim 3, we will determine whether activating β-catenin in HC will enhance transdifferentiation into fully- functional CC in the absence of a functional biliary system. First, we will determine the effect of inhibiting or overexpressing β-catenin on the formation of biliary structures in vitro (HC-derived organoid cultures). Next, we will exogenously activate β-catenin using a Wnt agonist and assess its efficacy in alleviating cholestatic injury in mice with biliary insufficiency. Finally, we will utilize an immune-deficient, bile duct deficient model of liver repopulation and determine if transplanted TG HC have an advantage over wild-type HC in rescuing the phenotype. Thus, the proposed studies will further our understanding of the role of β-catenin in HC reprogramming and biliary repair, and will provide highly significant information for therapeutic and translational use.

胆管病是胆汁树的慢性，进行性疾病，可以被获取或遗传。不管病因学，胆管疾病具有常见的病理机制，包括感染，异常导管增殖，随着时间的流逝，纤维化，凝发和胆汁淤积可能会导致肿瘤发生，肝硬化或肝衰竭。尽管最近在我们对这些疾病的理解和诊断方面的进步，对于大多数疾病没有可靠的治疗方法胆管疾病。迫切需要强调热发展的基于机械的研究。先前的工作已将Wnt/β-catenin信号通路确定为胆道模型中的模块化靶标损伤，例如3,5-二氧基碳酸1,4-二氢核苷（DDC）饮食。突变的不可降解形式的过表达长期DDC的转基因（TG）小鼠中的β-catenin的大量导致血清合金显着降低磷酸酶是胆道损伤的常见预后标记，并同时增加胆汁流量。值得注意的是改善与这些TG小鼠的肝细胞（HC）中胆道标记A6的宽度表达有关。进一步的分析表明，最早在DDC后1个月内，TG在HC中的表达增加。在胆道受伤期间，众所周知，HC会改变其表型并获得胆管细胞（CC）样特征，一个过程称为细胞重编程。 HC重编程可能会通过修改胆汁来减少胆汁修复有助于胆道修复毒性，作为从头CC修复胆道上皮的来源，或创建新的通道以促进胆汁流动。这是该提案的总体假设是胆汁淤积过程中HC中Wnt/β-catenin信号的激活将诱导重新编程到类似CC的表型，并且此过程将有助于恢复胆汁流并减少胆汁淤积性肝病的严重程度。在AIM 1中，我们将明确确定β-catenin过表达HC是否完全通过分离永久标记，分化为功能性CC或在胆汁淤积过程中维持中间表型 HC及其后代，并通过表型表征，功能测试和转录组来分析它们分析。在AIM 2中，我们将表征Wnt/β-catenin通过HC激活HC中的胆道表型的机制使用公正的方法来识别胆汁淤积中β-catenin下游的转录因子，并评估 YAP信号传导作为β-catenin的潜在下游效应子的贡献，使用体内两基因报告基因系统。在AIM 3中，我们将确定在HC中激活β-catenin是否会增强转变为完全在没有功能性胆道系统的情况下，功能性CC。首先，我们将确定抑制或过表达β-catenin在体外形成胆道结构（HC衍生的器官培养物）。接下来，我们会的使用Wnt激动剂外源激活β-catenin，并评估其在减轻小鼠胆汁淤积损伤方面的有效性胆道功能不全。最后，我们将利用免疫缺陷，胆管的肝脏再生模型不足和确定移植的TG HC是否比野生型HC具有优势。那，提议研究将进一步了解β-catenin在HC重编程和胆道修复中的作用，并将提供治疗和翻译使用的高度重要信息。