A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Abstract

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [³H]cholesterol and measuring release of the labeled sterol. Using [³H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [³H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [³H]cholesterol when apo A-I, HDL₃, or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [³H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-1 (r² = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r² = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [³H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.

The efflux of cholesterol from macrophages in the arterial wall is thought to be an early step in the process termed reverse cholesterol transport (RCT; 1–2). Efflux can be mediated by a number of pathways involving 1) aqueous diffusion, 2) scavenger receptor BI (SR-B1), 3) ABCG1, and 4) ABCA1 (3–5). Although all of these pathways can contribute to the removal of free cholesterol (FC) from cells, it is ABCA1 that has been shown to play a major role in the maintenance of normal cholesterol levels in tissues, as demonstrated by the accumulation of large amounts of cholesterol in macrophages in individuals with genetic mutations in ABCA1 (Tangier disease) or in mouse models in which this protein is genetically eliminated (6–8).

A link between the in vitro efflux of cholesterol from macrophages and atherosclerosis has recently been established by studies demonstrating a negative correlation between cholesterol efflux from J774 mouse cells and coronary artery disease (CAD) as measured by either carotid intima-media thickness (cIMT) or angiographic measurements (9). Although not as yet definitely established, preliminary data indicate that the primary efflux mechanism linking cholesterol efflux to arterial cholesterol deposition is ABCA1 (10). In contrast to the other efflux pathways, ABCA1 uses as a ligand lipid-free/lipid-poor apolipoproteins, primarily apo A-I, whereas other pathways employ mature, fully lipidated HDL as cholesterol acceptors (3, 4, 11). Regardless of the pathway, the standard protocol for quantitating cell cholesterol efflux employs cells labeled with radioactive cholesterol and determination of the fractional release of the labeled sterol to a variety of acceptors ranging from whole serum to isolated apoproteins (12). Although this approach to measuring cholesterol efflux has provided large amounts of data on both the efficiency of various extracellular acceptors and on different efflux pathways, a protocol using radiolabeled cholesterol does not lend itself to the development of a high-throughput assay that can efficiently screen large numbers of specimens such as serum or serum fractions.

In the present study we have substituted a fluorescent sterol, BODIPY-cholesterol, for the generally used radiolabeled cholesterol. BODIPY-cholesterol is a fluorescent analog of free cholesterol in which carbon-24 of the sterol side chain is linked directly to the dipyrromethene boron difluoride (“BODIPY”) moiety (13). In a series of experiments using J774 macrophages together with a variety of cholesterol acceptors, we have compared efflux of BODIPY-cholesterol and [³H]cholesterol. These studies have demonstrated that the use of the fluorescent-labeled sterol provides an efficient measurement of efflux when compared with radiolabeled cholesterol. We found that BODIPY-cholesterol is released from cells primarily via the ABCA1 pathway. Thus, the greater efficiency for efflux of the BODIPY-cholesterol compared with labeled cholesterol, together with the greater simplicity of the fluorescent assay, allows the development of efflux protocols suitable for rapid, high-throughput screening for efflux capacity of large numbers of sera or serum fractions.

MATERIALS AND METHODS

Reagents and Materials

RPMI 1640, Minimum Essential Medium Eagle (MEM), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Mediatech Cellgro (Manassas, VA). MEM buffered with 10 mM HEPES (pH 7.4) was prepared using HEPES purchased from Fisher Scientific (Newark, DE). FBS, gentamicin, trypsin EDTA, methyl-β-cyclodextrin (CD, substitution range 10.5–14.7), Cpt-cAMP, polyethylene glycol 8000 (PEG), probucol, and acyl-CoA cholesterol acyltransferase (ACAT) inhibitor (Sandoz 58-035) were purchased from Sigma-Aldrich (St. Louis, MO). BODIPY-cholesterol and egg phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (Alabaster, AL). Fatty acid free BSA was purchased from Millipore (Kankakee, IL) and [1,2-³H]cholesterol (specific activity is 4.7 Ci/mmol) was purchased from Perkin Elmer Analytical Sciences (Waltham, MA).

