In this study, we found that the ability of HDL to promote cholesterol efflux from macrophages was strongly and inversely associated with both subclinical atherosclerosis and obstructive coronary artery disease. These associations persisted after adjustment for traditional cardiovascular risk factors, including the levels of HDL cholesterol and apolipoprotein A-I.
Although cholesterol efflux from macrophages represents only a small fraction of overall flux through the reverse-cholesterol-transport pathway, it is probably the component that is most relevant to atheroprotection.19
We used an assay that integrates the pathways known to mediate cholesterol efflux from macrophages (i.e., ABCA1, ABCG1, scavenger receptor B1, and aqueous diffusion). Only a small part of the observed relationship between cholesterol efflux capacity and atherosclerosis was explained by variation in HDL cholesterol levels. Indeed, efflux capacity served as the stronger predictor of both carotid intima–media thickness and coronary disease status in regression models that included both variables.
Small studies conducted with the use of a rat hepatoma cell line have shown decreased efflux capacity of whole serum or plasma in patients with coronary disease, diabetes, or diabetic ne-phropathy.20–22
Similarly, an autopsy study of nonhuman primates showed an inverse correlation between atherosclerotic burden and efflux capacity.23
In contrast, analyses that used human skin fibroblasts as the cholesterol source showed no significant differences according to the presence or absence of diabetes24
or the presence or absence of the metabolic syndrome.25
An analysis of patients undergoing coronary angiography also showed no significant variation in efflux capacity from fibroblasts according to disease status, disease severity, or risk of future events.26
In contrast with our findings, those studies have shown no relationship or only minimal relationship between whole-serum efflux capacity and the level of HDL cholesterol or apolipoprotein A-I. These discrepancies may reflect the nature of the cell line and the acceptor chosen for the ex vivo assessment of cholesterol efflux capacity. In this study, our goal was to assess the capacity of the whole HDL fraction to promote cholesterol efflux from macrophages.
A substantial body of evidence suggests that cholesterol efflux capacity, an integrated measure of HDL quantity and quality, is reflective of the role of HDL in atheroprotection. Cholesterol efflux has been shown to protect macrophages from LDL-induced apoptosis and to enhance endothelial function.27–29
In vivo studies in mice have indicated that ABCA1 and ABCG1 play a key role in facilitating cholesterol efflux and reverse cholesterol transport.30
Mice that are deficient in these proteins have marked increases in foam-cell accumulation and atherosclerosis, providing compelling evidence that the macrophage efflux pathway is antiatherogenic in vivo.31,32
Similarly, a study of patients with rare ABCA1 mutations showed an inverse relationship between cellular cholesterol efflux and carotid intima–media thickness.33
Given the substantial heterogeneity in the particle size, charge, and protein composition of HDL, it may not be surprising that HDL cholesterol levels are a poor surrogate for cholesterol efflux capacity. A recent report suggests that interindividual differences in the pre-β
(lipid-poor) apolipoprotein A-I particle concentration explain some of the observed variation.17
In the present study, associations between multiple lipid-related variables and efflux capacity remained significant after adjustment for the HDL cholesterol level. These findings are consistent with previous analyses that implicate HDL-associated apolipoprotein E and phospholipids as mediators of cholesterol efflux.34,35
The attenuated efflux capacity noted in smokers is worthy of additional follow-up and may be related to apolipoprotein A-I oxidation.36
Future studies may prove fruitful in elucidating additional components of HDL that determine cholesterol efflux capacity.
These results could be important in the assessment of new therapies targeting HDL metabolism and reverse cholesterol transport. We have shown the feasibility of this approach in blood samples from two previously reported, small, placebo-controlled trials. The moderate relationships noted between changes in HDL cholesterol levels and changes in efflux capacity reinforce the idea that these two metrics provide complementary information. We noted increased efflux capacity after therapy with pioglitazone, a phenomenon that could be related to enhanced transcription of apolipoprotein A-I.37
In contrast, no such increase was noted after patients had been treated with statins, a finding that is consistent with the concept that statins most likely exert therapeutic benefit by means of a mechanism that is distinct from the promotion of cholesterol efflux. Our demonstration that cholesterol efflux capacity is associated with atherosclerosis in humans helps support the use of this measure in guiding the development of new HDL-targeted therapies for humans.
One limitation of this study is its cross-sectional approach. Another limitation is that although our assessment of cholesterol efflux capacity reflects the ability to mobilize free cholesterol from macrophages, it does not capture variation in the reverse-cholesterol-transport pathway in terms of cellular components (i.e., the hydrolysis of cholesteryl esters and the status of endogenous macrophage cholesterol transporters) or terminal components (i.e., uptake into the liver and biliary excretion).38
In conclusion, cholesterol efflux capacity, a key metric of HDL function, is not explained simply by circulating levels of HDL cholesterol or apolipoprotein A-I and is independently related to both the presence and the extent of atherosclerosis. These findings reinforce the concept that assessment of HDL function may prove informative in refining our understanding of HDL-mediated atheroprotection.