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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1258-1266.)
© 1997 American Heart Association, Inc.

Articles

Lysophosphatidylcholine Promotes Cholesterol Efflux From Mouse Macrophage Foam Cells

Seijiro Hara; Tsutomu Shike; Nobuo Takasu; ; Takuji Mizui

From Discovery Research Laboratories II, Shionogi & Co, Ltd, Osaka, Japan.

Correspondence to Seijiro Hara, PhD, Developmental Research Laboratories, Shionogi & Co, Ltd, 3-1-1, Futaba-Cho, Toyonaka, Osaka 561, Japan. E-mail seijiro.hara{at}shionogi.co.jp

	Abstract

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Abstract We examined the effects of lysophosphatidylcholine (lyso-PC) on promoting cholesterol efflux from macrophage foam cells. Mouse peritoneal macrophages were converted to foam cells by incubation with [³H]cholesteryl linoleate–labeled or unlabeled acetyl-LDL. When these cells were incubated with lyso-PC, [³H]cholesterol release was promoted in relation to both dose and time, and cellular cholesterol mass was decreased, while medium cholesterol mass was increased. These cholesterol efflux–promotive effects of lyso-PC were confirmed by the fact that the lyso-PC–treated cells showed less oil red O staining than the control cells. ApoE secretion, estimated by Western blotting of the medium, was also augmented by lyso-PC. Both the cholesterol and apoE released by lyso-PC treatment were floated by ultracentrifugation of the medium after its density had been adjusted to 1.210 g/mL. By electron microscopic analysis, vesicular lipoproteins were observed in ultracentrifugally concentrated conditioned medium of lyso-PC. Monensin, a protein secretion inhibitor, effectively inhibited [³H]cholesterol release induced by lyso-PC but not by apoA-I. These results suggest that lyso-PC may inhibit the development of atherosclerosis or enhance its regression by stimulating cholesterol efflux from macrophage foam cells.

Key Words: lysophosphatidylcholine • cholesterol efflux • apoE

	Introduction

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Macrophage-derived foam cells, which accumulate cholesteryl ester, have been recognized as a characteristic feature of atherosclerotic lesions in both experimental animals and human patients,^{1 2 3} and the presence of foam cells contributes to blood vessel narrowing in these lesions. HDL has been recognized as an antiatherogenic lipoprotein because of the inverse correlation observed between serum HDL levels and the incidence of coronary heart disease in many epidemiological studies.⁴ With respect to the antiatherogenic effects of HDL, intravenous injections of it have been found to cause regression of atherosclerosis,⁵ and transgenic mice overexpressing apoA-I, a major apolipoprotein of HDL, remain resistant to diet-induced atherosclerosis.⁶ These antiatherogenic actions of HDL are believed to occur via "reverse cholesterol transport," which mediates the movement of excess cholesterol from extrahepatic tissues to the liver.^{7 8} In in vitro experiments, HDL could promote cholesterol efflux, the initial step in reverse cholesterol transport, from macrophage-derived foam cells.^{9 10} This in vitro cholesterol efflux action of HDL is considered to contribute to the antiatherogenic effects of HDL in vivo.

Lyso-PC is a major lipid component of atherogenic lipoproteins such as oxidized LDL and ß-VLDL.^{11 12} In recent extensive studies, lyso-PC was demonstrated to be atherogenic because of its chemoattracting potency for monocytes,¹³ inhibition of arterial relaxation induced by endothelium-derived relaxing factor,¹⁴ stimulation of several adhesion molecule and growth factor gene expression in endothelial cells,^{15 16} stimulation of growth factor gene expression in monocytes,¹⁷ and contribution to macrophage proliferation.¹⁸

In this study we demonstrate that lyso-PC promotes cholesterol efflux from macrophage foam cells. We discuss this unique action of lyso-PC, focusing on the underlying mechanisms and the physiological role of lyso-PC in atherosclerotic lesions.

	Methods

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Materials
DMEM and Dulbecco's PBS were purchased from Nissui Pharmaceutical Co. L-Glutamine, streptomycin-penicillin solutions, and FCS were from ICN Biomedicals, Inc. FCS was inactivated at 56°C for 30 minutes before use. [Cholesteryl-1,2,6,7-³H(N)]-linoleate (3182 GBq/mmol) was purchased from New England Nuclear. Plastic tubes and pipettes were from Becton Dickinson. Plastic dishes were from Nunc. BSA and oil red O were obtained from Nacalai Tesque, Inc. Formalin was from Kanto Chemical Co, Inc. Lyso-PC (palmitoyl, stearoyl, myristoyl), lysophosphatidylserine (palmitoyl), lysophosphatidylinositol (from soybean), lysophosphatidylethanolamine (palmitoyl), PC (dipalmitoyl), human apoA-I, monensin, PMA, and dibutyryl cyclic AMP were from Sigma Chemical Co.