Preparation of lipoproteins and human sera

Human HDL₃ was isolated by sequential ultracentrifugation from plasma of healthy, normolipemic individuals, as previously described (HDL₃, d = 1.12–1.21 g/ml) (14). Apo A-I was purified from delipidated HDL using ethanol/diethyl ether followed by anion-exchange chromatography on a Q-Sepharose column (14). The blood samples used in these studies were collected, with informed consent, from normolipemic individuals (both males and females) ranging in age from 23 to 70 years. The blood samples were obtained in vacutainer tubes and allowed to clot at room temperature for 45 min. The sera were spun at 2400 rpm for 15 min after clotting and the samples were aliquoted and stored at −80°C for up to 2 years before use. The sera had an average level of HDL-cholesterol of 58 ± 15 mg/dl (35–97 mg/dl), LDL-cholesterol of 121 ± 27 mg/dl (66–180 mg/dl), and triglycerides of 115 ± 60 mg/dl (42–230 mg/dl). The human serum samples were used individually or combined to obtain a pool of serum. The sera used in the study had undergone one cycle of freezing and thawing. The effects of freezing on the efflux assay were tested by measuring both BODIPY-cholesterol and [³H]cholesterol efflux on fresh and frozen sera (n = 4). The BODIPY-and [³H]cholesterol efflux values of fresh sera did not differ significantly from those of frozen sera (paired t-test). To isolate the serum total HDL fraction (apoB-depleted serum), the pools of sera or individual serum samples were depleted of apoB-containing lipoproteins by precipitating them with PEG (MW = 8000) solution and diluted to either 1% or 2% (equivalent to 0.7% or 1.4% whole serum, respectively) as described previously (15, 16), and as indicated in the figure legends. The presence of PEG does not alter the efflux capacity of the extracellular acceptors. The concentrations of preβ-1 protein in selected sera were determined by two-dimensional gel chromatography as previously described (16).

Preparation of labeling media

Labeling medium containing BODIPY-cholesterol was prepared by complexing the sterols (unlabeled cholesterol and BODIPY-cholesterol) with CD at a molar ratio of 1:40 (cholesterol/CD). The BODIPY-cholesterol represented 20% of the total cholesterol in the labeling medium. Unlabeled cholesterol and BODIPY-cholesterol were dried under nitrogen in the dark to form a thin film in a round-bottom 100-ml glass bottle. The cholesterol mixture was solubilized by adding 20 mM CD in MEM-HEPES buffer. The suspension was sonicated in a water bath (37°C) for 30 min, placed in a shaking water bath for 3 h (37°C), and kept at 4°C until use. Before use, the suspension was sonicated for 30 min, filtered using a 0.45-μm syringe filter, and diluted with an equal volume of MEM-HEPES buffer containing 2 µg/ml ACAT inhibitor. There was no loss of fluorescent sterol upon filtration. The final concentrations of BODIPY-cholesterol, unlabeled cholesterol, and CD in the labeling medium were 0.025 mM, 0.1 mM, and 10 mM, respectively.

The [³H]cholesterol-labeling medium was prepared by drying [³H]cholesterol (4 µCi/ml) on the walls of a conical glass tube and then dissolving the residue in 50 µl of ethanol. The solution was then incubated with 5% FBS overnight and diluted with the desired volume of RPMI medium containing 2 µg/ml ACAT inhibitor; the final concentration of ethanol was <0.5%. The detailed protocol for preparing the [³H]cholesterol-labeling medium has been described previously (17).

Efflux of BODIPY-cholesterol and [3H]cholesterol

J774 cells were plated in RPMI supplemented with 10% FBS and gentamicin in 48-well plates at a density of 75,000 cells/well for 24 h. Cells were labeled with BODIPY-cholesterol by incubating the monolayers with 0.25 ml of labeling medium containing CD/BODIPY-cholesterol/unlabeled cholesterol for 1 h, followed by washing with MEM-HEPES. J774 cells were then equilibrated with RPMI containing 0.2% BSA and the ACAT inhibitor, with or without cAMP (0.3 mmol/L), for 18 h. After this equilibration period, the cells were washed with MEM-HEPES buffer and incubated with MEM-HEPES media containing the cholesterol acceptors as indicated in the figures. Incubation times were 4 h or as indicated in the figures. At the end of the incubation time, the efflux media were removed, filtered through a 0.45-μm filter, and the fluorescence intensity was recorded using a Molecular Devices M2 plate reader (excitation 482 nm, emission 515 nm). After equilibration, the time-zero (t₀) cell monolayers (cells not incubated with extracellular acceptors) were solubilized with 1% cholic acid and mixed well by shaking on a plate shaker for 4 h at room temperature; then the fluorescence intensity was recorded. The fluorescence intensity values obtained at this time were used as the t₀ measurements. Fractional efflux of BODIPY-cholesterol was calculated based on the fluorescence intensity of the media divided by the t₀ monolayer fluorescence values measured after cholic acid solubilization. BODIPY-cholesterol efflux to media containing no acceptors (“background efflux”) was 6.3 ± 0.6% and was subtracted from BODIPY-cholesterol effluxes of all the acceptors. ACAT inhibitor was present at all times during the experiment.

