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Karla Y Barbosa-Sabanero, Justin R Chang, Genqing Y Liang, Madhu Lal, Kapil Bharti; Unraveling the role of NOX4, a NADPH oxidase, in RPE Epithelial to Mesenchymal Transition. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3078. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal injury often triggers epithelial to mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells leading to a condition called proliferative vitreoretinopathy (PVR). The EMT process is characterized by the switch of epithelial phenotype of the RPE into fibroblast-like phenotype. The aim of this study is to discover key molecular players involved in EMT of the RPE, and identify their role with the goal to discover potential therapeutic targets.
We performed a siRNA screen to identify candidate genes required to maintain epithelial phenotype of iPSC-RPE. A reporter induced pluripotent stem (iPS) cell line that expresses GFP when differentiated into RPE cells was used for the siRNA screen. The screen identified NOX4, a NADPH oxidase enzyme, known to produce reactive oxygen species (ROS) as a potential candidate for inducing EMT in RPE cells. We validated the functional role of NOX4 in the RPE-EMT with an in vitro RPE injury model, using shRNA and pharmacological inhibitors by performing Western Blot, immunocytochemistry, and gene expression analysis.
Immunohistochemistry and Western blot analysis showed that NOX4 is present in the membrane and nucleus of healthy RPE cells. EMT of RPE changed NOX4 localization to predominantly membrane and the cytoskeleton. Mechanical injury induced EMT in RPE caused upregulation of EMT genes such as ZEB1, SMA, Vimentin, Rac1, TGF-Beta, and FOXO3a, which were suppressed by genetic knockdown or pharmacological inhibition of NOX4. Consistently, the levels of ROS were increased in the injured RPE cells undergoing to EMT.
Overall these findings suggest that oxidative stress plays a role in the RPE-EMT and PVR response. Our studies confirmed that NOX4 plays a role in EMT, inducing EMT markers and migratory behavior in RPE. If confirmed by in vivo studies we propose that NOX4 inhibition can be used as a novel treatment to suppress RPE-EMT and conditions such as PVR.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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