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Jiahn-Dar Huang, Jung Wha Lee, Ignacio M. Larrayoz, Ignacio R. Rodriguez; A Simple Compound LC01 Inhibits 7-ketocholesterol Mediated Inflammation And Cytotoxicity In ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):875.
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7-ketocholesterol (7KCh) is a highly toxic and pro-inflammatory oxysterol found in oxidized lipoprotein deposits in the sub-RPE region. This oxysterol may contribute to the pathogenesis of certain forms of age-related macular degeneration (AMD) by causing local chronic inflammation. This study investigates the 7KCh-mediated inflammatory pathways and the effects of a simple lipid compound LC01 that inhibits this inflammation.
Cultured ARPE-19 cells were treated with 7KCh dissolved in hydroxypropyl-β-cyclodextrin in serum-free medium. The effects of LC01 and a structurally similar control (SSC) were tested at different concentrations. Cell viability was measured using a commercially available cellular dehydrogenase assay. The mRNA expression for various cytokines was measured by qRT-PCR 24 hr after 7KCh treatment using GAPDH expression as an endogenous control. The levels of secreted VEGF, IL-6, and IL-8 proteins were determined by ELISA 48 hr after treatment. The expression of phosphorylated Akt, PKCζ, p38 MAPK, and ERK1/2 were measured by immunoblot 3 and 12 hr after treatment.
Treating ARPE-19 cells with 7KCh induced the expression of inflammatory and angiogenesis markers including VEGF, IL-1β, IL-6, IL-8, TNF-α, TGF-β1, IΚBα, RelA, NFkB1 but not CCL2. Addition of 1uM LC01 attenuated the expression of these factors and increased cell viability while the SSC had little or no effect. Previously we have demonstrated that 7KCh induces inflammation by activating three NF-ΚB related pathways: ERK1/2, p38 MAPK, and Akt-PKCζ- NF-ΚB. In this study the addition of LC01 and SSC had little or no effect on the phosphorylation of ERK1/2, p38 MAPK, and Akt. However, LC01 clearly inhibited the phosphorylation of PKCζ while SSC had no effect.
LC01 significantly abates the cell death and inflammation caused by 7KCh. Our data suggests LC01 works by inhibiting the activation of PKCζ. LC01 seems to be a potent anti-inflammatory/angiogenic agent for the treatment of 7KCh-mediated chronic inflammation. We are investigating LC01 as a potential pharmacological treatment for "wet" AMD.
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