Cellular Cholesterol Efflux Mediated by Cyclodextrins

Abstract

In this study, we compared the kinetics of cholesterol efflux from cells with 2-hydroxypropyl-␤-cyclodextrins and with discoidal high density lipoprotein (HDL) particles to probe the mechanisms governing the remarkably rapid rates of cyclodextrin-mediated efflux. The rate of cholesterol efflux was enhanced by shaking cells growing in a monolayer and further enhanced by placing cells in suspension to achieve maximal efflux rates. The extent of efflux was dependent on cyclodextrin concentration, and maximal efflux was observed at concentrations >50 mM. For several cell types, biexponential kinetics of cellular cholesterol efflux were observed, indicating the existence of two kinetic pools of cholesterol: a fast pool (half-time (t1 ⁄2 ) ϳ19 -23 s) and a slow pool with t1 ⁄2 of 15-30 min. Two distinct kinetic pools of cholesterol were also observed with model membranes (large unilamellar cholesterol-containing vesicles), implying that the cellular pools are in the plasma membrane. Cellular cholesterol content was altered by incubating cells with solutions of cyclodextrins complexed with increasing levels of cholesterol. The number of kinetic pools was unaffected by raising the cellular cholesterol content, but the size of the fast pool increased. After depleting cells of the fast pool of cholesterol, this pool was completely restored after a 40-min recovery period. The temperature dependence of cyclodextrinmediated cholesterol efflux from cells and model membranes was compared; the activation energies were 7 kcal/mol and 2 kcal/mol, respectively. The equivalent activation energy observed with apo-HDL-phospholipid acceptor particles was 20 kcal/mol. It seems that cyclodextrin molecules are substantially more efficient than phospholipid acceptors, because cholesterol molecules desorbing from a membrane surface can diffuse directly into the hydrophobic core of a cyclodextrin molecule without having to desorb completely into the aqueous phase before being sequestered by the acceptor.The first step in reverse cholesterol transport is the efflux of cellular cholesterol molecules to extracellular acceptors (1-3). This initial step is thought to be mediated by high density lipoproteins (HDL) 1 or by specific subpopulations of HDL (1-3). It is generally accepted that cholesterol efflux occurs by an aqueous diffusion mechanism whereby the cholesterol molecules desorb from the plasma membrane into the aqueous phase, diffuse, and are solubilized by an acceptor particle (2, 4).␤-Cyclodextrins are cyclic heptasaccharides consisting of ␤(1-4)-glucopyranose units (5). These water-soluble compounds contain a hydrophobic core capable of solubilizing nonpolar substances (5, 6). Thus, cyclodextrins have been used as vehicles to deliver hydrophobic drugs (5, 6). The ␤-cyclodextrins (7 glucose units), when compared with ␣ (6 glucose units) and ␥ (8 glucose units) cyclodextrins, have the highest affinity for encapsulating sterols, in particular cholesterol (7). Chemical modifications of the hydroxyl groups of cyclodextrins often enhance b...

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