The trabecular meshwork (TM) is believed to be a major site for the regulation of the aqueous outflow. Residing in this tissue are TM cells that are essential for the maintenance of normal outflow. Aberrations of cell integrity may be a key step toward obstruction of the aqueous outflow, intraocular pressure elevations, and glaucomatous conditions. Along with the cells, extracellular matrix (ECM) materials such as fibronectin, laminin, and collagens are also thought to be important for normal functioning of the TM. We propose, in this application, to examine a family of receptors for ECM proteins, the integrins, and the cell-matrix interactions in the human TM. The integrins are known to interact with cytoskeletal proteins, mediate cell attachment, and influence a variety of cellular activities. They are also crucial in the determination of growth, morphology, metabolism, and differentiation of cells. In the TM system, cells line the trabecular beams that are made up of connective tissues. The TM cells in vivo are continually subjected to flows of the aqueous humor and changes in the intraocular pressure. The lining integrity against stress is critically dependent on the adhesion of TM cells to the matrices through integrin receptors. Disruption in the adhesion of TM cells to the ECM would conceivably lead to cell loss, denudation of the beams, and pathologic consequences. Characterization of the integrins and elucidation of the cell-matrix interactions in the TM are therefore of fundamental significance. We have initiated investigations on integrins and have found that the integrin repertoire in cultured TM cells is similar to that in tissues. We will further our efforts to evaluate the roles of integrins in adhesion of cultured TM cells to the ECM and to determine the integrin-mediated signaling pathways by immunoprecipitation, immunofluorescence staining and other methods. Activators and inhibitors will be used to define further the potential signaling mechanisms involved. We propose to dissect systematically the information transduced by cell-ECM interactions using the in vitro human TM cultures coupled with defined matrices. The effects of individual ECM substrata on the integrin distribution and biologic characteristics of TM cells will be assessed by cell biology and molecular biology techniques. To probe the functional roles of integrins, human TM cells will be transfected to result in either enhanced or suppressed expression of specific integrins. The effects of gene alterations will be analyzed. In addition, the integrin distribution and expression in conditions that are known to induce ECM changes in TM cells, such as after phagocytic challenges and after glucocorticoid treatments, will be examined. Modulation in the integrins will be correlated with the ECM changes. The effects of growth factors on the production and expression of fibronectin, laminin, collagen, and integrins by TM cells in both cell culture and organ culture systems will also be investigated. Protocols of immunohistochemical staining, and dot blot, Western blot and Northern blot analyses will be followed. Our goal is to increase the understanding of the biologic characteristics of healthy as well as experimentally-altered TM cells, particularly with regard to the dynamics and control mechanisms involved in the cell-matrix interactions in the TM. This information will assist in uncovering the pathogenic processes of glaucomas.
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