©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of Acceptor Particle Size on the Efflux of Cellular Free Cholesterol (*)

(Received for publication, February 14, 1995; and in revised form, May 18, 1995)

W. Sean Davidson (§) , Wendi V. Rodrigueza (¶) , Sissel Lund-Katz , William J. Johnson , George H. Rothblat , Michael C. Phillips (**)

From the Department of Biochemistry, The Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 19129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several subspecies of human high density lipoprotein (HDL) have been shown to exist, and particle size is one parameter that can be used to distinguish them. Recently, a small HDL subspecies has been described that may be a particularly efficient acceptor of peripheral cell unesterified (free) cholesterol (FC). To address the effects of particle size on the ability of HDL to remove FC from cells, homogeneous, well defined HDL particles were reconstituted (rHDL) that varied in particle diameter within the size range of human HDL particles (7-13 nm). The abilities of each of these particles to remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were compared on the basis of their phospholipid (PL) content as well as on a per particle basis. The effect of particle size was also examined using small unilamellar vesicles (SUV) of 25 nm in diameter and large unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV were prepared by sonication, and the LUVs were prepared by extrusion techniques. The FC efflux efficiency of these particles (in order of decreasing efficiency) was: rHDL > SUV > LUV when compared on the basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes, smaller particles were more efficient). The FC efflux differences between the rHDL and the vesicles were not due to the absence of apolipoprotein in the vesicles. No difference was detected among the rHDL of varying size, nor was a difference detected among the LUVs of varying size when compared on the basis of PL content. When the FC efflux data for rHDL and LUVs were normalized on the basis of the number of acceptor particles present at a given PL concentration, a correlation was found between acceptor particle radius and the ability to accept cellular FC with larger particles being the most efficient. However, the dependence of the rate of FC efflux on acceptor particle size was not quantitatively the same within the rHDL and LUV classes of acceptor particles. The dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.


INTRODUCTION

The concept of high density lipoprotein (HDL)()heterogeneity has become a topic of great interest because of recent reports by several investigators that HDL subspecies may play a particularly important role in the various stages of reverse cholesterol transport(1, 2, 3, 4) , the postulated process by which excess peripheral cell cholesterol is transported to the liver for catabolism(5) . HDL subspecies have been characterized according to a variety of physical properties including apolipoprotein composition (6) , charge characteristics(7) , and particle size(8) . Of particular interest are the studies of Fielding et al.(9, 10) , which have demonstrated that a minor charge subspecies of human HDL may be a particularly important factor in the first step of reverse cholesterol transport, the removal of cellular unesterified (free) cholesterol (FC) from cells. One characteristic of this particle is its small size (70 kDa) (9) with respect to the bulk of human plasma HDL (180-390 kDa) (11) . It is conceivable that the size of an HDL particle may be an important mediator of reverse cholesterol transport because smaller particles are expected to more freely infiltrate the interstitial space, the site of the majority of peripheral cell FC removal(12) . Currently, the effect of particle size on this function of HDL has not been studied in detail. Such studies of HDL size subspecies have been hindered by the high degree of structural and compositional heterogeneity that is inherent in even highly purified preparations from human plasma.

To study the effect of HDL structure on the ability to remove cellular FC, we have taken the approach of reconstituting well defined HDL particles (rHDL) that are homogeneous in composition and structure and testing their abilities to remove cultured cell FC. We have previously studied the role of HDL apolipoprotein structure (13) and phospholipid composition (14) on the ability of the particles to remove cellular FC. The current study focuses on the effect of HDL particle size on this function. A previous study by Agnani and Marcel (15) took a similar approach to this problem. In that work, phosphatidylcholine-containing rHDL particles of increasing size that contained 2, 3, and 4 molecules of apolipoprotein AI (apoAI) were used in cultured cell FC efflux experiments. The authors concluded that the larger rHDL were the most effective FC acceptors and that the FC efflux to these particles could be correlated to the particle diameter, the number of apoAI molecules per particle, and the apoAI:phospholipid ratio. However, all of these parameters are interrelated, and the important characteristic among them could not be distinguished because the rHDL contained different amounts of apoAI per particle and different PL:apoAI ratios. A different conclusion about the effect of rHDL size on FC efflux was reported by Jonas et al.(16) . They concluded that smaller rHDL particles were more efficient acceptors of cell-derived and lipoprotein-derived FC than larger particles. However, this conclusion was reached when the data (initially based on apoAI content) were postexperimentally normalized to the phospholipid content of the acceptor particles. Since the dependence of FC efflux on the phospholipid content of HDL is not a linear relationship(13, 14, 17) , such an analysis is not straightforward and can lead to misleading results. Close inspection of the data reveals that the FC efflux promoted by the various particles in this study correlated to the amount of PL contained in the particle, in agreement with Agnani and Marcel(15) .

The objective in the current study was to test the hypothesis that the size of a FC acceptor particle can affect its ability to remove FC from cells. Since the size of a rHDL particle is related to its PL:apoAI ratio, it is important to understand the dependence of FC efflux to rHDL in terms of both the phospholipid concentration and the number of particles present in a FC efflux incubation. This can be done by preparing differently sized rHDL particles that contain the same number of molecules of apoAI per particle and comparing their abilities to remove cell FC when they are added to the extracellular medium at equal phospholipid concentrations. This allows a straightforward normalization of the FC efflux to the acceptor PL content, and the FC efflux can then be expressed in terms of the number of particles present at a given phospholipid concentration. FC efflux experiments of this type were performed with four highly homogeneous, well characterized rHDL that varied in particle diameter across the size range of human plasma HDL (7.3-12.8 nm). The smallest three of these particles contained two molecules of apoAI, with the largest containing three molecules. In addition, the ability of unilamellar vesicles ranging in diameter from 25 to 180 nm in diameter to remove FC was also assessed. The results indicate that, among rHDL, larger particles are more effective than smaller particles when compared on a per particle basis. A similar relationship was found for the large unilamellar vesicles although the vesicles were less efficient acceptors than rHDL. This effect was not due to the absence of apolipoproteins in the unilamellar vesicles, because vesicles that contained adsorbed apoAI were not able to remove more cellular FC than unmodified vesicles. The results from this study suggest that the dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.


