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Enrique Salero, James Moroney, Jeffrey L Goldberg; RPE dedifferentiation can be inhibited by small molecules. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2970.
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Retinal degeneration is characterized for progressive loss of photoreceptor cells that largely are responsible for vision loss. The etiology is attributed primarily to retinal pigment epithelium (RPE) cells that are a monolayer of pigmented cells underlying and supporting the neural retina. We have described that under some conditions RPE cells undergo EMT and exhibit mesenchymal properties. Ensuring that RPE cells are maintained as a cobblestone and do not undergo EMT is critical for photoreceptor survival. Small molecule has an effect on protein function and can inhibit a specific function of a multifunctional protein and disrupt protein-protein interactions. The purpose of this work is to study whether RPE dedifferentiation can be inhibited or reverse through the use of small molecules that allow us to identify the molecular pathways involve in such mechanisms.
Human RPE cells from cadaveric donors were isolated and cultured. To activate EMT process, we use different cell passages (P0-P14) of hRPE cells in culture and test with EMT markers, then we added individually or cocktail of small molecules that inhibit the signaling pathways (TGF-β, Wnt/β-catenin, FGFR, MEK1/2) involve in EMT process. Transepithelial electrical resistance (TEER) was performed to analyze tight junction before and after treatment. Western blot and immunostaining were performed before and after treatments. Total RNA was extracted from hRPE at different passages before and after treatment. Gene expression assay was carried out by qRT-PCR analysis.
Our results demonstrate that hRPE cells decrease the expression of RPE markers and EMT markers activates over the passages in culture. Our results suggest that inhibition by specific small molecules targeting TGFβ, Wnt/β-catenin signaling pathway decrease the expression of EMT markers at early passages rather than late passages in RPE cells. Immunostaining corroborates the observation made from gene expression and protein analysis. We observe morphological changes in all RPE passages after one week of treatment, cells become more epithelial.
The protection of RPE cells against EMT process is afforded by the inhibition of TGFβ and Wnt/β-catenin pathways to prevent dedifferentiation of RPE in early passages, however in late passages other signaling pathway could be involve and need to be study. The use of small molecules may be a novel therapeutic target in retinal degenerative diseases.
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