©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cellular Cholesterol Efflux Mediated by Cyclodextrins (*)

(Received for publication, April 11, 1995; and in revised form, May 17, 1995)

Elisabeth P. C. Kilsdonk (1)(§), Patricia G. Yancey (1), Genevieve W. Stoudt (1), Faan Wen Bangerter (2), William J. Johnson (1), Michael C. Phillips (1), George H. Rothblat (1)(¶)

From the  (1)Department of Biochemistry, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19129 and (2)Pfizer Central Research, Groton, Connecticut 06340

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In this study, we compared cholesterol efflux mediated by either high density lipoproteins (HDL) or -cyclodextrins, cyclic oligosaccharides that are able to dissolve lipids in their hydrophobic core. -Cyclodextrin, 2-hydroxypropyl--cyclodextrin, and methyl--cyclodextrin at 10 mM induced the release of 50-90% of L-cell [H]cholesterol after 8 h of incubation, with a major portion of this cholesterol being released in the first 1-2 h of incubation. The cholesterol efflux kinetics are different if cells are incubated with HDL, which induces a relatively constant rate of release of cholesterol throughout an 8-h incubation. Cholesterol efflux to cyclodextrins was much greater than phospholipid release. To test the hypothesis that maximal efflux rate constants for a particular cell are independent of the type of acceptor, we estimated the maximal rate constants for efflux (V) of cellular cholesterol from L-cells, Fu5AH cells, and GM3468A fibroblasts. The rate constant for HDL-mediated efflux varied among cell lines in the order Fu5AH > L-cells > fibroblasts. However, these differences were not evident when cyclodextrins were used as cholesterol acceptors. The estimated V values for cyclodextrin-mediated efflux were 3.5-70-fold greater than for HDL for the three cell lines. The very high efficiency of cyclodextrins in stimulating cell cholesterol efflux suggests that these compounds can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the atherosclerotic plaque.


INTRODUCTION

The first step in reverse cholesterol transport is the efflux of cellular cholesterol to a suitable acceptor, and this process is thought to be mediated by HDL()or specific HDL subclasses(1, 2, 3) . Although extensively investigated, the exact mechanism underlying the movement of cholesterol molecules from the plasma membrane to extracellular acceptors remains controversial. Although the generally accepted mechanism is now thought to involve the movement of cholesterol molecules from the cell membrane through the aqueous phase to the acceptor particle (i.e. aqueous diffusion mechanism)(2) , other mechanisms have been proposed, including collision (4) or receptor-mediated mechanisms(5) .

-Cyclodextrins, cyclic oligosaccharides consisting of 7 (1-4)-glucopyranose units, are water-soluble compounds with a hydrophobic cavity capable of dissolving hydrophobic compounds and thus enhancing their solubility in aqueous solutions(6, 7) . Cyclodextrins have been used extensively as drug delivery vehicles(6, 8) , and derivatives of -cyclodextrins have been made by chemical modifications of the hydroxyl groups which greatly improve their solubility and their ability to dissolve hydrophobic compounds, as well as reducing their toxicity(9, 10, 11) . In vitro, -cyclodextrins have a high affinity for sterols as compared to other lipids(11, 12) , and, because of the relatively high specificity of -cyclodextrins for cholesterol, it has been suggested that these compounds might be effective in modifying cholesterol metabolism in vivo(11, 13) . It can be proposed that cyclodextrins can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the atherosclerotic plaque. Although there is extensive literature on cyclodextrins, relatively little has been published on their ability to influence cholesterol metabolism, either in vitro or in vivo. It has been demonstrated by Frijlink et al.(10) that injection of either -cyclodextrin or 2OHpCD (200 mg/kg dose) resulted in transient decreases in both serum-unesterified and total cholesterol. It has been proposed that the cholesterol-lowering effect of the cyclodextrins was due to their ability to efficiently cross the capillary wall and to function as cholesterol carriers that redistribute cholesterol from the interstitial space to the plasma compartment(10) , after which it was rapidly cleared in the urine. It is believed that the cyclodextrin-cholesterol complexes are processed within the kidneys. Within the kidneys, the dissociation of cholesterol from the carrier cyclodextrin can result in the deposition of cholesterol and the associated nephrotoxicity sometimes associated with cyclodextrins(10) .

