Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells

Abstract

Apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein (HDL), has many protective functions against atherosclerosis, one of them being cholesterol efflux capacity. Although cholesterol efflux capacity measurement is suggested to be a key biomarker for evaluating the risk of development of atherosclerosis, the assay has not been optimized till date. This study aims at investigating the effect of different states of cells on the cholesterol efflux capacity. We also studied the effect of apoA-I modification by homocysteine, a risk factor for atherosclerosis, on cholesterol efflux capacity in different states of cells. The cholesterol efflux capacity of apoA-I was greatly influenced by the extent of differentiation of THP-1 cells and attenuated by excessive foam cell formation. N-Homocysteinylated apoA-I indicated a lower cholesterol efflux capacity than normal apoA-I in the optimized condition, whereas no significant difference was observed in the cholesterol efflux capacity between apoA-I in the excessive cell differentiation or foam cell formation states. These results suggest that cholesterol efflux capacity of apoA-I varies depending on the state of cells. Therefore, the cholesterol efflux assay should be performed using protocols optimized according to the objective of the experiment.

Validation and application of a novel cholesterol efflux assay using immobilized liposomes as a substitute for cultured cells

“…Cell culture was carried out as described previously [ 12 ]. Briefly, THP-1 cells were maintained in RPMI-1640 media (Sigma-Aldrich) containing 10% fetal bovine serum (FBS), 0.1% penicillin/streptomycin, and 0.1% nonessential amino acids.…”

“…A cholesterol efflux assay using THP-1 cells was carried out as described previously [ 12 ]. Briefly, THP-1 cells (2.5 × 10 5 cells/well) were differentiated into macrophages via culture in RPMI-1640 medium containing 100 ng/ml of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) supplemented with 0.2% bovine serum albumin (BSA) for 2 days.…”

“…On the other hand, recent research suggests that it is necessary to determine CEC of HDL for a more reliable assessment of cardiovascular risk [ 11 ]. Most CEC assay methods require cells, radioisotope-labeled cholesterol as a tracer, and HDL as a cholesterol acceptor [ 12 ]. Recently, a simple method was reported using fluorescently labeled cholesterol instead of radioisotope-labeled cholesterol and apolipoprotein B-100 depleted serum (BDS) obtained by polyethylene glycol (PEG) treatment rather than HDL obtained by ultracentrifugation [ 13 ].…”

Correlates and Predictors of Cerebrospinal Fluid Cholesterol Efflux Capacity from Neural Cells, a Family of Biomarkers for Cholesterol Epidemiology in Alzheimer’s Disease

“…We surveyed the literature to identify immortalized, commonly used, readily available and phenotypically stable neuronal, microglial, and astrocytic cell lines to be used as the cholesterol source cells. Another requirement was that the cells need not further differentiate to the target cell type, as this may introduce variability into the assay [24]. Preference was given to human over mouse lines, but a human microglial cell line matching the requirements could not be identified.…”

Comparison of a novel cholesterol efflux assay using immobilized liposome-bound gel beads with the conventional method

“…Cholesterol efflux assay using cultured cells was performed as reported previously [18]. Briefly, THP-1 cells were differentiated into macrophages by the stimulation of 100 ng/ml phorbol 12-myristate 13 acetate in RPMI-1640 supplemented with 0.2% bovine serum albumin (BSA) in 24-well cell culture plate at a density of 2.5 × 10 5 cells/well for 2 days.…”