EXPERIMENTAL PROCEDURES

Materials

Sodium cholate and bovine serum albumin were purchased from Sigma. 1-Palmitoyl, 2-oleoyl phosphatidylcholine (POPC) was purchased from Avanti Polar Lipids (Birmingham, AL) (+99% grade). [1,2-H]cholesterol was obtained from DuPont NEN. Minimal essential medium and bovine calf and fetal serum were from Life Technologies, Inc. Media were supplemented with 50 µg/ml of gentamicin (Sigma). All other reagents were analytical grade.

Methods

Purification of Human Apolipoproteins

Human HDL was isolated from the fresh plasma of normolipidemic subjects by sequential ultracentrifugation as described previously(18) . HDL was delipidated (apoHDL) in ethanol:diethyl ether as described by Scanu and Edelstein (19) , and purified apoAI was isolated by anion exchange chromatography on Q-Sepharose (20) and stored in lyophilized form at -70 °C. Prior to use, apoAI was resolubilized in 6 M guanidine HCl and dialyzed extensively against standard Tris buffer (10 mM Tris, 150 mM NaCl, 1.0 mM EDTA, pH 8.2).

Preparation and Characterization of Discoidal Reconstituted HDL Particles

All discoidal particles were reconstituted using the sodium cholate removal method described in detail elsewhere (21) using the starting PL to protein stoichiometries shown in Table 1. Intact rHDL were separated from unreacted lipid and protein by gel filtration chromatography on a (2.5 100 cm) Superose 6 column eluted with Tris buffer (0.7 ml/min at 25 °C). The homogeneity and hydrodynamic diameters of rHDL particles were estimated by Phast native polyacrylamide gradient gel electrophoresis using precast, 8-25% gradient gels (Pharmacia Biotech Inc.)(21) . The particle migrations were determined to within 0.1 mm by digitizing the gel with a Mustek 105 scanner followed by computer image analysis (Jandel Scientific, San Rafael, CA). Intact rHDL particles were chemically analyzed using the Markwell modification of the Lowry protein assay(22) , while PL were determined as inorganic phosphorus by the method of Sokoloff and Rothblat(23) . The number of protein molecules per particle was determined by cross-linking the apoAI with dimethyl suberimidate(24) . The electrophoretic mobilities of native and reconstituted HDL were measured on Beckman Paragon preformed 0.5% agarose gels, and the potential at the particle surface of shear (surface potential, S) was calculated as described by Sparks and Phillips(25) . The average percentage of -helix content was determined by circular dichroism (CD) spectroscopy on a Jasco J41A spectropolarimeter at 25 °C in a 0.1-cm path length quartz cuvette. The percent -helix was determined from the molar ellipticities at 222 nm(21) .



Preparation and Characterization of PL Unilamellar Vesicles

POPC small unilamellar vesicles (SUV) with estimated diameters of 25 ± 5 nm were made using the sonication technique of Barenholz et al.(26) . The SUV that had been reisolated by ultracentrifugation at 40,000 rpm in a 50-Ti rotor for 2 h at 25 °C appeared homogeneous by gel filtration chromatography. SUV preparations in Tris buffer were mixed with apoAI to make SUV with adsorbed apoAI (SUV-apoAI) as described by Yokoyama et al.(27) . The initial preparation contained SUV and apoAI at a 170:1 mol POPC:mol apoAI ratio, and this mixture was incubated for 16 h at 25 °C. Using gel filtration chromatography, SUV-apoAI fractions that were similar in size to protein-free SUV (25 nm) were combined and concentrated. Analysis of the PL and protein content of four independent preparations resulted in SUV-apoAI that contained 4-5 molecules of apoAI per SUV. The isolated SUV-apoAI appeared to be stable for at least 3 days at 4 °C; no lipid-free apoAI was detectable by gel filtration chromatography during this period (data not shown). LUVs were prepared by the extrusion technique of Hope et al.(28) . Briefly, POPC multilamellar liposomes were placed into an extrusion device (Lipex Biomembranes Inc., Vancouver, B.C.), which passed the mixture through polycarbonate filters of varying pore size under pressures of 100-500 lb/in. The resulting LUVs were classified according to the pore size of the filter through which they were passed (for example, LUVs were created by passage through a 50-nm filter). The characteristics of the LUVs made with 50-, 100-, and 200-nm filters are shown in Table 2.



Efflux of Plasma Membrane Cholesterol

Mouse L-cell fibroblast or rat Fu5AH hepatoma cells were used to monitor the FC efflux efficiencies of the various vesicles and rHDL. The methods for the efflux assay as well as the postexperiment work-up were as previously reported (13) with the following exceptions. The L-cells were plated 6 days prior to the experiment in 12-well cell plates (22 mm) at 75-100 10 cells/well. 1.0-1.5 µCi/well [H]FC in bicarbonate-buffered minimal essential medium with 3% fetal calf serum was used for the cell-labeling procedure. After a brief wash, the efflux measurements were initiated by the application of 1.0 ml/well of the test medium containing 0.5% bovine serum albumin and the rHDL complex at the appropriate concentration. Typical cell protein values were between 250 and 300 µg of protein/well. Efflux experiments that used rat Fu5AH hepatoma cells were done exactly the same as the L-cell efflux experiments except that only 0.5-1.0 µCi of labeled FC was used to label the cells.

Data Analysis

The fractional release of cellular FC determined experimentally was analyzed as described in detail for these systems previously(13) . Briefly, the kinetic analysis assumes a closed system in which FC exists in one of two kinetic pools, either the cellular FC pool or the acceptor FC pool. The equilibration of FC between these pools is fit to the single exponential equation Y = He + H (see (5) for a detailed description of this equation), which has been shown to fit the data better than a double exponential equation by comparison of the F statistic (13) . Y represents the fraction of radiolabeled FC remaining in the cells, t is the incubation time in h, H is a preexponential term that reflects the fraction of cellular FC that exists in the medium at equilibrium, g is the sum of the rate constants for efflux (k) and influx (k), and H is a constant that represents the fraction of labeled cell FC that remains associated with the cells at equilibrium due to a constant retrograde flux of FC from the extracellular acceptor to the cell. H, g, and H are variables that can be fit to the experimental data by computer (Jandel Scientific, San Rafael, CA). The apparent rate constant for the efflux process (k) is the product of H and g. This value should be considered ``apparent'' because it is dependent on acceptor particle concentration under some conditions. The apparent tvalue in hours is then calculated as follows: t = ln 2/k.


