June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Protection of RPE cells against oxidative stress-induced cell death by choroidal melanocytes and their secreted factors
Author Affiliations & Notes
  • Julien Blouin
    CHU de Quebec-Université Laval Research Center, section of regenerative medicine; and LOEX Research center of Université Laval, Universite Laval, Quebec, Quebec, Canada
    Departement of ophtalmologie, Universite Laval Faculte de medecine, Quebec, Quebec, Canada
  • Julie Bérubé
    CHU de Quebec-Université Laval Research Center, section of regenerative medicine; and LOEX Research center of Université Laval, Universite Laval, Quebec, Quebec, Canada
  • William Gauthier-Naud
    CHU de Quebec-Université Laval Research Center, section of regenerative medicine; and LOEX Research center of Université Laval, Universite Laval, Quebec, Quebec, Canada
    Departement of ophtalmologie, Universite Laval Faculte de medecine, Quebec, Quebec, Canada
  • Kim Santerre
    CHU de Quebec-Université Laval Research Center, section of regenerative medicine; and LOEX Research center of Université Laval, Universite Laval, Quebec, Quebec, Canada
    Departement of ophtalmologie, Universite Laval Faculte de medecine, Quebec, Quebec, Canada
  • Solange Landreville
    CHU de Quebec-Université Laval Research Center, section of regenerative medicine; and LOEX Research center of Université Laval, Universite Laval, Quebec, Quebec, Canada
    Departement of ophtalmologie, Universite Laval Faculte de medecine, Quebec, Quebec, Canada
  • Stephanie Proulx
    CHU de Quebec-Université Laval Research Center, section of regenerative medicine; and LOEX Research center of Université Laval, Universite Laval, Quebec, Quebec, Canada
    Departement of ophtalmologie, Universite Laval Faculte de medecine, Quebec, Quebec, Canada
  • Footnotes
    Commercial Relationships   Julien Blouin, None; Julie Bérubé, None; William Gauthier-Naud, None; Kim Santerre, None; Solange Landreville, None; Stephanie Proulx, None
  • Footnotes
    Support  FRQS 283517
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2972. doi:
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      Julien Blouin, Julie Bérubé, William Gauthier-Naud, Kim Santerre, Solange Landreville, Stephanie Proulx; Protection of RPE cells against oxidative stress-induced cell death by choroidal melanocytes and their secreted factors. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2972.

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

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Abstract

Purpose : The goal of this project is to study the involvement of the choroid and its most abundant cells, the melanocytes, in the protection of retinal pigment epithelial (RPE) cells against oxidative stress.

Methods : RPE were seeded on a choroidal substitute that was produced using the self-assembly approach of tissue engineering (3D models). Choroidal melanocytes were added (or not) to the choroidal stromal substitutes. These different cell combinations of 3D models were exposed to oxidative stress (tert-butyl hydroperoxide: TBHP, 250 μM, 2h). Levels of reactive oxygen species (CellRox) and cellular death (Ethidium-homodimer) were then assessed. In 2D models, RPE cells were exposed to oxidative stress (500, 1000 μΜ, 2h), then incubated with either fresh media or media conditioned by melanocytes that were previously exposed to oxidative stress (500μΜ, 2h) using different dilutions. Death of the RPE cells was measured using LDH release assay and by viability assays (Annexin/ Propium iodide; Caspase 3/7). Proteins and cytokines secreted by melanocytes, fibroblasts and RPE cells in 2D cultures were identified and measured (Human XL Cytokine Array Kit; ELISA).

Results : The presence of melanocytes in the engineered tissues reduced CellRox intensity by 45% and decreased RPE cell death by 90%. Oxidative stress-induced cell death of RPE cells was reduced by 5% when the RPE cells were left to recover in the media conditioned by melanocytes (1:1 ratio with fresh culture media). Compared to choroidal fibroblasts and RPE cells, melanocytes secreted respectively 49 and 46-times more pigment epithelium-derived factor (PEDF) and 6 and 4-times more osteopontin (OPN).

Conclusions : Overall, our results demonstrate that choroidal melanocytes protect RPE cells against oxidative stress-induced cell death. PEDF and OPN were identified as proteins mainly secreted by melanocytes, and may be involved in this protection. The identification of factors that prevent RPE cell death could help to find a cure for diseases where RPE needs protection, such as dry AMD.

This is a 2021 ARVO Annual Meeting abstract.

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