In vitro studies have demonstrated that exposure of fibroblasts to cyclodextrins can produce cell toxicity, and that the extent of this toxicity is reduced by the presence of serum(6) . Exposure of erythrocytes to cyclodextrins results in hemolysis in the order of > > (14) . This hemolysis may be attributed to the removal of erythrocyte membrane components, particularly cholesterol. Extensive release of erythrocyte cholesterol can be achieved upon incubation of the cells with the acceptor(11) . What is particularly intriguing about the interaction of cyclodextrins with erythrocytes is the rapidity of the movement of cholesterol from the cells to the incubation medium, with half-times (t) of apparently less than 1 min(11) . This rapid exchange is in contrast to that previously reported for the exchange or transfer of cholesterol from erythrocytes to serum or isolated lipoproteins, which is in the range of 1-8 h (for a review, see (15) ). It was this very fast exchange rate that prompted us to conduct the present detailed study of the characteristics of cholesterol movement between cells in culture and cyclodextrins.


MATERIALS AND METHODS

Cell Culture

MEM, DMEM, and Ham's F12 were obtained from Mediatech (Herndon, VA). Calf serum, heat-inactivated fetal bovine serum (FBS), and gentamicin were purchased from Sigma. Media were buffered with sodium bicarbonate (2 g/liter), and cells were cultured in a humidified incubator at 37 °C with 5% CO. All media were supplemented with 2 mML-glutamine and 50 µg of gentamicin/ml. Mouse L-cell fibroblasts and GM3468A human fibroblasts were cultured in MEM with 10% FBS, and Fu5AH cells were cultured in MEM supplemented with 5% calf serum.

Efflux of Cell [H]Cholesterol

Cells were plated in 16- or 22-mm wells 3 to 7 days prior to efflux experiments (1 10 cells per well for 12-well plates and 0.5 10 per well for 24-well plates). Pretreatments were started when the cells were about 90% confluent, except for GM3468A fibroblasts, which were used 2 to 4 days after reaching confluency. Cells were labeled for 24 h with medium containing 1 or 2 µCi of [H]cholesterol/ml (0.1% ethanol final concentration) and 2.5% FBS (1 ml/well). [1,2-H]Cholesterol was obtained from DuPont NEN and checked for purity by thin layer chromatography prior to use(16) . Subsequently, cells were incubated with medium containing 0.2% bovine serum albumin for 24 h to allow for equilibration of the radioactive isotope in the various cellular cholesterol pools. An acyl-CoA:cholesterol O-acyltransferase inhibitor, Sandoz 58-035, was added during both labeling and equilibration (1 µg/ml, 0.1% dimethyl sulfoxide final concentration, Sandoz compound 58-035 was a gift from Dr. John Heider). After labeling and equilibration, cells were rinsed three times with medium supplemented with only gentamicin and were then switched to medium containing various cholesterol acceptors. This medium was buffered with 50 mM Hepes (Life Technologies, Inc.) instead of sodium bicarbonate. Cells were incubated for 10 min to 8 h in a shaking water bath (40 rpm) at 37 °C, and 50-µl aliquots were taken out for the liquid scintillation counter at various time points. Cholesterol acceptors used in these studies were varying concentrations of either HDL, -cyclodextrin (CD, Sigma, C-4805), 2-hydroxypropyl--cyclodextrin (2OHpCD, Sigma, C-0926), or methyl--cyclodextrin (MCD, Sigma, C-4555). HDL (d = 1.125-1.21 g/ml) was isolated by sequential ultracentrifugation of human plasma according to Hatch and Lees(17) . Cell monolayers (before and after the incubation with acceptors) were rinsed three times with phosphate-buffered saline, and lipids were extracted with 2-propanol overnight(18) . An internal standard (cholesteryl methyl ether, Sigma) was added to the wells prior to extraction, and cholesterol was measured by gas-liquid chromatography using a 50% phenylmethyl polysiloxane column(19) . An extra set of cells was harvested at the beginning of the efflux period to determine initial cellular [H]cholesterol and cholesterol mass. Cell protein was determined on the remaining monolayer using a modification of the method of Markwell et al.(20) . Cell protein was dissolved in a solution of 0.1 N NaOH and 1% SDS, and duplicate aliquots were taken out for protein determinations.