RESULTS

FC Efflux to Discoidal rHDL Particles Compared on the Basis of PL Concentration

To determine the effect of particle size on FC efflux within the size range of human HDL particles, homogeneous, discoidal rHDL that varied in size from 7 to 13 nm in diameter were reconstituted with apoAI and POPC. Table 1shows clear increases in the particle hydrodynamic diameter as the rHDL phospholipid to protein ratio was increased. All rHDL contained 2 molecules of apoAI except for the 12.8-nm particle, which contained 2 or 3 molecules. The particles appeared homogeneous on native polyacrylamide gradient gel electrophoresis after reisolation by gel filtration chromatography (data not shown). All rHDL exhibited surface potential values in the pre- migration range according to the classification scheme proposed by Sparks and Phillips (-7.0 to -10.5 mV)(25) . In addition, the -helix content of the associated apoAI molecules increased with the increasing PL:apoAI ratio in agreement with previous studies in this laboratory (refer to (21) for a detailed discussion of the effects of PL:apoAI ratio on the structure of rHDL particles). These data show that the conformation of apoAI changes in response to increases in the PL content of rHDL particles.

The ability of these particles to remove tritiated FC from mouse L-cells was determined. Previous studies demonstrated that decreases in cellular FC mass closely paralleled decreases in cellular FC radioactivity upon exposure to acceptor-containing medium, indicating that the radiolabel correctly reflected movement of FC mass(13) . In addition, the PL concentration of the extracellular medium has been shown to be constant over the 6-h time course for apoAI/POPC rHDL particles, indicating that the particles did not precipitate out of solution during the experiment(14) . Gel filtration chromatography of test medium recovered after an efflux incubation showed that the FC radioactivity appearing in the medium eluted at a volume that was characteristic of the discoidal rHDL particle that was present initially in the medium(14) . This indicated the following: (a) the acceptor particles sequestered cellular FC, and (b) the acceptor particles did not significantly change size during the efflux assay (i.e. there was no fusion or aggregation of rHDL). Fig. 1shows the fraction of cellular FC remaining in the cell over a 6-h FC efflux incubation with each of the four rHDL particles present at 50 µg/ml of PL. The tvalues for the 7.3-, 9.3-, 10.0-, and 12.8-nm rHDL were 12.6 ± 1.3, 12.0 ± 2.0, 12.5 ± 1.6 and 13.5 ± 2.2 h, respectively, indicating that the FC efflux to each of these complexes was similar despite the differences in PL:apoAI ratio and apoAI conformation. To determine the dependence of FC efflux on acceptor particle concentration, the FC efflux rate constants were measured for each particle over a range of 10-200 µg of POPC/ml (Fig. 2). No significant differences were detectable at low or high acceptor particle concentrations. Analysis of the kinetic data provides information about the predicted equilibrium distribution of FC between the cell and acceptor pools. At low rHDL concentrations (10 µg of PL/ml) the distribution was similar for all four rHDL with 7-10% of cell FC present in the acceptor pool at equilibrium. At 200 µg of PL/ml this increased to 75-80% of cellular FC in the medium (data not shown). To estimate the maximal FC efflux rate that can be attained by these particles, the initial rates of cellular FC efflux were plotted as a function of particle concentration as proposed by Hofstee(29) . This linearizes the data so that a maximal velocity (V) can be estimated by extrapolating the data to the condition of infinitely high acceptor concentration(13, 14) . It should be noted that the V values as expressed below in percentage of FC released per h are actually the maximum k. However, in the experiments reported in this study, all of the cell FC behaved as a single kinetic pool for efflux. Thus, if the cell concentration of FC is taken as unity throughout, then V equals the maximum k. The actual V can be obtained by multiplying the maximal k by the FC mass contained in the cells at the initiation of the experiment (e.g. at 40 µg of FC/mg of cell protein a V value of 10%/h corresponds to a mass release of 4 µg of FC/h). All rHDL particles exhibited a similar V of 10-11% of cell FC released/h. A similar independence of FC efflux on rHDL particle size was found in experiments using rat Fu5AH hepatoma cells as the FC donors (data not shown). These results indicate that, when compared on the basis of PL content, no difference in FC efflux is observed between rHDL of varying PL:apoAI ratio.


Figure 1: Time course of H-labeled free cholesterol efflux from mouse L-cell fibroblasts to apoAI/POPC rHDL of increasing particle hydrodynamic diameter. Mouse L-cell fibroblasts that had been trace-labeled with H-labeled free cholesterol in 22-mm tissue culture wells were incubated for 6 h at 37 °C in a humidified incubator (5% CO) with 1.0 ml of test medium containing 0.5% bovine serum albumin, 1.0 µg/ml Sandoz compound 58035 (to prevent intracellular esterification of the label), and the apoAI/POPC rHDL (see Table 1) at 50 µg of POPC/ml. The acceptors shown are POPC:apoAI 37:1 (), 75:1 (), 107:1 (▾), and 156:1 () (mol:mol). The verticalaxis indicates the fraction of initial labeled FC that remained in the cell at the designated times. Each point represents the mean of six cell wells. The errorbars represent one S.D. All curves were obtained by fitting the entire time course to the model for tracer equilibration between two pools (see ``Methods'').




Figure 2: The concentration dependence of the rate for cholesterol efflux from mouse L-cell fibroblasts to apoAIPOPC complexes of increasing particle hydrodynamic diameter. The incubation conditions were the same as those for Fig. 1except that the acceptor concentration was varied as shown. The acceptor particles are the same as in Fig. 1with the same symbols. Each point represents six cell wells, and the errorbars represent one S.D. The values shown are corrected for the FC efflux to control medium.



