The long term objective of the laboratory is to examine the physiology of microvessel endothelial cells (MEC) as it relates to the post-translational regulation of microvascular paracellular permeability. The underlying premise is that interendothelial junctional pores are not fixed, and the degree of patency is modulated by actin and actin-associated proteins. The applicant has developed an in vitro physiological model using cultured pulmonary MEC to investigate signal transduction cascades and to identify potential cytoskeletal targets associated with regulation of the microvascular barrier. Hypoxia/reoxygenation (H/R) injury mimicking, in part, ischemia-reperfusion injury is used to create an inflammatory setting. The overall hypothesis to be tested is that reactive oxygen species generated in reoxygenation injury act principally through activation of inflammatory second messenger pathways (e.g. protein kinase C, phosphatidylinositol 4,5-biphosphate) or inhibition of barrier promotion pathways (cAMP-dependent protein kinase) and not merely as nonspecific toxins. Through these pathways, the reactive oxygen species disrupt membrane-cytoskeleton interactions. This in turn destabilizes the plasma membrane and by so doing not only increases paracellular permeability but also increases MEC intracellular susceptibility to the cascade of reactive oxygen species. Conversely, the restorative response of junctional integrity, primarily acting through increases in cAMP, reestablishes membrane-cytoskeleton linkages. In parallel with a bubble chamber model for H/R injury in MEC, a simple chemical model is used to produce injury employing ATP-depleting agents and exogenously added reactive oxygen species, as well as inhibitors and activators of second messenger cascades. The question of whether repeated, minor exposures to H/R protect MEC against a major H/R event by the production of specific cytoskeletal proteins will also be examined. Lastly, assays will be performed to: (1.) Compare pulmonary MEC to other MEC phenotypes to ascertain unique injury responses; and (2.) Determine if the induced H/R injuries and repair coincide with changes in MEC shape and contractility associated with monolayer junctional permeability to macromolecules and neutrophil diapedesis. Hence, the regulation of paracellular permeability by membrane- cytoskeletal proteins may provide for the development of novel strategies for therapeutic or prophylactic intervention.
