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Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol

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Abstract

Background and aims

Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7β-hydroxycholesterol (7β-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 μM), a polyphenol from red wine.

Methods

ARPE-19 cells were treated with 7β-OH, 7KC, or 25-OH (5–40 μg/mL; 24–48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1β, IL-6, IL-10, IL-12p70, TNF-α, MCP-1) and VEGF.

Results

7β-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7β-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-α, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERK½ signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion.

Conclusion

7β-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.

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Acknowledgments

This work was supported by grants from the INSERM, and the University Hospital of Dijon (CHU de Dijon). The authors are indebted to Ms. Linda Northrup for reviewing the English version of the manuscript.

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Authors and Affiliations

  1. Centre de Recherche INSERM 866 (Lipides, Nutrition, Cancer)–Equipe Biochimie Métabolique et Nutritionnelle, Université de Bourgogne, GDR CNRS 2583, 21000, Dijon, France

    M. Baarine

  2. Service d’Ophtalmologie, CHU/Hôpital Général, 21000, Dijon, France

    B. Dugas, L. Malvitte, A. Bron & C. Creuzot-Garcher

  3. INRA U 1129, UMR FLAVIC, 21000, Dijon, France

    F. Ménétrier

  4. Service Commun de Microscopie Electronique, INRA, 21065, Dijon, Cedex, France

    J. Lherminier

  5. Groupe de Recherche Å’il et Nutrition, INRA, UMR1129 FLAVIC, 21000, Dijon, France

    A. Bron & C. Creuzot-Garcher

  6. INSERM 866, Faculté des Sciences Gabriel, 6 Bd Gabriel, 21000, Dijon, France

    Gérard Lizard

  7. Université de Bourgogne and INSERM 866, Faculté des Sciences Gabriel, 21000, Dijon, France

    B. Dugas, S. Charbonnier, K. Ragot, D. Delmas, L. Malvitte, T. Khalfaoui, N. Latruffe & Gérard Lizard

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  1. B. Dugas
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  2. S. Charbonnier
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  3. M. Baarine
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  4. K. Ragot
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  5. D. Delmas
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  8. L. Malvitte
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  9. T. Khalfaoui
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  10. A. Bron
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  11. C. Creuzot-Garcher
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Correspondence to Gérard Lizard.

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Dugas, B., Charbonnier, S., Baarine, M. et al. Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol. Eur J Nutr 49, 435–446 (2010). https://doi.org/10.1007/s00394-010-0102-2

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  • DOI: https://doi.org/10.1007/s00394-010-0102-2

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