July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Nicotinamide reverses RPE contractility in a model of PVR
Author Affiliations & Notes
  • Timothy A Blenkinsop
    Cell Development Regenerative Biology, MSSM, New York, New York, United States
  • Marie Fernandes
    Cell Development Regenerative Biology, MSSM, New York, New York, United States
  • Nathan Boles
    Eye Group, Neural Stem Cell Institute, Rensselaer, New York, United States
  • Lauren Schiff
    Cell Development Regenerative Biology, MSSM, New York, New York, United States
  • Bar Nachmani
    Cell Development Regenerative Biology, MSSM, New York, New York, United States
  • Footnotes
    Commercial Relationships   Timothy Blenkinsop, None; Marie Fernandes, None; Nathan Boles, None; Lauren Schiff, None; Bar Nachmani, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5806. doi:
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      Timothy A Blenkinsop, Marie Fernandes, Nathan Boles, Lauren Schiff, Bar Nachmani; Nicotinamide reverses RPE contractility in a model of PVR. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5806.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : In proliferative vitreoretinopathy (PVR), the retinal pigmented epithelium (RPE) undergoes an epithelial to mesenchymal transition (EMT) and becomes contractile, leading to retinal detachment and vision loss. EMT is associated with RPE exposure to cytokines. Here, we model the mechanisms of RPE transformation using primary adult human RPE (ahRPE). RNAseq comparison of in vitro transformed RPE to patient-dissected PVR samples led to identify networks enriched in both. We then tested whether treatment with nicotinamide (NAM) is sufficient to prevent RPE contractility in vitro.

Methods : AhRPE are isolated from human donor globes obtained from eye banks. Passage 0 RPE monolayers are replated and treated for 5 days with 10ng/ml TGFβ1/TNFα and compared to patient dissected PVR membranes containing RPE. RNA is used for sequencing using the Illumina HiSeq platform. 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 TGFβ1/TNFα are analyzed by Enrichr to identify pathways. NAM was identified as a potential inhibitor of EMT and was used to treat RPE induced by TGFβ1/TNFα. RPE cells were subjected to immunofluorescence (IF), imaged by confocal, time-lapse microscopy and upon RNA isolation, quantitative RT-PCR was performed.

Results : Primary ahRPE treated in vitro with TGFβ1/TNFα undergo EMT and create contractile membranes resembling by gene and protein expression comparisons those observed in patients with PVR. RNAseq comparison between patient-dissected PVR membranes, native RPE and contractile RPE obtained in vitro upon TGFβ1/TNFα treatment showed that datasets from in vitro PVR membranes clustered with patient specimens and away from native RPE. This suggests that the in vitro model may mimic in vivo PVR. When cultured in presence of NAM, RPE retained their native phenotype (tight junctions, expression of RPE markers) whereas without NAM they expressed EMT markers. We also found that NAM can prevent RPE to develop contractile membranes when exposed to TGFβ1/TNFα.

Conclusions : In vitro treatment of RPE with the combination of TGFβ1 and TNFα induces RPE to contract into membranes replicating the gene expression profile found in patient-dissected PVR membranes. NAM prevents RPE to undergo EMT and in vitro contractile membranes formation, suggesting that NAM is a promising therapeutic agent to treat patients with PVR.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.


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