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: 10019522
• 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/10019522
• 关键词: Agonist; Alkaline Phosphatase; Animal Model; Bile fluid; Biliary; Cell Nucleus; Cells; ChIP-seq; Cholestasis; Chronic; Cirrhosis; Defect; Development; Diet; Disease; Epithelial; Epithelium; Etiology; Exposure to; Fibrosis; Gene Expression; Genetic; Genotype; Hepatic; Hepatobiliary; Hepatocyte; Immune; Impairment; In Vitro; Inflammation; Injury; Intrahepatic Cholestasis; Knock-out; Knockout Mice; Label; LacZ Genes; Life; Ligation; Liver; Liver Failure; Location; Mediating; Medical; Methods; Modality; Modeling; Mus; Mutate; Organoids; Pathologic; Pathway interactions; Patients; Phenotype; Physiology; Process; Prognostic Marker; Progressive Disease; 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; base; beta catenin; bile duct; biliary tract; cholangiocyte; cholestatic injury; cholestatic liver disease; 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.

胆管病是一种慢性、进行性胆道疾病，既可以是后天的，也可以是遗传的。不管 在病因学上，胆管疾病有共同的病理机制，包括炎症，异常的胆管增殖， 纤维化、乳管减少和胆汁淤积，随着时间的推移可导致肿瘤形成、肝硬变或肝功能衰竭。尽管最近 在我们对这些疾病的了解和诊断方面的进展，目前还没有被证明有效的治疗方法来治疗大多数疾病 胆管病。因此，迫切需要强调治疗发展的机械论研究。 先前的工作已经发现Wnt/β-连环蛋白信号通路在胆道疾病小鼠模型中是一个可调节的靶点 损伤，如3，5-二乙氧基-1，4-二氢可乐定(DDC)饮食。一种突变的不可降解形式的过表达 β-catenin转基因(TG)小鼠长期服用DDC后血清碱性显著降低 磷酸酶是胆道损伤的常见预后标记物，同时胆汁流量增加。值得注意的是，这 改善与胆汁标志物A6在这些TG小鼠的肝细胞(HC)中的广泛表达有关。 进一步分析表明，TG早在DDC后1个月就增加了HC胆汁标志物的表达。 在胆管损伤过程中，HC会改变其表型，并获得胆管细胞(CC)样特征，这是一个过程 也就是所谓的细胞重编程。HC重新编程可能通过修改胆汁以减少 毒性，作为一种新的CC的来源，以修复胆管上皮，或创造新的渠道，促进胆汁流动。 因此，该方案的主要假设是，在胆汁淤积期间，Hc中Wnt/β-catenin信号被激活。 将诱导重新编程为CC样表型，这一过程将有助于恢复胆汁流量和减少 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-过度表达HC是否完全 分化为功能性CC或在胆汁淤积期间保持中间表型 通过表型鉴定、功能测试和转录本对其进行分析 分析。在目标2中，我们将描述Wnt/β-catenin激活HC胆汁表型的机制。 用无偏倚方法鉴定胆汁淤积症β-连环蛋白下游转录因子 利用体内双基因报告基因研究YAP信号作为β-连环蛋白潜在下游效应因子的作用 系统。在目标3中，我们将确定激活HC中的β-连环蛋白是否会促进转分化为完全- 在没有功能胆道系统的情况下，功能CC。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来源的器官培养)。接下来，我们将 用Wnt激动剂外源性激活β-catenin并评价其减轻胆汁淤积性损伤的作用 胆道功能不全。最后，我们将利用免疫缺陷、胆管缺陷的肝脏再繁殖模型和 确定移植的TG-HC在挽救表型方面是否比野生型HC更有优势。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的作用的理解，并将提供 对于治疗和翻译使用具有重要意义的信息。