Hepatic Stabilin-2 Mechanisms of Clearance

摘要 人肝脏清除细胞外基质材料，包括透明质酸、硫酸软骨素和胶原蛋白。 死亡的红细胞，从循环，除了作为一个代谢动力室。这种拾荒行为 活性由表达稳定蛋白-2受体的肝窦内皮细胞（LSEC）进行。后 稳定蛋白-2通过一系列配体激活，诱导MAP激酶和NF-kB通路，所有LSEC “感知”他们的环境并使用一氧化氮等小分子进行回报。LSEC充当接口 在血液和肝细胞之间，含有调节大分子的窗孔（筛板） 灌注到肝细胞。LSEC的损伤通常会减少窗孔的数量和大小， 影响整个肝脏代谢肝脏疾病，如脂肪肝，影响超过三分之一的成年人， 这些疾病直接影响LSEC的清除/传感能力。损坏的LSEC 导致细胞外基质物质在血液中积聚，然后对肾功能产生负面影响。的 肝脏清除血液中的死细胞，但它也是转移性癌症的一个部位，两者密切相关 对LSEC的健康至关重要。为了更充分地了解LSEC的清除作用，项目负责人建议 在模拟肝窦的剪切应力条件下检查它们。本研究将 这是第一次在不同流量下审查LSEC的清除活动，以评估 稳定素-2作为主要受体。需要解决的问题包括：剪切应力对 简单配体或复杂配体，如细胞周HA包被细胞？LSEC如何对它们的配体做出反应 以及脂肪肝这种常见疾病对LSEC Stabilin-2介导的 内吞作用？根据他以前对稳定蛋白-2的研究，项目负责人假设特定的配体 在剪切应力下，循环中富含透明质酸的细胞与稳定蛋白-2的结合增强，使它们能够 在肝脏中停止循环和增殖，稳定蛋白-2的表达水平与肝脏 清除功能这项研究的中心假设是，配体-稳定蛋白-2相互作用， 随后是胞吞作用和细胞信号传导的激活，受到窦状隙处的剪切应力的影响， 肝代谢状态（例如，脂肪肝），其影响血液清除和癌细胞转移到肝脏。 项目负责人将使用稳定的重组细胞和来自正常人和正常人的原代LSEC来检验这一假设。 大鼠肝脏疾病模型。将在流动细胞培养下评价来自这些肝脏的纯化LSEC 条件以监测由稳定蛋白-2传播的配体结合和信号传导事件。为实现项目 为了实现这一目标，项目负责人将追求三个具体目标：1）确定稳定蛋白-2结合的配体下流动 2）确定剪切应力对LSEC中细胞信号传导的影响，和3）确定稳定蛋白-2 在正常和脂肪肝中的表达和内吞活性。

ABSTRACT The human liver scavenges extracellular matrix material, including hyaluronic acid, chondroitin sulfate, and dead red blood cells, from circulation, in addition to serving as a metabolic power house. This scavenging activity is performed by liver sinusoidal endothelial cells (LSECs) expressing the Stabilin-2 receptor. Upon activation by a subset of ligands, Stabilin-2 induces the MAP kinase and NF-kB pathways, by which all LSECs "sense" their environment and reciprocate using small molecules like nitric oxide. LSECs serve as the interface between the blood and hepatocytes and contain fenestrae (sieve plates) that regulate macromolecules perfusing through to the hepatocytes. Damage of LSECs often reduces the number and size of the fenestrae, affecting overall liver metabolism. Liver diseases, such as fatty liver, affect more than a third of the adult population, and these diseases directly affect the scavenger/sensing capacity of LSECs. Damaged LSECs lead to a build-up of extracellular matrix material in the blood that then negatively affects kidney function. The liver clears dead cells from blood, but it is also a site for metastatic cancer, both of which are intimately related to the health of LSECs. To more fully understand the clearance role of LSECs, the project leader proposes to examine them under shear stress conditions that mimic conditions in the hepatic sinusoid. This study will be the first time that the clearance activity of LSECs has been examined under variant flow to assess activity of Stabilin-2 as the major receptor. Questions to be addressed include: What effect does shear stress have on simple ligands or complex ligands like pericellular HA coated cells? How do LSECs respond to their ligands during shear stress, and what effect does fatty liver, a common ailment, have on LSEC Stabilin-2-mediated endocytosis? From his previous studies with Stabilin-2, the project leader hypothesizes that specific ligand binding to Stabilin-2 by circulating hyaluronan-rich cells is enhanced under shear stress, allowing them to cease circulation and proliferate in liver, and that expression levels of Stabilin-2 directly correlates with liver clearance function. The central hypothesis of the proposed research is that ligand-Stabilin-2 interactions, followed by endocytosis and activation of cell signaling, are influenced by shear stress at the sinusoids and hepatic metabolic status (e.g., fatty liver), which affects blood clearance and cancer cell metastasis to liver. The project leader will test this hypothesis using stable recombinant cells and primary LSECs from normal and disease models of rat livers. Purified LSECs from these livers will be evaluated under flow cell culture conditions to monitor ligand binding and signaling events propagated by Stabilin-2. To accomplish the project goal, the project leader will pursue three specific aims: 1) Determine Stabilin-2 binding of ligands under flow conditions, 2) Determine the effects of shear stress on cellular signaling in LSECs, and 3) Determine Stabilin-2 expression and endocytic activity in normal and fatty liver.