Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages

Abstract

Objective: Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro.

Methods and results: Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously. Blood was drawn before and at several time points after injection for apolipoprotein A-I, phosphatidylcholine, and FC measurement. In WT mice, serum FC peaked at 20 minutes and rapidly returned toward baseline levels by 24 hours. Unexpectedly, ABCA1 KO and ABCG1 KO mice did not differ from WT mice regarding the kinetics of FC mobilization. In contrast, in SR-BI KO mice the increase in FC level at 20 minutes was only 10% of that in control mice (P<0.01). Bone marrow-derived macrophages from WT, SR-BI O, ABCA1 KO, and ABCG1 KO mice were incubated in vitro with rHDL and cholesterol efflux was determined. Efflux from SR-BI KO and ABCA1 KO macrophages was not different from WT macrophages. In contrast, efflux from ABCG1 KO macrophages was approximately 50% lower as compared with WT macrophages (P<0.001).

Conclusions: The bulk mobilization of FC observed in circulation after rHDL administration is primarily mediated by SR-BI. However, cholesterol mobilization from macrophages to rHDL is primarily mediated by ABCG1.

Figure 1

Human apolipoprotein A-I (apoA-I, panel A), phosphatidylcholine (PC, panel B), and free cholesterol (FC, panel C) levels in serum from C56BL/6 female mice before and 20 minutes, 2, 6 and 24 hours after i.v administration of either PBS (n=4, dashed line) or rHDL (n=8, solid line). See methods section for details.

Figure 2

Human apolipoprotein A-I (apoA-I, panel A), phosphatidylcholine (PC, panel B), and free cholesterol (FC, panel C) levels in serum from SR-BI KO (n=3) and control (n=4) male mice before and 20 minutes, 2, 6 and 24 hours after i.v. administration of rHDL. PC and FC levels are expressed as change from baseline. See methods section for details. Solid line: KO mice; dashed line: control mice.

Figure 3

Human apolipoprotein A-I (apoA-I, panel A), phosphatidylcholine (PC, panel B), and free cholesterol (FC, panel C) levels in serum from ABCA1 KO (n=4) and control (n=4) female mice before and 20 minutes, 2, 6 and 24 hours after i.v. administration of rHDL. PC and FC levels are expressed as change from baseline. See methods section for details. Solid line: KO mice; dashed line: control mice.

Figure 4

Human apolipoprotein A-I (apoA-I, panel A), phosphatidylcholine (PC, panel B), and free cholesterol (FC, panel C) levels in serum from ABCG1 KO (n=5) and control (n=5) male mice before and 20 minutes, 2, 6 and 24 hours after i.v. administration of rHDL. PC and FC levels are expressed as change from baseline. See methods section for details. Solid line: KO mice; dashed line: control mice.

Figure 5

In vitro cholesterol efflux from bone marrow macrophages obtained from SR-BI (panel A), ABCA1 (panel B) and ABCG1 (panel C) deficient mice and respective controls in the presence of rHDL as acceptor. Cells were labeled with [³H]-cholesterol as described in the methods and incubated for 4 h with rHDL (25 μg/ml) and either free human apoA-I (10 μg/ml) or HDL3 (25 μg/ml) as controls. See methods section for details.