The long term goals of this project ar to understand, at the molecular level, the adhesive interactions at the intercellular junctions of the vascular endothelium between endothelial cells (EC) and leukocytes that pass through the junction (specifically at the level of the postcapillary venule) en route to a site of inflammation. Tight membrane contacts are made between these different cell types suggesting that specific ligand/receptor type binding interactions mediate this movement. An understanding of the molecular mechanisms governing this stage of inflammation might provide insights into ways to inhibit this process (e.g. in an arthritic joint) or augment it (e.g. in an immunocompromised host).
The specific aims of this project are shorter term goals relating to characterizing two integral membrane proteins of the EC intercellular junction that have been described recently in my lab. One of these, Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM) is not known to mediate adhesive interactions between cells bearing it (See PRELIMINARY STUDIES). The other, HEC-1 is a presumptive cell adhesion molecule now being characterized. Monocytes and nurtrophils also express PECAM, and one of the hypotheses to be tested in this proposal is whether binding of PECAM on the leukocyte to PECAM on the EC during transendothelial migration allows this interaction to be so tight, yet transient. The first specific aim of the project is to purify, clone, and sequence HEC-1. Structural information will provide insights to its function. A cDNA clone would be transfected into mammalian cells for direct functional studies.
The second aim i s to characterize the adhesive interactions mediated by these transfected proteins. Reagents generated to specifically block this adhesion would be used to identify the ligands for these molecules.
The third aim i s to use the reagents known to block adhesion via these molecules to see whether they are involved in EC-leukocyte binding during transendothelial migration in an in vitro assay we have developed. The question of whether these molecules acts as a membrane-bound gradient of 0chemoattractant will also be tested by immunoelectron microscopy. The 1fourth aim is to study the changes in surface expression, and possibly the 2changing role of these molecules in vitro in response to defined cytokines, 3in situ by immunohistochemistry in acute and chronically inflamed human 4tissue, and in vivo animal models that test the ability of the transfected cells to participate in, and the blocking reagents to interfere with, the inflammatory response.
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