Efflux was calculated from the fraction of initial [H]cholesterol remaining in the cells at each time point. For time course experiments, these data were fitted to a single exponential model using nonlinear regression as described previously (21) . The equation used was y = Ae + C, in which y is the fraction of initial cell [H]cholesterol remaining in the cells and t is the incubation time in hours. The apparent rate constant for efflux of cellular cholesterol, k, is the initial slope of the efflux curve and can be calculated using k = AB, and the values are the average of triplicate determinations. Approximations of maximal efflux rate constants were derived from the kversus k/[S] plot, in analogy with a Hofstee plot used to determine maximal velocities for enzyme reactions. In this plot, the intercept with the y axis is the maximal k, and the substrate concentration at which half of the maximal rate constant for efflux is reached, the K, is the slope of the regression line. The nonparametric Mann-Whitney test was used to compare treatments.

Efflux of Cell Phospholipid

L-cells were plated at 1 10 cells per well (35-mm wells) in 2 ml of MEM containing 10% FBS and 1 µCi of [methyl-H]choline chloride/ml (NET-109) and grown to confluency for 4 days. Subsequently, cells were rinsed three times with MEM and incubated with MEM plus 0.2% bovine serum albumin for 1 h. The cells were then rinsed again, and efflux was started by incubating the cells with 1 ml of medium per well for 2 h at 37 °C. Separate wells were used to determine initial cell H-phospholipid and cell phospholipid mass. Incubation media were MEM alone as a control, MEM containing HDL (0.2 or 0.5 mg of phospholipid/ml), or cyclodextrins (CD, 2OHpCD, or MCD at 2 or 5 mM). At the end of the incubation, media were collected and 800 µl was used for phospholipid extraction. For both the t cellular H-phospholipid and the medium H-phospholipid counts at t = 2 h, samples were extracted according to Bielicki et al.(22) to remove non-phospholipid [H]choline counts. Phospholipid efflux was expressed as the percentage release of initial cell H-phospholipid to the medium after 2 h of incubation. Initial cell phospholipid mass was used to calculate the specific activity of the labeled phospholipids to predict the released phospholipid mass.

Direct mass determinations of released cell cholesterol and phospholipid were conducted on the media from L-cells exposed to 10 mM 2OHpCD for 2 h at 37 °C. There was a tendency of the cyclodextrin to co-extract with lipids if the traditional chloroform-methanol extraction procedures were employed, and the presence of cyclodextrins complicated the subsequent mass determinations. To circumvent this problem, media containing the 2OHpCD was extracted twice with 2 volumes of methylene chloride, followed by 2 additional extractions with 1 volume of ethyl ether. After combining and drying the extraction solvents, the cholesterol was determined as described previously(23) . Phospholipid mass was determined by the method described by Sokoloff and Rothblat (24) and was sensitive to at least 0.1 µg of phosphorus. Isotopic amounts of radiolabeled cholesterol and phospholipid were added to the media prior to extraction to monitor lipid recoveries. The recovery of cholesterol was 105% ± 3%, the recovery of phospholipid from the incubation media was 81% ± 6%.

Toxicity Assay

The release of cellular adenine, as described by Reid et al.(25) , was used to monitor membrane integrity. To label the intracellular adenosine pool of L-cells, cells were pretreated the same as described above for efflux experiments, and, in addition, 0.5 µCi of [8-C]adenine (Amersham) per ml was added to the equilibration medium. Before efflux medium was added, the wells were rinsed extensively with MEM/gentamicin to remove extracellularly bound [C]adenine. The release of cellular adenine was monitored by removing 75-µl medium aliquots, filtering through 0.45-µm Multiscreen filtration plates (Millipore), and counting 50-µl aliquots. The initial amount of cellular [C]adenine was quantified by dissolving the cells in 1 ml of 0.5% Triton X-100.

Binding of -Cyclodextrins to Cells

The total cell association of [C]2-hydroxypropyl--cyclodextrin ([C]2OHpCD; CTD Inc., Gainesville, FL) to L-cells was determined by incubation with 0-20 mM 2OHpCD (specific activity between 70 and 1960 dpm/nmol) for 2 h at 37 °C. The cells were then rinsed, and cell monolayers were dissolved in 0.1 N NaOH. Aliquots were used for liquid scintillation counting and protein determinations.


