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Karla Yadira Barbosa, Justin Chang, Madhu Lal, Kapil Bharti; The Role of Oxidative Stress Pathway in RPE Epithelial to Mesenchymal Transition. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3010.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Retinal injury often triggers retinal pigment epithelial (RPE) cells to undergo epithelial to mesenchymal transition (EMT) leading to a condition called proliferative vitreoretinopathy (PVR). PVR causes fibrosis and can cause retinal detachment leading to severe vision loss. The EMT process is characterized by the switch of epithelial phenotype of the RPE into fibroblast phenotype. The aim of this study is to determine key molecular players involved in EMT of the RPE with the goal to discover potential therapeutic targets.
Methods: We performed a siRNA screen to identify candidate genes and pathways 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. An in vitro RPE injury model was developed to validate the identified pathways. Human iPS cell derived RPE cultured in monolayer were subject to mechanical injury to induce EMT. Gene expression was confirmed by immunocytochemistry and qRT-PCR. Various signaling pathways were manipulated using pharmacological and genetic inhibitors.
Results: siRNA screen identified several previously known pathways including tight junction and MAP kinase pathways critical for regulating EMT process in RPE cells. In addition, this screen identified oxidative stress as a potentially novel pathway in regulating epithelial phenotype of iPSC-RPE. Results from siRNA screen were validated in our in vitro RPE injury model using immunohistochemistry and gene expression for key oxidative stress & EMT pathway components (FOXO, TGF-beta, BMP1, ZEB1). FOXO, a key oxidative stress response transcription factor was localized to the nucleus in injured RPE cells during EMT. This was co-incidental with increased levels of ROS in the injured RPE cells undergoing EMT.
Conclusions: Overall these findings suggest that oxidative stress plays a role in the RPE-EMT and PVR response. These findings contribute to the knowledge about the multiple signaling pathways are involved in EMT and provide the possibility of identifying potential therapeutic targets for PVR.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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