The [³H]cholesterol labeling of the cells was accomplished as previously described (17). Briefly, J774 cells were incubated for 24 h in 0.25 ml of RPMI media supplemented with 5% FBS and 4 µCi/ml of [³H]cholesterol. Radiolabel present in the cells was determined by extracting cell lipids of t₀ wells (cells not incubated with extracellular acceptors) with 2-propanol and measuring [³H]cholesterol cpm in the lipid extract by liquid-scintillation counting. The efflux of [³H]cholesterol was calculated as the percentage of radiolabel in the media compared with that present in the cells (t₀). The background efflux of [³H]cholesterol was 1.4 ± 0.2% and was subtracted from the [³H]cholesterol effluxes of all acceptors. ACAT inhibitor was present at all times during the experiment.

To determine if hemolysis would affect the BODIPY-cholesterol efflux assay, J774 cells treated with cAMP were incubated with 1% plasma or hemolysed plasma (10–100% lysed red blood cells) for 4 h. BODIPY-cholesterol efflux to plasma with moderate hemolysis (<10% hemolysis) did not differ from that of plasma with no hemolysis. However, BODIPY-cholesterol efflux to plasma with 100% lysed red blood cells had a significantly lower efflux compared with that of plasma with no hemolysis. We also examined if the presence of bilirubin in the serum affected the BODIPY-cholesterol efflux assay. BODIPY-cholesterol efflux to 1% serum spiked with bilirubin added at different concentrations (0.25–2.5 mg/dl) was not significantly different from that of control serum (without bilirubin).

Statistical analysis

All statistical analyses were performed using GraphPad Prism (San Diego, CA) software. Data are presented as mean ± SD. Statistical significance was determined by unpaired t-tests unless otherwise indicated. Coefficient of variation (CV) was calculated by dividing the SD with the mean of that variable. Deming regression was used to assess the relationship between the [³H]cholesterol and BODIPY-cholesterol efflux. Significance was assessed at p ≤ 0.05.

RESULTS

General protocol for determining % efflux

Labeling cells with [3H]cholesterol and BODIPY-cholesterol.

The protocols for labeling cells with BODIPY-cholesterol differed from those of labeling with [³H]cholesterol. Isotopic labeling used previously developed methods employing the delivery of the radiolabeled cholesterol solubilized in FBS followed by a 24 h labeling period. In contrast, labeling cells with the fluorescent sterol employed a 1 h exposure to BODIPY-cholesterol complexed with CD as described in Methods.

For both fluorescence and radiolabel, the % efflux values can be determined by direct measurement of either the loss of the BODIPY-cholesterol/[³H]cholesterol from the cell monolayer or by the accumulation of these labeled sterols in the incubation medium. To establish which of these approaches was more reliable, we ran a control study of BODIPY-cholesterol and [³H]cholesterol efflux in which 20 replicate wells of cAMP-treated J774 cells were exposed to 20 µg/ml of lipid-free apo A-I or to 2% apoB-depleted pooled human serum for 4 h. BODIPY-cholesterol efflux based on media values yielded a CV of 8.2% and 9.8% for 2% apoB-depleted serum and apoA-I, respectively, whereas the efflux based on the residual fluorescence in the cells had a CV of 21.9% (2% apoB-depleted serum) and 23.7% (20 µg/ml of lipid-free apo A-I). Thus, accumulation of BODIPY-cholesterol in the media gave more reproducible values than assaying for the loss of the compound from the cells. These are similar to the values for [³H]cholesterol efflux to 2% apoB-depleted serum (CV = 7.2%) and apo A-I (CV = 13.2%). In the present experiments, percent efflux for both [³H]cholesterol and BODIPY-cholesterol was determined using ((media / time zero values) × 100).

BODIPY-cholesterol efflux from control and cAMP- treated cells.

J774 cells were used throughout this study because the efflux pathways in these cells, particularly ABCA1-mediated efflux, are easily upregulated by exposure of the cells to cAMP (3, 18). Our initial experiment compared the efflux of BODIPY-cholesterol and [³H]cholesterol from J774 macrophage cells treated with or without cAMP to 10 µg/ml of lipid-free apo A-I, 25 µg/ml human HDL_3, and 2% apoB-depleted pooled human serum. As shown in Fig. 1A and B, treatment of J774 cells with cAMP stimulated release of both fluorescent cholesterol and radiolabeled cholesterol with all extracellular acceptors. With each of the acceptors, the efflux patterns were similar; however, the fractional release of BODIPY-cholesterol was substantially greater than that of [³H]cholesterol. The difference in fractional efflux between the cAMP-treated and control cells is generally accepted to represent the contribution of ABCA1 to efflux (3). In the J774 cell system, treatment with cAMP or cholesterol enrichment has also been shown to upregulate the expression of ABCG1 in addition to ABCA1 (3). However, ABCG1 does not release cholesterol to lipid-free apolipoproteins whereas apo A-I serves as a primary ligand for the ABCA1 efflux pathway. The observation that cAMP treatment enhanced efflux of BODIPY-cholesterol to apo A-I is consistent with a substantial amount of efflux of the fluorescent sterol occurring via the ABCA1 pathway. In addition, cAMP-treated J774 cells were used in a recent study that demonstrated a negative association between cholesterol efflux and plaque burden (9). This recent study used apoB-depleted human serum as the acceptor; this total HDL preparation has been used in several studies because the presence of apoB-containing lipoproteins can confound the measurement of cellular cholesterol flux (19).