FC Efflux to Unilamellar Vesicles Compared on the Basis of PL Concentration

To further examine the effect of acceptor particle size on FC efflux efficiency, unilamellar vesicles of varying diameters (25-185 nm) were produced by sonication and extrusion techniques (Table 2). The abilities of these vesicles to accept cellular FC from L-cells and rat Fu5AH hepatoma cells were compared over a range of acceptor PL concentrations (Fig. 3). As for the rHDL, all of the unilamellar vesicles were shown by gel filtration chromatography to not significantly change size or precipitate out of solution during a 6-h FC efflux incubation (data not shown). The FC efflux to a 90:1 (mol:mol) PL:apoAI discoidal rHDL is also shown. In all cases, the rHDL was substantially more efficient than the same amount of PL present as an SUV. This was consistent with previous observations in this laboratory and others(13, 14, 16) . Furthermore, the SUV were more efficient than the various LUVs, which were not significantly different from each other. The FC efflux results were similar for both cell types studied (Fig. 3, A and B). The equilibrium distribution of FC at each concentration was similar for all unilamellar vesicles; about 75-80% of the FC was in the acceptor pool at equilibrium at high concentrations. The equilibrium distributions exhibited a different concentration dependence in the case of the unilamellar vesicles compared with the discoidal rHDL; the vesicles exhibited a higher FC capacity at low acceptor concentrations (data not shown). This result is likely due to differences in the retrograde FC flux from the acceptor to the cell. The tvalues for FC desorption from rHDL, SUV, and LUV particles are about 15, 45, and 235 min, respectively(5, 30, 50) . As an additional comparison, isolated human LDL was used as an acceptor of L-cell FC (Fig. 3A). LDL is of comparable diameter to the SUV (20-35 nm), but it contains a nonexchangeable apolipoprotein (apoB-100). Despite this, the LDL and SUV exhibited similar abilities to remove labeled FC (the FC mass distributions were different because LDL contains FC initially). The order of FC efflux efficiency as reflected in the k values was rHDL > SUV = LDL > LUV, indicating a general correlation between FC efflux efficiency and particle size, with smaller particles being more efficient than larger ones when compared on the basis of PL concentration. However, this relationship does not hold for particles with diameters larger than 70 nm and, as shown in the previous section, also does not hold for rHDL particles below 12 nm in diameter.


Figure 3: The concentration dependence of the rate of cholesterol efflux from mouse L-cell fibroblasts and rat FU5AH hepatoma cells to a discoidal rHDL particle and unilamellar vesicles of varying size. The incubation conditions were the same as those for Fig. 1except that the acceptor concentration is varied as shown. The particles shown are as follows: 90:1 (PL:apoAI, mol ratio) POPC/apoAI discoidal rHDL particle (), SUV (), LDL isolated from human plasma at d = 1.019-1.066 g/ml (filledhexagon), LUV (), LUV (), and LUV (). PanelA shows the results from a FC efflux incubation with mouse L-cells, and panelB shows the same experiment performed with rat Fu5AH hepatoma cells. Except for the rHDL data with the L-cells (n = 9 wells), each point represents three cell wells, and the errorbars represent one S.D. The values shown are corrected for the FC efflux to control medium.



The differences in FC efflux efficiency noted at low acceptor particle concentrations persisted at high concentrations. The average V values for the L-cell FC efflux experiment (in terms of percentage of cellular FC released per h) for the discoidal rHDL, SUV, LDL, LUV, LUV, and LUV were 12.0 ± 2.2, 3.0 ± 0.1, 3.0 ± 0.1, 1.0 ± 0.1, 1.0 ± 0.1, and 1.0 ± 1.0, respectively. The V values from the rat Fu5AH hepatoma cell FC efflux experiment for the discoidal rHDL, SUV, LUV, LUV, and LUV were 20.0 ± 1.3, 10.4 ± 0.2, 1.2 ± 0.1, 1.5 ± 0.1, 1.0 ± 0.1, and 1.2 ± 0.1%/h, respectively. Thus, the V values for the rHDL, SUV, and LUV acceptor particles did not converge to a common value.

The difference in V between an SUV and a rHDL made with the same PL has been reported previously(13, 14) , and it was hypothesized that the rHDL apolipoproteins were capable of interacting with the cell plasma membrane (PM) to increase the intrinsic rate of FC desorption from the lipid surface into the aqueous medium(13) . Since discoidal rHDL and SUV have significantly different particle diameters (and, therefore, different diffusion coefficients (see Tables I and II)), a direct test of this hypothesis requires the comparison of acceptor particles of similar size and composition that either do or do not contain apolipoproteins(31) . To this end, SUV were prepared with five molecules of apoAI adsorbed to the vesicle surface without significantly changing the size of the original SUV (see ``Methods''). The abilities of the SUV and SUV-apoAI to remove cellular FC from mouse L-cells and Fu5AH cells were compared on the basis of acceptor PL content. Gel filtration chromatography demonstrated that the FC counts appearing in the SUV-apoAI particles over a 6-h FC incubation had a similar elution volume to that of the unmodified SUV, indicating that they were stable during the time course of the incubation and were of similar size to the SUV. Fig. 4shows the FC efflux rate constants for efflux from mouse L-cells found with a low (50 µg of acceptor particle POPC/ml) and a high (500 µg of POPC/ml) concentration of SUV, SUV-apoAI, and a 90:1 (POPC:apoAI (mol:mol)) discoidal rHDL particle. The discoidal rHDL was substantially more efficient at both concentrations than the same amount of PL present in an SUV. It is clear that the presence of five molecules of apoAI on the surface of particles of otherwise similar size and PL content did not result in an increase in the particle FC efflux efficiency. Similar results were obtained using Fu5AH hepatoma cells (data not shown).


Figure 4: The concentration dependence of the rate constants for cholesterol efflux from mouse L-cell fibroblasts to a discoidal rHDL particle and SUV alone or with adsorbed apoAI. The incubation conditions were the same as those for Fig. 1except that the acceptor concentration was varied as indicated below. The SUV and rHDL were the same as shown in Fig. 2. The SUV-apoAI contained five molecules of apoAI per vesicle. The openbars represent the FC efflux at 50 µg POPC/ml, and the filledbars represent the FC efflux at 500 µg of POPC/ml. Each point represents three cell wells, and the errorbars represent one S.D. The values shown are corrected for the FC efflux to control medium.



