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

• 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
• 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

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.

胆管病是胆道系统的慢性进行性疾病，可以是后天性的，也可以是遗传性的。无论 胆管病具有共同的病理机制，包括炎症，异常的胆管增生， 肝纤维化、胆管减少和胆汁淤积，其可随时间推移导致肿瘤发生、肝硬化或肝衰竭。尽管最近 尽管我们对这些疾病的理解和诊断取得了进展，但大多数疾病仍没有经过证实的治疗方法 胆管疾病因此，迫切需要强调治疗发展的机制研究。 先前的工作已经确定Wnt/β-catenin信号通路是胆道梗阻小鼠模型中的可调节靶点。 损伤，如3，5-二乙氧羰基-1，4-二氢可力丁（DDC）饮食。突变的不可降解形式的过表达 β-连环蛋白在转基因（TG）小鼠中长期DDC导致血清碱性磷酸酶（ALP）显著降低， 磷酸酶，一种常见的胆道损伤的预后标志物，同时增加胆汁流量。值得注意的是， 改善与这些TG小鼠肝细胞（HC）中胆汁标志物A6的广泛表达相关。 进一步分析显示，TG早在DDC后1个月就增加了HC中胆汁标志物的表达。 在胆道损伤过程中，HC的表型发生改变，获得胆管细胞（CC）样特征，这是一个过程， 称为细胞重编程。HC重编程可能通过修饰胆汁以减少胆汁分泌而促进胆管修复。 毒性，作为新生CC的来源以修复胆管上皮，或创建新的通道以促进胆汁流动。 因此，该提案的总体假设是，胆汁淤积期间HC中Wnt/β-连环蛋白信号传导的激活 将诱导重编程为CC样表型，并且该过程将有助于恢复胆汁流动并减少胆汁分泌。 胆汁淤积性肝病的严重程度。在目标1中，我们将明确地确定β-连环蛋白过表达的HC是否完全 在胆汁淤积期间，通过分离永久标记的CC， HC及其后代，并通过表型表征、功能测试和转录组学分析它们。 分析.在目标2中，我们将通过以下方法来表征Wnt/β-catenin激活HC胆汁表型的机制： 使用无偏的方法来鉴定胆汁淤积中β-catenin下游的转录因子，以及评估 利用体内双基因报告基因研究雅普信号作为β-catenin潜在下游效应子的作用 系统在目标3中，我们将确定激活HC中的β-连环蛋白是否会增强向完全- 功能性胆囊切除术后的胆系功能首先，我们将确定抑制或 过表达β-连环蛋白对体外胆管结构形成的影响（HC衍生的类器官培养物）。接下来我们就 使用Wnt激动剂外源性激活β-连环蛋白，并评估其在减轻患有胆汁淤积性损伤的小鼠中的功效。 胆功能不全最后，我们将利用免疫缺陷、胆管缺陷的肝脏再增殖模型， 确定移植的TG HC在挽救表型方面是否优于野生型HC。因此，拟议的 研究将进一步加深我们对β-连环蛋白在HC重编程和胆道修复中作用的理解，并将提供 非常重要的信息用于治疗和翻译用途。