RESULTS

The high efficacy of three types of -cyclodextrin for stimulating the efflux of L-cell [H]cholesterol is shown in Fig. 1. -Cyclodextrin (CD), 2-hydroxypropyl--cyclodextrin (2OHpCD), and methyl--cyclodextrin (MCD) all induced a concentration-dependent release of cellular cholesterol. MCD was more efficient than the two other cyclodextrins. Nearly 90% of cellular [H]cholesterol was released after 8 h of incubation with 10 mM MCD. Table 1gives the sterol mass of L-cells after 8 h of incubation with 2OHpCD and MCD. Since L-cells cannot synthesize cholesterol(26) , the fractional release of labeled cholesterol is equal to the decrease of cellular cholesterol mass (compare Fig. 1and Table 1). MCD also produced a larger fractional release of cellular desmosterol than the other cyclodextrins.


Figure 1: Dose-response curve for L-cell [H]cholesterol efflux by -cyclodextrin, 2-hydroxypropyl--cyclodextrin and methyl--cyclodextrin. [H]Cholesterol-labeled L-cells were incubated with CD (), 2OHpCD (), and MCD () at 0, 2, 5, or 10 mM. The percent of [H]cholesterol released to the medium was measured after 8 h of incubation at 37 °C. Values are means ± S.D. (n = 3). Error bars are within the markers if not apparent. FC, cell-free cholesterol.





A comparison of the time course of cellular [H]cholesterol efflux mediated by HDL or by various concentrations of 2OHpCD is shown in Fig. 2. There was no significant efflux of [H]cholesterol when cells were incubated with medium without any cholesterol acceptor. HDL induced efflux of cholesterol at a relatively constant rate during the 8-h time course. However, 2OHpCD induced a more rapid rate of cholesterol release than HDL, even when present at the lowest concentration of 2 mM. The initial fast rate of [H]cholesterol release diminished after 1 to 2 h, until an apparent equilibrium was reached between the labeled cholesterol in the medium and in the cells. Thus, the kinetics for the efflux of cellular cholesterol are very different when cyclodextrin is compared to HDL.


Figure 2: Time course for efflux of L-cell [H]cholesterol by MEM, HDL, or 2OHpCD. [H]Cholesterol-labeled L-cells were incubated with MEM alone (), 0.2 mg of HDL-phospholipid/ml (), or 2OHpCD (2 mM (), 5 mM (), 10 mM ()). Medium aliquots were removed at the indicated times, counted by a liquid scintillation counter, and efflux is expressed as the fraction of [H]cholesterol remaining in the cells. Values are means ± S.D. for triplicate wells. Error bars are within the markers if not apparent.



The kinetics of the cholesterol efflux time course studies (Fig. 2) suggested that incubation of L-cells with cyclodextrin resulted in the rapid equilibration of labeled cholesterol between cells and medium. Table 2shows the estimated molar ratio of cell free cholesterol to cyclodextrin in the incubation medium after 8 h exposure of L-cells to the cholesterol acceptors. The molar cell-free cholesterol:cyclodextrin ratio was approximately 1:1900 for 2OHpCD, and 1:1300 for CD, respectively. These ratios were obtained at both 2 mM and 5 mM concentrations of the cyclodextrins. These results indicate that cholesterol equilibrates between the cells and the available cyclodextrin molecules in the medium, and that the final ratio is dependent on the affinity of cholesterol for the type of cyclodextrin that is present.



The cyclodextrin-mediated release of a major portion of cell cholesterol mass, as shown in Table 1, might be expected to deplete plasma membrane cholesterol and result in cell toxicity. To test for the presence of CD-induced cell toxicity, cells were preincubated with labeled adenine, the subsequent release of which serves as a measure of cell membrane integrity(25, 27) . The release of cellular [C]adenine after an 8-h incubation of L-cells with MEM alone or various concentrations of cyclodextrins was inversely correlated with the cell total sterol content (Fig. 3). The higher the concentrations of cyclodextrins the greater the depletion of cell sterol and the greater the leak of [C]adenine (Fig. 3). At equivalent concentrations, MCD was more toxic than 2OHpCD, consistent with its greater ability to promote cholesterol efflux. The results shown in Fig. 3were obtained from studies in which cells were extensively depleted of cell cholesterol by long incubations with CD, therefore, a time course for the release of labeled adenine was done using various acceptors. Fig. 4shows the results for incubations of 2 and 8 h. The pattern for the release of labeled adenine obtained after 8 h of incubation was similar to that previously observed (Fig. 3); however, release of adenine above background levels was not evident after a shorter exposure of cell to the acceptors (2 h), even at the highest concentration of -cyclodextrins (10 and 20 mM). This indicates that toxicity occurs only after prolonged incubations with cyclodextrins, but not during the first initial rapid phase of efflux.


