Increased endothelial permeability is commonly associated with leukocyte accumulation at sites of vascular trauma and inflammation. The regulatory pathways associated with vascular permeability during endothelial-leukocyte interactions are not well understood at the molecular level. We have identified a factor (or series of factors) termed Endothelial Permeability Factor (EPF) which promotes endothelial permeability during interactions with polymorphonuclear leukocytes (PMN). EPF is a small, hydrophilic compound(s) released into soluble supernatants derived from activated PMN. Isolation and partial purification of EPF indicates that PMN-derived 5'-adenosine monophosphate (5'AMP) accounts for greater than 50 percent of this activity. From these data, we hypothesize that biochemical pathways exist to dampen permeability changes brought about during leukocyte transendothelial migration. The overall goal of this proposal is characterize, both structually and biologically, 5'AMP and other EPF actions on endothelial permeability.
Three specific aims are proposed to accomplish this overall goal. As a first specific aim, using physical means, we will purify and elucidate the structural nature of EPF's in addition to 5'AMP, and examine activation specificity for release of these factors. Initial steps have been taken to provide feasability for such identification. The role of the endothelial cytoskeleton in EPF-elicited barrier function will be established. As a second specific aim, we will define details of PMN-derived 5'AMP - endothelial crosstalk, endothelial extracellular signaling pathways, and the role of ecto-5'-nucleotidase (CD73) in regulation of endothelial permeability by 5'AMP and other EPF. These experiments will be extended into an in vivo model of increased pulmonary vascular permeability.
In specific aim three, we will study details of endothelial lateral membrane protein organization during PMN transmigration and relate these findings to the action of 5'AMP and other EPF identified herein. Such experiments should yield insight into the regulation of endothelial permeability during a number of acute disorders, and could provide the basis for development of novel therapeutics for disorders of vascular permeability.
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