FC Efflux to Discoidal rHDL and Unilamellar Vesicles Compared on a Per Particle Basis

Since the PL:apoAI ratios differed among the rHDL (Table 1), the number of particles per ml of extracellular medium at a given concentration of PL also varied. At a given PL concentration, for every one 12.8-nm rHDL particle there were 5.0 times as many 7.3-nm particles, 2.2 times as many 9.3-nm particles, and 1.4 times as many 10.0-nm particles. When the data from Fig. 2are replotted correcting for the particle number, it is clear that the FC efflux efficiency increased with particle size (Fig. 5A). These results are consistent with those of Agnani and Marcel(15) . A similar analysis is shown in Fig. 5B for the unilamellar vesicles based on the number of PL molecules present in each vesicle (Table 2). The efflux efficiency slightly increased with vesicle size. Fig. 6shows the rate constant for FC efflux for all of the particles in this study at a constant number of acceptor particles (4.4 10 particles/ml). The rate constant for FC efflux exhibited a linear dependence on the radius of both the rHDL (Fig. 6, inset) and the LUV (Fig. 6). The slope of the regression line through the rHDL data was significantly steeper than that for the LUV, indicating that a change in size of the rHDL improved the ability of the particle to remove cell FC to a greater extent than the same change in size of the LUV. The FC efflux to the SUV did not fit either of these relationships. Relative to the rHDL discs, the SUV were less efficient than predicted by their size, whereas, relative to the LUV, the SUV were more efficient than predicted by their size. Apparently, the rate of FC efflux is proportional to acceptor particle size when particles of similar structure are compared but not when structurally dissimilar particles are compared.


Figure 5: The dependence on acceptor particle number of the rate of FC efflux from mouse L-cell fibroblasts to discoidal rHDL particles and unilamellar vesicles of increasing hydrodynamic diameter. The incubation conditions were the same as those for Fig. 1. The data from the L-cell experiments in Fig. 2and Fig. 3have been expressed in terms of the number of particles present at each PL concentration (see Table 1and Table 2). PanelA shows efflux to the discoidal rHDL of various size compared with the SUV. The acceptors shown are POPC:apoAI 37:1 (), 75:1 (), 107:1 (▾), 156:1 (), and SUV (). PanelB compares efflux to the LUV; the particles shown are as follows: LUV (), LUV (), and LUV (). The data in panelA are from six wells, whereas the data in panelB are from three cell wells. The errorbars represent one S.D. The values shown are corrected for the FC efflux to control medium.




Figure 6: The relationship of rHDL and unilamellar vesicle size and FC efflux efficiency at a fixed number of acceptor particles. The FC efflux rate was estimated for each type of acceptor particle used in this study at a particle number of 4.4 10 per ml of medium by analyzing the data in Fig. 2and 3 by the method proposed by Hofstee (29) and extrapolating to the appropriate PL concentration for each of the different particles at this particle number. This particle number was selected because it was the maximum number of the LUV preparation studied (i.e. at 10,000 µg of POPC/ml there are 4.4 10 LUV particles/ml present). The symbols are the same as in Fig. 5.




DISCUSSION

There is general agreement that the PM pool of FC leaves the cell via the aqueous diffusion mechanism(5, 32) . FC transfer by this mechanism occurs by a two step process that involves the desorption of a FC molecule from the PM and subsequent diffusion through the aqueous phase until it incorporates into a PL-containing acceptor particle (33) . At dilute acceptor concentrations, the rate of cellular FC transfer to an extracellular particle is first order with respect to the concentration of acceptor particle [A] and depends on the rate constants for (a) desorption from the PM (k) and (b) collision with and incorporation into the acceptor particle (k). Under these conditions, the rate of FC efflux (v) = k[A] where k is a function of the rate constants k and k (see the Appendix in (14) ). In contrast, at high acceptor concentrations, the FC efflux rate depends on the rate constant for FC desorption from the PM (k) as well as the concentration of PM FC that is available for desorption [DC] so that v = V = k[DC](14) . If it is assumed that [DC] is the same for any two given acceptor particles, it follows that both particles should exhibit a common V at sufficiently high acceptor particle concentrations. The possible explanations for the observed differences in FC efflux for variously sized FC acceptor particles at low and high acceptor concentrations are considered below.

FC Transfer under Conditions of Low Acceptor Particle Concentration

At low acceptor particle concentrations (<100 µg of POPC/ml), the rate of FC efflux depends on k, which includes the term describing the propensity of FC to collide with and incorporate into the acceptor particle (k). Under these conditions, this term can be affected by (a) different diffusion coefficients of the various acceptor particles and/or (b) changes in the percentage of FC-acceptor collisions that result in successful incorporation into the acceptor particle(14) . The collision frequency between dissolved FC molecules and extracellular acceptor particles per unit volume is directly proportional to the product (number of acceptor particles present particle radius)(31) . Thus, a given number of LUV make better ``targets'' for diffusing FC molecules than the same number of small rHDL, despite slower LUV diffusion rates (see Tables I and II for the respective diffusion coefficients). However, at a given PL concentration, the relative number of particles can differ by up to 2 orders of magnitude, with many more rHDL particles present than LUV particles (see Table 1and Table 2). Relative to LUV, the small target size of rHDL is offset by their greater numbers at a given PL concentration, explaining the ranking of acceptor efficiencies depicted in Fig. 3. As predicted by DeLamatre et al.(31) , when compared at a fixed particle number concentration, rHDLs of varying sizes exhibit a linear relationship between the degree of FC efflux and particle radius, with larger rHDL being more efficient than smaller ones (Fig. 6). A linear relationship between these parameters also exists for the various LUVs. Interestingly, the slope of the rHDL line is significantly steeper than that of the LUV line, indicating that small increases in rHDL size have larger effects on the number of rHDL-FC effective collisions than similar increases in LUV size. Furthermore, the FC efflux to the SUV does not appear to fit on either the rHDL or the LUV line. These observations may be due to differences in the ability of colliding FC molecules to incorporate into the particle (effective collision frequency). We have proposed that FC acceptor particles containing more loosely packed PL molecules exhibit a higher effective collision rate than those with relatively ordered, tightly packed PL(14) . The PL bilayer of an LUV is essentially a planar surface in which PL acyl chains are motionally restricted (34) compared with the curved surface of the SUV(33, 35) . An increased FC-SUV effective collision frequency relative to the LUV may explain the higher FC efflux efficiency of the SUV than predicted from its size by the LUV data (Fig. 6). In the case of the rHDL particles, the presence of the apolipoprotein may facilitate the incorporation of incoming FC molecules by creating PL packing defects in the particle surface(30) , contributing to the increased efficiency of the rHDL over the vesicles. Although the presence of apoAI on the surface of an SUV does not increase its ability to accept cell FC (Fig. 4), it may be that apoAI facilitates FC incorporation into discoidal particles more efficiently than into spherical particles. More FC efflux studies using spherical and discoidal acceptor particles are required to address this issue.