Fig. 1.

BODIPY-cholesterol and [³H]cholesterol efflux to different acceptors. For BODIPY-cholesterol efflux, J774 cells were labeled with medium containing cyclodextrin:BODIPY-cholesterol mixture for 1 h (see Materials and Methods). For [³H]cholesterol efflux, J774 cells were labeled with medium containing 5% FBS and [³H]cholesterol for 24 h. Both sets of cells were equilibrated with or without cAMP for 16 h before incubating 4 h with the extracellular acceptors. The acceptors were apo A-I (10 µg/ml), HDL₃ (25 µg/ml), or 2% apoB-depleted pooled human serum. BODIPY-cholesterol efflux (A) was significantly higher than the [³H]cholesterol efflux (B) for the corresponding acceptor. The values are mean ± SD (n = 3). *The efflux values for both BODIPY-cholesterol and [³H]cholesterol efflux were significantly higher for the cAMP treated cells (closed bars) than nontreated cells (open bars) for the same acceptor (p < 0.01).

BODIPY-cholesterol efflux to lipid-free apo A-I and phospholipid vesicles.

It has been repeatedly demonstrated that lipid-free or lipid-poor apolipoproteins, particularly apo A-I, are particularly efficient acceptors of cholesterol released from cells via the ABCA1 pathway (11, 20). Using J774 cells that are upregulated for ABCA1 by treatment with cAMP, we have quantitated and compared BODIPY-cholesterol and [³H]cholesterol efflux to increasing concentrations of apo A-I (Fig. 2). The V_max (16.1 ± 0.3%/4 h) for efflux of BODIPY-cholesterol is greater than that for radiolabeled cholesterol (V_max = 10.2 ± 0.5%/4 h). In contrast, the K_m values are similar (K_m = 2.3 ± 0.2 µg apo A-I / ml for BODIPY-cholesterol; K_m = 5.3 ± 0.8 µg apo A-I/ml for [³H]cholesterol). The efflux from control cells not exposed to cAMP of both BODIPY-cholesterol (2.4 ± 0.6%) and [³H]cholesterol (0.20 ± 0.1%) were much lower than that obtained with cAMP treatment, consistent with the data shown in the previous figures. On the other hand, egg PC small unilamellar vesicles (SUVs) that are free of apolipoproteins are very inefficient cholesterol acceptors. In contrast to the efflux obtained with apo A-I, efflux of both BODIPY - and [³H] - cholesterol to SUVs were low and similar, even at a very high concentration of 400 µg phospholipid/ml (BODIPY-cholesterol = 8.3 ± 4.1%/4 h; [³H]cholesterol = 6.6 ± 1.8%/4 h).

Fig. 2.

BODIPY-cholesterol and [³H]cholesterol efflux to different concentrations of apo A-I. J774 cells were labeled with either BODIPY-cholesterol for 1 h or [³H]cholesterol for 24 h, before the cells were equilibrated with cAMP for 16 h. The cells were then incubated with increasing concentrations of apo A-I for 4 h and BODIPY-cholesterol and [³H]cholesterol efflux were measured as indicated in Materials and Methods. The curves were fitted to the Michaelis-Menten Equation (hyperbola). The data points are mean ± SD (n = 3) and the error bars are contained within the symbols. BODIPY-cholesterol efflux, ■; [³H]cholesterol efflux, ○.

BODIPY- cholesterol efflux to apoB-depleted serum.

Figure 3 illustrates the efflux of BODIPY- and [³H]cholesterol obtained with increasing concentrations of apoB-depleted human serum. At all concentrations, efflux of BODIPY-cholesterol was substantially greater than that of radiolabeled cholesterol. We also examined the time course of BODIPY-cholesterol and [³H]cholesterol efflux from J774 cells using 2% apoB-depleted pooled human serum as an acceptor. BODIPY-cholesterol and [³H]cholesterol efflux were linear for 6 h with 2% apoB-depleted serum. BODIPY-cholesterol had a rate of release of 11.8 ± 0.6%/h with 2% apoB-depleted serum (Y = (11.8 ± 0.6)*X + (−4.8 ± 2.2); r² = 0.99). The release of [³H]cholesterol was considerably lower with a rate of 2.7 ± 0.3%/h with 2% apoB-depleted serum (Y = (2.7 ± 0.3)*X + (−1.7 ± 1.3); r² = 0.96).

