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␤-1 HDL. The same result was obtained when these sera were similarly analyzed based on similar apoA-I. Key Words: ABCA1 Ⅲ apolipoprotein A-I Ⅲ cholesterol efflux Ⅲ high-density lipoprotein cholesterol Ⅲ macrophages E pidemiological and interventional studies 1-4 demonstrate an inverse relationship between high-density lipoprotein cholesterol (HDL-C) levels and coronary heart disease, which is an observation also supported by animal studies. 5 Thus, high HDL-C levels are thought to independently reduce coronary heart disease risk. Although HDL has been shown to have both antioxidant and antiinflammatory properties, 6 its beneficial antiatherogenic effect is likely attributable to its central role in reverse cholesterol transport, ie, the transport of cholesterol from peripheral tissues to the liver for excretion to reduce its accumulation in tissue cells such as vessel wall macrophages. 7 Because both HDL metabolism and cholesterol transport are complex processes, it has been difficult to obtain in vivo evidence that modulating HDL levels can affect removal of cholesterol from macrophage foam cells in the vessel wall and reduce atherosclerotic lesions. Overexpression of apolipoprotein (apo) A-I in mice can reduce progression of atherosclerotic lesions, 8 and infusion of apoA-I/phospholipid complexes in humans promotes lesion regression and increases fecal excretion of bile acids. 9 However, results of studies in subjects with monogenic disorders of HDL metabolism 10,11 and post hoc analyses of epidemiological studies raise questions regarding the mechanism underlying the association between HDL-C levels and coronary heart disease. 12 We recently demonstrated that in healthy individuals having a wide range of HDL-C and apoA-I levels, the capacity of serum HDL to promote cholesterol efflux from macrophages in vitro is negatively correla...

Variants at the APOE/C1/C2/C4 Locus Modulate Cholesterol Efflux Capacity Independently of High‐Density Lipoprotein Cholesterol

“…CEC of apolipoprotein B–depleted (HDL‐enriched) plasma was measured essentially as described by de la Llera‐Moya et al 10. Immediately after thawing and gentle mixing of plasma, apolipoprotein B–containing lipoproteins were precipitated by the addition of 0.4 volume of PEG 6000 20% in 200 mmol/L glycine at pH 7.4, thorough mixing by 5‐s vortexing, and incubation on ice for 20 minutes 15.…”

“…Because the transporters involved vary between cell types, we conducted our study with 2 different models representing 4 different conditions. Specifically, CEC was measured from J774 mouse macrophages in which cholesterol efflux is dependent mostly on SR‐B1 and passive diffusion as well as mouse ABCA1 after its overexpression on overnight treatment with cAMP10; and baby hamster kidney (BHK) cells that overexpress human ABCA1 after overnight treatment with mifepristone 11. The ABCA1 transporter is especially important because it allows unidirectional flux of cholesterol to lipid‐free apolipoprotein A‐I and small HDL particles,12 and was tested with mifepristone‐stimulated BHK and cAMP‐stimulated J774 cells.…”

Effects of Dietary Flavonoids on Reverse Cholesterol Transport, HDL Metabolism, and HDL function

“…The cholesterol efflux capacity of HDL was shown to be a significant predictor of CHD status even after plasma HDL cholesterol and apoA-I were adjusted for (19). Importantly, differences in cholesterol efflux capacity have been reported in humans with similar HDL cholesterol concentrations (58), supporting the need to examine metrics of HDL function beyond HDL cholesterol. Other quantitative measures of HDL, such as HDL particle number, may explain some of this variation (59).…”

Premature and severe cardiovascular disease in a Mexican male with markedly low high-density-lipoprotein-cholesterol levels and a mutation in the lecithin:cholesterol acyltransferase gene: A family study

“…The capacity of total isolated HDL to promote cell cholesterol efflux was tested for each individual. cAMP-stimulated J774 murine macrophages and Fu5AH hepatoma cells were used to assess ABCA1 and SR-BI-mediated cholesterol efflux, respectively, following the procedure described by de la Llera-Moya et al (21)(22), with slight modifications. Briefly, total isolated HDL fractions as estimated by phospholipids (100 µg of phospholipids/ml) were incubated with 1,2-3H cholesterol labeled Fu5AH or J774 cells at 37˚C for 4 h. The percentage of cholesterol efflux was calculated comparing radioactivity measurements in the medium and cells.…”