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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:600-605.)
© 1996 American Heart Association, Inc.

Articles

Lysophosphatidylcholine Potentiates the Mitogenic Activity of Modified LDL for Human Monocyte–Derived Macrophages

Masakazu Sakai; Akira Miyazaki; Hideki Hakamata; Yoshihiro Sato; Takeshi Matsumura; Shozo Kobori; Motoaki Shichiri; Seikoh Horiuchi

From the Departments of Biochemistry (A.M., H.H., S.H.) and Metabolic Medicine (M. Sakai, Y.S., T.M., S.K., M. Shichiri), Kumamoto University School of Medicine, Kumamoto, Japan.

Correspondence to Seikoh Horiuchi, MD, PhD, Department of Biochemistry, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860, Japan.

	Abstract

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Abstract The growth of murine peritoneal macrophages is induced by oxidized LDL (Ox-LDL), and lysophosphatidylcholine (lysoPC) plays an important role in its mitogenic activity. In the present study, Ox-LDL–induced macrophage growth was examined with human monocyte–derived macrophages. The cell growth of human macrophages was significantly induced by Ox-LDL but not by acetylated LDL (acetyl-LDL). The treatment of acetyl-LDL with phospholipase A₂, however, led to a marked increase in its mitogenic activity, with a concomitant conversion of 75% of its phospholipids to lysoPC. The growth-stimulating activity became positive only when both acetyl-LDL and lysoPC were coincubated, although neither of them exhibited cell growth–promoting activity. These results suggest that Ox-LDL could stimulate the growth of human monocyte–derived macrophages, and lysoPC may play an essential role in the mitogenic activity of Ox-LDL.

Key Words: human monocyte–derived macrophage • foam cell • oxidized LDL • lysophosphatidylcholine • scavenger receptor

	Introduction

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Macrophage-derived foam cells play an essential role in the progression of the early stage of atherosclerosis.¹ Through the scavenger receptor pathway, macrophages take up chemically modified LDLs, such as acetyl-LDL, malondialdehyde-modified LDL, and Ox-LDL, and become foam cells in vitro.² Among these modified LDLs, Ox-LDL is emphasized as a likely candidate for an atherogenic lipoprotein in vivo because of its presence in human atherosclerotic plaques.^{3 4} Ox-LDL can induce proliferation of murine peritoneal resident macrophages⁵ in which the lysoPC of Ox-LDL plays a crucial role.⁶ Since several lines of evidence suggest that macrophage-derived foam cells proliferate in situ in atherosclerotic lesions,^{7 8 9} it seems reasonable to expect that the stimulation of macrophage growth by Ox-LDL might enhance the progression of atherosclerosis in addition to foam cell formation by Ox-LDL. Our demonstration of Ox-LDL–induced macrophage growth was derived from experiments using mouse^{5 6} rather than human macrophages. Since the cellular functions of human macrophages are not necessarily identical to those of their mouse counterparts, experiments with human macrophages to test whether Ox-LDL can stimulate their growth are crucial to generalize the significance of Ox-LDL–induced macrophage growth in human pathology.

There are at least two different subpopulations of local macrophages that differ in their origins. One is monocyte-derived macrophages; the other, resident macrophages, includes peritoneal macrophages, alveolar macrophages, and Kupffer cells in hepatic sinusoid. Monocyte-derived macrophages are in the terminal stage of differentiation in the mononuclear phagocyte system, in which hematopoietic stem cells in bone marrow differentiate to colony-forming unit granulocyte/macrophages, to promonocytes, and finally to monocytes. Differentiated monocytes are released into the circulation and migrate into the subendothelial space by the action of various chemotactic factors, followed by further differentiation to macrophages (exudate macrophages).¹⁰ In the early stage of differentiation in bone marrow, colony-forming unit granulocyte/macrophages migrate to peripheral tissues and become resident macrophages.¹¹ Resident macrophages are characterized by their capacity to proliferate by themselves in peripheral tissues to supply local macrophages.^{12 13 14 15} In contrast, monocyte-derived macrophages do not proliferate any longer without growth stimulation.¹⁰

