CELLULAR MODULATION OF MICROVESSEL PERMEABILITY IN VIVO

本项目的总体目标是研究细胞 在完整的微血管中的渗透性调节机制。 拟议的研究有两个具体目标。 在具体目标1下， 待检验的假设是：（1）内皮细胞[Ca 2 +]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;和（2）基础 NO释放水平提供了氧化剂清除剂功能， 在控制条件下微血管的完整性。 除了 作为一种血管扩张剂，NO被认为是一种重要的 微血管通透性的内在调节剂。 进一步调查 NO在调节微血管通透性中的作用 不同的条件具有重要意义。 在拟议的研究中， 内皮细胞中NO依赖性信号转导途径将 改变内皮细胞[Ca 2 +]I和微血管 在相同的实验条件下测定渗透率。 为了进一步研究NO的作用机制， 鸟苷酸环化酶调节渗透性，研究人员已经开发出 一种检测形成细胞的氧化剂水平变化的方法， 在NO抑制后使用荧光探针检测微血管壁。 的 该方法的独特优点是，所有的实验将 在具有正常渗透性的完整微血管中进行 特性，并且单微血管灌注技术将 使NO对渗透性变化的直接影响， 与其作为血管扩张剂的血液动力学效应分离。 根据具体 目的2，待检验的假设是内皮细胞的增加， [Ca2+]I与白细胞迁移的相关性与那些 引起的炎症介质，白细胞迁移，可 通过减弱钙内流和增加屏障而减少 内皮细胞的功能。 调查员写了一本小说 用银沉淀描绘内皮细胞边界的方法， vivo，它为钙信号传导的研究提供了一个绘图工具 在完整微血管中的单个内皮细胞中。 使用 共聚焦显微镜将使微血管的局部变化 渗透性和个体内皮细胞[Ca 2 =]I， 同时在体内识别血管结构。 这些是 最直接的实验方法来调查 内皮细胞[Ca 2 +]I、白细胞 迁移和个体血管通透性的局部变化- 细胞基础 这种程度的细胞定位在完整的微血管 这是以前的方法无法实现的。

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.