CELLULAR MODULATION OF MICROVESSEL PERMEABILITY IN VIVO

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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.