HDL from CETP-deficient subjects shows enhanced ability to promote cholesterol efflux from macrophages in an apoE- and ABCG1-dependent pathway

Abstract

Genetic deficiency or inhibition of cholesteryl ester transfer protein (CETP) leads to a marked increase in plasma levels of large HDL-2 particles. However, there is concern that such particles may be dysfunctional in terms of their ability to promote cholesterol efflux from macrophages. Recently, the ATP-binding cassette transporter ABCG1, a macrophage liver X receptor (LXR) target, has been shown to stimulate cholesterol efflux to HDL. We have assessed the ability of HDL from subjects with homozygous deficiency of CETP (CETP-D) to promote cholesterol efflux from macrophages and have evaluated the role of ABCG1 and other factors in this process. CETP-D HDL-2 caused a 2- to 3-fold stimulation of net cholesterol efflux compared with control HDL-2 in LXR-activated macrophages, due primarily to an increase in lecithin:cholesterol acyltransferase-mediated (LCAT-mediated) cholesteryl ester formation in media. Genetic knockdown or overexpression of ABCG1 showed that increased cholesterol efflux to CETP-D HDL was ABCG1 dependent. LCAT and apoE contents of CETP-D HDL-2 were markedly increased compared with control HDL-2, and increased cholesterol esterification activity resided within the apoE-HDL fraction. Thus, CETP-D HDL has enhanced ability to promote cholesterol efflux from foam cells in an ABCG1-dependent pathway due to an increased content of LCAT and apoE.

Figure 1. HDL-2–mediated mass cholesterol efflux from macrophages.

Mouse peritoneal macrophages (MPMs) or THP-1 cells were incubated for 24 hours in DMEM containing AcLDL (50 μg protein/ml) with T0 (3 μM), then cholesterol efflux was performed for 8 hours with HDL (50 μg/ml HDL protein) added to media. (A) HDL-2–mediated cholesterol efflux from MPMs. The data show the increase in TC, FC, and CE mass in media and represent mean ± SEM of 9 independent experiments. *P < 0.01, ^#P < 0.05 versus control HDL-2. (B) Cholesterol removal from MPMs by HDL-2. The data show the mass of cholesterol remaining in cells at the end of the efflux period and represent mean ± SEM of values from 9 independent experiments. *P < 0.01, ^#P < 0.05 versus no addition of HDL-2; ^†P < 0.05 versus control HDL-2. (C) HDL-2–mediated cholesterol efflux from MPMs treated with or without T0. The data show the increase in TC mass in media and represent mean ± SD of an experiment performed in triplicate. *P < 0.05, versus control HDL-2. (D) HDL-2–mediated cholesterol efflux from THP-1 macrophages. The data represent mean ± SD of an experiment performed in triplicate. *P < 0.01, versus control HDL-2.

Figure 2. Effect of suppression of ABCG1 expression by siRNA on HDL-2–mediated cholesterol efflux from macrophages.

Mouse macrophages (MPMs) were transfected with siRNA against ABCG1 or scrambled control siRNA (160 nM) for 48 hours. Cholesterol efflux was performed for 8 hours in DMEM containing HDL-2 (50 μg/ml HDL protein). (A) HDL-2–mediated cholesterol efflux from cells. The data show the increase in TC mass in media and represent mean ± SD of an experiment performed in triplicate. *P < 0.05 versus control HDL-2, in cells transfected with scrambled control siRNA. (B) The cholesterol removal from cells by HDL-2 was estimated by changed intracellular cholesterol mass in the cells. The data show TC mass remaining in cells at the end of the incubation period and represent mean ± SD of an experiment performed in triplicate. *P < 0.05 versus cells transfected with scrambled control siRNA. Protein level of ABCG1 normalized against β-actin was determined by Western blot analysis (inset).

Figure 3. Analysis of LCAT, apoA-I, and apoE mass in HDL-2, and the effect of LCAT inhibitor on net cholesterol efflux to HDL-2 from macrophages and isotopic CE formation in media containing HDL-2.

(A) Twenty micrograms HDL protein was analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with indicated antibodies. C1–4, control HDL-2; P1–P4, CETP-D HDL-2. (B) Isotopic CE formation in media containing HDL-2. THP-1 macrophages were incubated in RPMI-1640 containing [³H]AcLDL with T0 for 24 hours, then cholesterol efflux was performed for 8 hours in RPMI-1640 containing HDL-2 (50 μg/ml HDL protein) preincubated with or without 2 mM of LCAT inhibitor (LCAT-I; E600). The data represent mean ± SD of an experiment performed in triplicate. *P < 0.005, versus control HDL-2. (C) Net cholesterol efflux to HDL-2 treated with E600 from THP-1 macrophages. Macrophages were treated with T0 and AcLDL for 24 hours, then cholesterol efflux was performed for 8 hours in RPMI-1640 containing HDL-2 (50 μg/ml HDL protein) pretreated with or without E600. The data represent mean ± SD of an experiment performed in triplicate. *P < 0.005, **P < 0.0005, versus control HDL-2. Similar data were obtained in 2 separate experiments.

Figure 4. Analysis of SM mass in HDL-2 and HDL-3 and SMase-treated HDL-2–mediated cholesterol efflux from macrophages.

(A) SM/PC ratio of HDL-2 and HDL-3. The incubation of normal HDL-2 with SMase was carried out for 1 hour at 37°C using 1.4 U SMase/mg HDL protein. The data represent mean ± SD. *P < 0.05, versus control HDL-2. (B) SMase-treated HDL-2–mediated cholesterol efflux from T0-stimulated mouse macrophages. Macrophages were treated with T0 (3 μM) and with AcLDL (50 μg protein/ml) for 24 hours, and then cholesterol efflux was performed for 8 hours in DMEM containing HDL-2 (50 μg/ml HDL protein). The data represent mean ± SD of an experiment performed in triplicate. ^#P < 0.01, versus normal control HDL-2. Similar data were obtained in 2 separate experiments.

Figure 5. Net cholesterol efflux to apoE-free HDL-2 from macrophages and CE formation in media containing apoE-free HDL-2.

ApoE was removed from HDL by heparin-sepharose chromatography, then the cholesterol efflux studies were using the holo–HDL-2 fraction or HDL-2 depleted of apoE. (A) Immunoblot analysis of LCAT, apoE, and apoA-I in the holo–HDL-2 fraction and the apoE-free HDL-2. (B) Isotopic CE formation in media containing holo–HDL-2 or apoE-free HDL-2. THP-1 macrophages were incubated in RPMI-1640 containing [³H]AcLDL with T0 for 24 hours, then cholesterol efflux was performed for 8 hours in RPMI-1640 containing HDL-2 (50 μg/ml HDL protein). The data represent mean ± SD of an experiment performed in triplicate. ^†P < 0.001, versus apoE⁺ control HDL-2. (C) Net cholesterol efflux to holo–HDL-2- and apoE-free HDL-2 from THP-1 macrophages. Macrophages were treated with T0 and AcLDL for 24 hours, and then cholesterol efflux was performed for 8 hours in RPMI-1640 containing HDL-2 (50 μg/ml HDL protein). The data represent mean ± SD of the experiment performed in triplicate. *P < 0.05, **P < 0.01, versus apoE⁺-control HDL-2. (D) LCAT activity of HDL-2 particles. FC content of HDL-2 fraction was measured using the enzymatic method before and after incubation in plastic tubes for 8 hours at 37°C with gentle shaking. The reduced FC mass of HDL-2 was estimated as the actual LCAT activity. The data represent mean ± SD of an experiment performed in triplicate. *P < 0.05, ^#P < 0.005 versus 0 hours.