Oxysterols Incorporate in Primary Porcine Retinal Pigment Epithelial Cells and Induce Inflammation and Oxidative Stress

Abstract

Purpose: : Aging is associated with an accumulation of cholesterol in the Bruch’s membrane. Cholesterol is prone to undergo oxidation and to generate oxysterols that have cytotoxic properties. The aim of this study was to evaluate the effects of oxysterols on cell viability, oxidative stress and inflammation and to measure oxysterol incorporation in primary porcine retinal pigment epithelial (RPE) cell cultures.

Methods: : Porcine RPE cells were incubated with oxysterols (24S–hydroxycholesterol, 24OH ; 25–hydroxycholesterol, 25OH and 7–ketocholesterol, 7K) (100µg, 50µM) for 24 and 48h. Cell viability was evaluated by measuring mitochondrial succinate dehydrogenase (SDH) activity. The content of 24OH, 25OH and 7K was determined in cells and media by gas chromatography. The intracellular formation of reactive oxygen species (ROS) was detected by using the fluorescent probe dichlorofluorescein diacetate (DCFH₂–DA). IL–8 was assayed in the media by ELISA and its cellular expression was evaluated by RT–PCR.

Results: : Incorporation of 24OH in RPE cells was higher than that of 25OH and 7K (27.9µg vs 10.8 and 6.8µg at 24h; 29.0µg vs 20.8 and 10.3µg at 48h). In the media, 25OH was present in higher amount than 24OH and 7K (31.4µg vs 12.5 and 17.9µg at 24h; 14.1µg vs 7.4 and 10.0µg at 48h). SDH activity increased with 25OH and 24OH compared to the control cells (+35% and +20% with 24h; +25% and +9% with 48h) whereas it decreased for the cells incubated with 7K (–17% with 24h). All oxysterols induced a significant 2–4 fold increase in ROS production compared to the control. Oxysterols induced IL–8 expression (x 2–10) and production in the following decreasing order of magnitude: 25OH (529 pg/mL) > 24OH (211 pg/mL) > 7K (38 pg/mL) > control (4 pg/mL).

Conclusions: : These data consistently suggest that the cytotoxicity of 24OH and 25OH in RPE cells may be related to the induction of oxidative and inflammatory reaction that may be involved in the development of age–related diseases.

Keywords: oxidation/oxidative or free radical damage • inflammation • aging