Glaucoma is a blinding eye disorder which in many cases is due to elevated intraocular pressure (IOP); elevated IOP and glaucoma can be caused by administration of ocular corticosteroids such as dexamethasone (DEX). This grant request is to apply gene cloning methods to make specific molecular probes to characterize the glucocorticoid-regulated species in the human trabecular meshwork (HTM) of the eye which may be involved in development of these conditions. The HTM cell culture system would be the initial source to produce the probes and to test the effects of sustained DEX treatment, since prior work showed the induction of a potentially unique class of cellular and secreted proteins in these cells. The induced proteins appear related in dose and time to clinical steroid effects on IOP, and can also be seen in non-cultured trabecular meshwork tissues dissected from organ cultures perfused for 2 weeks with DEX. The applicant's cell-free translation studies demonstrated the presence of showed a marked progression with time suggesting that molecular biology approaches could be used to obtain relevant cDNA clones. During pilot studies over the past year the applicant and his coworkers have used subtraction screening approaches to isolate and partially characterize an initial group of high interest clones which show the distinctive time and dose characteristics characteristic of in steroid-induced IOP changes. The applicant and his coworkers are currently employing quantitative PCR approaches to examine these and other potentially important probes in the steroid-treated HTM cells and tissues. The applicant and his coworkers also plan to use the probes they are developing to examine in situ models using perfusion organ culture and pathological specimens from patients. It is possible that applications of the approaches outlined in this proposal may provide clues to the changes which occur in the outflow pathway in steroid glaucoma and perhaps in primary open-angle glaucoma (POAG).
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