Figure 3: Relation between cell sterol mass and [C]adenine release after 8 h of incubation of L-cells with various acceptors. [C]Adenine-labeled L-cells were incubated with MEM (), 2OHpCD (2 mM (), 4 mM (), 10 mM ()), or MCD (2 mM ()) for 8 h. After the incubation, the amount of cellular [C]adenine release was measured in the efflux media, and monolayers were used to assay L-cell protein and sterol (cholesterol plus desmosterol) mass. The correlation between toxicity and total cell sterol mass is presented. Each point represents a single well.




Figure 4: Release of L-cell [C]adenine after 2 or 8 h of incubation with various acceptors. [C]Adenine-labeled L-cells were incubated with either MEM alone, HDL (0.2 mg of phospholipid/ml), or cyclodextrins at the indicated concentrations. The amount of cellular [C]adenine released to the medium was measured by removing and counting medium aliquots after 2 and 8 h of incubation.



Efflux of Cellular Phospholipids

Although -cyclodextrins are reported to have a relatively high specificity for sterols compared to phospholipids(11, 12, 28) , it is important to know the amount of cyclodextrin-mediated release of cellular phospholipids in order to acquire information on the mechanism by which cyclodextrins promote cellular cholesterol efflux. To quantitate the fractional release of cell phospholipids, L-cells were grown in [H]choline as described under ``Materials and Methods'' to label phosphatidylcholine and sphingomyelin, the 2 major phospholipids in cell plasma membranes. Subsequent incubation in medium supplemented with cyclodextrins or HDL provided an estimate of the fractional release of phospholipids from the cells (Fig. 5). The fractional release of cellular phospholipids was small; maximally, 2% of cellular phospholipids were released after 4 h of incubation with 5 mM MCD. This value is similar to that obtained with high concentrations of HDL and considerably lower than that observed for cholesterol efflux. An attempt was made to quantitate the actual mass of phospholipid present in the medium after a 2-h incubation of L-cells with 10 mM 2OHpCD. Under conditions where from 38% to 64% of the cell pool of sterol (average 9.5 µg of sterol/well; 80% cholesterol, 20% desmosterol) was recovered, no measurable phospholipid was detected in the media (sensitivity of assay = 2.5 µg of phospholipid) from monolayers having 115 ± 8 µg of phospholipid/well.


Figure 5: Efflux of [H]choline-labeled L-cell phospholipids. L-cell phospholipids were labeled with [H]choline, and efflux of cellular H-phospholipids was measured after a 2-h incubation of the cells with 1 ml of MEM per well containing HDL (0.2 and 0.5 mg of phospholipid/ml), 2OHpCD, or MCD (at 2 and 5 mM). Cellular lipids at the beginning of the experiments and medium aliquots after 2 h of incubation at 37 °C were extracted to determine phospholipid-derived H counts. Efflux is expressed as the percent of initial cell H-phospholipid released to the medium. Values are means ± S.D. of triplicate wells.



Cyclodextrin-mediated Cholesterol Efflux from Different Cell Types

Previously, it has been observed that the rate of cholesterol release from various cells differs considerably depending on the individual tissue culture cell line(29, 30) . The differences in the relative rates of cholesterol release among cells is observed with any phospholipid-containing acceptor including vesicles, reconstituted and native lipoproteins, and whole serum(29, 31, 32, 33) . To determine if cell-specific fractional efflux values persisted when the very efficient and phospholipid-free cyclodextrins served as cholesterol acceptors, a series of acceptor-dose experiments were conducted comparing L-cell mouse fibroblasts, Fu5AH rat hepatoma cells, and GM3468A human skin fibroblasts. HDL, 2OHpCD, and MCD served as acceptors. Incubation times of 10 min were used for the cyclodextrins and 30 min for the HDL in order to approximate the initial rates of cell cholesterol release. Fig. 6shows the dose-response curves for the three cell types on three different acceptors. It is clear from the data in Fig. 6A that with HDL as the acceptor the differences in k among cell types was as previously reported (Fu5AH L-cell > fibroblasts)(29) . However, in contrast to the HDL data, the same cell specificity, in terms of cholesterol efflux, was not apparent when either 2OHpCD (Fig. 6B) or MCD (Fig. 6C) served as acceptors. With the cyclodextrins, cholesterol efflux from the human fibroblasts and Fu5AH hepatoma cells was similar and slightly greater than that for L-cells.


