Cholesterol Efflux

Quantification of cholesterol efflux was performed, as described previously.51, 55, 56 THP‐1 cells (ATCC) were maintained in RPMI‐1640 medium (Lonza) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin, and 2 mmol/L l‐glutamine (Invitrogen) at 37°C and 5% CO₂. THP‐1 cells were seeded at 0.2×10⁶ cells per well in 24‐well plates and differentiated into macrophages for 72 hours with 200 nmol/L phorbol‐12‐myristate‐13‐acetate (catalog no. P1585; Sigma‐Aldrich). Cells were washed twice with PBS, then labeled by incubation in RPMI‐1640 medium supplemented with 0.1% (wt/vol) fatty acid‐free bovine serum albumin and [³H]‐cholesterol (0.5 μCi/mL, catalog no. NET139001MC; PerkinElmer) for 24 hours.

To evaluate cholesterol efflux mediated by plasma from human participants and rats, labeled cells were washed and then incubated with fresh serum‐free medium containing plasma (3%, vol/vol) from human participants or rats for 4 hours. To evaluate an effect of added bilirubin on cholesterol efflux to normobilirubinemic plasma, labeled cells were washed and then incubated with fresh serum‐free medium containing plasma (3%, vol/vol) in the absence or presence of UCB IXα (17.1 μmol/L) for 4 hours. For the concentration‐ and time‐dependent experiments, labeled cells were washed with PBS and treated with different concentrations of UCB (3, 10, or 17.1 μmol/L) or solvent vehicle (0.1% dimethyl sulfoxide) for different time periods (4, 8, 16, or 24 hours). Cells were washed again with PBS and then incubated with fresh serum‐free medium containing human plasma (3%, vol/vol) or apo A1 (10 μg/mL, catalog no. SRP4693; Sigma‐Aldrich) for 4 hours. To differentiate whether UCB inhibits cholesterol efflux by interacting with THP‐1 macrophages or with cholesterol acceptors, labeled cells were washed and then treated with UCB (17.1 μmol/L) or solvent vehicle (0.1% dimethyl sulfoxide) for 4 hours. Cells were washed again with PBS and then incubated with fresh serum‐free medium containing human plasma (3%, vol/vol) or apo A1 (10 μg/mL; Sigma‐Aldrich) in the absence or presence of UCB (17.1 μmol/L) for 4 hours. Effluxed (medium supernatant) and intracellular (cell lysate) [³H]‐cholesterol were counted by liquid scintillation. Cholesterol efflux (percentage of total cholesterol) was determined by the ratio of radio‐labeled cholesterol in the medium to that of both medium and cells.