FC Transfer under Conditions of High Acceptor Particle Concentrations

At high concentrations of acceptor particle (>500 µg of POPC/ml), the FC efflux differences already noted between the various particle types persist, resulting in a range of V values. This result is unexpected because the V for different particles should converge to a single value that reflects the rate-limiting step of FC desorption from the cell PM. The factors responsible for the V differences can be examined by assuming two limiting situations: a) the experimentally derived V values represent the true maximal rate of FC efflux to the various acceptor particles or, b) these V values do not represent the true maximal rate of FC efflux to the acceptor particles. Each of these situations is addressed in turn below.

VValues Represent True Maximal FC Efflux Rate

If the observed V values are reliable expressions of the true maximal FC efflux velocity (v) to the various particles then, as discussed above, v = V = k[DC](14) . It follows that particles exhibiting different V values must differentially affect either the intrinsic tendency of FC to desorb from the PM (k) or the concentration of cell PM FC that is available for desorption [DC] (or both). k can be affected by a modification of the PM either via the exchange of acceptor-derived PL into the PM or through an interaction of acceptor particle-derived apolipoproteins with the cell PM(13, 14) . Since all of the particles in this study contain the same type of PL and it has been demonstrated that the exchange rate of PL is significantly slower than that for FC(32) , it is unlikely that exchange of acceptor PL can account for the V differences. Similarly, the demonstration that SUV of otherwise similar structure and differing only in the presence or absence of apoAI, can remove cellular FC at similar rates (Fig. 4) suggests that the observed V differences between discoidal rHDL and SUV (13, 14) are not due to an apolipoprotein-PM interaction. Although the possibility exists that SUV-associated apoAI is not in the correct conformation to interact with the cell surface, the observation in Fig. 3that unilamellar vesicles that lack apolipoproteins also exhibit different V values (e.g. LUV versus SUV) argues that the V differences are not due to the presence of an apolipoprotein.

Apart from acceptor particle-induced changes in k, changes in the concentration of cell FC available for efflux ([DC]) could also affect the V if the acceptor particles have limited access to regions of the plasma membrane that may be particularly important for FC efflux. It can be proposed that small particles have access to PM regions that may be obscured by extracellular matrix or to highly curved surfaces located within cell surface folds or invaginations(36) , whereas large particles may be restricted from these sites. The observation of monoexponential FC efflux kinetics appears to preclude the existence of such regions, although the time frame of the FC efflux studies may not be sufficient for their detection. PM FC heterogeneity has been proposed in a number of cell types(37) , and kinetic studies have suggested that certain regions of the membrane may release FC faster than others(38) , although the structural characteristics of these regions remain unclear. This issue will require further study.

VValues Do Not Represent True Maximal FC Efflux Rate

Alternatively, it can be proposed that a complication inherent in the cell FC efflux assay prevents the measurement of V values that represent true maximal rates of FC efflux to the various acceptor particles. Since the FC efflux differences that are noted at low acceptor concentrations persist at high concentrations, a simple explanation is that the same rationale is responsible for the observations at both concentrations (14) . It was proposed above that different effective collision frequencies between dissolved FC molecules and the various acceptor particles can account for the observed differences in FC efflux under conditions of low acceptor concentrations. Ordinarily, this explanation would not be expected to account for FC efflux differences at high acceptor particle concentrations because a surplus of acceptor particles should compensate for a low effective collision frequency (5) . Since FC efflux differences are observed at high acceptor particle concentrations, it follows that the concentration of acceptor particles at the surface of the cell PM (where most of the FC transfer is expected to occur) is limited to a concentration that is a) distinct from the bulk medium concentration and b) below that required to make FC desorption from the PM rate-limiting(14) . In this case, the rate of FC efflux is described by a similar equation as that for low acceptor particle concentrations. Thus, the FC efflux rate is governed by the k term as well as the concentration of acceptor at the cell surface [A]. The k term can be affected by the effective collision rate of the various acceptor particles as in the case of low acceptor concentration. Furthermore, since small rHDL exhibit more efficient FC efflux than LUV, it is logical to propose that the cell surface exhibits a characteristic that limits the [A] of large particles at the cell surface and allows a higher [A] for smaller particles. Thus the V differences among the various particles may arise from different FC-acceptor effective collision frequencies as well as different concentrations of the various sized particles at the cell surface. An observation that supports this idea is that, in L-cells, the rHDL exhibits a 4-fold higher V value than the SUV. The same comparison in rat Fu5AH hepatoma cells yields only a 2-fold difference. These discrepancies may be explained by differential partitioning of the particles into the proximal and distal regions of the extracellular medium, presumably in response to the variations in cell surface complexity between the two cell types.

