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
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.
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.
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.
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.
KeywordsARPE-19 cells Caspase-independent cell death Inflammatory cytokines Oxysterols Phospholipidosis Resveratrol Reactive oxygen species VEGF
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.
- 1.Klein R, Klein BE, Linton KL (1992) Prevalence of age-related maculopathy. The Beaver dam eye study. Ophthalmology 99:933–943Google Scholar
- 7.Malvitte L, Montange T, Vejux A, Joffre C, Bron A, Creuzot-Garcher C, Lizard G (2008) Activation of a caspase-3-independent mode of cell death associated with lysosomal destabilization in cultured human retinal pigment epithelial cells (ARPE-19) exposed to 7beta-hydroxycholesterol. Curr Eye Res 33:769–781Google Scholar
- 8.Elner SG, Elner VM, Field MG, Park S, Heckenlively JR, Petty HR (2008) Retinal flavoprotein autofluorescence as a measure of retinal health. Trans Am Ophthalmol Soc 106:215–222Google Scholar
- 9.Dunaief JL, Dentchev T, Ying GS, Milam AH (2002) The role of apoptosis in age-related macular degeneration. Arch Ophthalmol 120:1435–1442Google Scholar
- 14.Luthra S, Fardin B, Dong J, Hertzog D, Kamjoo S, Gebremariam S, Butani V, Narayanan R, Mungcal JK, Kuppermann BD, Kenney MC (2006) Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 47:5569–5575CrossRefGoogle Scholar
- 17.Lemaire S, Lizard G, Monier S, Miguet C, Gueldry S, Volot F, Gambert P, Néel D (1998) Different patterns of IL-1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α-, 7β-hydroxycholesterol, or 7-ketocholesterol. FEBS Lett 440:434–439CrossRefGoogle Scholar
- 18.Ramos MA, Kuzuya M, Esaki T, Miura S, Satake S, Asai T, Kanda S, Hayashi T, Iguchi A (1998) Induction of macrophage VEGF in response to oxidized LDL and VEGF accumulation in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol 18:1188–1196Google Scholar
- 20.Prunet C, Montange T, Vejux A, Laubriet A, Rohmer JF, Riedinger JM, Athias A, Lemaire-Ewing S, Néel D, Petit JM, Steinmetz E, Brenot R, Gambert P, Lizard G (2006) Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients. Cytometry A 69:359–373Google Scholar
- 25.Pedruzzi E, Guichard C, Ollivier V, Driss F, Fay M, Prunet C, Marie JC, Pouzet C, Samadi M, Elbim C, O’dowd Y, Bens M, Vandewalle A, Gougerot-Pocidalo MA, Lizard G, Ogier-Denis E (2004) NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol 24:10703–10717CrossRefGoogle Scholar
- 26.Scherle PA, Jones EA, Favata MF, Daulerio AJ, Covington MB, Nurnberg SA, Magolda RL, Trzaskos JM (1998) Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes. J Immunol 161:5681–5686Google Scholar
- 35.Lizard G, Monier S, Cordelet C, Gesquière L, Deckert V, Gueldry S, Lagrost L, Gambert P (1999) Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall. Arterioscler Thromb Vasc Biol 19:1190–1200Google Scholar
- 40.Schmitz G, Grandl M (2009) Endolysosomal phospholipidosis and cytosolic lipid droplet storage and release in macrophages. Biochim Biophys Acta 1791:524–539Google Scholar
- 41.Lizard G, Miguet C, Bessede G, Monier S, Gueldry S, Neel D, Gambert P (2000) Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occurring during 7-ketocholesterol-induced apoptosis. Free Radic Biol Med 28:743–753CrossRefGoogle Scholar
- 43.Lemaire-Ewing S, Berthier A, Royer MC, Logette E, Corcos L, Bouchot A, Monier S, Prunet C, Raveneau M, Rébé C, Desrumaux C, Lizard G, Néel D (2009) 7beta-Hydroxycholesterol and 25-hydroxycholesterol-induced interleukin-8 secretion involves a calcium-dependent activation of c-fos via the ERK1/2 signalling pathway in THP-1 cells: oxysterols-induced IL-8 secretion is calcium-dependent. Cell Biol Toxicol 25:127–139CrossRefGoogle Scholar
- 47.Yoshida A, Yoshida S, Khalil AK, Ishibashi T, Inomata H (1998) Role of NF-kappaB-mediated interleukin-8 expression in intraocular neovascularization. Invest Ophthalmol Vis Sci 39:1097–1106Google Scholar
- 49.Ziegler CC, Rainwater L, Whelan J, McEntee MF (2004) Dietary resveratrol does not affect intestinal tumorigenesis in ApcMin/+ mice. J Nutr 134:5–10Google Scholar