It is well documented that cell-matrix interactions play an important regulatory role in controlling cell behavior (growth, differentiation, gene expression, and organization of cytoskeleton) and the organization of the extracellular matrix. The importance of cell-matrix interactions on the trabecular meshwork (TM) and aqueous outflow, however, is not known. Preliminary studies suggest that integrin/syndecan mediated signaling events could control aqueous outflow, since the heparin II (Hep II) domain of fibronectin that contains alpha4 beta1 integrin and syndecan binding sites lowers intraocular pressure in human eye organ cultures. The goal of this grant is to determine (1) whether outflow facility is increased because the Hep II domain affects the formation of cell contacts (cell-cell and cell-matrix) interactions and /or the organization of the extracellular matrix in the TM and (2) if the Hep II domain uses interactions with alpha4 beta1 integrins and syndecans to modulate outflow facility. The effect of the Hep II domain of fibronectin on aqueous outflow will be determined by perfusing the anterior chamber of primates or human eye organ cultures with recombinant Hep II domains and then analyzing its effects on aqueous outflow and the ultrastructure of the TM (cell contacts and/or matrix formation). Recombinant Hep II domains containing mutations in the alpha4 beta1 and /or syndecan binding sites will be used to determine the role of integrins and syndecans in aqueous outflow. The effect of the Hep II domain on cell contacts and the formation of matrix will be done by using various biochemical (zymograms, immunoprecipitation, Northern and Western blots) and microscopy (immunofluorescence, electron, and in situ hybridization) procedures to determine the effect of the Hep II domain on the localization of proteins in cell contacts (FAK, paxillin, cadherins, catenins and vinculin) and the expression of matrix proteins (stromelysin, gelatinases, fibronectin, laminin, and collagen). These studies should demonstrate how matrix-mediated interactions may influence aqueous outflow. Ultimately, such information should prove useful in the development of therapeutic peptides to control glaucoma.
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