Mechanisms of synergistic regulation of biliary inflammation and fibrosis

 描述(由申请人提供)： 背景：在美国退伍军人中，由于酒精和毒素滥用以及肝炎病毒引起的肝脏疾病的风险越来越高，是导致住院和死亡的最常见原因之一。慢性肝病的特征是特定的肝细胞受损，胶原和基质堆积，纤维化，最终形成疤痕。胆管细胞是原发性硬化性胆管炎(PSC)、原发性胆汁性肝硬化症(PBC)等胆管病的靶细胞，其特点是胆管细胞的增殖/缺失导致纤维化。胆管病的管理是退伍军人面临的主要挑战之一。这项建议旨在增加我们对调节肝纤维化的因素的了解，并改善患者护理，从而为这些疾病提供更有效的治疗。组胺(HA)不仅由肥大细胞(MCs)等多种细胞分泌，也可由胆管细胞合成。我们已经证明：(I)HA通过与特定的HA受体相互作用来刺激胆管的增殖/丢失 组氨酸脱羧酶(HDC)是负责合成HA的酶，通过与miR-125b和血管内皮生长因子(VEGF)相互作用和调节，调节胆管生长。我们建议的理由是建立在以前发表的数据基础上的，这些数据表明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(HSCs)相互作用的MCs是HA的主要来源，可能导致瘙痒等情况。我们这个项目的总体目标是证明：(I)HA(通过特定的HR相互作用)是一种来自多种细胞来源的纤维化促进剂，包括胆管细胞和MCs；(Ii)HA介导的纤维化是由miR-125b/VEGF轴驱动的；(Iii)胆管细胞、MCs和HSCs协同调节纤维化。我们将利用这两种方式 在我们的建议中建立了体外和体内模型。我们所有已建立的动物模型都与这一提议相关，并支持以VA为中心的研究，通过模拟在急性和慢性肝损伤患者中看到的人类肝纤维化。我们的初步数据表明：(I)HA促进纤维化，(Ii)抑制肥大细胞来源的HA可减少纤维化和肝星状细胞的激活。我们提出了一个新的假设，即HA的协同调节通过miR-125b/HDC/VEGF轴促进了肝脏炎症和纤维化的进展。我们提出了以下目标：特定目标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和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的效果，以及通过阻断HA受体激活而获得的潜在治疗方法。特定目的2-确定体内和体外HA介导的肝纤维化的细胞机制和潜在的治疗方法。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和特定的HA受体，并评估参与肝纤维化的信号通路。在体外，我们将利用成熟的分子生物学技术来评估调控HA诱导的纤维化的途径。具体目的3：采用不同的体内外肝纤维化模型，研究HA介导的肝纤维化在胆管细胞、MCs和HSCs之间的细胞间相互作用。我们的工作假设是，在肝损伤后，胆管增殖诱导MC募集，导致HA释放和HSC激活增加。我们将在相关的体内纤维化模型中评估这些事件，并使用体外细胞培养直接研究细胞间的相互作用。成功完成这些研究所获得的信息有望为组胺在肝纤维化进展中所起的作用提供重要的见解，这最终将有助于确定重要的信号通路，这些信号通路可以作为开发纤维化治疗干预措施的靶点。

 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.