Figure 6: Dose-response of cholesterol efflux from Fu5AH hepatoma cells, L-cells, and human skin fibroblasts exposed to HDL, MCD, and 2OHpCD. [H]Cholesterol-labeled cells in 12-well plates were incubated with 2 ml of medium containing one of the following: HDL (A), 2OHpCD (B), or MCD (C). Incubation with HDL was for 30 min, and incubations with cyclodextrins were for 10 min. Medium aliquots were removed and counted, and efflux is expressed as the fraction of initial cell [H]cholesterol released from the cells and presented as fractions/h. Data are mean ± S.D. of triplicate wells. , Fu5AH cells; , L-cells; , fibroblasts.



The data presented in Fig. 6can be used to obtain estimates of the maximum rate of cellular cholesterol release for each cell type-acceptor combination. This estimate of V for cholesterol efflux is derived from V versus V/[S] plots, where V is the fractional release of cholesterol per h and [S] is the concentration of acceptor, in analogy with a Hofstee plot used to determine maximal velocities for enzyme reactions(34) . Estimated V values for the 3 cell types are shown in Fig. 7and illustrate that in contrast to the results with HDL as an acceptor, the 2 cyclodextrins yielded similar V values that did not differ between the fibroblasts and hepatoma cells (>3 fractions/h, Fig. 7). L-cells were somewhat slower (2.4-2.8 fractions/h). Thus: 1) the maximum efflux from cells exposed to cyclodextrins is extremely rapid with values of approximately 3 fractions/h, 2) V values for cells exposed to OHpCD and MCD are the same, 3) there is little or no difference between V for cholesterol efflux among cell types exposed to cyclodextrins, and 4) V values differ considerably among cells when HDL is the acceptor, and, even with the fastest cell type (Fu5AH, 1 fraction/h), the maximum rate of release to HDL is much slower than to cyclodextrins.


Figure 7: V values for cholesterol efflux from Fu5AH hepatoma cells, L-cells, and human skin fibroblasts exposed to HDL, MCD, and 2OHpCD. Cholesterol efflux values from the dose experiment shown in Fig. 6were used to estimate the V for cholesterol release. , MCD; , 2OHpCD; , HDL.



Binding of Cyclodextrin to Cells

To test whether efflux mediated by cyclodextrins was linked to the direct association of the cyclodextrins (i.e. binding and/or internalization) with cells, we measured the association of [C]2OHpCD to L-cells over a wide concentration range (1 to 20 mM). Subsequently, the cell association data were compared with the dose-response curve for cholesterol efflux at the same concentrations (Fig. 8). As was previously demonstrated, cholesterol efflux approached a maximal value, consistent with a saturable process; however, binding/internalization of labeled 2OHpCD at 37 °C was linear over the whole concentration range. In addition, the amount of labeled cyclodextrin associated with the cells was consistently very low (0.5% of the amount present in the extracellular medium).


Figure 8: Binding of [C]2OHpCD to L-cells. L-cells were incubated with the indicated concentrations of [C]2OHpCD for 2 h at 37 °C. After washing the cell monolayers, the amount of cell-associated radiolabeled cyclodextrin was determined. Efflux of cholesterol was determined from separate wells using unlabeled 2OHpCD as described under ``Materials and Methods.'' Values are the means of 3 wells ± S.D.




DISCUSSION

Because of their ability to encapsulate and solubilize hydrophobic molecules, cyclodextrins have been used extensively as drug delivery systems(6, 28) . The three general types of cyclodextrins are distinguished by the number of glucose units in these cyclic oligomers and are: = 6, = 7, and = 8 glucose units, respectively. The physicochemical properties of the cyclodextrins can be influenced by a number of different structural modifications involving the additions of functional groups to the molecule(7, 28) . The ability to solubilize guest molecules is, in part, a function of the size of the hydrophobic cavity within cyclodextrins, and this internal cavity has been estimated to range in diameter between 5 Å and 8 Å, depending on the specific cyclodextrin(28) . Clearly, the three cyclodextrins used in this investigation have an enormous capacity for removing cholesterol from cells in culture. The efflux of cholesterol is concentration-dependent, and, at the end of an 8-h incubation period with 10 mM cyclodextrin, from 50% to 80% of the labeled cholesterol has been removed from L-cell mouse fibroblasts (Fig. 1). In this experimental system in which the cyclodextrins are present in the culture medium in the absence of any source of exogenous cholesterol, efflux of labeled cholesterol is an accurate reflection of the depletion of cellular sterol (Table 1). The extensive removal of cell sterol results in membrane instability, as measured by the leakage of [C]adenine, and a strong inverse correlation is obtained between cell sterol content and adenine release (Fig. 3). Although this toxic response could be a complicating factor in some cell culture studies, shorter incubations which do not result in as extensive cell sterol depletion do not result in any detectable increase in adenine leakage (Fig. 4).