Lipoproteins
Human LDL (d=1.019 to 1.063 g/mL) and HDL₃ (d=1.125 to 1.210 g/mL) were prepared from the plasma of healthy human subjects and were isolated by differential ultracentrifugation.¹⁹ Acetyl LDL was prepared by repeated addition of acetic anhydride to LDL as described elsewhere.²⁰ Lipoproteins were dialyzed against buffer A (150 mmol/L NaCl, 0.24 mmol/L EDTA, pH 7.4). [³H]Cholesteryl linoleate–labeled acetyl LDL was prepared as described elsewhere.²¹ Briefly, 3.7 MBq of [³H]cholesteryl linoleate in 150 µL of DMSO was added to buffer A and sonicated for 30 seconds. After preheating the mixture (37°C, 10 minutes), 2 mg protein of acetyl LDL was added and the heating (37°C) continued for 4 hours. Final concentration of DMSO was 10%. After that, the reaction mixture was dialyzed against buffer A at 4°C, followed by centrifugation to remove aggregates, and the specific activity was adjusted to 40 000 dpm/µg protein.

96-Well [³H]Cholesterol-Release Experiments
Peritoneal macrophages were harvested from unstimulated female ICR mice weighing 25 to 30 g obtained from SLC Co, Shizuoka, Japan, in PBS as described.^{22 23} The peritoneal perfusate was collected by centrifugation at 400g at 4°C for 10 minutes. After washing once with PBS, the cells were suspended in the culture medium (DMEM containing 10% FCS by volume, 100 U/mL penicillin, and 100 µg/mL streptomycin) at a density of 2x10⁶/mL. Aliquots (0.1 mL) of this suspension were dispersed onto 96-well plastic dishes with 50 µg/mL of [³H]cholesteryl linoleate–labeled acetyl LDL, and the cells were cultured in humidified air containing 5% CO₂ at 37°C for 20 hours. The washing procedure to remove nonadherent cells after plating was skipped because no changes in the promotion of [³H]cholesterol release induced by both apoA-I and lyso-PC were observed between samples prepared with and without this washing procedure. In this experimental condition, 39 000±6000 dpm of radioactivities per well (mean±SD, three separate experiments) was incorporated into the cells. The resultant mouse macrophage foam cells were washed twice with 0.15 mL DMEM containing 0.1% BSA, and further incubation was performed in 0.15 mL DMEM containing 0.1% BSA with lyso-PC for 24 hours. Lyso-PC was added as an ethanolic solution, and the final ethanol concentration in the medium was 0.5%. We determined biological effects of lyso-PC by using palmitoyl–lyso-PC, except for the study determining the effects of fatty acid chain length. After 24 hours' incubation, aliquots (0.05 mL) of the medium were taken and their radioactivities measured by liquid scintillation counting. Centrifugation of the medium was skipped because no changes in medium radioactivities were observed between samples with and without centrifugation. Next, 5 µg/mL apoA-I was added to the existing medium and further incubation performed for 5 hours; then aliquots (0.05 mL) of the medium were again taken to measure their radioactivity. [³H]Cholesterol efflux during the final 5-hour incubation with apoA-I was calculated by subtracting the radioactivities released during the initial 24-hour period from those released over the entire experimental period. Lyophilized apoA-I was reconstituted by the buffer containing 20 mmol/L Tris-HCl, 50 mmol/L NaCl, and 1 mmol/L CaCl₂, pH 8.0. In our preliminary experiments, the cholesterol efflux ability of apoA-I reached nearly maximum at the dose of 5 µg/mL, which was comparable to that of 50 µg/mL of HDL₃. In the experiments using monensin, it was added to the medium as ethanolic solution, and the final ethanol concentration in the medium was 0.005%. The concentration of monensin was chosen by referring to the former study,²⁴ in which 0.1 µmol/L monensin was clearly shown to inhibit apoE secretion in mouse macrophages.

6-Well Nontracer Experiments
Mouse peritoneal cells, collected as described above, were suspended in the culture medium at a density of 3x10⁶/mL. Aliquots (2 mL) of this suspension were dispersed onto 6-well plastic dishes and the cells cultured for 2 hours. Cells were then washed twice with culture medium to remove nonadherent cells, and further incubation was performed in 2 mL DMEM containing 10% FCS with 50 µg/mL acetyl LDL for 20 hours. The resultant mouse macrophage foam cells were washed twice with 2 mL DMEM containing 0.1% BSA, and further incubation was performed in 3 mL DMEM containing 0.1% BSA with lyso-PC for 24 to 48 hours. Lyso-PC was added as an ethanolic solution as described above. We determined biological effects of lyso-PC by using palmitoyl–lyso-PC. After incubation, the cells and the medium were analyzed as follows.

