Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells.
OBJECTIVE
Aging is associated with an accumulation of cholesterol esters in the Bruch membrane. Cholesterol esters are prone to undergo oxidation and generate oxysterols that have cytotoxic and proinflammatory properties. We investigated the effects of three oxysterols on mitochondrial dysfunctions, inflammation, and oxidative stress in primary cultures of porcine retinal pigment epithelial (RPE) cells.


METHODS
RPE cells were incubated with oxysterols (50 micro M of 24-hydroxycholesterol, 25-hydroxycholesterol, or 7-ketocholesterol) for 24 hr and 48 hr. Oxysterol content was determined in cells and in corresponding media by gas chromatography. Mitochondrial activity was measured by mitochondrial dehydrogenase activity. The intracellular formation of reactive oxygen species in RPE cells was detected by using the fluorescent probe DCFH-DA. IL-8 was assayed in the supernatants by ELISA, and the corresponding cellular transcripts were semiquantified by RT-PCR.


RESULTS
Analyses of the oxysterols content in the RPE cells and corresponding media suggested a high rate of cellular uptake, although some differences were observed between 7-ketocholesterol on the one hand and 24-hydroxycholesterol and 25-hydroxycholesterol on the other hand. All oxysterols induced slight mitochondrial dysfunctions but a significant 2- to 4-fold increase in reactive oxygen species (ROS) production compared with the control. They also enhanced IL-8 gene expression and IL-8 protein secretion in the following decreasing order: 25-hydroxycholesterol > 24-hydroxycholesterol > 7-ketocholesterol.


CONCLUSIONS
We conclude that in confluent primary porcine RPE cells, 24-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol are potent inducers of oxidation and inflammation.
Joffre
Corinne
C
Eye and Nutrition Research Group, INRA, UMR1129 FLAVIC, Dijon, France. cjoffre@dijon.inra.fr
Leclère
Laurent
L
Buteau
Bénédicte
B
Martine
Lucy
L
Cabaret
Stéphanie
S
Malvitte
Laure
L
Acar
Niyazi
N
Lizard
Gérard
G
Bron
Alain
A
Creuzot-Garcher
Catherine
C
Bretillon
Lionel
L
D000818:Animals; D002849:Chromatography, Gas; D004797:Enzyme-Linked Immunosorbent Assay; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006888:Hydroxycholesterols / Q000633:toxicity*; D007249:Inflammation / Q000139:chemically induced*; D016209:Interleukin-8 / Q000235:genetics; D007653:Ketocholesterols / Q000633:toxicity*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D018384:Oxidative Stress / Q000187:drug effects*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D005452:Fluoresceins; D005456:Fluorescent Dyes; D006888:Hydroxycholesterols; D016209:Interleukin-8; D007653:Ketocholesterols; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C029569:diacetyldichlorofluorescein; C044563:24-hydroxycholesterol; C007997:25-hydroxycholesterol; C003001:7-ketocholesterol