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N. Fatma, E. Kubo, C. B. Toris, W. D. Stamer, D. P. Singh; PRDX6 Attenuates Oxidative Stress- and TGFβ-Induced Abnormal Expression of Extracellular Matrix Proteins in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1646. doi: https://doi.org/.
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Oxidative stress-and TGFβ-induced insult to trabecular meshwork (TM) cells has been shown to cause abnormal accumulation of extracellular matrix protein (ECM) that may lead to resistance to aqueous outflow. Peroxiredoxin 6 (PRDX6) protects cells by removing reactive oxygen species (ROS) and mediating survival signaling. Using primary human TM cells as a model, we demonstrated the ability of PRDX6 in attenuating the oxidative stress- and TGFβ-induced damage or overstimulation of ECM gene/protein expression.
TM cells from normal and glaucomatous human subjects were cultured in complete Opti-MEM medium. To deliver PRDX6 to cells, a full length of Prdx6 cDNA was cloned into Nco1 and EcoR1 sites of pTAT-HA prokaryotic vector and protein was purified. TM cells were subjected to H2O2-induced oxidative stress or treated with variable concentrations of TGFβ1/TGFβ2 for variable times in presence or absence of TAT-HA-PRDX6 protein (2-5ug/ml) to evaluate its protective efficacy. Cell viability was measured with MTS assay and type of cell death was determined with TUNEL and DAPI staining. Western blot and real time PCR were done to assess the level of endogenous/exogenous PRDX6 as well as intracellular levels of ECM proteins; αsm-actin, βigh3, fibronectin, fibulin5, thrombospondin1, PAI1 and tropomyocin1 and 2. Intracellular ROS level was measured using H2-DCFH-DA dye, and activation of TGFβ with Emax Immunoassay. 8-OHdG immunochemistry was performed to monitor oxidative stress-induced DNA damage.
PRDX6 was highly expressed in TM cells; however a reduced PRDX6 mRNA and protein expression was observed in cells derived from glaucomatous subjects. These cells possessed increased expression of ECM proteins like βig-h3, α-sm-actin, and Tmp1 and reduced expression of MMP2. Importantly, normal TM cells exposed to H2O2 showed features similar to glaucomatous TM cells, specifically reduced PRDX6 levels with increased ROS levels, and remarkable DNA damage, and the abnormalities were reversed by addition of TAT-HA-PRDX6. Also, PRDX6 supply to TM cells attenuated TGFβ1/β2 induced excessive accumulation of ECM proteins.
Our findings provide evidence that oxidative stress-induced deleterious changes in TM may be related to reduced PRDX6 expression, and a supply of an antioxidant such as PRDX6 should be an effective means of reducing oxidative stress-mediated progression of glaucoma.
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