The kinetics of cholesterol efflux to cyclodextrins are different from those observed with a physiological cholesterol acceptor such as the HDL used in the current experiment. Previously we have demonstrated that cell cholesterol efflux generally follows first order kinetics (2) and that in some cells cholesterol appears to be released from 2 kinetic pools(35, 36, 37) . As can be seen from Fig. 2, the efflux observed with cyclodextrins is much more rapid than with HDL and appears to reach equilibrium quickly between cells and medium. That equilibrium conditions are approached is demonstrated by the fact the estimated molar ratio of cholesterol to cyclodextrin molecules after 8 h of incubation with 2 mM concentrations of CD and 2OHpCD are approximately 1:1300 and 1:2000, and these ratios are similar when cyclodextrin concentrations are raised to 5 mM (Table 2). Although it has been suggested that cyclodextrins can form 1:1 molar complexes with cholesterol(28) , the high molar ratio observed in this study probably reflects an equilibrium distribution between cell and acceptor, as well as the fact that other compounds derived from cells or medium may compete with cholesterol for encapsulation within the cyclodextrins.

Of particular interest is the observation that the cell specificity of the cholesterol release rate that has been observed consistently with native and reconstituted acceptor particles (29, 36) is not evident when different cell types are exposed to cyclodextrins ( Fig. 6and Fig. 7). As illustrated in Fig. 6A, the rates of release from the three cell types exposed to HDL are Fu5AH hepatoma > L-cells > human skin fibroblasts, consistent with previously published data(29) . In contrast, all of these cells exhibited similar rates of release to either 2OHpCD or MCD (Fig. 6, B and C). The data from the dose-response studies (Fig. 6) were used to calculate an approximate V value (see ``Materials and Methods'' and (33) ) for each acceptor/cell combination. When we compare the fastest (Fu5AH) and slowest cells (fibroblasts) (Fig. 7), cyclodextrin produced V values for cholesterol efflux that were 3.5-fold greater than HDL for the Fu5AH and 70-fold greater for fibroblasts.

Two closely related questions that arise from the present investigation on cyclodextrin-mediated cholesterol efflux are as follows. 1) What is the mechanism underlying efflux to phospholipid-containing acceptors whose rate is specific for each cell type? 2) Is the mechanism for cholesterol efflux to cyclodextrins similar to that of lipoproteins such as HDL? We have previously proposed that the efflux or exchange of cholesterol between cells and lipoproteins proceed primarily by an aqueous diffusion mechanism in which cholesterol molecules desorb from the cell membrane and are subsequently incorporated into the lipoprotein acceptor particle(2, 15) . We also proposed that the efficiency of the efflux process was influenced by the presence of an unstirred water layer surrounding cells which reduced diffusion and mixing of the desorbed cholesterol molecules with acceptor particles, particularly at low concentrations of acceptors(2, 15) . A prediction of this model was that the rate-determining step in cholesterol efflux was the desorption of cholesterol from the plasma membrane and thus: 1) at infinite acceptor concentrations the V for efflux (i.e. minimum t) would be the same for all types of acceptor particles (2, 15) and 2) the differences in efflux among cell types was a function of the composition of the plasma membrane that modulated the rate of desorption of cholesterol molecules(2, 15) . This latter prediction was confirmed by studies demonstrating that the difference in efflux between 2 cell types was maintained when efflux was measured using plasma membrane vesicles as cholesterol donors(30) . Although we have consistently observed a difference in efflux among cell types(29, 36) , a series of recent studies has indicated that the estimated maximum rate of cell cholesterol release is not the same for all acceptor particles and can be modified by acceptor composition and size. For example, the estimated V value for cholesterol efflux from L-cells to reconstituted HDL particles containing phospholipid (dioleoylphosphatidylcholine or 1-palmitoyl, 2-oleoylphosphatidylcholine) in the liquid crystalline state ranged between 0.12 and 0.14 fraction/h, whereas V with reconstituted particles with phospholipid in the gel phase (dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine) yielded values of 0.03 fraction/h(33) . Another recent investigation of the effect of acceptor size has demonstrated that the estimated V values for efflux from L-cells were: 0.12 fraction/h for reconstituted HDL (13 nm diameter), 0.03 fraction/h for 100 nm large unilamellar vesicles and 0.01 fraction/h for 200 nm large unilamellar vesicles(40) . The very high efficiency of cyclodextrins for cholesterol efflux extends these observations and re-emphasizes the importance of acceptor size on the V for cell cholesterol efflux.

