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B. A. Pfeffer, M. Salvador-Silva, C. Agarwal, D. Meyer, M. Cavet, K. W. Ward; Dose-Response Effects of Dexamethasone on Myocilin Protein and Gene Expression in Cultured Monkey Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1629.
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Among the side effects of corticosteroid (CS) therapy, whether systemic or directly to the eye, is an increase in intraocular pressure (IOP) culminating in drug-induced glaucoma. In these cases, the protein myocilin (MYOC) is thought to be secreted in excess, and to accumulate and obstruct aqueous outflow in CS responders in the human population. We assessed the dose-response to dexamethasone (DEX) with respect to MYOC at the protein and gene expression levels, using a non-human primate in vitro model.
Confluent cultures of monkey trabecular meshwork (TM) cells, at 3rd or 4th passage, were maintained from their initial seeding in multiwell plates using media supplemented with 10% fetal bovine serum, but no other added CS. Cell strains derived from individual animals were tested separately. TM cells were incubated for 5 days (including a change of experimental media at 3 days) with DEX in a concentration range of 3 - 300 nM. Following the treatments, conditioned media (CM) and cell lysates were harvested. MYOC in CM was assessed by PAGE-Western blot (WB), followed by quantitative densitometry of specific chemiluminescence. RNA purified from lysates was analyzed by quantitative RT-PCR using MYOC-specific primers.
Under control conditions, MYOC, both as protein and message, was detected at measurable levels that were used to normalize DEX-induced changes in expression. Individual TM strains exhibited differences in the maximal response to DEX (ranging from at least 3-fold to as high as 30-fold) compared to control. In WBs as well as by qRT-PCR, up-regulation of MYOC was evident for all cultures even at the lowest DEX concentration, and maximal responses were achieved with 100 to 300 nM DEX, depending on the strain of cells examined. These results are in agreement with a previously reported EC50 of 30 nM for DEX-induced MYOC expression in human TM. The dose-response to DEX observed for MYOC protein suggests a direct correlation with its effects on message abundance, without intervening posttransciptional regulation. DEX treatment did not compromise cell viability or integrity.
Increased MYOC expression in vitro may be an indicator for gene transactivation-mediated IOP increases associated with chronic CS administration. The techniques reported here can be used to screen GCs and also novel GC receptor ligands in the effort to identify agents with the antiinflammatory potency of conventional GCs while minimizing adverse ocular events associated with their use.
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