Development of a Cell-Based, High-Throughput Screening Assay for Cholesterol Efflux Using a Fluorescent Mimic of Cholesterol

Abstract

Reverse cholesterol transport is the process by which extrahepatic cells, including macrophage-derived foam cells in arterial atherosclerotic plaque, transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Cholesterol efflux from peripheral cells is the first step in this process, and finding drugs and interventions that promote this event is an important endeavor. Radioisotope-labeled cholesterol traditionally has been employed in measuring efflux efficiency, but this reagent has limitations for high-throughput screening. We developed an alternative method to measure cholesterol efflux in macrophage-derived foam cells using a novel fluorescent cholesterol mimic comprising the Pennsylvania Green fluorophore, attached by a linker containing a glutamic acid residue, to a derivative of N-alkyl-3β-cholesterylamine. Compared with the traditional radioisotope-based assay, this fluorescence-based assay gave similar results in the presence of known modulators of cholesterol efflux, such as cyclic AMP, and different cholesterol acceptors. When the fluorescent probe was employed in a high-throughput screening format, a variety of chemicals and bioactive compounds with known and unknown effects on cholesterol efflux could be tested simultaneously by plate-reader in a short period of time. Treatment of THP-1-derived macrophages with inhibitors of the membrane transporter ATP-binding cassette A1, such as glyburide or a specific antibody, significantly reduced the export of this fluorescent compound, indicating that ATP-binding cassette A1 represents the primary mediator of its cellular efflux. This fluorescent mimic of cholesterol provides a safe, sensitive, and reproducible alternative to radioactive assays in efflux experiments and has great potential as a valuable tool when incorporated into a drug discovery program.

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

“…This assay uses the Pennsylvania Green fl uorophore attached by a linker containing a glutamic acid residue to a derivative of N-alkyl-3 ␤ -cholesterylamine ( 32 ). The fractional release of this compound is less than that obtained with [use of the BODIPY-cholesterol effl ux assay, as described in this study, has the potential of streamlining studies linking cell cholesterol effl ux to clinical endpoints such as IMT and angiography.…”

Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

“…These fluorescent cholesterol analogs have also demonstrated significant potential as substitutes for traditional radioisotope-labeled cholesterol for investigating lipid trafficking, including cholesterol efflux (7,22). Cholesterol efflux may prevent the development of atherosclerosis by reducing the accumulation of cholesterol in the artery wall (2,23) and is a key step in RCT.…”

“…Previously, Zhang et al developed fluorescent cholesterol bearing the Pennsylvania Green fluorophore for use as a molecular probe for the cholesterol efflux assay. This fluorescent sterol exhibits good correlation with radioisotope-labeled cholesterol in the efflux assay in THP-1 cells (24). Sankaranarayanan et al demonstrated that another fluorescent sterol, bearing a BODIPY fluorophore, can be used as a sensitive probe in the cholesterol efflux assay in J774 mouse macrophages (11).…”

“…However, the procedure is lengthy and labor-intensive, and the radioactivity disposal procedure can limit the use of this assay in high-throughput screening. By contrast, fluorescent cholesterol analogs have demonstrated potential as suitable alternatives for radioisotope-labeled cholesterols (7). For decades, fluorescent cholesterol analogs have been used in membrane biophysics for the investigation of lipid trafficking and the membrane organization of native cholesterol (8,9).…”

“…Thus, these findings support NBD-cholesterol as a sensitive and specific probe, which may be used efficiently for cholesterol efflux measurement. Several other studies have reported that fluorescent BODIPY-cholesterol and Pennsylvania Green-cholesterol can also be used in cholesterol efflux assay as substitutes for [ 3 H]-cholesterol (11,24). Further investigation to compare different fluorescent analogs in parallel with [ 3 H]-cholesterol may assist in determining which fluorescent analog can mimic native cholesterol efflux most closely in the assay.…”