CELLULAR MODULATION OF MICROVESSEL PERMEABILITY IN VIVO

这个项目的总体目标是研究细胞 参与完整微血管通透性调节的机制。 拟议中的研究有两个具体目标。在具体目标1下， 有待检验的假设是：(1)内皮细胞[钙]i增加 触发的一氧化氮(NO)释放是增加 炎症条件下的微血管通透性；(2)基础 NO的释放水平提供了一种氧化清除剂功能，以维持 在控制条件下微血管的完整性。除了……之外 其作为血管扩张剂的作用，NO已被公认为一种重要的 微血管通透性的内在调节剂。进一步调查 一氧化氮在微血管通透性调节中的作用 不同的条件意义重大。在拟议的研究中， 内皮细胞中的NO依赖的信号转导通路将 内皮细胞[Ca~(2+)]i和微血管的修饰和变化 渗透率将在相同的实验条件下进行测定。 为了进一步研究NO的作用机制，除了激活 鸟苷环化酶在调节通透性方面的研究人员已经开发出来 一种从形成细胞中检测氧化剂水平变化的方法 微血管壁用荧光探头后无抑制。这个 这种方法的独特优势是，所有的实验都将 在通透性正常的完整微血管中进行 特性，并且单血管灌注技术将 使一氧化氮对通透性变化的直接影响 从其作为血管扩张剂的血液动力学作用中分离出来。在特定项下 目标2，需要检验的假设是内皮细胞的增加 与白细胞迁移相关的[Ca2+]i类似于 由炎性介质引发，白细胞迁移可以 通过减弱钙离子内流和增加屏障而减少 内皮细胞的功能。这位调查员写了一本小说 血管内皮细胞边界的银沉淀法 Vivo，这为钙信号的研究提供了一种作图工具 在完整的微血管中的单个内皮细胞中。对.的使用 共聚焦显微镜将使微血管的局部变化成为可能 要测量的通透性和单个内皮细胞[Ca~(2+)]i，以及 血管结构需在体内同时鉴定。这些是 最直接的实验方法来研究 内皮细胞[Ca~(2+)]_i与白细胞变化的关系 迁移，以及个体血管渗透性的局部变化- 单元格基础。在完整的微血管中这种程度的细胞定位 以前的方法是无法实现的。

The overall aim of this project is to investigate the cellular mechanisms involved in permeability modulation in intact microvessels. The propose studies have two specific aims. Under Specific Aim 1, the hypotheses to be tested are that (1) increased endothelial [Ca2+]I triggered nitric oxide (NO) release is a necessary step for increasing microvessel permeability under inflammatory conditions; and (2) basal levels of NO release provide an oxidant scavenger function to maintain the integrity of microvessels under control conditions. In addition to its role as a vasodilator, NO has been recognized as an important intrinsic modulator of microvessel permeability. Further investigation of the roles of NO in modulating microvessel permeability under different conditions is of great significance. In the proposed studies, the NO-dependent signal transduction pathway in endothelial cells will be modified and, changes, in endothelial [Ca2+]I and microvessel permeability will be determined under the same experimental conditions. To further investigate mechanisms of NO besides the activation of guanylate cyclase in regulating permeability, investigator has developed a method to detect he changes in oxidant levels from cells forming the microvessel wall using a fluorogenic probe after NO suppression. The unique advantages of the approach are that all of the experiments will be conducted in intact microvessels, which have normal permeability properties, and that the single microvessel perfusion technique will enable the direct effect of NO on changes in permeability to be separated from its hemodynamic effect as a vasodilator. Under Specific Aim 2, the hypotheses to be tested are that the increases in endothelial [Ca2+]I associated with leukocyte migration are similar to those elicited by inflammatory mediators, and leukocyte migration can be reduced by attenuating calcium influx and increasing the barrier function of endothelial cells. The investigator has developed a novel method to delineate endothelial boundaries with silver precipitation in vivo, which provides a mapping tool for the study of calcium signaling in individual endothelial cells in intact microvessels. The use of confocal microscopy will enable local changes in microvessel permeability and individual endothelial [Ca2=]I to be measured and the vascular structure to be identified in vivo simultaneously. These are the most director experimental approaches to investigating the relationship between changes in endothelial cell [Ca2+]I, leukocyte migration, and local changes in vascular permeability on an individual- cell basis. This degree of cellular localization in intact microvessels could not be achieved by previous methods.