Mechanisms of synergistic regulation of biliary inflammation and fibrosis

 描述（由申请人提供）： 胆道炎症和纤维化的协同调节机制背景：美国退伍军人因酒精和毒素滥用以及肝炎病毒导致的肝病风险越来越高，并且是住院和死亡的最常见原因之一。慢性肝病的特征是特定肝细胞的损害-胶原和基质积聚纤维化和最终瘢痕形成。胆管细胞是诸如原发性硬化性胆管炎（PSC）和原发性胆汁性肝硬化（PBC）的胆管病中的靶细胞，其特征在于胆管细胞的增殖/损失导致纤维化。胆管疾病的管理是退伍军人面临的主要挑战之一。该提案旨在增加我们对调节肝纤维化的因素的理解，并改善患者护理，从而为这些疾病提供更有效的治疗方法。组胺（HA）由包括肥大细胞（MC）在内的许多细胞分泌，但也由胆管细胞合成。我们已经表明：（i）HA通过与特异性HA受体相互作用刺激胆汁增殖/丢失 (HRs)和（ii）负责HA合成的酶，组氨酸脱羧酶（HDC）通过与miR-125 b和血管内皮生长因子（VEGF）相互作用和调节miR-125 b和VEGF来调节胆汁生长。我们的建议的基本原理是建立在以前发表的数据，显示PBC和PSC患者的HA水平增加，MC浸润增加与纤维化增加呈正相关。与胆管细胞和肝星状细胞（HSC）相互作用的MC是HA的主要来源，并可能导致瘙痒等疾病。本项目的总体目标是证明（i）HA（通过特异性HR相互作用）是一种源自包括胆管细胞和MC在内的几种细胞来源的纤维化促进剂;（ii）HA介导的纤维化由miR-125 b/VEGF轴驱动;（iii）胆管细胞、MC和HSC协同调节纤维化。我们将利用两者 体外和体内模型。我们建立的所有动物模型都与该提案相关，并通过模拟急性和慢性肝损伤患者中观察到的人类肝纤维化来支持以VA为中心的研究。我们的初步数据表明，（i）HA促进纤维化和（ii）抑制肥大细胞衍生的HA减少纤维化和肝星状细胞活化。我们提出了新的假说，即HA的协同调节通过miR-125 b/HDC/VEGF轴促进肝脏炎症和纤维化进展。我们提出以下目的：具体目的1：证明HA促进肝脏炎症和纤维化。我们将通过用HA和HA受体激动剂治疗啮齿动物模型并评估纤维化的进展来实现这一目标。此外，在模拟人类肝纤维化的模型中，我们将测量阻断肥大细胞来源的HA和阻断HA受体活化对纤维化的潜在治疗的影响。具体目标2 -确定HA介导的肝纤维化的细胞机制和潜在的治疗方法，在体内和体外。为了实现这一目标，我们将使用药理学和分子方法来阻断HDC和特异性HA受体，并评估参与肝纤维化的信号通路。在体外，我们将利用已建立的分子生物学技术来评估调节HA诱导的纤维化的途径。具体目标3：采用各种肝纤维化的体外和体内模型，确定HA介导的肝纤维化在胆管细胞、MC和HSC之间的细胞间相互作用。我们的工作假设是，肝损伤后，胆管增殖诱导MC募集，导致HA释放和HSC活化增加。我们将在相关的体内纤维化模型和使用体外细胞培养直接研究细胞间相互作用中评估这些事件。这些研究成功完成后获得的信息有望为组胺在肝纤维化进展中的作用提供重要的见解，这将最终有助于识别重要的信号通路，这些信号通路可用于开发纤维化治疗的治疗干预措施。

 DESCRIPTION (provided by applicant): Mechanisms of synergistic regulation of biliary inflammation and fibrosis Background: The risk of liver diseases due to alcohol and toxin abuse and hepatitis viruses in US Veterans is increasingly high and is one of the most common reasons for hospitalization and mortality. Chronic liver disease is characterized by damage to specific liver cellsècollagen and matrix accumulation fibrosis and eventual scarring. Cholangiocytes are the target cells in cholangiopathies such as Primary Sclerosing Cholangitis (PSC) and Primary Biliary Cirrhosis (PBC), which are characterized by the proliferation/loss of cholangiocytes leading to fibrosis. Management of cholangiopathies represents one of the major challenges for Veterans. This proposal aims to increase our understanding of factors that regulate liver fibrosis and to improve patient care leading to more effective treatments for these disorders. Histamine (HA) is secreted by many cells including mast cells (MCs), but also is synthesized by cholangiocytes. We have shown that: (i) HA stimulates biliary proliferation/loss via interaction with specific HA receptors (HRs) and (ii) the enzyme responsible for HA synthesis, histidine decarboxylase (HDC) regulates biliary growth via interaction with and modulation of miR-125b and vascular endothelial growth factor (VEGF). The rationale for our proposal is built upon previously published data showing that HA levels increase in PBC and PSC patients and increased MC infiltration positively correlates with increased fibrosis. MCs, which interact with both cholangiocytes and hepatic stellate cells (HSCs) are the main source of HA, and may contribute to conditions like pruritus. Our overall goals of this project are to demonstrate that (i) HA (via specific HR interaction) is a fibrosis-promoting agent derived from several cellular sources including cholangiocytes and MCs; (ii) HA-mediated fibrosis is driven by the miR-125b/VEGF axis and (iii) cholangiocytes, MCs and HSCs synergistically regulate fibrosis. We will utilize both in vitro and in vivo models in our proposal. All of our established animal models are relevant to the proposal and support VA-centered research by mimicking human liver fibrosis that is seen in patients with both acute and chronic liver injury. Our preliminary data demonstrates that (i) HA promotes fibrosis and (ii) inhibition of mast cell-derived HA decreases fibrosis and hepatic stellate cell activation. We propose the novel hypothesis that synergistic regulation of HA contributes to hepatic inflammation and fibrosis progression via the miR-125b/HDC/VEGF axis. We propose the following aims: Specific Aim 1: To demonstrate that HA promotes hepatic inflammation and fibrosis. We will develop this aim by treating rodent models with HA and HA receptor agonists and evaluating the progression of fibrosis. Further, in models that mimic human liver fibrosis, we will measure the effects of blocking mast cell-derived HA and the potential therapies derived from blocking HA receptor activation on fibrosis. Specific Aim 2 - To determine the cellular mechanisms and potential therapies of HA-mediated liver fibrosis, in vivo and in vitro. To achieve this, we will use pharmacological and molecular approaches to block HDC and the specific HA receptors and evaluate the signaling pathway involved in hepatic fibrosis. In vitro, we will utilize established molecular biology techniques to evaluate the pathways that regulate HA-induced fibrosis. Specific Aim 3: To determine the cell-to-cell interaction of HA-mediated hepatic fibrosis between cholangiocytes, MCs and HSCs using various in vitro and in vivo models of hepatic fibrosis. Our working hypothesis is that following liver injury, bile ducts proliferate inducing MC recruitment, leading to an increase in HA release and HSC activation. We will evaluate these events in both relevant in vivo models of fibrosis and using in vitro cell cultures to study cell-to-cell interaction directly. The information gained upo the successful completion of these studies are expected to provide important insights into the role that histamine plays during hepatic fibrosis progression, which will ultimately help in the identification of important signaling pathways that can be targeted for the development of therapeutic interventions for fibrosis treatment.