The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages

Abstract

Objective: We measured efflux from macrophages to apolipoprotein B-depleted serum from 263 specimens and found instances in which serum having similar high-density lipoprotein cholesterol (HDL-C) differed in their efflux capacity. Thus, we wanted to elucidate why efflux capacity could be independent of total HDL-C or apolipoprotein A-I (apoA-I).

Methods and results: To understand why sera with similar HDL-C or apoA-I could differ in total efflux capacity, we assessed their ability to promote efflux via the pathways expressed in cAMP-treated J774 macrophages. Briefly, macrophages were preincubated with probucol to block ABCA1, with BLT-1 to block SR-BI, and with both inhibitors to measure residual efflux. ABCG1 efflux was measured with transfected BHK-1 cells. We used apolipoprotein B-depleted serum from specimens with similar HDL-C values at the 25(th) and 75(th) percentiles. Specimens in each group were classified as having high or low efflux based on total efflux being above or below the group average. We found that independently of HDL-C, sera with higher efflux capacity had a significant increase in ABCA1-mediated efflux, which was significantly correlated to the concentration of pre beta-1 HDL. The same result was obtained when these sera were similarly analyzed based on similar apoA-I.

Conclusions: Sera with similar HDL-C or apoA-I differ in their ability to promote macrophage efflux because of differences in the concentration of pre beta-1 HDL.

Figure 1. Contribution of Efflux Pathways to Total Cholesterol Efflux

Efflux from control and cAMP treated J774 macrophages was measured after 2h pretreatment with inhibitors to block specific cholesterol transporters. All cells were incubated for 4h with apoB-depleted serum isolated from aliquots of a human serum pool at 2.8% (equivalent to 2% serum). All procedures as in methods. Results are the average of 3 independent experiments.

Figure 2. Efflux from cAMP Treated J774 Cells to Serum Pairs with Similar HDL-C

Total cholesterol efflux from cAMP treated J774 macrophages was measured after 4h incubation with 2.8% apoB-depleted serum as in methods. There were significant differences in efflux (p<0.05) between all pairs with the same or similar HDL-C as determined by unpaired, 2-tailed t tests.

Figure 3. Efflux from cAMP Treated J774 Cells to Serum Pairs with Similar ApoA-I

Total cholesterol efflux from cAMP treated J774 macrophages was measured as in figure 2. There were significant differences in efflux (p<0.05) between all pairs with the same or similar apoA-I as determined by unpaired, 2-tailed t tests.

Figure 4. Correlation of ABCA1 Efflux to the Concentration of preβ-1 HDL in Serum with Similar High or Low HDL-C

ABCA1 efflux from cAMP J774 cells was the Probucol sensitive efflux measured after 4h incubation with 2.8% apoB-depleted serum from specimens with similar HDL-C (HDL-C±6%) at either the 25^th percentile (open circles: HDL-C=48, range=45-51, n=22) or the 75^th percentile (open triangles: HDL-C=73, range 69-77, n=18). The specific contribution of ABCA1 to cholesterol efflux from cAMP treated J774 macrophages was significantly associated to the serum level of preβ-1 HDL (r²=0.425, p=0.0002, n=29) measured by 2D gels. All procedures as in methods.

Figure 5. Correlation of ABCA1 efflux to preβ-1 HDL Levels in Serum with High or Low Efflux Capacity

ABCA1 efflux was measured as in figure 4 using apoB-depleted serum form male and female donors. Levels of preβ-1 HDL in all these specimens (n=63) were measured using a commercial ELISA (see methods) and are reported as ng/ml/aliquot assayed. These values were significantly correlated to ABCA1-mediated efflux (r²=0.102, p=0.011, n=63). Open squares= specimens with high efflux. Closed squares=specimens with low efflux.