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Justin Ren Yuan Chang, Karla Yadira Barbosa, Madhu Lal, Marc Ferrer, Kapil Bharti; mTOR and Rho family of GTPases regulate epithelial phenotype in injured retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):594.
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© ARVO (1962-2015); The Authors (2016-present)
Maintaining healthy epithelial phenotype of retinal pigment epithelium (RPE) is important in alleviating poor prognosis of RPE-related diseases including proliferative vitreoretinopathy (PVR) and age-related degeneration (AMD). The goal of this study is to validate potential therapeutic targets which were previously identified by high-throughput screening (HTS) assays.
Using a human reporter induced pluripotent stem (iPS) cell line that expresses GFP when cells are differentiated into RPE, we developed a robust in vitro RPE injury model. In this model, GFP expression declines as trypsinized RPE cells lose epithelial phenotype, while GFP expression increases in confluent, mature RPE cells. With GFP as readout, we performed HTS using miRNA mimics and siRNA libraries, in combination with bioinformatics analyses, to discover genes that affect the ability of injured RPE cells to re-attain the epithelial phenotype. Immunofluorescence was used to discern the activity of candidate pathways in the atrophied RPE. Epithelial-to-mesenchymal transition (EMT) gene expression array was utilized to investigate the effect of small molecule modulators on RPE undergoing wound healing following mechanical injury to cells.
HTS assays and bioinformatics analyses indicated that mTOR and the Rho family of GTPases, i.e. RhoA, Cdc42, Rac1, are involved in regulating the epithelial phenotype in injured RPE. siRNA screening and small molecule modulators targeting mTOR complexes and the GTPases suggested that mTOR activity is required for epithelial phenotype maintenance, while Rho GTPases suppress epithelial character of RPE cells. Immunofluorescence demonstrated that Rho GTPases are active after RPE injury. RT-qPCR of injured RPE treated by Rho GTPase modulators showed that these small molecules regulate genes involved in EMT and RPE epithelial fate.
Small molecules or biologics that target the expression of mTOR and Rho-family of GTPases are promising therapeutic agents for RPE-related diseases, such as PVR and AMD.
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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