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Marie Fernandes, Lauren Schiff, Tomasz Swigut, Nathan Boles, Rajini Srinivasan, Alvaro Rada-Iglesias, Qingjie Wang, Janmeet Saini, Tom Kiehl, Jeffrey Stern, Joanna Wysocka, Sally Temple, Timothy A Blenkinsop; Nicotinamide protects against PVR by preventing RPE cells to undergo EMT. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2477. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In proliferative vitreoretinopathy (PVR), the retinal pigmented epithelium (RPE) undergoes an epithelial to mesenchymal transition (EMT) and becomes contractile leading to retina detachment and vision loss. EMT is associated with RPE exposure to cytokines. We model this using primary adult human RPE (ahRPE) to study the mechanisms of RPE transformation. We compare in vitro transformed RPE to patient-dissected PVR samples by RNA seq and identify networks enriched in both. We then test the efficacy of inhibitors to proteins in these shared networks in their ability to prevent and or reverse the RPE transformation into contractile membranes.
ahRPE are isolated from human donor globes obtained from eye banks. Passage 0 RPE monolayers are replated and treated 5 days with 10ng/ml TGFb1 or TNFα alone, or TGFb1/TNFa. RNA is reverse transcribed and cDNA is used for sequencing with the Illumina HiSeq. Reads are analyzed with tophat v2.0.14 to the gencode 23 transcriptome model and htseq. Differential analysis is performed with DESeq2. Gene networks enriched in RPE treated with TGFb1/TNFa are analyzed by Enrichr to identify pathways. Nicotinamide (NIC) was identified as a potential inhibitor and used to treat RPE that undergoes EMT induced by TGFb1/TNFa. RPE cells are subjected to immunofluorescence (IF), imaged by confocal, time-lapse microscopy and their RNAs subjected to qPCR.
Primary ahRPE treated with TGFb1/TNFa undergo EMT and create contractile membranes resembling those observed in patients with PVR, as shown by gene and protein expression data. RNA seq comparison between patient PVR membranes, native RPE and contractile RPE obtained in vitro showed that datasets from in vitro PVR membranes clustered with patient specimens and away from native RPE. This suggests that our in vitro model mimics in vivo PVR. When cultured in the presence of NIC, RPE retained their native phenotype (tight junctions, expression of RPE markers) whereas without NIC they express EMT markers. We also found that NIC can prevent RPE to develop contractile membranes when exposed to TGFb1/TNFa.
We can model PVR and recapitulate molecular changes similar to those observed in patient dissected PVR membranes. NIC prevents RPE to undergo EMT and in vitro contractile membranes formation suggesting that NIC is a promising therapeutic agent to treat patients with 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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