Cell Analyses
After incubation, the cells were washed twice with PBS and their lipids extracted with hexane-isopropanol (3:2, vol/vol) to determine cholesterol mass. After that, the cells were dissolved with 0.2N NaOH and protein contents were measured. For oil red O staining, the cultures were kept on chamber slides (Nunc Inc) and the cells fixed with formalin.

Medium Analyses
After incubation, the culture medium was centrifuged (3000 rpm, 15 minutes, 4°C) followed by passage through 0.45-µm filter (MILLEX-HV, Millipore Products Division). Ultracentrifugation of the medium was performed after its density had been adjusted to 1.210 g/mL with KBr. To determine cholesterol mass, lipids in the medium and the ultracentrifuged samples were extracted with chloroform-methanol (2:1, vol/vol).²⁵ LDH activity in the medium was measured. ApoE contents in the medium and the ultracentrifuged samples were estimated using Western blotting. The ultracentrifuged samples were negatively stained and analyzed by electron microscopy.

Measurement of Cholesterol Mass
Lipid extracts from the cells and the medium were evaporated by N₂ gas and dissolved with isopropanol, and then the cholesterol mass was quantified by enzyme fluorometry.²⁶ The amount of esterified cholesterol was calculated by subtracting the free cholesterol from total cholesterol.

Measurement of Protein Contents
The protein contents of the lipoproteins and cells were determined as described²⁷ using BSA as a standard.

Oil Red O Staining
After washing the cells with PBS, they were air dried, fixed with 6% formalin for 5 minutes, stained with a saturated concentration of oil red O for 60 minutes, and counterstained with Meyer's hematoxylin for 5 minutes. All procedures were performed at room temperature.

Measurement of LDH Activity
LDH activities in the medium were determined using the standard kit purchased from Wako Pure Chemical Industries, using a Cobas-Fara centrifugal analyzer (Roche Diagnostics). The LDH activity observed when the cells were lysed by 0.1% Triton X 100 was taken as 100%. In our preliminary experiments, LDH activity of 0.5% ethanol conditioned medium was exactly the same as that of conditioned medium without ethanol.

ApoE Western Blotting
To prepare ultracentrifuged samples for Western blotting, the density of the medium was adjusted to 1.210 g/mL and the samples (3 mL per tube) were ultracentrifuged (100 000 rpm, 3.5 hours, 4°C) using a TLA100.3 rotor (Beckman Instruments, Inc). Next, 0.6 mL of the upper fraction was taken, and samples from two tubes were combined and dialyzed against buffer A. Fifteen microliters of the medium and the ultracentrifuged samples were electrophoresed on 4% to 20% SDS-PAGE under reducing conditions. The proteins were electrophoretically transferred to nitrocellulose sheets. The nitrocellulose strips were blocked with PBS containing 3% BSA and allowed to react with goat anti-human apoE (1/500, Chemicon). The bands were visualized by Vectastain ABC-PO kit (Vector Laboratories, Inc) using 4-chloro-1-naphthol (Amresco) as a substrate. Band intensities were quantified by densitometry with a flying spot scanner (Shimadzu Corporation).

Electron Microscopy
For electron microscopic analysis, we used two ultracentrifuged samples, one being the same as that used for Western blotting, and the other prepared by ultracentrifugation of the medium twice. Medium density was adjusted to 1.210 g/mL, the samples (38 mL per tube) were ultracentrifuged (49 000 rpm, 20 hours, 4°C) using a 50.2Ti rotor (Beckman Instruments), and the upper fraction (8 mL) was taken. The density of this sample was adjusted to 1.210 g/mL again, and the samples (3 mL per tube x2) were ultracentrifuged (100 000 rpm, 3.5 hours, 4°C). After the second ultracentrifugation, 0.8 mL of the upper fraction was taken, and samples from two tubes were combined and dialyzed against buffer A. Ten-microliter portions of these ultracentrifuged samples were negatively stained with 10 µL of saturated uranyl acetate and the mixtures applied to a carbon-stabilized copper grid and left there for awhile. Excess staining solution was removed with a filter paper, and the grids were viewed with a JEM-1200EX electron microscope (Jeol).

Statistical Analysis
Statistical significance among the experimental groups was evaluated by Tukey's method after one-way analysis of variance.

