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I. M. Larrayoz, I. Pascual, J. W. Lee, I. R. Rodriguez; 7-Ketocholesterol Induces VEGF and Other Cytokines via Multiple Inflammatory Pathways. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1846.
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7-Ketocholesterol (7kCh) is a highly toxic oxysterol that has been implicated in some of the major aging diseases such as atherosclerosis, Alzheimer's, Parkinson’s and age-related macular degeneration (AMD). Recently, 7kCh was localized in the primate retina associated with lipid deposits in the choriocapillaris, Bruch’s membrane and the vascular endothelial cells of the neural retina (Moreira et al. IOVS, 2008). 7-Ketocholesterol was also shown to be a potent inducer of VEGF and IL-8 in cultured RPE cells. The purpose of this study is to characterize the pathways by which 7kCh induces inflammatory responses in RPE cells.
ARPE19 cells were treated with sublethal doses of 7kCh complexed in hydroxypropyl beta-cyclodextrin. Cell viability was determined using a cellular dehydrogenase activity assay. Real-time qRT-PCR was performed with SYBR green or specific Taqman probes using an ABI 7500 instrument. Proteins were quantified by commercially available ELISA kits or by immunoblotting.
In cultured ARPE19 cells 7kCh induced the mRNA expression of cytokines, VEGF, IL-6 and IL-8 as well as IΚB in a dose and time dependent manner. In concentrations up to 15 µM for 24 h, 7kCh did not affect the cell viability but upregulated the NF-ΚB, ERK, PI3K-AKT and P38 pathways. Specific inhibitors for the above mentioned pathways blocked the 7kCh-mediated induction of VEGF, IL-6 and IL-8. Interestingly, N-acetyl cysteine was also able to attenuate the cytokine induction. This suggests that reactive oxygen species (ROS) may be involved in the 7kCh-mediated cytokine induction.
7-Ketocholesterol induces inflammation by ROS formation and by interactions with liver X receptors which result in the activation of intracellular kinases and NF-kB. Understanding the inflammatory pathways activated by 7kCh may be important in understanding the pathogenesis mechanisms of AMD and other aging diseases.
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