Abstract
Purpose: :
7-Ketocholesterol (7KCh) is a highly toxic oxidized form of cholesterol that has been implicated in mediating inflammatory responses and cell death in cultured RPE cells. Previous studies have suggested the involvement of ATP-binding cassette (ABC) transporters in 7KCh efflux. Other studies have implicated the formation of 7KCh-fatty acid esters, by acyl-coenzyme A: cholesterol acyltransferase (ACAT/SOAT), in mediating 7KCh inflammatory responses. In this study, we investigated both of these mechanisms to determine their role in eliminating and/or neutralizing 7KCh-mediated cytotoxicity and inflammation.
Methods: :
7KCh was delivered to ARPE-19 or D407 cells complexed with either LDL (7KLDL) or hydroxypropyl-b-cyclodextrin (7K-HPBCD). Efflux studies were performed by treating the cells with 7KLDL and 7K-HPBCD in serum-free media for 24 hr. Cells were then washed with PBS and incubated for 20 hr in fresh serum-free media in the presence of HDL (100 ug/mL). Cell pellets and conditioned media were analyzed. Identification and quantification of 7KCh, cholesterol and 7KCh metabolites was performed by HPLC-UV and LCMS. The mRNA expression of ABCA1/G1 and ACAT1 was measured in human tissues and cultured cells by qRT-PCR. ABCA1 and ABCG1 expression was increased using 22R-hydroxycholesterol (10 uM), a potent agonist of liver-X-receptors, or knockdown using siRNAs.
Results: :
In the presence of HDL, 22R-hydroxycholesterol pretreated cells only increased 7KCh efflux by 10% of over HDL alone. Knockdown of ABCA1 and ABCG1 expression using siRNAs had no effect on 7KCh efflux. Analyses of ARPE-19 cells treated with 7KCh by LCMS failed to detect any hydroxylated or sulfated forms of 7KCh in the cells or in the conditioned media. The analyses detected four different 7KCh-esters which accounted for 5-10% of the internalized 7KCh. qRT-PCR analyses detected a significant presence of ACAT1 mRNA in human retina and ARPE-19 cells similar to the levels in human liver. The expression of ACAT1 mRNA is 2-fold greater in human retina than in brain. ACAT1 mRNA was detected at 5-fold greater levels in ARPE-19 cells than in D407 cells.
Conclusions: :
Our results indicate that 7KCh efflux is mostly independent of ABCA1/G1 but dependent on the presence of HDL. No hydroxylated or sulfated forms of 7KCh were detected but four 7KCh-esters were detected and partially identified. The formation of these esters suggests the involvement of ACATs. The nature of the 7KCh-esters and their role in metabolism, efflux and/or inflammation is under investigation.
Keywords: retinal pigment epithelium • lipids • inflammation