Fig. 3.

BODIPY-cholesterol and [³H]cholesterol efflux to different concentrations of apoB-depleted human serum. After labeling J774 cells with either BODIPY-cholesterol for 1 h or [³H]cholesterol for 24 h, the cells were equilibrated with cAMP for 16 h. The cells were then incubated with increasing concentrations of apoB-depleted human serum for 4 h and both BODIPY-cholesterol and [³H]cholesterol efflux were measured. The curves were fitted to the Michaelis-Menten Equation (hyperbola). The data points are mean ± SD (n = 3) and the error bars are contained within the symbols. BODIPY-cholesterol efflux, ■; [³H]cholesterol efflux, ○.

BODIPY-cholesterol efflux via the ABCA1 pathway.

The data illustrated in suggest that the primary efflux pathway by which the fluorescent sterol is released from cells involves ABCA1. Thus the efflux of BODIPY-cholesterol is considerably faster than that of radiolabeled cholesterol, and the efflux is enhanced by exposure of J774 cells to cAMP. To further establish the role of ABCA1 in BODIPY-cholesterol efflux, we compared fractional efflux values for both BODIPY-cholesterol and [³H]cholesterol with and without probucol pretreatment, as probucol has been shown to inhibit ABCA1-mediated efflux of cholesterol (21). In this study, cAMP-treated J774 cells were exposed to media containing 50 µg/ml of apo A-I or 2% apoB-depleted human serum. The efflux of BODIPY-cholesterol to apoA-I was almost totally inhibited by probucol and efflux to apoB-depleted serum was greatly reduced (Table 1). Probucol treatment also reduced the efflux of [³H]cholesterol; however, the magnitude of the inhibition was not as great as that observed with the fluorescent sterol (Table 1). The enhanced stimulation of efflux by cAMP, the release of the fluorescent sterol to lipid-free apoA-I, and the inhibition by probucol are consistent with the primary pathway for efflux being via ABCA1.

TABLE 1.

Contribution of ABCA1 to BODIPY-cholesterol and [³H]cholesterol efflux in J774 macrophage cells

	% inhibition by probucol
	BODIPY-cholesterol efflux	[33H]cholesterol efflux
Apo A-I (50 µg/ml)	95.3 ± 0.4	80.7 ± 0.4
2% apoB-depleted serum	70.7 ± 2.3	50.7 ± 0.4

For BODIPY-cholesterol efflux, J774 cells were labeled with medium containing cyclodextrin:BODIPY-cholesterol mixture for 1 h (see Materials and Methods). For [³H]cholesterol efflux, J774 cells were labeled with medium containing 5% FBS and [³H]cholesterol for 24 h. The cells were then equilibrated with or without cAMP for 16 h followed by pretreatment with or without probucol (10 µM) for 2 h. Efflux was measured after incubating the cells with either apo A-I (50 µg/ml), or 2% apoB-depleted pooled human serum for 4 h. Probucol was not present during efflux phase. % of inhibition by probucol (ABCA1 contribution) was calculated by subtracting the efflux values obtained from cells treated with cAMP and probucol from efflux values of cells treated with cAMP but not probucol.

BODIPY-cholesterol and 3[H]cholesterol efflux to human sera.

The goal of developing a BODIPY-cholesterol efflux system is to provide a high-throughput assay for comparing the efflux efficiency of individual serum donors, particularly because our recent studies have demonstrated a significant negative association between efflux and atherosclerosis (9). Many efflux studies have used apoB-depleted sera as cholesterol acceptors (16). The removal of apoB-containing lipoproteins has been employed to obtain a reliable measure of HDL efflux capacity in the absence of any potential efflux to apoB-containing lipoproteins. The precipitation of apoB-lipoproteins with PEG yields a total HDL preparation that avoids the potential changes in HDL that can occur during isolation of HDL particles by centrifugation. To determine whether the precipitation protocol was necessary in studies using BODIPY-cholesterol, we conducted a preliminary study in which efflux from whole serum was compared with efflux obtained with a comparable concentration of apoB-depleted serum from the same individual. We found that BODIPY-cholesterol efflux obtained using whole serum was reduced by 25 ± 6% (n = 11) upon removal of apoB lipoproteins, whereas the contribution of apoB-lipoproteins to [³H]cholesterol efflux was greater (42 ± 8%, n = 11). The contribution of apoB-containing lipoproteins to BODIPY-cholesterol efflux to whole serum was less than that to radiolabeled cholesterol, which is consistent with efflux of BODIPY-cholesterol being a more specific probe for ABCA1-mediated efflux.