Restricted Access of Acceptor Particles to the Cell Surface

It is not yet clear whether the surface structure of living cells restricts large acceptor vesicles from specific regions of the PM that are important for FC efflux (i.e. effectively changing [DC]) or if it restricts them from closely approaching the cell surface altogether (change in [A]). The most obvious cellular characteristics that may restrict acceptor particle access to the cell surface include the unstirred water layer (USWL) at the cell surface(39) , the convoluted nature of the PM(36) , and the extracellular matrix. The USWL is a layer of fluid over a planar cell surface, which is not subject to convective currents found in the bulk medium, and passage though it occurs solely by passive diffusion(40) ; its thickness for most cells present in a planar cell monolayer is between 300 and 800 µm(39) . The USWL is known to dramatically alter the cell surface concentration of solutes that are destined for passage through the cell PM(39) . However, in the case of an extracellular FC acceptor particle that is not metabolized by the cell, the concentration of acceptor particles at the cell surface should ultimately equilibrate with the bulk medium particle concentration. The tfor this equilibration is proportional to the square of the USWL thickness and inversely proportional to the diffusion coefficient of the particular solute (39) (see Table 1and Table 2). Assuming an USWL thickness of 300 µm, it can be calculated that the t for equilibration of the the largest particles (LUVs) across the USWL is about 3 h, whereas the tfor the smallest rHDL is about 6 min. Therefore, during the 6-h FC efflux experiments the bulk medium concentration of LUV does not fully equilibrate across the USWL because only two half-times have elapsed. This is a possible explanation for the different V values obtained for the different size particles. Another cell surface complication is the presence of an extracellular matrix composed of proteoglycans(41) . This matrix is 50-200 nm thick in vascular endothelial cells (42) and may be a selective barrier for the passage of various materials(43) . It is possible that this matrix can exclude extracellular particles on the basis of size, operating like a filter over the cell surface. For example, it has been demonstrated that arterial endothelial cells can take up LDL faster when sialic acid residues are removed from the cell glycocalyx(44) , and albumin uptake by cells is more efficient at cell surface sites that are less glycosylated than at heavily glycosylated sites(45) . In addition to the cell glycocalyx, the convoluted nature of the cell surface may also contribute to the exclusion of large acceptor particles from the cell surface. Bovine aortic endothelial cells have been shown to exhibit ridges and depressions that can vary from the plane of the cell surface by 50-100 nm(36) . Determination of the cellular characteristic(s) that are responsible for this effect requires further studies in which the complexity of the cell surface is manipulated.

In summary, the differences in FC efflux efficiency noted for the variously sized acceptor particles in this study may be explained by two factors. The first is the differential effective FC-acceptor collision frequencies induced by the PL packing characteristics of the various particles. The second factor is the different concentrations of the variously sized particles at the cell surface (or at specific cell surface regions) due to the complexity of the cell surface. It appears that particles in the size range of human plasma HDL can traverse the cell surface barrier to a similar extent and that, on a per particle basis, large rHDL particles are more efficient than smaller ones because they provide a larger target for diffusing FC molecules. Interestingly, large (11-25-nm) discoidal complexes have been documented in the peripheral lymph of dogs in response to hypercholesterolemia(46) . The presence of such particles in this locus, which is important for FC removal from peripheral cells, may be significant for the process of reverse cholesterol transport.


FOOTNOTES

*
This work was supported by Program Project Grant HL22633, National Institutes of Health Training Grant HL07443, and a predoctoral fellowship from the American Heart Association, Southeastern Pennsylvania Affiliate (to W. S. D.). 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.

§
Current address: Dept. of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, 506 S. Mathews Ave., Urbana, IL 61801.

Dr. Rodrigueza is an International Research Fellow of the American Heart Association.

**
To whom correspondence should be addressed: Dept. of Biochemistry, The Medical College of Pennsylvania and Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129.

The abbreviations used are: HDL, high density lipoprotein; apoAI, apolipoprotein AI; FC, free cholesterol; rHDL, reconstituted HDL; LUV, large unilamellar vesicle; PL, phospholipid; POPC, 1-palmitoyl, 2-oleoyl phosphatidylcholine; S, surface potential in millivolts (mV); SUV, small unilamellar vesicle; PM, plasma membrane; USWL, unstirred water layer.


ACKNOWLEDGEMENTS

We thank Faye Baldwin, Sheila Benowitz, and Margaret Nickel for expert technical assistance.


