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.

胆管病是胆管系统的慢性进行性疾病，可以是获得性的，也可以是遗传性的。不管 病因学上，胆管病具有共同的病理机制，包括炎症、异常的导管增殖、 纤维化、胆管减少和胆汁淤积，随着时间的推移，可能导致肿瘤发生、肝硬化或肝衰竭。尽管最近 我们对这些疾病的理解和诊断取得了进展，但大多数疾病还没有经过证实的治疗方法 胆管病。因此，迫切需要强调治疗开发的基于机制的研究。 先前的工作已将 Wnt/β-catenin 信号通路确定为小鼠胆道模型中的可调节靶点 损伤如3,5-二乙氧基羰基-1,4-二氢可力丁(DDC)饮食。突变的不可降解形式的过度表达 长期接受 DDC 治疗的转基因 (TG) 小鼠中的 β-连环蛋白导致血清碱性显着降低 磷酸酶是胆道损伤的常见预后标志物，并且胆汁流量同时增加。值得注意的是，这 改善与这些 TG 小鼠肝细胞 (HC) 中胆道标志物 A6 的广泛表达有关。 进一步分析显示，早在 DDC 后 1 个月，TG 就增加了 HC 中胆道标志物的表达。 在胆道损伤期间，HC 会改变其表型并获得胆管细胞 (CC) 样特征，这是一个过程 称为细胞重编程。 HC 重编程可能通过修改胆汁来减少胆汁修复，从而有助于胆道修复 毒性，作为从头 CC 的来源来修复胆管上皮，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-catenin 信号的激活 将诱导重编程为类似 CC 的表型，并且该过程将有助于恢复胆汁流量并减少 胆汁淤积性肝病的严重程度。在目标 1 中，我们将明确确定 β-catenin 过度表达 HC 是否完全 通过分离永久标记的细胞，分化为功能性 CC 或在胆汁淤积期间维持中间表型 HC 及其后代，并通过表型表征、功能测试和转录组学对其进行分析 分析。在目标 2 中，我们将通过以下方式描述 Wnt/β-catenin 激活 HC 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 使用体内双基因报告基因，Yap 信号传导作为 β-连环蛋白的潜在下游效应子的贡献 系统。在目标 3 中，我们将确定激活 HC 中的 β-catenin 是否会增强转分化为完全- 缺乏功能性胆道系统的功能性 CC。首先，我们将确定抑制或 过度表达 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，我们将 使用 Wnt 激动剂外源性激活 β-连环蛋白，并评估其减轻小鼠胆汁淤积损伤的功效 胆道功能不全。最后，我们将利用免疫缺陷、胆管缺陷的肝脏再生模型和 确定移植的 TG HC 在挽救表型方面是否比野生型 HC 有优势。因此，建议的 研究将进一步了解 β-连环蛋白在 HC 重编程和胆道修复中的作用，并将提供 对于治疗和转化用途非常重要的信息。