	Results

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[³H]Cholesterol Release Induced by Lyso-PC
In our [³H]cholesterol-release experiments using 96-well plates, the test materials were first incubated with mouse macrophage foam cells for 24 hours, and a second 5-hour incubation was performed after addition of apoA-I. This procedure allowed us to evaluate two independent characteristics, one being the stimulating activity of cholesterol release (first 24-hour incubation) and the other being the modulating activity against apoA-I–induced cholesterol release (second 5-hour incubation). In this system, lyso-PC promoted significant [³H]cholesterol release dose dependently; on the other hand, PC promoted weak but significant [³H]cholesterol release at the dose of 50 µmol/L; however, its dose dependency was not clear (Table). Lyso-PC showed mild but significant enhancing effects on apoA-I–induced cholesterol release from mouse macrophage foam cells (Table). This enhancing effect of lyso-PC may reflect the operation of lyso-PC–mediated cholesterol efflux in addition to apoA-I–mediated cholesterol efflux. In the time course experiments, [³H]cholesterol release induced by apoA-I was saturated at 24 hours of incubation, but that induced by lyso-PC continued linearly to at least 48 hours of incubation (Fig 1).

View this table: [in this window] [in a new window]   Table 1. Effects of Various Doses of Lyso-PC and PC on the Promotion of [3H]Cholesterol Efflux and on ApoA-I–Induced [3H]Cholesterol Efflux in Mouse Macrophage Foam Cells

View larger version (20K): [in this window] [in a new window]   Figure 1. Time course of [3H]cholesterol efflux induced by apoA-I (left), lysophosphatidylcholine (lyso-PC; middle), and phosphatidylcholine (PC; right) in mouse macrophage foam cells. Mouse macrophage foam cells were prepared as described in "Methods" (96-well [3H]cholesterol-release experiments) with [3H]cholesteryl linoleate–labeled acetyl-LDL and incubated with the indicated doses of apoA-I (left: 1 µg/mL, ; 5 µg/mL, ), lyso-PC (middle: 20 µmol/L, ; 40 µmol/L, ), and PC (right: 20 µmol/L, ; 40 µmol/L, ). Medium radioactivity was counted after 24- and 48-hour incubations. At each time point, medium radioactivity observed in vehicle incubation was subtracted. Data represent mean±SD of quadruplicate determinations representative of two similar experiments.

Effects of Lyso-PC on Cellular Cholesterol Contents
To confirm the activity of lyso-PC in promoting cholesterol release from mouse macrophage foam cells, the cellular cholesterol mass was determined. Treatment with apoA-I or lyso-PC but not PC for 48 hours resulted in a decrease of cellular total cholesterol mass (Fig 2A). The cells incubated with lyso-PC for 48 hours had decreased contents of esterified cholesterol but similar contents of free cholesterol compared with control cells (Fig 2B). These characteristics of lyso-PC action resembled those of apoA-I. The promotion of cholesterol efflux by lyso-PC was further confirmed by the fact that lyso-PC–treated cells (40 µmol/L, 48 hours) showed less oil red O staining than control cells (Fig 3).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effects of apoA-I, lyso-PC, and PC on the cholesterol mass in mouse macrophage foam cells. Mouse macrophage foam cells were prepared as described in "Methods" (6-well nontracer experiments) with acetyl-LDL and incubated with the indicated doses of apoA-I, lyso-PC, and PC for 48 hours. After washing the cell monolayers, the cell-associated lipids were extracted with hexane-isopropanol (3:2 vol/vol), and the cholesterol mass was determined by enzyme fluorometry. A, Changes of total cholesterol mass before and after incubation with apoA-I (left: 1 µg/mL, ; 5 µg/mL, ), lyso-PC (middle: 20 µmol/L, ; 40 µmol/L, ), and PC (right: 20 µmol/L, ; 40 µmol/L, ). B, Esterified (left) and free (right) cholesterol mass observed after 48-hour incubation under the indicated conditions. Data represent mean±SD of triplicate determinations representative of two similar experiments.

View larger version (154K): [in this window] [in a new window]   Figure 3. Oil red O staining of mouse macrophage foam cells treated with lyso-PC. Mouse macrophage foam cells were prepared as described in "Methods" with acetyl-LDL using chamber slides and incubated with 40 µmol/L lyso-PC (right) or vehicle (left) for 48 hours. After washing of the cell monolayers, oil red O staining was performed as described in "Methods" and photographed (x400).

