Our long-term objective in this proposal is to evaluate the hypothesis that the sialated macromolecules of the extracellular matrix in the trabecular meshwork/Schlemm's canal (TM/SC) system have a major role in determining the resistance to the outflow of aqueous humor in normal and glaucomatous eyes.
The specific aims are: (1) To document changes, as a function of age, in the amounts and glycoforms of fibronectin, thrombospondin, and a 140 kD glycoprotein (140 kD-GP); these are 3 of 7 sialated molecules that we have identified in the TM/SC system. (2) To investigate the effect of fibronectin and thrombospondin on cytokinesis of trabecular cells in vitro as function of the amount and activity of tissue plasminogen activator (tPA) produced by these cells. (3) To characterize the recently discovered 56 kD sialated glycoprotein (Cort-GP) induced by the administration of -corticosteroids to trabecular cells in vitro. (4) To document the changes that occur in sialated macromolecules of the aqueous outflow pathway in eyes with. glaucomatous disease, with particular emphasis on primary open-angle glaucoma (POAG). The experimental design is to use microanalytical methods that will enable us to study selected macromolecules in the TM/SC system. We will utilize the observed changes in sialic acid moieties in aging eyes and in eyes with POAG as a means for obtaining more information about the structure and expression of specific polypeptides and RNA molecules that are involved in the normal and abnormal functioning of the TM/SC system. The methods to be used are: (1) isoelectric focusing, lectin and antibody affinities, PCR amplification and quantitative analysis to detect changes in the amounts and glycoforms of fibronectin, thrombospondin, and 140 KD-GP; (2) cell culture, ELISA, and a chromogenic substrate assay to determine the effect of fibronectin and thrombospondin on trabecular cells as a function of the amount of tPA produced by these cells; (3) SDS-PAGE, lectin affinity, as well as isolation of polysomes and mRNA to characterize Cort-GP; (4) immunohistochemistry and immunoblotting, with antisera against fibronectin, thrombospondin, type VI collagen, and Cort-GP to document changes that occur in these glycoproteins in tissue from glaucomatous eyes.
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