Mechanisms for Arsenic-Induced Vascular Disease

描述（由申请人提供）：众所周知，长期暴露于三价砷（As III）会导致心血管疾病。在人类肝脏中，As（III）促进血管重塑、门静脉纤维化和高血压，但这些As III诱导的血管变化的分子病理生理学尚不清楚。在一般情况下，三价砷引起的血管疾病的发病机制已研究不足，部分原因是缺乏相关的动物模型敏感的慢性低剂量三价砷的影响。我们的初步研究结果表明，完整的成年小鼠暴露于低至10 ppb的As III在他们的饮用水引起的肝窦内皮细胞（LSEC）的开窗和毛细血管化。此外，我们还表明，主要的，短期培养的小鼠或人类LSEC是有用的，在揭示功能作用的As（III）-刺激的NADPH氧化酶（NOX）的活性氧物质的产生在早期信号事件影响表型（如开窗），这一重要的靶细胞。拟议的研究的目的是使用这些在体内和离体模型，以调查的机制，通过它作为（III）启动LSEC重塑和As（III）诱导的血管疾病的分子病理学。这些研究的总体假设表明，As（III）在g-蛋白偶联细胞信号传导的水平上起作用，以促进NOX氧化剂的产生，从而破坏LSEC开窗的维持和对毛细作用的抑制。因此，本提案的具体目标是确定：As（III）引起完整小鼠肝窦毛细血管化和重塑的分子机制。将野生型和NOX缺陷（p47 phox-/-）小鼠暴露于As（III）（10-50 ppb）2周，并定量SEC脱窗和毛细作用的形态测定（光和电子显微镜水平）决定因素。药理学（血管内皮细胞生长因子受体或百日咳毒素的抗体）修饰的小鼠将用于评估VEGF受体和Gi蛋白相关信号传导对As III诱导的血管变化的贡献。二. NOX产生的超氧化物在介导As III诱导的原代鼠和人LSEC表型转化中的作用。将从人肝或野生型和p47 phox-/-小鼠分离的LSEC离体暴露于As（III）以证明活性氧和氮物质产生的不平衡介导VEGF受体维持的LSEC开窗的AsIII-失调的机制。三.如果LSEC GT Pase活性的不平衡介导As（III）刺激LSEC的重塑。RhoA或Rac 1-GTdR siRNA的靶向递送和选择性GTdR活性测定将用于剖析Rho家族成员在As（III）刺激的LSEC毛细作用中的作用。

DESCRIPTION (provided by applicant): Chronic exposure to trivalent arsenic (As III) is well known to cause cardiovascular diseases. In the human liver, As (III) promotes vascular remodeling, portal fibrosis, and hypertension, but the molecular pathophysiology of these As III-induced vascular changes is unknown. In general, the pathogenesis of As III induced vascular diseases has been understudied, in part, due to a lack of relevant animal models sensitive to chronic low dose As III effects. Our preliminary results show that exposure of intact adult mice to as low as 10 ppb of As III in their drinking water caused defenestration and capillarization of liver sinusoidal endothelium (LSEC). Furthermore, we also show that primary, short term cultures of murine or human LSEC are useful in revealing functional roles for As (III)-stimulated NADPH oxidase (NOX) generation of reactive oxygen species in the early signaling events affecting phenotype (e.g. fenestration) of this important target cell. The objective of the proposed studies is to use these in vivo and ex vivo models to investigate the mechanisms through which As (III) initiates LSEC remodeling and the molecular pathology of As (III)-induced vascular diseases. The global hypothesis for these studies states that As (III) acts at the level of g-protein coupled cell signaling to promote NOX oxidant generation that disrupts maintenance of LSEC fenestrations and suppression of capillarization. Accordingly, the specific aims of this proposal are to determine: I. the molecular mechanism by which As (III) causes liver sinusoidal capillarization and remodeling in intact mice. Wildtype and NOX deficient (p47phox -/-) mice will be exposed to As (III) (10-50 ppb) for 2 weeks and morphometric (light and electron microscopic level) determinants of SEC defenestration and capillarization will be quantified. Pharmacologically (antibodies to vascular endothelial cell growth factor receptor or Pertussis toxin) modified mice will be used to assess the contribution VEGF receptor and Gi-protein linked signaling to As III-induced vascular changes. II. the role of NOX generated superoxide in mediating As III-induced phenotypic conversion of primary murine and human LSEC. LSEC isolated from human liver or wildtype and p47phox -/- mice will be exposed to As (III) ex vivo to demonstrate mechanisms through which an imbalance of reactive oxygen and nitrogen species generation mediates AsIII-dysregulation of VEGF receptor maintained LSEC fenestration. III. if an imbalance in LSEC GT Pase activity mediates As (III) stimulated remodeling of the LSEC. Targeted delivery of RhoA or Rac1-GTPase siRNA and selective GT Pase activity assays will be used to dissect the roles of Rho family members in As (III)-stimulated LSEC capillarization.