Comparison of Cholesterol Release and LDH Release Induced by Lyso-PC
Total cholesterol mass and LDH activity in the 48-hour conditioned medium of lyso-PC were determined. Lyso-PC (10 to 40 µmol/L) increased medium total cholesterol mass dose dependently, but its effects on medium LDH activity were very small (Fig 4A). ApoA-I also increased medium total cholesterol mass, with small effects on medium LDH activity (Fig 4B). On the other hand, higher doses of lyso-PC (60 to 80 µmol/L) clearly caused LDH release from mouse macrophage foam cells, but medium total cholesterol mass observed under this condition was lower than that observed with lower doses of lyso-PC (Fig 4A). To establish the 100% LDH release, the cells were lysed with 0.1% Triton X 100. In this case, cholesterol was recovered from the medium because the cells were completely lysed (Fig 4B). However, a lower dose of Triton X 100 (0.01%) could not lyse the cells completely, and only LDH activity was observed in the medium (Fig 4B). When 0.01% Triton X 100 was applied, cholesterol was recovered from the cell ghosts attached to the plate. Total cholesterol mass and protein contents of the cells or cell ghosts with the incubation of control, lyso-PC (40 µmol/L), apoA-I (5 µg/mL), or Triton X 100 (0.01%) were 23.9, 12.6, 8.6, and 26.2 µg cholesterol per well and 174, 177, 167, and 52 µg protein per well, respectively.

View larger version (39K): [in this window] [in a new window]   Figure 4. Effects of lyso-PC, apoA-I, and Triton X 100 on cholesterol and LDH release from mouse macrophage foam cells. Mouse macrophage foam cells were prepared as described in "Methods" (6-well nontracer experiments) with acetyl-LDL and incubated with (A) various doses of lyso-PC and (B) lyso-PC, apoA-I, and Triton X 100 for 48 hours. The total cholesterol mass (open bars) and LDH activity (hatched bars) in the medium were determined as described in "Methods." The control group in B shows 0.5% ethanol treatment. Data represent the mean of duplicate determinations representative of two similar experiments.

Analysis of Cholesterol Released by Lyso-PC
The medium cholesterol increased by lyso-PC treatment was the free-form type (Fig 5A) and floated by ultracentrifugation of the medium, whose density had been adjusted to 1.210 g/mL (Fig 5B). These results suggest that the cholesterol in the medium exists in the form of lipoprotein. To confirm this hypothesis, ultracentrifuged samples of lyso-PC–treated (40 µmol/L, 48 hours) conditioned medium were negatively stained and analyzed by electron microscopy, revealing the existence of vesicular lipoproteins (Fig 6).

View larger version (19K): [in this window] [in a new window]   Figure 5. Analysis of cholesterol released from mouse macrophage foam cells by lyso-PC. Mouse macrophage foam cells were prepared as described in "Methods" (6-well nontracer experiments) with acetyl-LDL and incubated with or without lyso-PC (40 µmol/L) for 48 hours. A, Medium free (open bars) and esterified (solid bars) cholesterol mass were determined. B, Medium was separated by ultracentrifugation after density adjustment to 1.210 g/mL. Total cholesterol mass in the upper half fraction (open bars) and the lower half fraction (closed bars) were determined. Data represent mean±SD of triplicate determinations (A) or duplicate determinations (B) representative of two similar experiments.

View larger version (202K): [in this window] [in a new window]   Figure 6. Electron microscopic analysis of negatively stained vesicles from ultracentrifugally concentrated culture medium of mouse macrophage foam cells treated with lyso-PC. Mouse macrophage foam cells were prepared as described in "Methods" (6-well nontracer experiments) with acetyl-LDL and incubated with lyso-PC (40 µmol/L) for 48 hours. Pooled medium was concentrated by ultracentrifugation once (left) or twice (right), as described in "Methods." After dialysis, each sample was negatively stained and examined with an electron microscope. Bar=100 nm.

ApoE Western Blotting
To estimate apoE secretion from mouse macrophage foam cells, Western blotting was performed. The apoE band was detected in 48-hour conditioned medium, and the band intensity was augmented twofold on treatment with 40 µmol/L lyso-PC (Fig 7). ApoA-I (5 µg/mL) treatment did not have a clear effect on the apoE band intensity, and apoE in the medium was floated by ultracentrifugation of medium after its density had been adjusted to 1.210 g/mL in the same manner as for the medium cholesterol experiment (data not shown). Analysis of ultracentrifuged samples from several incubation conditioned media revealed that lyso-PC treatment augments apoE secretion time dependently, accompanied by an increase of total cholesterol mass (Fig 8).

