July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
p38 signaling activates quiescent primary adult human RPE into contractile membranes modeling Proliferative Vitreoretinopathy
Author Affiliations & Notes
  • Lauren Schiff
    Cell, Development and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Nathan Boles
    Neural Stem Cell Institute, Rensselaer, New York, United States
  • Bar A Nachmani
    Cell, Development and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Marie Fernandes
    Cell, Development and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Timothy A. Blenkinsop
    Cell, Development and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Footnotes
    Commercial Relationships   Lauren Schiff, None; Nathan Boles, None; Bar Nachmani, None; Marie Fernandes, None; Timothy Blenkinsop, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5260. doi:
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      Lauren Schiff, Nathan Boles, Bar A Nachmani, Marie Fernandes, Timothy A. Blenkinsop; p38 signaling activates quiescent primary adult human RPE into contractile membranes modeling Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5260.

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

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Abstract

Purpose : Proliferative vitreoretinopathy (PVR) is a metaplasia of the eye, manifested by the transformation of retinal pigment epithelial (RPE) cells into contractile membranes, leading to vision loss. Previously, we reported our discovery that TGFβ1 and TNFα evoke a contractile phenotype in RPE. When compared to patient dissected PVR membranes, we hypothesize that this model will allow evaluation of the molecular changes underpinning RPE transformation into a myocontractile phenotype.

Methods : RPE were dissected from human eye globes (N=72) and cultured for one month to allow formation of an epithelial monolayer that exhibit native physiology. RPE cultures were trypsinized, replated and after 24 hours, treated with 10ng/ml TGFβ1 and/or TNFα for 5 days (N=26). For p38 inhibition studies, cells were additionally treated with 10ng/ml SB 202190. Cells were analyzed using immunofluorescence, RT-qPCR, Western blot, time-lapse microscopy and scratch wound healing assays.

Results : TGFβ1 and TNFα (TNT) invokes changes in the human RPE that resemble properties of PVR, including the production of contractile 3D membranes (363.7 ± 61.8) and expression of periretinal membrane and EMT-associated genes Col1a1 (629.9 ± 219.7, P ≤ 0.01), Col1a2 (472.9 ± 204.4, P ≤ 0.05) and Snai1 (122.0 ± 40.7, P ≤ 0.01). RNA-seq between TNT treatment and patient dissected PVR samples revealed a specific p38-MAPK signaling signature. p38 is transiently activated upon TNT treatment and inhibition of p38 prevents a TNT induced PVR phenotype. Treatment with TNT and SB 202190 resulted in decreased gene expression of Col1a1 (0.03 ± 0.01, P ≤ 0.001), Col1a2 (0.04 ± 0.002, P ≤ 0.001) and Snai1 (0.02 ± 0.006, P ≤ 0.001). Cell migration was increased upon treatment with TNT (38.6 ± 2.1, P ≤ 0.001) and decreased (72.9 ± 1.9, P ≤ 0.001) upon treatment with TNT and SB 202190 in a wound healing assay. Additionally, contractile membrane formation by RPE was reversed by treatment with a p38 inhibitor.

Conclusions : These results suggest that when RPE are exposed to both TGFβ1 and TNFα simultaneously, RPE activate myocontractile programs that get activated through p38-MAPK signaling. Our discovery that inhibition of p38 not only prevents contractile PVR-like membrane formation, but also reverses contractility suggests inhibitors of this pathway may have therapeutic potential for patients with Proliferative Vitreoretinopathy.

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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