Paradoxical association of enhanced cholesterol efflux with increased incident cardiovascular risks

Abstract

Objective: Diminished cholesterol efflux activity of apolipoprotein B (apoB)-depleted serum is associated with prevalent coronary artery disease, but its prognostic value for incident cardiovascular events is unclear. We investigated the relationship of cholesterol efflux activity with both prevalent coronary artery disease and incident development of major adverse cardiovascular events (death, myocardial infarction, or stroke).

Approach and results: Cholesterol efflux activity from free cholesterol-enriched macrophages was measured in 2 case-control cohorts: (1) an angiographic cohort (n=1150) comprising stable subjects undergoing elective diagnostic coronary angiography and (2) an outpatient cohort (n=577). Analysis of media from cholesterol efflux assays revealed that the high-density lipoprotein fraction (1.063<d<1.21) contained only a minority (≈ 40%) of [(14)C]cholesterol released, with the majority found within the lipoprotein particle-depleted fraction, where ≈ 60% was recovered after apolipoprotein A1 immunoprecipitation. Albumin immunoprecipitation recovered another ≈ 30% of radiolabeled cholesterol within this fraction. Enhanced cholesterol efflux activity from ATP-binding cassette transporter A1-stimulated macrophages was associated with reduced risk of prevalent coronary artery disease in unadjusted models within both cohorts; however, the inverse risk relationship remained significant after adjustment for traditional coronary artery disease risk factors only within the outpatient cohort. Surprisingly, higher cholesterol efflux activity was associated with increase in prospective (3 years) risk of myocardial infarction/stroke (adjusted hazard ratio, 2.19; 95% confidence interval, 1.02-4.74) and major adverse cardiovascular events (adjusted hazard ratio, 1.85; 95% confidence interval, 1.11-3.06).

Conclusions: Heightened cholesterol efflux to apoB-depleted serum was paradoxically associated with increased prospective risk for myocardial infarction, stroke, and death. The majority of released radiolabeled cholesterol from macrophages in cholesterol efflux activity assays does not reside within a high-density lipoprotein particle.

Keywords: cholesterol efflux; high-density lipoprotein-cholesterol; macrophage; prognosis.

Figure 1

A, Distribution of radiolabeled choles terol found in various cholesterol acceptor serum fractions, RAW264.7 macrophages were incubated in the absence (basal) or presence (ATP-binding cassette transporter A1 [ABCA1] stimulated) of 8-Br-cAMP as indicated with media containing apoB-depleted serum (1.4% vol/vol) in cholesterol efflux assays, as described in Methods in the online-only Data Supplement. Radiolabeled cholesterol in the indicated lipoprotein and lipoprotein-depleted fractions (densities are indicated) after stepwise buoyant density ultracentrifugation was quantified as described in Methods in the online-only Data Supplement. The distribution of radiolabeled-cho-lesterol in lipoprotein-depleted media as assessed after immunoprecipitation (IP) of apolipoprotein A1 (apoA1) and albumin using anti-apoA1 mouse monoclonal antibodies and antialbumin antibodies as described in Methods in the online-only Data Supplement. Results represent mean±SD for 4 separate experiments. B, (Left) Dose–response curves of various sources of albumin (bovine and human albumin from Sigma; fast performance liquid chro-matography (FPLC) -isolated human serum albumin [HSA] and FPLC-isolated and delipidated HSA) on total cholesterol efflux in [³H]-cholesterol-loaded RAW cells, and (right) isolated human apoA1. HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; and VLDL, very low–density lipoprotein.

Figure 2

Cholesterol efflux assay methodology and characterization. A, General scheme of cholesterol efflux assay used. B, Autoradiogram and quantification of cellular [¹⁴C] free cholesterol (FC) and [¹⁴C]cholesterylester (CE) found in [¹⁴C] cholesterol-loaded macrophage RAW cells. C, Time course of total cholesterol efflux to 2% serum, vol/vol (pool made from random sampling of the indicated number [n] of patient samples from the outpatient cohort and control serum pool, high-density lipoprotein-cholesterol [HDLc] 59 mg/dL, used). D, Dose-dependent efflux for total cholesterol from RAW264.7 cells incubated with the indicated final concentration (vol/vol) of apolipoprotein B (apoB)–depleted human serum as cholesterol acceptor. Serum pools with different HDLc levels as indicated were used. Values shown are an average of at least triplicate measurements. E, Intra batch apoB-depleted serum total cholesterol efflux assay reproducibility was plotted as even plates vs odd plates in the same batch, Spearman correlation coeffi-cient (R=0.89; P<0.001). ABCA1 indicates ATP-binding cassette transporter A1.

Figure 3

Forrest plot indicating prevalent coronary artery disease (CAD) risk in stable angiographic and outpatient cohorts. Apolipoprotein B–depleted serum as the cholesterol acceptor in cholesterol efflux assays. A, Relationship between total cholesterol efflux activity and prevalent CAD plotted from low to high tertiles in the angiographic cohort. B, Outpatient cohort. Multilogistic regression model included adjustments for age, sex, smoking, diabetes mel-litus, hypertension, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol levels. Patient sample size (n) is indicated. The 5% to 95% confidence interval (CI) is indicated by line length.

Figure 4

Forrest plot indicating incident cardiovascular risks for the stable angiographic cohort. Relationship between total cholesterol efflux activity and incident (3 years) risk of myocardial infarction (MI)/stroke (left) or major adverse cardiovascular event (MACE; MI, stroke, or death; right) plotted from low to high tertiles. Multilogistic regression model included either no adjustments or adjustments for age, sex, smoking, diabetes mellitus, hypertension, low-density lipoprotein-cholesterol, and high-density lipoprotein-cholesterol levels as indicated. The 5% to 95% confdence interval (CI) is indicated by line length.