Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol

The effect of oxysterols on efflux of cholesterol from mouse L-cell fibroblasts, rat Fu5AH hepatoma cells, J774 macrophages, and human EA.hy 926 endothelial cells was studied. Cells were preincubated with 25-hydroxycholesterol (25-OHC) either during labeling of the cells with [3H]cholesterol or during equilibration after labeling. Subsequently, the release of [3H]cholesterol into medium containing 0.2 mg HDL3/ml was measured and the fractional release of cellular [3H]cholesterol was calculated. Pretreatment with 25-OHC (1 microgram/ml) caused a 30% reduction in [3H]cholesterol efflux from L-cells during 8 h of incubation with HDL3. 25-OHC also inhibited cholesterol efflux from Fu5AH and J774 cells, but the effect was less marked. There was only a small, nonsignificant reduction of efflux from EA.hy 926 cells. The mechanisms of 25-OHC-induced inhibition of cellular cholesterol efflux was further studied in L-cells, because of their sensitivity to 25-OHC treatment. The effect of 25-OHC on cholesterol efflux was dose-dependent, with significant effects seen at 25-OHC concentrations as low as 50 ng/ml. The half-time for cholesterol efflux from 25-OHC-treated cells (5 micrograms/ml) was 13.0 +/- 3.3 h compared to 5.7 +/- 1.0 in control cells, corresponding to a 55% reduction in the rate of cholesterol release. Other oxysterols, including 7-ketocholesterol, 7 alpha- and 7 beta-hydroxycholesterol, and 22(S)-hydroxycholesterol also inhibited [3H]cholesterol efflux from L-cells significantly, but to a lesser degree. 25-Hydroxycholesterol (5 micrograms/ml) reduced efflux from both normal and cholesterol-enriched cells by 31 and 14%, respectively. Inhibition of efflux was similar when reconstituted HDL3-apolipoprotein/phosphatidylcholine particles or small unilamellar phosphatidylcholine vesicles were used as cholesterol acceptors instead of HDL3. The content of phospholipids, cholesterol and the FC/PL ratio of intact cells and from isolated plasma membrane vesicles were the same for control and 25-OHC-treated cells. Efflux of [3H]cholesterol from plasma membranes isolated from 25-OHC-treated cells was 20% less than efflux from membranes from control cells. The difference in efflux observed in intact cells is partially explained by the reduction in efflux from the plasma membrane. In conclusion, our studies suggest that oxysterols, especially 25-hydroxycholesterol, can reduce cellular cholesterol efflux in vitro. Therefore oxysterols, either endogenous or derived from the diet, may influence cellular cholesterol efflux in vivo, the first step in reverse cholesterol transport.