The plasma membrane pool of cholesterol leaves the cell by the aqueous diffusion mechanism which involves the initial desorption of cholesterol molecules from the membrane(2, 15) . At high acceptor concentrations where V = V, the cholesterol efflux rate depends on the rate constant for cholesterol desorption from the plasma membrane (k) as well as the concentration of plasma membrane cholesterol that is available for the desorption ([DC]) so that V = V = k[DC]. It follows that the high values of V observed with the cyclodextrins could arise because of alterations in k and/or [DC]. At this time is not possible to distinguish these possibilities. Increases in k could perhaps arise from cyclodextrin-membrane interactions that facilitate cholesterol desorption. Alternatively, increases in the concentration of plasma membrane cholesterol available for desorption could arise because, relative to HDL particles, small cyclodextrin molecules can access more of the cell surface. The modulation of acceptor concentration at the cell surface based on size could be a reflection of the complexity of the surface in terms of either the unstirred water layer, the cell glycocalyx or membrane domains caused by plasma membrane ridges, depressions, or vacuoles. Of particular importance is the observation that very high rates of cholesterol release can be obtained from all cells with the appropriate acceptor. It has previously been assumed that one of the major rate-limiting steps in cell cholesterol efflux was the desorption of cholesterol from the plasma membrane, since in most cells efflux appeared to be slower than intracellular transport steps(2, 19, 38) . If physiological acceptors with efflux potentials approaching that of cyclodextrins exist, efflux would no longer be rate-limiting, and intracellular metabolic events would become more important in determining overall rates of cholesterol release.

Sufficient information is not currently available to permit a definitive mechanistic explanation of how cyclodextrins remove cell cholesterol. However, some possibilities can be eliminated. The fact that there is a very large difference in the release of cholesterol and phospholipid indicates that membrane solubilization or vesiculation is not occurring. If this were the case, the relative release of cholesterol and phospholipid should correspond to their abundance in the plasma membrane. In addition, the very low association of cyclodextrins with cells (Fig. 8) indicates that irreversible binding and/or internalization events are probably not playing a significant role in cholesterol efflux. Further studies will be necessary to establish the mechanism(s) underlying the highly efficient removal of cholesterol from cells by cyclodextrins.

In addition to the use of cyclodextrins as valuable tools for studies on cholesterol transport and membrane lipid flux, the cyclodextrins and related compounds (39) have the potential to serve as model agents that could impact on either the progression or regression of the atherosclerotic plaque. Administration of large doses of these compounds could shift the distribution of cholesterol between the tissue and plasma compartments by having the acceptor molecules acting as a sink for cholesterol. On the other hand, it is possible that low concentrations of cyclodextrins could function as catalysts, shuttling cholesterol between compartments and enhancing rates of exchange, while not being present in sufficient quantities to shift equilibria. It is this latter mode that is particularly intriguing and justifies further investigation.


FOOTNOTES

*
This research was funded by Program Project Grant HL22633 and Training Grant HL07443 (to P. Y.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Niels Stensen Foundation, Amsterdam, The Netherlands and by an International Research Fellowship from the American Heart Association.

To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, Medical College of Pennsylvania and Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129. Tel.: 215-991-8308; Fax: 215-843-8849.

The abbreviations used are: HDL, high density lipoprotein; DMEM, Dulbecco's modified Eagle's medium; MEM, Eagle's minimum essential medium; FBS, fetal bovine serum; CD, -cyclodextrin; MCD, methyl--cyclodextrin; 2OHpCD, 2-hydroxypropyl--cyclodextrin.


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