The intent of this investigation was to develop a cholesterol efflux assay in which the use of radiolabeled cholesterol could be eliminated by substituting a fluorescent-labeled cholesterol analog for the radiolabeled cholesterol. Thus, it was necessary to demonstrate a correlation between fractional efflux of BODIPY-cholesterol and [³H]cholesterol. Figure 4 demonstrates a significant relationship (P < 0.0001, Deming regression) between the measurement of efflux from J774 cells based on the release of radiolabel and fluorescent cholesterol to 23 apoB-depleted human sera. The reference range for BODIPY-cholesterol efflux assay using 23 healthy subjects is 38.9–48.1% (95% CI).

Fig. 4.

Relationship between BODIPY-cholesterol efflux and [³H]cholesterol efflux. After labeling J774 cells with either BODIPY-cholesterol for 1 h or [³H]cholesterol for 24 h, the cells were equilibrated with cAMP for 16 h. The cells were then incubated with apoB-depleted serum samples prepared from serum of 23 healthy individuals. Efflux was for 4 h. The relationship between the BODIPY-cholesterol efflux and [³H]cholesterol efflux was assessed using Deming regression (p <0.0001). Combined results of four independent experiments each done in triplicate (n = 12).

There are a number of different HDL subfractions that contribute to the efflux of cell cholesterol. Spherical, mature HDL has been shown to promote cholesterol efflux via SR-BI, ABCG1, and aqueous transfer, whereas lipid-free/poor apoproteins function as the acceptor of cholesterol released via the ABCA1 pathway, resulting in the formation of nascent HDL particles (22, 23). The data presented in Figs. 1 and 2 demonstrate that lipid-free apo A-I promotes the release of BODIPY-cholesterol via the ABCA1 pathway. Thus, it was of interest to determine the relationships between both serum HDL cholesterol and preβ-HDL concentrations and the efflux of BODIPY-cholesterol and [³H]cholesterol. As illustrated in Fig. 5A, efflux of BODIPY-cholesterol correlated with the concentration of preβ-1 (r² = 0.6) and had no significant correlation to total HDL-C (n = 23 individual sera, combined results of four independent experiments in triplicate; Fig. 5B). In contrast, the efflux of [³H]cholesterol had a significant correlation to HDL-C levels (r² = 0.6, Fig. 5D) and an association that exhibited a trend between [³H]cholesterol efflux and preβ-1 that did not reach statistical significance (r² = 0.2, n = 23 individual sera, combined results of four independent experiments in triplicate; Fig. 5C). These associations are consistent with BODIPY-cholesterol release occurring, to a large extent, by the ABCA1 pathway and responding more specifically to serum preβ-HDL levels than does the release of [³H]cholesterol.

Fig. 5.

Correlations of BODIPY-cholesterol efflux and [³H]cholesterol efflux to HDL-C and preβ-HDL. J774 cells were labeled with either BODIPY-cholesterol for 1 h or [³H]cholesterol for 24 h, before the cells were equilibrated with cAMP for 16 h. The cells were then incubated with apoB-depleted serum samples from 23 healthy individuals for 4 h and the effluxes were measured. The correlations between BODIPY-cholesterol efflux or [³H]cholesterol efflux to HDL-C and preβ-HDL were assessed using linear regression. BODIPY- cholesterol efflux significantly correlated with preβ-1 (r² = 0.6, p < 0.001; ○), but did not correlate with HDL-C (•). In contrast [³H]cholesterol efflux had a significant correlation with HDL-C (r² = 0.6, p < 0.0001; ▪), but had no significant correlation with preβ-1 (□). Combined results of 4 independent experiments each done in triplicate (n = 12). Data are presented as mean ± SEM.

In the experiments described above, apoB-depleted sera were used at a concentration of 2% to act as the acceptors for both fluorescent and radiolabeled cholesterol. This concentration is similar (2.8%) to that which has been used for a number of efflux studies in the past (9, 10, 19). To establish how changing the concentration of apoB-depleted serum influences BODIPY-cholesterol efflux, efflux values were obtained using apoB-depleted sera at three different concentrations (0.5%, 1%, and 2%). Fluorescent sterol efflux obtained using these three different concentrations had the same rank order. However, BODIPY-cholesterol values obtained using 1% sera had the best spread among individual serum samples. In addition, 1% apoB-depleted serum will require smaller amounts of serum to be collected in future screening studies and is less likely to be toxic to the cells (24). In subsequent experiments, we used 1% apoB-depleted sera becausee this concentration yielded the most consistent values when multiple serum samples were tested. The reproducibility of the fluorescent assay is illustrated in Fig. 6. In this study, six individual serum samples were tested for efflux capacity at three different efflux times and on subsequent weeks. The agreement between fractional efflux values determined 1 week apart was excellent and all of the fractional efflux values obtained at 2 h, 4 h, and 6 h fit a straight line (r² = 0.98). The inter-assay CV of the two experiments was 3.3% (1 week apart). In a larger study using 18 apoB-depleted sera at 1% concentration, the agreement between two assays conducted a week apart had an r² = 0.7 (efflux time, 4 h).

