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D.–J. Oh, R.E. Peck, J.L. Martin, D.M. Hoberman, D.J. Rhee; Effects of Dexamethasone and TGF–ß2 on SPARC Expression in Cultured Human Trabecular Meshwork and Ciliary Body . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1875.
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The matricellular protein, SPARC, modulates cellular interaction with extracellular matrix (ECM) and influences cell behavior during ECM remodeling. Dexamethasone (DEX) has been shown to cause an increase in ECM in trabecular meshwork (TM). Elevated levels of transforming growth factor–ß2 (TGF–ß2) were found in the aqueous humor of glaucomatous eyes. We investigated the possible involvement of SPARC in intraocular pressure regulation in cultured human TM and ciliary body smooth muscle (CBSM) cells incubated with DEX and TGF–ß2.
Cultures of TM endothelial and CBSM cells were incubated with DEX (100 nM) and TGF–ß2 (1.0 ng/ml) for 1, 3, and 7 days. Changes in mRNA expression of SPARC were assessed using quantitative reverse transcription–polymerase chain reaction (qRT–PCR) in control and treated cultures of TM endothelial and CBSM cells. Changes in SPARC at protein level were examined using western blotting and enzyme–linked immunosorbent assay (ELISA).
In TM cells, DEX did not change SPARC mRNA at day 1, but decreased 32% at day 3 and 37% at day 7. DEX decreased immunoblot staining 30% at day 1, 20% at day 3, and 18% at day 7. DEX did not change free SPARC levels compared to controls (13 ng/ml/106cells) at 1–, 3–, or 7–days. TGF–ß2 induced a significant increase in SPARC mRNA 51% at day 1, 125% at day 3, and 200% at day 7. Immunoblot staining increased 60% at day 1, 110% at day 3, and 65% at day 7. Free SPARC levels increased 11% at day 1, 30% at day 3, and 43% at day 7. In CBSM cells, DEX did not change SPARC mRNA compared to control at 1–, 3–, or 7–days. Immunoblot staining decreased 23% at day 1, 35% at day 3, and 22% at day 7. Free SPARC levels did not change compared to controls (130 ng/ml/106cells). TGF–ß2 increased SPARC mRNA 130% at day 1, 240% at day 3, and 350% at day 7. Immunoblot staining increased 168–175% at 1–, 3–, and 7–days. Free SPARC levels increased 13% at day 1, 25% at day 3, and 10% at day 7.
DEX causes a decrease in SPARC in TM cells with minimal effect in CBSM cells. TGF–ß2 induces a significant increase in SPARC in both cell types. ECM changes in DEX–treated eyes are different than POAG eyes. SPARC’s differential response may indicate its involvement in the differing ECM response.
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