REFERENCES
  1. Rader, D. J., Castro, G., Zech, L. A., Fruchart, J., and Brewer, H. B.(1991) J. Lipid Res. 32, 1849-1859 [Abstract]
  2. Jonas, A., Kézdy, K. E., and Wald, J. H.(1989)J. Biol. Chem. 264, 4818-4824 [Abstract/Free Full Text]
  3. Huang, Y., von Eckardstein, and Assmann, G.(1993)Arterioscler. Thromb. 13, 445-448 [Abstract]
  4. Huang, Y., von Eckardstein, A., Wu, S., Maeda, N., and Assmann, G.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1834-1838 [Abstract]
  5. Phillips, M. C., Johnson, W. J., and Rothblat, G. H.(1987)Biochim. Biophys. Acta 906, 223-276 [Medline] [Order article via Infotrieve]
  6. Cheung, M. C., and Albers, J. J.(1984)J. Biol. Chem. 259, 12201-12209 [Abstract/Free Full Text]
  7. Marcel, Y. L., Weech, P. K., Nguyen, T., Milne, R. W., and McConathy, W. J.(1984) Eur. J. Biochem. 143, 467-476 [Abstract]
  8. Havel, R. J., Eder, H. A., and Bragdon, J. H.(1955)J. Clin. Invest. 34, 476-482
  9. Castro, G., and Fielding, C. J.(1988)Biochemistry 27, 25-29 [Medline] [Order article via Infotrieve]
  10. Francone, O. L., and Fielding, C. J.(1990)Eur. Heart J.11,Suppl. E, 218-224 [Medline] [Order article via Infotrieve]
  11. Skipski, K. P. (1972) in Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism (Nelson, G. J., ed.) pp. 471-583, WileyInterscience, New York
  12. Reichl, D. (1990) European Heart J.11, Supplement E, 230-236 [Medline] [Order article via Infotrieve]
  13. Davidson, W. S., Lund-Katz, S., Johnson, W. J., Anantharamaiah, G. M., Palgunachari, M. N., Segrest, J. P., Rothblat, G. H., and Phillips, M. C.(1994) J. Biol. Chem. 269, 22975-22982 [Abstract/Free Full Text]
  14. Davidson, W. S., Gillotte, K. L., Lund-Katz, S., Johnson, W. J., Rothblat, G. H., and Phillips, M. C.(1995)J. Biol. Chem. 270, 5882-5890 [Abstract/Free Full Text]
  15. Agnani, G., and Marcel, Y. L.(1993)Biochemistry 32, 2643-2649 [Medline] [Order article via Infotrieve]
  16. Jonas, A., Bottum, K., Theret, N., Duchateau, P., and Castro, G.(1994)J. Lipid Res. 35, 860-870 [Abstract]
  17. Johnson, W. J., Mahlberg, F. H., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1988)J. Biol. Chem. 263, 14099-14106 [Abstract/Free Full Text]
  18. Lund-Katz, S., and Phillips, M. C.(1986)Biochemistry 25, 1562-1568 [Medline] [Order article via Infotrieve]
  19. Scanu, A. M., Edelstein, C.(1971)Anal. Biochem. 44, 576-588 [Medline] [Order article via Infotrieve]
  20. Weisweiler, P., Friedl, C., and Ungar, M.(1987)Clin. Chim. Acta 169, 249-254 [CrossRef][Medline] [Order article via Infotrieve]
  21. Sparks, D. L., Phillips, M. C., and Lund-Katz, S.(1992)J. Biol. Chem. 267, 25830-25838 [Abstract/Free Full Text]
  22. Markwell, M. A., Haas, S. M., Bieber, L. L., and Tolbert, N. E.(1978)Anal. Biochem. 87, 206-210 [Medline] [Order article via Infotrieve]
  23. Sokoloff, L., and Rothblat, G. H.(1974)Proc. Soc. Exp. Biol. Med. 146, 1166-1172
  24. Swaney, J. B. (1986)Methods Enzymol. 128, 613-626 [Medline] [Order article via Infotrieve]
  25. Sparks, D. L., and Phillips, M. C.(1992)J. Lipid Res. 33, 123-130 [Abstract]
  26. Barenholz, Y., Gibbes, D., Litman, B. J., Goll, J., Thompson, T. E., and Carlson, F. D. (1977)Biochemistry 16, 2806-2810 [Medline] [Order article via Infotrieve]
  27. Yokoyama, S., Fukushima, D., Kupferberg, J. P., Kezdy, F. J., and Kaiser, E. T.(1980) J. Biol. Chem. 255, 7333-7339 [Free Full Text]
  28. Hope, M. J., Bally, M. B., Webb, G., and Cullis, P. R.(1985)Biochim. Biophys. Acta 812, 55-65
  29. Christensen, H. N., and Palmer, G. A. (1974) Enzyme Kinetics, pp. 50-60, W. B. Saunders Co., Philadelphia
  30. Letizia, J. Y., and Phillips, M. C.(1991)Biochemistry 30, 866-873 [Medline] [Order article via Infotrieve]
  31. DeLamatre, J., Wolfbauer, G., Phillips, M. C., and Rothblat, G. H.(1986) Biochim. Biophys. Acta 875, 419-428 [Medline] [Order article via Infotrieve]
  32. Johnson, W. J., Mahlberg, F. H., Rothblat, G. H., and Phillips, M. C.(1991) Biochim. Biophys. Acta 1085, 273-298 [Medline] [Order article via Infotrieve]
  33. Thomas, P. D., and Poznansky, M. J.(1988)Biochem. J. 254, 155-160 [Medline] [Order article via Infotrieve]
  34. Huang, C. H. (1969)Biochemistry 8, 344-352 [Medline] [Order article via Infotrieve]
  35. McLean, L. R., and Phillips, M. C.(1984)Biochim. Biophys. Acta 776, 21-26 [Medline] [Order article via Infotrieve]
  36. Barbee, K. A., Davies, P. F., and Ratneshwar, L.(1994)Circ. Res. 74, 163-171 [Abstract]
  37. Schroeder, F., Jefferson, J. R., Kier, A. B., Knittel, J., Scallen, T. J., Wood, W. G., and Hapala, I.(1991)Proc. Soc. Exp. Biol. Med. 195, 235-252
  38. Rothblat, G. H., Mahlberg, F. H., Johnson, W. J., and Phillips, M. C.(1992) J. Lipid Res. 33, 1091-1097 [Abstract]
  39. Barry, P. H., and Diamond, J. M.(1984)Physiol. Rev. 64, 763-871 [Medline] [Order article via Infotrieve]
  40. Westergaard, H., and Dietschy, J. M.(1974)J. Clin. Invest. 54, 718-732 [Medline] [Order article via Infotrieve]
  41. Clough, G.(1991) Prog. Biophys. Mol. Biol. 55, 47-69 [CrossRef][Medline] [Order article via Infotrieve]
  42. Haldenby, K. A., Chappell, D. C., Winlove, C. P., Parker, K. H., and Firth, J. A. (1994)J. Vasc. Res. 31, 2-9 [Medline] [Order article via Infotrieve]
  43. Ryan, U. S. (1986)Fed. Proc. 45, 101-108 [Medline] [Order article via Infotrieve]
  44. Gorog, P., and Born, G. V.(1982)Br. J. Exp. Pathol. 63, 447-451 [Medline] [Order article via Infotrieve]
  45. Gerrity, R. G., and Schwartz, C. J.(1977)Prog. Biochem. Pharmacol. 14, 134-137
  46. Lefevre, M., Sloop, C. H., and Roheim, P. S.(1988)J. Lipid Res. 29, 1139-1148 [Abstract]
  47. Weast, R. C. (ed) CRC Handbook of Chemistry and Physics, 63rd Ed. (1983) p. F-40, CRC Press, Boca Raton, FL
  48. Rodrigueza, W. V., Pritchard, P. H., and Hope, M. J.(1993) Biochim. Biophys. Acta 1153, 9-19 [Medline] [Order article via Infotrieve]
  49. Barenholz, Y. (1984) in Physiology of Membrane Fluidity, Vol I (Shinitzky, M., ed) pp. 134-149, CRC press, Boca Raton, FL
  50. Rodrigueza, W. V., Wheeler, J. J., Klimuk, S. K., Kitson, C. N., and Hope, M. J. (1995)Biochemistry 34, 6208-6217 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.