Fig. 6.

Reproducibility of the BODIPY-cholesterol efflux assay between experiments and at different time points. J774 cells were labeled with BODIPY-cholesterol for 1 h before equilibrating the cells with cAMP for 16 h. The cells were then incubated with 1% apoB-depleted serum samples and efflux is measured at 2 h (□), 4 h (◆), and 6 h (○). The BODIPY-cholesterol efflux values significantly correlated between experiments conducted one week apart and at all time points (r² =0.98, p < 0.0001). The inter-assay CV (Experiment 1 vs. Experiment 2) was 3.3%.

DISCUSSION

Experiments to study cell cholesterol efflux generally use cells prelabeled with radioactive cholesterol as donors, and the release of isotope provides values from which fractional efflux (% efflux) can be determined. This approach has been productive in supplying information on both the efflux pathways available to the cell being studied and the serum apolipoproteins and lipoproteins that serve as the most efficient acceptors. A variety of donor cells have been used, with recent emphasis on macrophages, including the established cell lines such as J774 mouse macrophages. In J774 macrophages, the extent to which an efflux pathway contributes to efflux to either serum or HDL can be measured by determining the difference in fractional efflux between control cells and cells pretreated with cAMP because exposure to cAMP upregulates ABCA1 and ABCG1. Another approach to determining the contribution of each pathway to efflux is to use inhibitors such as probucol, which has been shown to inhibit ABCA1 efflux (21), and BLT-1, which inhibits SR-BI efflux (3). All of these approaches utilize cells that contain radiolabeled cholesterol, and the protocols require a number of manipulations, including 1) removal of the acceptor medium, 2) filtration or centrifugation of the media to remove floating cells, 3) determination of the total amount of labeled cholesterol in the cells at the time of exposure (t₀), a process that requires lipid extraction of the cells, and 4) liquid-scintillation counting of both the cell extracts and the efflux medium. The present study was designed to determine whether a cholesterol efflux assay could be developed that avoided the need to use donor cells that contained radiolabeled cholesterol and eliminated some of the steps needed to establish the efflux capacity of serum, HDL, or apolipoproteins. To accomplish this, we labeled the cells with a fluorescent analog of cholesterol, BODIPY-cholesterol, and used the release of this fluorescent sterol as a surrogate measure of cell cholesterol efflux.

BODIPY-cholesterol is a fluorescent analog of free cholesterol in which carbon-24 of the sterol side chain is linked directly to the dipyrromethene boron difluoride (“BODIPY”) moiety (13). BODIPY-cholesterol has many attractive photophysical properties, such as a high fluorescence quantum yield, high photostability, and insensitivity to pH and polarity (13, 25–27). It partitions into the liquid-disordered and liquid-ordered phases of model bilayer membranes and cell membranes (28), with a preference for the liquid-ordered phase in giant unilamellar vesicles (29). BODIPY-cholesterol has been used for live-cell monitoring of cholesterol trafficking (30) and for analysis of the kinetics of transfer of self-quenched BODIPY-cholesterol from donor to acceptor vesicles in the presence of StAR protein (31).

Recently, another protocol designed to measure cell cholesterol efflux using a fluorescent mimic of cholesterol has been published. This assay uses the Pennsylvania Green fluorophore attached by a linker containing a glutamic acid residue to a derivative of N-alkyl-3β-cholesterylamine (32). The fractional release of this compound is less than that obtained with [³H]cholesterol, whereas the fractional efflux value obtained with BODIPY-cholesterol is greater than that of [³H]cholesterol. Although this fluorescent assay has the potential of being used for high-throughput screening for efflux, it was designed to be used as a screen for bioactive compounds that modulate cell cholesterol efflux. In contrast, the primary application of the BODIPY-cholesterol efflux assay will be to screen large numbers of sera or serum HDL fractions for efflux capacity, because efflux capacity has recently been shown to have a negative association with atherosclerosis (9).

