Mechanisms for Arsenic-Induced Vascular Disease

描述（由申请人提供）：众所周知，慢性暴露于三价砷（As III）会导致心血管疾病。在人类肝脏中，As （III）促进血管重塑、门脉纤维化和高血压，但这些As III诱导的血管改变的分子病理生理尚不清楚。总的来说，由于缺乏对慢性低剂量砷致血管疾病敏感的相关动物模型，对砷致血管疾病的发病机制研究不足。我们的初步研究结果表明，在正常成年小鼠的饮用水中暴露于低至10 ppb的砷会导致肝窦内皮（LSEC）脱壳和毛细血管化。此外，我们还表明，小鼠或人类LSEC的初级、短期培养有助于揭示As （III）刺激的NADPH氧化酶（NOX）在影响这一重要靶细胞表型（例如开窗）的早期信号事件中产生活性氧的功能作用。拟议研究的目的是利用这些体内和离体模型来研究As （III）启动LSEC重塑的机制和As （III）诱导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蛋白偶联细胞信号水平上起作用，促进NOX氧化剂的产生，从而破坏LSEC开窗的维持和抑制毛细化。因此，本提案的具体目的是确定：1 . As （III）在完整小鼠中引起肝窦毛细血管化和重塑的分子机制。野生型和氮氧化物缺乏（p47phox -/-）小鼠将暴露于（10-50 ppb） As （III）中2周，并对SEC脱窗和毛细管化的形态计量学（光和电镜水平）决定因素进行量化。药理学（血管内皮细胞生长因子受体或百日毒抗体）修饰小鼠将用于评估VEGF受体和gi蛋白相关信号在As iii诱导的血管改变中的作用。2。NOX生成的超氧化物在介导As iii诱导的小鼠和人原发性LSEC表型转化中的作用。从人肝脏或野生型和p47phox -/-小鼠中分离的LSEC将在体外暴露于As (III)，以证明活性氧和氮物种生成失衡介导asii - VEGF受体失调维持LSEC开孔的机制。3。如果LSEC GT Pase活性失衡介导As （III）刺激LSEC重塑。RhoA或Rac1-GTPase siRNA的靶向递送和选择性GTPase活性测定将用于分析Rho家族成员在As （III）刺激的LSEC毛细管化中的作用。

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.