CONTROL OF MICROCIRCULATORY EXCHANGE FUNCTION

描述(申请人提供)：炎症过程的特征是微血管对大分子的渗透性增加(高通透性)。血管壁和血细胞之间的信号相互作用提供了一种独特的方式来沟通、协调和整合对体内不断变化的组织环境的适当的生理反应。在体内微循环的复杂细胞环境中，关于通透性相关的信号机制的信息很少。了解体内这些重要的相互作用是了解微血管运输及其在血管疾病中的功能变化的综合调控的基础。尤其是一氧化氮(NO)在微血管通透性控制中的作用仍存在争议。我们建议在体内验证三个主要假说：1)eNOS相关的信号级联调节微血管运输。2)内皮型一氧化氮合酶的分子运动(移位、转运)在内皮细胞调节机制中是一个重要的功能步骤。3)eNOS信号机制调节微血管通透性对缺血再灌注(I-R)的反应。为了验证这些假说，我们将A)确定eNOS-/-小鼠体内eNOS活性和高通透性之间是否存在因果关系，以及它们的野生型eNOS/对照组之间是否存在因果关系；B)测试纯血管扩张剂(ACh)和非血管扩张剂(PAF)刺激后eNOS从膜到胞浆的移位；C)确定eNOS//-小鼠和eNOS-/-小鼠微血管通透性反应是否存在差异，以及eNOS-/-小鼠在基线和I-R后eNOS的亚细胞定位。此外，我们还将确定在ACh、PAF和I-R反应中eNOS磷酸化是否发生变化，并评估PKB和MAP激酶在这些过程中的意义。 免疫沉淀、免疫印迹、活体和共聚焦显微镜以及NO测量和计算机辅助数字图像分析方法将用于评估体内微血管通透性的调节。

DESCRIPTION (provided by applicant): Inflammatory processes are characterized by an increase in microvascular permeability (hypermeability) to macromolecules. Signaling interactions between vascular wall and blood cells provide a unique way of communicating, coordinating, and integrating an appropriate physiologic response to the changing tissue environment in vivo. There is a paucity of information on permeability-related signaling mechanisms in the complex cellular environment of the in vivo microcirculation. Knowledge of these important interactions in vivo is fundamental to understand the integrated regulation of microvascular transport and its functional alterations in vascular disease. In particular, the role of nitric oxide (NO) in the control of microvascular permeability remains controversial. We propose to test in vivo three major hypotheses: 1) an eNOS-assoctated signaling cascade regulates microvascular transport. 2) molecular movement (translocation, trafficking) of eNOS from membrane to other cellular compartments is a functionally and differentially important step in the endothelium-mediated regulatory mechanisms. 3) eNOS signaling mechanisms regulate microvascular permeability responses to ischemia-reperfusion (I-R). To test these hypotheses, we will A) determine whether or not a cause-effect relationship exists between eNOS activity and hyperpermeability in vivo in eNOS -/- mice and their wild-type eNOS +/+ control; B) test translocation of eNOS from membrane to cytosol after stimulation with a pure vasodilator (ACh) and a non-vasodilator that causes hyperpermeability (PAF); C) determine whether or not differences in microvascular permeability responses exist in eNOS +/+ and eNOS -/- mice and the subcellular location of eNOS under baseline and after I-R. In addition, we will determine whether or not changes in eNOS phosphorylation occur in response ACh, PAF and to I-R, and assess the significance of PKB and MAP kinases in these processes. Methods of immunoprecipitation, western blotting, intravital and confocal microscopy, as well as NO measurements and computer-assisted digital image analysis will be applied to evaluate the regulation of microvascular permeability in vivo.