The fact that BODIPY-cholesterol efflux is saturatable (Fig. 3) suggests that the release of the fluorescent sterol may occur preferentially via the ABCA1 pathway. Such a specificity is consistent with the enhanced ABCA1-mediated efflux of the BODIPY-cholesterol when compared with [³H]cholesterol, as determined by comparing efflux from cAMP ± cells (Figs. 1A, B). Studies have demonstrated that probucol is an effective inhibitor of ABCA1-mediated lipid efflux (21). The inhibition of efflux of BODIPY-cholesterol by probucol further documents the importance of ABCA1 in mediating BODIPY-cholesterol efflux (Table 1). Because of the proven importance of ABCA1 in promoting cholesterol efflux (20, 33) and the recent results linking efflux to plaque burden in humans (9), we used probucol inhibition of efflux to examine if ABCA1 is the major cell protein promoting efflux of BODIPY-cholesterol. As presented in Table 1, pretreatment of cells for 2 h with probucol resulted in a reduction of both fluorescent and radiolabeled cholesterol efflux, effectively eliminating BODIPY-cholesterol efflux to apo A-I and greatly reducing efflux to apoB-depleted human serum. Efflux to the apoB-depleted serum that remained following probucol treatment can be attributed to efflux mediated by the non-ABCA1 pathways that are known to be present in J774 cells (3, 34) and represents a lower fraction of total efflux in BODIPY-cholesterol labeled cells.

The fact that the release of BODIPY-cholesterol from cells is mediated primarily by ABCA1 suggests that this fluorescently-labeled sterol partitions into the pool of membrane lipid that is incorporated into the nascent HDL particles created by the activity of the transporter. The cellular cholesterol released via ABCA1 originates from the plasma membrane (35, 36) as well as from late endosomes (37). The preference of BODIPY-cholesterol to partition into the liquid-ordered phase of membranes (29) is consistent with BODIPY-cholesterol originating from plasma membrane raft domains being incorporated efficiently into nascent HDL particles (36). Because [³H]cholesterol is released from cells by ABCA1 much more slowly than BODIPY-cholesterol, it seems that incorporation of the BODIPY moiety into the cholesterol molecule enhances partitioning into the substrate pool for nascent HDL formation by ABCA1. The enhanced sensitivity of BODIPY-cholesterol to ABCA1-mediated efflux accounts for the relative effluxes of BODIPY-cholesterol and [³H]cholesterol to the plasma HDL fraction (apoB-depleted serum) observed with ABCA1-expressing cells (Fig. 3). However, as has been observed with other cell types (30), BODIPY-cholesterol also undergoes efflux faster than [³H]cholesterol from J774 cells in which ABCA1 is not upregulated with cAMP. In this situation, cholesterol efflux occurs primarily by diffusion-mediated pathways (11), and thus, it follows that BODIPY-cholesterol desorbs more readily from the plasma membrane than does [³H]cholesterol. This effect is presumably a consequence of the presence of the BODIPY group in the cholesterol molecule, which weakens interactions between cholesterol and the fatty acyl chains of neighboring phospholipid molecules in the membrane. Such an effect has been observed in mixed phospholipid/BODIPY-cholesterol monolayer experiments (13) and in calculations of acyl chain order in model membranes containing BODIPY-cholesterol (30).

The demonstration that the efflux of radiolabeled cholesterol from J774 cells has an inverse association with both cIMT and angiographic CAD (9) suggests that the quantitation of cholesterol efflux, as a measure of HDL functionality, may be useful in predicting cardiovascular risk. The protocol for determining serum efflux capacity has until now relied on the release of radiolabeled cholesterol from cell monolayers (12). For efflux to be used as a probe for the presence of atherosclerosis, or for the effectiveness of pharmacological agents in enhancing RCT, it will be necessary to test large numbers of sera. Because the use of radiolabeled cholesterol for this kind of testing has obvious limitations, the availability of a fluorescent efflux assay generally will be preferable. It should be noted that the use of the BODIPY-cholesterol efflux assay, as described in this study, has the potential of streamlining studies linking cell cholesterol efflux to clinical endpoints such as IMT and angiography. However, an association of BODIPY-cholesterol efflux and atherosclerosis has not yet been established. Thus, the “gold standard” for determining the association of cholesterol efflux to clinical endpoints remains the measurement of the efflux of [³H]cholesterol from cAMP treated J774 macrophages. The association between efflux and atherosclerosis appears to be linked to the level of ABCA1 in the cholesterol donor cells and the level of preβ-HDL in the serum. The present study indicates that BODIPY-cholesterol efflux is a more sensitive probe for ABCA1-mediated efflux than radiolabeled cholesterol. If so, then the increased sensitivity, coupled with the ability to quantitate efflux by fluorescent measurements, will result in a sensitive, high-throughput assay that will establish the relationships between ABCA1-mediated efflux and atherosclerosis.