Quantifying Cholesterol Efflux Pathways

J774 cells, maintained in RPMI plus 10% FBS and antibiotics in 5% CO₂, were plated in 24-multi well plates (70,000 cells/well) and labeled for 24h in the presence of an ACAT inhibitor (2μg/ml CP113,818, a gift from Pfizer) using 0.5ml/well of 2μCi/ml [1,2-³H] cholesterol (Perkin Elmer) in RPMI plus 1% FBS. To up-regulate ABCA1 in J774 cells we incubated an additional 16h with 0.5ml/well medium containing 0.3mM Cpt-cAMP (Sigma) and 0.2% BSA in RPMI. The relative contribution of various efflux pathways was measured by 2h pretreatment of replicate wells of cAMP treated J774 cells with RPMI-0.2% BSA alone or this medium plus 20μM Probucol, 1μM BLT-1 or both to specifically inhibit ABCA1 and SR-BI15 (link). The contribution of ABCG1 to total efflux capacity was directly measured by 4h incubation of ABCG1 transfected BHK-1 cells with the same specimens used with J774 cells. In BHK-I cells transfected with ABCG1, receptor expression is regulated by mifepristone; the difference between cells treated overnight with mifepristone (10nM) and untreated cells represents ABCG1 efflux19 (link). Sera for these studies were aliquots stored at -70°C, used after a single thaw, and chosen to have similar HDL-C (HDL-C± 6%) at either the 25^th (low HDL=45, n=22) or 75^th (high HDL=73, n=18) percentile of the HDL-C distribution. Efflux was the fraction of total cellular cholesterol released in 4h to apoB-depleted serum, obtained after removal of apoB lipoproteins with Polyethylene Glycol (PEG, MW 8000, Sigma)18 (link), and diluted to 2.8% (equivalent to 2% serum) in MEM-HEPES (0.5ml/well). We have routinely used this dilution to promote release of radioactive cholesterol to the medium in 4h that is well above background. Supplementary figure I shows the dependence of the various receptor-mediated efflux pathways expressed in J774 cells on the dose of apoB-depleted serum. In this figure, ABCA1 efflux is the difference between cAMP treated and control J774 cells, SR-BI is the efflux measured from Fu5AH cells where SR-BI is the major efflux pathway and ABCG1 efflux is the difference between mifepristone treated and untreated transfected BHK-1 cells. SR-BI and ABCG1 efflux show linear dose dependence; the dose-dependence for ABCA1 mediated efflux fits a non-linear regression that tends to plateau but a concentration of 2.8% apoB-depleted serum is below this point (GraphPad Prism 5, GraphPad Software Inc., San Diego, CA). To further validate our macrophage model, we measured ABCA1 efflux using BHK-1 cells transfected with ABCA1 where receptor expression is regulated with mifepristone and found that the dose dependence of ABCA1 efflux measured with BHK-1 cells was identical to that measured with J774 cells (Supplementary Fig. I). In every experiment, we monitored up-regulation of ABCA1 in J774 cells as increased efflux to apoA-I (20μg/ml) from cells treated with cAMP compared to untreated cells (11±4 fold stimulation, n=10 experiments) and used an aliquot of a standard serum pool at 2% to monitor inter-assay variability in total efflux from cAMP J774 cells and ABCG1 efflux from BHK-1 cells. Transfected BHK-1 cells were a kind gift from Dr. J. Oram. All cellular incubations were done at 37°C and expression of cholesterol transporters was confirmed by western blot in initial experiments. Sera from subjects with similar high or low HDL-C were defined as having low or high efflux capacity based on total J774 efflux below or above the average efflux for each group. The same analysis was done using a subset of these specimens chosen to have similar apoA-I at either the 25^th (low apoA-I 127mg/dl, n=11) or 75^th (high apoA-I=163mg/dl, n=10) percentile of the apoA-I distribution.