View larger version (31K): [in this window] [in a new window]   Figure 7. Western blot analysis of 48-hour conditioned medium from mouse macrophage foam cells treated with lyso-PC. Mouse macrophage foam cells were prepared as described in "Methods" (6-well nontracer experiments) with acetyl-LDL and incubated with or without lyso-PC (40 µmol/L) for 48 hours. A, Conditioned medium from vehicle (left lane) or lyso-PC (middle lane) incubated and nonconditioned medium (right lane) were analyzed by Western blotting from SDS-PAGE gels. A typical lane from triplicate lanes is shown. B, Results from densitometric analysis. Data represent mean±SD of triplicate determinations representative of three similar experiments.

View larger version (39K): [in this window] [in a new window]   Figure 8. Western blot analysis of ultracentrifugally concentrated sample from 24- or 48-hour conditioned medium of mouse macrophage foam cells treated with lyso-PC. Mouse macrophage foam cells were prepared as described in "Methods" (6-well nontracer experiments) with acetyl-LDL and incubated with the indicated doses of lyso-PC for 24 or 48 hours. Pooled medium was concentrated by ultracentrifugation as described in "Methods." A, Dialyzed samples were analyzed by Western blotting from SDS-PAGE gels. B, Results of densitometric analysis () and the total cholesterol mass determination of ultracentrifugal samples () in vehicle (left), 20 µmol/L of lyso-PC (middle), and 40 µmol/L of lyso-PC (right) incubation are shown. One of two similar experiments is shown.

Effects of Monensin on Lyso-PC–Induced Cholesterol Release
To evaluate the importance of apoE secretion in the cholesterol efflux induced by lyso-PC, we performed experiments using a protein secretion inhibitor, monensin. In the [³H]cholesterol-release experiments, coexistence of monensin did not affect apoA-I–induced cholesterol release from mouse macrophage foam cells; however, it effectively inhibited lyso-PC–induced cholesterol release (Fig 9).

View larger version (24K): [in this window] [in a new window]   Figure 9. Effects of monensin on [3H]cholesterol efflux induced by apoA-I (left) and lyso-PC (middle and right) in mouse macrophage foam cells. Mouse macrophage foam cells were prepared as described in "Methods" (96-well [3H]cholesterol-release experiments) with [3H]cholesteryl linoleate–labeled acetyl-LDL and incubated with 5 µg/mL apoA-I (left), 20 µmol/L lyso-PC (middle), or 40 µmol/L lyso-PC (right) in the presence of monensin (0 µmol/L, ; 0.02 µmol/L, ; 0.1 µmol/L, ). Medium radioactivity was counted after 24 and 48 hours of incubation. For each time point, medium radioactivity observed in vehicle or vehicle plus monensin incubation was subtracted. Data represent mean±SD of quadruplicate determinations representative of two similar experiments.

Effects of Lyso-PC–Related Compounds
To determine the effects of fatty acid chain length in lyso-PC, we studied [³H]cholesterol release using myristoyl (C 14:0), palmitoyl (C 16:0), and stearoyl (C 18:0)–lyso-PC. At the dose of 20 µmol/L, the longer chain length tended to be more effective for releasing cholesterol; however, 40 µmol/L stearoyl–lyso-PC failed to show further increase of activity (Fig 10A). Lysophosphatidylinositol and lysophosphatidylserine had some cholesterol-releasing activities, but they were both much weaker than lyso-PC (Fig 10B). We could not test lysophosphatidylethanolamine because of its insolubility in ethanol and PBS.

View larger version (35K): [in this window] [in a new window]   Figure 10. Effects of fatty acid chain length of lyso-PC and lyso-PC derivatives on the promotion of [3H]cholesterol efflux in mouse macrophage foam cells. Mouse macrophage foam cells were prepared as described in "Methods" (96-well [3H]cholesterol-release experiments) with [3H]cholesteryl linoleate–labeled acetyl-LDL and incubated with (A) the indicated doses of myristoyl–lyso-PC (left: 20 µmol/L, ; 40 µmol/L, ), palmitoyl–lyso-PC (middle: 20 µmol/L, ; 40 µmol/L, ), and stearoyl–lyso-PC (right: 20 µmol/L, ; 40 µmol/L, ) and (B) the indicated doses of lyso-PC (PC; left: 20 µmol/L, ; 40 µmol/L, ), lysophosphatidylinositol (PI; middle: 20 µmol/L, ; 40 µmol/L, ), and lysophosphatidylserine (PS; right: 20 µmol/L, ; 40 µmol/L, ). Medium radioactivity was counted after 24 and 48 hours of incubation. For each time point, medium radioactivity observed with vehicle incubation was subtracted. Vehicle in A was 0.5% ethanol and in panel B 0.5% PBS. Data represent mean±SD of quadruplicate determinations representative of two similar experiments.

