application) Post-capillary venules are the primary sites of extravasation of water, proteins, and blood borne cells, especially during inflammation and injury. Recent advances in microvascular techniques and cell biology have facilitated the evolution of new signaling paradigms to explain how chemical and mechanical stimulation of the postcapillary venules leads to overall increases in permeability to water and proteins such as albumin. In addition, progress in our understanding of the molecular basis of endothelial cell adhesion and vesicular transport provides an exciting opportunity to revisit fundamental issues of microvascular exchange with new tools and concepts. The overall objective of this proposal is to determine the role of its different structures, transport proteins and signaling mechanisms in governing venular exchange of water and proteins. The interendothelial junction and caveolae represent two different structures involved in venular exchange. The width of the interendothelial junction is dependent on the balance of adhesive (cell/cell and cell/matrix) and tensile (cytoskeletal contraction) forces. Direct activation of the contractile machinery serves as one means to test the strength of the tethering sites. Insight into the role of adhesion molecules in establishing normal venular permeability is gained by using antibodies and other reagents to reduce the number of tethering sites. Vesicular transport of albumin is dependent on receptor-mediated endocytosis and is likely modulated by intracellular signaling mechanisms. Using reagents that block vesicular transport allows partition of total transvenular albumin transport into intercellular and transcellular routes. In the high permeability postcapillary venules of the heart, the basement membrane is likely to play an important role in restricting movement of water and proteins. Removal of endothelial cells, application of enzymes and enzyme inhibitors can provide insight into this issue. Specific signaling pathways governing vesicular paracellular and basement membrane transport are determined by measuring specific second messengers of phosphorylated proteins and using known inhibitors of enzymes located in known signaling pathways. The isolated perfused coronary venule is the primary biological preparation used to address these issues. However, single cell and monolayer studies are also proposed using endothelial cells derived from the coronary venules. Fluorescence microscopy, ratio imaging, confocal microscopy, immunocytochemistry, western blots, and phosphorylation assays are some of the techniques employed to achieve the aims of the proposal.
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