Mechanism of cholesterol efflux in humans after infusion of reconstituted high-density lipoprotein

Abstract

Objectives: Infusion of reconstituted HDL (rHDL) leads to changes in HDL metabolism as well as to an increased capacity of plasma to support cholesterol efflux providing an opportunity to investigate mechanisms linking cholesterol efflux to changes in plasma HDL.

Methods and results: Patient plasmas after infusion of rHDL were tested ex vivo for their capacity to stimulate cholesterol efflux. Reconstituted HDL enhanced mobilization of cholesterol from tissues in vivo as shown by rising HDL cholesterol concentrations over the infusion period. Infusion of rHDL in vivo led to increased cholesterol efflux ex vivo; surprisingly, removing apoB-containing lipoproteins while preserving all HDL subfractions eliminated this increase. Infusion of rHDL led to the remodelling of plasma HDL; however, the capacity of plasma to support cholesterol efflux did not correlate with changes in the concentrations of any of HDL subfractions. Unmodified rHDL accounted for only a proportion of the increment in cholesterol efflux capacity. Furthermore, studies using HeLa and BHK cells overexpressing ABCA1, ABCG1, and SR-B1 showed that the contribution of these cellular mediators of cholesterol efflux to the enhanced capacity of plasma for the efflux was minimal.

Conclusion: Enhanced cholesterol efflux from tissues requires the presence of apoB-containing lipoproteins and may involve enhanced flow of cholesterol through multiple components of the reverse cholesterol transport pathway rather than being determined by a specific HDL subfraction.

Figure 1

Changes in lipid and lipoprotein levels and capacity of plasma to support cholesterol efflux during and after reconstituted HDL infusion. (A) Changes in plasma concentration of apolipoprotein A-I. (B) Changes in plasma concentrations of HDL-C. (C) Changes in plasma concentrations of LDL-C. (D) Changes in plasma concentrations of VLDL-C. (E) Changes in plasma total cholesterol concentrations. (F) Changes in the capacity of patient plasma to support cholesterol efflux. Closed symbols, reconstituted HDL infusion arm; open symbols, placebo infusion arm. Mean ± SD are shown; n = 13; *P < 0.050; **P < 0.005; ***P < 0.001 (vs. pre-infusion values). Data presented at panels A, B and F were reported previously.

Figure 2

Contribution of apoB-containing lipoproteins to enhanced capacity of plasma to support cholesterol efflux. (A) Changes in the capacity of whole patient plasma and apoB-depleted plasma to support cholesterol efflux. Mean ± SD are shown; n = 13. *P < 0.050, **P < 0.005. (B) Distribution of apoA-I among HDL subfractions before (1) and after (2) precipitation of apoB-containing lipoproteins. (C) Dose dependence of cholesterol efflux from THP-1 cells to whole human plasma (closed symbols) or apoB-depleted plasma (open symbols) supplemented with indicated concentrations of rHDL. Mean ± SD of quadruplicate determinations are shown. *P < 0.050, **P < 0.015, ***P < 0.001 (vs. before infusion). (D) The distribution of labelled cholesterol between lipoprotein fractions after pulse-chase experiment. The values shown are percentages of labelled cholesterol in each fraction relative to the total amount of released [³H]cholesterol (this normalization was necessary to permit comparisons among samples with a wide range of total labelled cholesterol). Means ± SD are shown; n = 13.

Figure 3

Contribution of lipid-free apoA-I and preβ₁-HDL to the enhanced capacity of plasma to support cholesterol efflux. (A) Changes in plasmas concentrations of preβ₁-HDL. Mean ± SD are shown; n = 13; *P < 0.001 (vs. pre-infusion values). (B) Cholesterol efflux from HeLa-ABCA1 cells (closed symbols) and HeLa-mock cells (open symbols) to lipid-free apoA-I; mean ± SD of quadruplicate determinations are shown; *P < 0.001 (vs. mock). (C) Cholesterol efflux from HeLa-ABCA1 cells (closed symbols) and HeLa-mock cells (open symbols) to rHDL; mean ± SD of quadruplicate determinations are shown. (D) Changes in the capacity of patient plasma to support cholesterol efflux from HeLa-ABCA1 cells (closed symbols) and HeLa-mock cells (open symbols); triangles denote an ‘ABCA1-dependent’ component of the efflux (i.e. the difference in the efflux from HeLa-ABCA1 and HeLa-mock cells). Mean ± SD are shown; n = 13.

Figure 4

Dynamics of remodelling of HDL subfractions after infusion of reconstituted HDL. (A) Distribution of apoA-I in HDL subfraction in a sample of pooled plasma. (B) Time course of changes in the abundance of HDL subfractions after rHDL infusion. The concentration of individual subfractions were calculated from relative abundance of the subfraction and apoA-I concentration in the sample. Means are shown; n = 13.

Figure 5

Contribution of mature HDL to enhanced capacity of plasma to support cholesterol efflux. (A) Cholesterol efflux from HeLa-ABCG1 cells (circles) and HeLa-mock cells (triangles) to reconstituted HDL; mean ± SD of quadruplicate determinations are shown; squares denote an ‘ABCG1-dependent’ component of the efflux (i.e. the difference in the efflux from HeLa-ABCG1 and HeLa-mock cells). (B) Changes in the capacity of patient plasma to support cholesterol efflux from HeLa-ABCG1 cells and HeLa-mock cells. Mean ± SD are shown; n = 13. (C) Cholesterol efflux from BHK-SR-B1 cells (circles) and BHK-mock cells (triangles) to rHDL; mean ± SD of quadruplicate determinations are shown; squares denote an ‘SR-B1-dependent’ component of the efflux (i.e. the difference in the efflux from BHK-SR-B1 and BHK-mock cells). (B) Changes in the capacity of patient plasma to support cholesterol efflux from BHK-SR-B1 cells and BHK-mock cells. Mean ± SD are shown; n = 13. *P < 0.010; **P < 0.001 (vs. before infusion).

Figure 6

Contribution of rHDL to enhanced capacity of plasma to support cholesterol efflux. (A) Dose dependence of cholesterol efflux from THP-1 cells to rHDL. (B) Dose dependence of cholesterol efflux from THP-1 cells to human plasma supplemented with indicated concentrations of rHDL; mean ± SD of quadruplicate determinations is shown in panels A and B. (C) Time course of remodelling of HDL subfractions after addition of rHDL to plasma in vitro. (D) Time course of redistribution of rHDL between HDL subfractions after addition of rHDL to plasma in vitro.