Effects of PMA and Dibutyryl Cyclic AMP
To investigate the involvement of PKC and PKA in the cholesterol efflux from mouse macrophage foam cells, we performed some experiments using a PKC activator, PMA, and a PKA activator, dibutyryl cyclic AMP. In the 96-well [³H]cholesterol-release experiments, PMA treatment did not promote cholesterol efflux. Radioactivities of [³H]cholesterol released during 24 hours in control (0.2% DMSO) and PMA at 10 nmol/L, 100 nmol/L, 500 nmol/L, 1000 nmol/L, and 2000 nmol/L were 1557±68, 1524±165, 1284±67, 1267±60, 1377±114, and 1300±100 dpm/well (mean±SD of triplicate assays), respectively. Lyso-PC–induced [³H]cholesterol release was not affected by depletion of PKC with 24 hours' PMA (200 nmol/L) pretreatment (data not shown). Lyso-PC–induced [³H]cholesterol release was also not affected by the coexistence of 1 mmol/L dibutyryl cyclic AMP (data not shown).

	Discussion

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The prominent finding in the current study is the demonstration that lyso-PC promotes cholesterol efflux from macrophage foam cells as does HDL or apoA-I. Lyso-PC promoted [³H]cholesterol release (Table, Fig 1), decreased cellular cholesterol mass (Fig 2), and increased medium cholesterol mass (Fig 4). Because lyso-PC is known to have detergent action, the released cholesterol could have been due to leakage after membrane damage. Thus, we compared cholesterol release with LDH release induced by lyso-PC and Triton X 100. Lyso-PC at 10 to 40 µmol/L promoted efficient cholesterol release without marked LDH release, while 60 to 80 µmol/L lyso-PC caused marked LDH release, accompanied by limited cholesterol release (Fig 4A). On the other hand, the lower dose of Triton X 100 (0.01%) treatment resulted in 100% LDH release but no cholesterol release (Fig 4B). These results suggest that cholesterol release is clearly dissociated from LDH release, and cholesterol release induced by lyso-PC is not due to simple membrane damage. Thus, the action of lyso-PC stimulation of cholesterol efflux may be unique.

The medium cholesterol increased by lyso-PC treatment was floated by ultracentrifugation of the medium after its density had been adjusted to 1.210 g/mL (Fig 5B), and electron microscopic analysis of ultracentrifuged samples clarified the existence of vesicular lipoproteins (Fig 6). These results suggest that cholesterol in the medium released by lyso-PC exists in the form of lipoproteins. In human monocyte-derived macrophage foam cells, it was reported that spontaneous cholesterol release from cells occurred during 2 to 6 days' incubation without any stimulation, and the released cholesterol formed discoidal lipoprotein particles containing apoE.²⁸ ApoE is an important molecule in cholesterol transportation²⁹ and has been reported to be synthesized and secreted by macrophages, especially macrophage foam cells.^{30 31} We performed apoE Western blotting using conditioned medium (Fig 7) and ultracentrifuged samples (Fig 8) and revealed that lyso-PC treatment augmented apoE accumulation in the medium and medium apoE was floated by ultracentrifugation of the medium after its density had been adjusted to 1.210 g/mL. Thus, the cholesterol released by lyso-PC treatment could exist as apoE-containing lipoproteins. We are now investigating the effects of lyso-PC on the synthesis and secretion of apoE and on the gene expression of apoE.

With respect to the relationship between cholesterol efflux and apoE secretion from macrophage foam cells, cholesterol efflux has been reported to be induced by serum independently of apoE secretion for two major reasons.²⁴ First, the cells secreted apoE but not cholesterol in the absence of serum and second, in its presence, the cells secreted cholesterol but little apoE in the presence of monensin (an inhibitor of protein secretion). These results agreed with the report that cholesterol efflux induced by HDL normally occurred in apoE knockout mouse macrophage foam cells.³² On the other hand, J774 macrophages, a murine macrophage cell line lacking expression of an endogenous apoE gene, did not show stimulation of cholesterol efflux by HDL but did after transfection of human apoE cDNA.³³ In the current study, we showed that monensin inhibited lyso-PC–induced cholesterol release but did not affect apoA-I–induced cholesterol release (Fig 9). These results suggest that apoE secretion may be important in lyso-PC–induced cholesterol efflux, and the mechanisms involved in cholesterol efflux by lyso-PC and apoA-I might be different. Because monensin nonspecifically blocks much of the transport of secretory proteins, lyso-PC–mediated cholesterol efflux may depend on the fundamental transport process, which is inhibited by monensin. The study using human monocyte-macrophage derived foam cells suggested the spontaneous production of cholesterol-enriched lipoproteins containing apoE²⁸ ; lyso-PC may stimulate the production of some kinds of lipoproteins, resulting in cholesterol efflux.

