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J. W. Lee, I. R. Rodriguez; 7-Ketocholesterol Efflux is Mediated by HDL and Independent of ABCG1 in RPE-Derived D407 Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):459.
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Recent studies have shown that 7-ketocholesterol (7KCh) efflux was dependent on the expression of ABCG1 and the presence of HDL in media in Abcg1+/+ macrophages. The purpose of this study is to test whether 7KCh efflux is dependent on ABCA1 or ABCG1 expression in D407 cultured RPE cells.
7KCh-enriched human LDL (7KLDL) was used to deliver 7KCh to the cells without interference from other oxidized lipids. 7KCh and cholesterol were quantified by HPLC-UV 24 h post-treatment. Efflux studies were performed by treating the cells with 7KLDL (50 µg/mL) in serum-free media for 24 h. Cells were then washed with PBS and incubated for 20 h in fresh serum-free media in the absence and presence of apoA-1 (10 µg/mL), HDL (100 µg/mL) or LDL (100 µg/mL). Cell pellets and conditioned media were collected and analyzed for 7KCh and cholesterol. ABCA1 and ABCG1 expression were suppressed using specific siRNAs before 7KLDL treatment.
In D407 cells, 7KCh uptake had increased in a dose-dependent manner after 7KLDL treatment. 7KCh was not detectable in the conditioned media without acceptors. By adding 100 µg/mL of HDL or LDL to the media, 7KCh efflux from D407 cells was increased 40% and 25%, respectively. ApoA1 did not promote 7KCh efflux. ABCA1 and ABCG1 siRNAs suppressed ABCA1 and ABCG1 mRNA levels 65% and 85%, respectively. A double ABCA1 and ABCG1 knockdown decreased cholesterol efflux by 10% when compared to the negative siRNA control. 7KCh was not detectable in the media from any of siRNA knockdown samples. Interestingly, the knockdown of ABCA1 and ABCG1 did not alter the 7KCh efflux in the presence of HDL or LDL in the media.
Our data suggests that 7KCh efflux is dependent on lipoprotein acceptors in the media but independent of ABCA1 and ABCG1. This indicates that 7KCh efflux is occurring either via other transporters or by mass-action exchange with lipoproteins.
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