Development of a Cell-Based, High-Throughput Screening Assay for Cholesterol Efflux Using a Fluorescent Mimic of Cholesterol
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Issue Date
2011-04Author
Zhang, Jun
Cai, Sutang
Peterson, Blake R.
Publisher
Mary Ann Liebert, Inc.
Type
Article
Article Version
Scholarly/refereed, publisher version
Metadata
Show full item recordAbstract
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
Description
This is the publisher's version, also available electronically from http://online.liebertpub.com/doi/abs/10.1089/adt.2010.0288
ISSN
1540-658XCollections
Citation
Zhang, Cai, Peterson. (2011). Development of a Cell-Based, High-Throughput Screening Assay for Cholesterol Efflux Using a Fluorescent Mimic of Cholesterol. ASSAY and Drug Development Technologies 9(2):136-146. http://www.dx.doi.org/10.1089/adt.2010.0288
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