New Strategies to Promote Macrophage Cholesterol Efflux

Abstract

The capacity of macrophages to dispose of cholesterol deposited in the atherosclerotic plaque depends on their ability to activate cholesterol efflux pathways. To develop athero-protective therapies aimed at promoting macrophage cholesterol efflux, cholesterol metabolism in THP-1 monocyte-derived macrophages has been extensively studied, but the intrinsic sensitivity of monocytes and the lack of a standardized procedure to differentiate THP-1 monocytes into macrophages have made it difficult to utilize THP-1 macrophages in the same or similar degree of differentiation across studies. The variability has resulted in lack of understanding of how the differentiation affects cholesterol metabolism, and here we review and investigate the effects of THP-1 differentiation on cholesterol efflux. The degree of THP-1 differentiation was inversely associated with ATP binding cassette A1 (ABCA1) transporter-mediated cholesterol efflux. The differentiation-associated decrease in ABCA1-mediated cholesterol efflux occurred despite an increase in ABCA1 expression. In contrast, DSC1 expression decreased during the differentiation. DSC1 is a negative regulator of the ABCA1-mediated efflux pathway and a DSC1-targeting agent, docetaxel showed high potency and efficacy in promoting ABCA1-mediated cholesterol efflux in THP-1 macrophages. These data suggest that pharmacological targeting of DSC1 may be more effective than increasing ABCA1 expression in promoting macrophage cholesterol efflux. In summary, the comparison of THP-1 macrophage subtypes in varying degrees of differentiation provided new insights into cholesterol metabolism in macrophages and allowed us to identify a viable target DSC1 for the promotion of cholesterol efflux in differentiated macrophages. Docetaxel and other pharmacological strategies targeting DSC1 may hold significant potential for reducing atherogenic cholesterol deposition.

Keywords: ATP binding cassette A1 transporter; cholesterol; desmocollin 1; docetaxel; macrophages.

Figure 1

Four main cholesterol efflux pathways in macrophages. The ABCA1-mediated cholesterol efflux pathway plays the primary role in removing excess cholesterol from macrophages. ABCA1 creates a plasma membrane microdomain to facilitate efflux of phospholipids and cholesterol to lipid-free or lipid-poor apolipoproteins such as apoA-I. An apoA-I binding protein DSC1 sequesters apoA-I and prevents the lipidation of apoA-I by ABCA1. The lipidated apoA-I is called a nascent HDL particle. The nascent, immature HDL acquires additional cholesterol via the ABCG1-mediated efflux pathway so as to form a mature HDL particle. It seems likely that ABCG1 function is to increase the pool of cholesterol available for efflux. Mature HDL particles bind to SR-BI on the cell surface, which facilitates diffusion of cholesterol molecules between the plasma membrane and the HDL particle bound. The extracellular domain of SR-BI forms a non-polar channel to mediate the cholesterol exchange. Mature HDL particles are also capable of acquiring cholesterol via aqueous diffusion (AD). AD is a simple diffusion process in which cholesterol molecules desorb from the plasma membrane or HDL particles, move down its concentration gradient in the extracellular aqueous space, and incorporate into the plasma membrane or HDL particles. LD, lipid droplet.

Figure 2

New strategies to promote ABCA1-mediated cholesterol efflux in differentiated macrophages. (A) THP-1 monocytes were cultured at the density of 2.0 × 10⁶/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 IU penicillin and 100 mg/ml streptomycin. The culture medium was refreshed every 2–3 days (Mondays, Wednesdays, and Fridays) with counting/reseeding the cells at the density of 2.0 × 10⁶/ml every 3–4 days (Mondays and Fridays). The cells cultured for 2, 16, or 37 days were treated with 100 ng/ml of PMA for 3 days to differentiate monocytes into macrophages, washed with RPMI 1640 three times, rested for 2 days in the PMA-free culture medium, and lysed to determine protein expression levels by immunoblotting. Numeric values shown below the CD14, ABCA1, and DSC1 blots represent band densities normalized to actin and relative to the 2-day cultured cells (lane 1). (B) At the end of the 2 days resting period, THP-1 cells were incubated with 50 μg/ml of acetylated LDL and 1 μCi/ml of [³H]-cholesterol for 24 h, equilibrated for 24 h, and treated with 5 μg/ml of apoA-I for 6 h to measure ABCA1-mediated cholesterol efflux to apoA-I. The indicated concentrations of docetaxel were added during the equilibration and apoA-I treatment period (35). Results were expressed as percentage of total (cell plus medium) [³H]-sterol appearing in the medium. Data values were displayed by using a box and whisker plot (n = 4). One-way analysis of variance with Dunnett's post-hoc correction was performed to calculate multiplicity-adjusted p-values. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 compared with the untreated control in each group. (C) At the end of the 2 days resting period, THP-1 cells were treated with indicated concentrations of docetaxel for 3 days prior to performing the sulforhodamine B cytotoxicity assay (35). Values are the mean ± standard deviation of triplicate determinations. Statistical analysis was performed as described above. ^*p < 0.05; ^****p < 0.0001 compared with the untreated control in each group. (D) DSC1 targeting strategies. The binding of apoA-I to DSC1 prevents ABCA1-mediated cholesterol efflux to apoA-I, thus small molecules blocking apoA-I-DSC1 interactions may hold therapeutic potential for reducing atherogenic cholesterol toxicity. In addition to small molecules such as docetaxel, peptide- and antibody-based targeting strategies may also be developed.