The extracellular matrix (ECM) has been studied primarily for its ability to promote cell adhesion. It is becoming apparent that ECM components can regulate cell adhesion in a negative manner as well. Negative modulation of cell adhesion is important in events such as proliferation, development, wound healing, and metastasis in which changes in cell shape and increased motility are critical. Studies by myself and others indicate that thrombospondin (TSP) and tenascin (TN) are two such ECM molecules which destabilize cell adhesion. This proposal seeks a better understanding of how TSP and TN regulate cell adhesion, especially in bovine aortic endothelia cells.
The first aim of this proposal is to determine the mechanism whereby TSP modulates focal adhesions. Initial studies will determine if TSP is present at adhesion plaques to see if adhesions are accessible to TSP. TSP heparin-binding and non-binding fragments, alone and in combination, and synthetic peptides will be tested in focal adhesion assays for activity to determine which domains of TSP are active. Since TSP is clustered at the cell surface and endocytosed, a cross-linked fusion protein of the heparin-binding domain linked to Protein A will be used to determine if multivalency of this domain is required for TSP activity. TSP's effect on metabolism of cell surface proteoglycans will be assessed by cetyl pyridinium chloride precipitation studies to determine if TSP-cell binding causes loss of cell surface HSPGs, potentially disrupting focal adhesions. Time-lapse IRM will be used to determine if TSP prevents the re-formation of focal adhesions or causes their disassembly. Focal adhesion assays on synchronized cells will determine if the effect of TSP is cell cycle specific.
The second aim of this proposal is to probe the functional significance of TSP modulation of focal adhesions by studying the effect of TSP on cell migration, proliferation and DNA synthesis, and on strength of attachment.
The third aim i s to characterize the TN-mediated loss of adhesions in comparison to TSP. TN will be studied for its cell and substrate specificity and the ability of a C-6-S proteoglycan to neutralize TN activity. Monoclonal antibodies to domains of TN will help assess which parts of TN are active. These studies have potential significance for the study of vascular disease, particularly in reendothelialization of vessels and in the development of atherosclerotic disease.
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