Pericytic venules are composed of an endothelial monolayer partially covered interdigitating pericytes (PC). Although much work has been done on EC in culture, we are uniquely positioned to study isolated and cultured venular EC. In addition, our laboratory is the first to isolate and perfuse single pericytic venules. Finally, in the past 2 years, we have implemented several new technologies for studying venular EC and PC, including imaging of cytosolic calcium, pH, and membrane potential. In addition, with our Meridian ACAS 570 system we are also able to study cell/cell communication using fluorescence recovery after photobleaching to probe intracellular signalling mechanisms with microinjection of caged compounds such as cyclic AMP, IP3, and calcium and to obtain cloned vascular cells from explanted pericytic venules using laser destruction of unwanted cells. Finally, we have developed the techniques for quantifying cyclic AMP, inositol phospholipids and arachidonate metabolites and for probing specific G proteins. The aforementioned cell biology technologies plus the isolated venule and intact microvascular bed will allow us to explore the following issues. 1. Venular hyperpermeability to water is achieved by one of 3 mechanisms; a) activation of the EC cytoskeleton and associated small to moderate widening of the interendothelial junction, b) detachment of EC form its basement membrane secondary to release of enzymes by EC; this mechanism mediates large increases in venular permeability to water and macromolecules, c) hyperpermeabilization of the EC plasma membrane. Alterations in F-actin content and organization will provide a measure of cytoskeletal activation and presence of tissue plasminogen activator, collagenase and other EC-secreted enzymes in the absence of cytoskeletal activation will indicate simple detachment. Hyperpermeabilization of endothelial cell will be monitored by cytosolic accumulation of normally-excluded fluorescent probes. Histamine, bradykinin, epinephrine, atrial natriuretic peptide, platelet-activating factor, tumor necrosis factor, and fibroblast growth factor will serve as primary modulators of venular permeability. 2. Receptor-mediated changes in venular permeability are transduced by G proteins that interact with adenylate cyclase (hypopermeability) or phospholipase A2/guanylate cyclase (hyperpermeability). The specific signaling pathway responsible for permeability alterations will be dissected using the techniques described above. In addition, we plan to use alpha-toxin to permeabilize the vascular cells and clamp the intracellular contents of specific signalling molecules at any desired level. 3. Pericytes modulate the permeability of the endothelial layer of exchange venules. This project will examine transmembrane signalling in pericytes, communication between venular EC and PC, the impact of adding pericytes on venular endothelial monolayer permeability and differences in permeability responses observed when the mediator acts on the luminal versus the pericyte surface of isolated venules. The proposed research program should yield new insights into the function of the major gatekeeper for water and macromolecular transport in the microcirculation. Because of the pivotal role of the pericytic venule in blood/tissue fluid exchange under normal and pathophysiological conditions the research program should serve as a foundation for elucidating the basic mechanisms of edema formation.
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