With respect to the signal-transduction mechanisms induced by lyso-PC, some investigators reported the involvement of PKC activation,^{34 35} and other investigators showed the inhibition of the lyso-PC effect by the elevation of cyclic AMP levels.³⁶ In our experiments, PMA did not cause [³H]cholesterol release, and PKC downregulation by 24 hours' PMA pretreatment did not inhibit lyso-PC–induced [³H]cholesterol release (data not shown). From these data, we consider that the activation of PKC is not involved in lyso-PC–induced cholesterol efflux. PKC activation in mouse macrophages was reported to cause the posttranscriptional inhibition of apoE expression.^{37 38} We showed the augmented medium apoE accumulation by lyso-PC, the opposite effect of PKC activation. More detailed experiments are necessary to determine the effects of lyso-PC on PKC activity. Concerning the effect of 1 mmol/L dibutyryl cyclic AMP, it did not inhibit lyso-PC–induced cholesterol efflux (data not shown), although the same concentration of dibutyryl cyclic AMP was shown to inhibit the biological effect of lyso-PC.³⁶ Thus, PKA activation may not inhibit lyso-PC–induced cholesterol efflux in our experimental system.

Lyso-PC is a major lipid component of atherogenic lipoproteins^{11 12} and is reported to increase in atherosclerotic lesions.³⁹ In recent extensive in vitro studies, lyso-PC has been demonstrated to be atherogenic because of its chemoattracting potency for monocytes,¹³ inhibition of arterial relaxation induced by endothelial-derived relaxing factor,¹⁴ stimulation of several adhesion molecule and growth factor gene expression in endothelial cells,^{15 16} stimulation of growth factor gene expression in monocytes,¹⁷ and contribution to macrophage proliferation.¹⁸ These findings seem to point toward lyso-PC's contributing to the development of atherosclerotic lesions in vivo. However, some antiatherogenic actions of lyso-PC have also been reported; for example, endothelial-dependent arterial relaxation⁴⁰ and induction of nitric oxide synthase^{41 42} and cyclooxygenase-2⁴³ in endothelial cells. The promotion of cholesterol efflux from macrophage foam cells demonstrated in the current study is also considered to be antiatherogenic, and we speculate that lyso-PC in atherosclerotic lesions may have multiple effects of stimulating or inhibiting the development of atherosclerosis. In addition, the action of lyso-PC of stimulating cholesterol efflux may be related to the finding that oxidized LDL with enriched lyso-PC accumulated less cholesterol ester in macrophages compared with acetyl-LDL.⁴⁴

With respect to the cholesterol efflux from macrophage foam cells, HDL has been investigated extensively because of its high potency for stimulating cholesterol efflux. ApoA-I, a major apolipoprotein of HDL, was reported to stimulate cholesterol efflux even in the free form,⁴⁵ and further study has revealed that synthetic peptides (37 amino acids) containing the amphipathic helix observed in HDL apolipoprotein also had the cholesterol-releasing potency.^{46 47} More recently, cyclodextrin was reported to stimulate cholesterol efflux, although the cell type used was not macrophage foam cells.⁴⁸ In the current study, we showed the ability of lyso-PC to stimulate cholesterol efflux from macrophage foam cells. This finding is considered interesting from the viewpoint of cholesterol efflux induced by a low-molecular-weight substance. We also investigated several lyso-PC derivatives. Comparing lyso-PC of various fatty acid chain lengths (C 14:0 to C 18:0) showed that the longer the chain length the more effective the lyso-PC at the dose of 20 µmol/L. However, 40 µmol/L stearoyl–lyso-PC (C 18:0) showed no further increase of activity (Fig 10A). More detailed experiments may be necessary to elucidate the real effects of fatty acid chain lengths of lyso-PC. The activities of lysophosphatidylinositol and lysophosphatidylserine were weaker than lyso-PC (Fig 10B). Thus, palmitoyl–lyso-PC was the most effective compound in our system. Using this compound in further studies should elucidate the mechanisms of cholesterol efflux induced by lyso-PC.

In conclusion, we demonstrated that lyso-PC promotes cholesterol efflux from macrophage foam cells. This finding suggests that lyso-PC may inhibit the development of atherosclerosis or enhance its regression. It is important to elucidate the underlying mechanisms of this phenomenon.

	Selected Abbreviations and Acronyms

 

DMEM = Dulbecco's modified Eagle's medium FCS = fetal calf serum lyso-PC = lysophosphatidylcholine PAGE = polyacrylamide gel electrophoresis PC = phosphatidylcholine PK = protein kinase PMA = phorbol 12-myristate 13-acetate

Received April 17, 1996; accepted September 24, 1996.

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