It is generally accepted that macrophage-derived foam cells in the early stage of atherosclerosis originate from monocyte-derived but not resident macrophages. In fact, circulating monocytes adhere to endothelial cells, migrate into the subendothelial space, and differentiate into macrophages, which leads to foam cell formation.^{16 17} To elucidate the pathophysiological significance of macrophage growth induced by Ox-LDL in vivo, it is important to know whether Ox-LDL can also induce the growth of monocyte-derived macrophages. To address this issue, the present study was undertaken to examine the mitogenic activity of Ox-LDL for human monocyte–derived macrophages. The results demonstrate the growth-stimulating activity of Ox-LDL for human monocyte–derived macrophages in which the lysoPC of Ox-LDL plays a key role.

	Methods

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Chemicals
Oleoyl-lysoPC, palmitoyl-lysoPC, stearoyl-lysoPC, PLA₂, and MTT were purchased from Sigma Chemical Co. [methyl-³H]thymidine (80 Ci/mmol) and [¹⁴C]palmitoyl-lysoPC (56 mCi/mmol) were purchased from New England Nuclear. Other chemicals were the best grade available from commercial sources.

Lipoproteins and Their Modifications
Human LDL (d=1.019 to 1.063 g/mL) was isolated by sequential ultracentrifugation from the plasma of normolipidemic subjects after overnight fasting.¹⁸ LDL was dialyzed against 0.15 mol/L NaCl and 1 mmol/L EDTA, pH 7.4. Acetyl-LDL was prepared by chemical modification of LDL with acetic anhydride.¹⁹ Ox-LDL was prepared by incubation of LDL with 5 µmol/L CuSO₄ for 20 hours at 37°C followed by the addition of 1 mmol/L EDTA and cooling.²⁰ LDL and acetyl-LDL were dialyzed against PBS and treated with PLA₂ as described by Quinn et al.²¹ Ox-LDL was labeled with [¹⁴C]palmitoyl-lysoPC by using the method of Albers et al.²² Briefly, 10 µCi [¹⁴C]lysoPC was dried under nitrogen and redissolved in 5 µL ethanol. This solution was slowly added to the surface of 1 mL Ox-LDL (1 mg/mL) with gentle stirring on a vortex mixer. The mixed solution was incubated at 37°C for 2 hours for equilibration of [¹⁴C]lysoPC with Ox-LDL and then dialyzed extensively against 0.15 mol/L NaCl and 1 mmol/L EDTA, pH 7.4. The specific radioactivity of [¹⁴C]lysoPC-labeled Ox-LDL was 2600 cpm/nmol lysoPC (1690 cpm/µg protein). [¹⁴C]Palmitoyl-lysoPC liposomes were prepared by mixing 5 µCi [¹⁴C]lysoPC (90 nmol) with 810 nmol cold palmitoyl-lysoPC. The mixture was dried under nitrogen and resolved in 5 µL ethanol. PBS (1 mL) was added to this solution, which was then sonicated. The specific radioactivity of the [¹⁴C]lysoPC liposomes was 3400 cpm/nmol lysoPC. The level of thiobarbituric acid–reactive substances in Ox-LDL was 85 nmol malondialdehyde/mg protein, whereas those of LDL, acetyl-LDL, PLA₂-treated LDL, and PLA₂-treated acetyl-LDL were within 3.0 nmol malondialdehyde/mg protein.²³ The levels of endotoxin associated with these lipoproteins were <1 pg/µg protein; these were measured by a kit (Toxicolor system, Seikagaku Corp). Moreover, macrophage growth was not induced by endotoxin at a concentration <1 ng/mL in our experimental system. Protein concentrations were determined by the bicinchoninic acid protein assay reagent (Pierce) with bovine serum albumin as a standard.²⁴

Cell Culture
Human peripheral blood monocytes were isolated by using the method of Fogelman et al²⁵ with Ficoll/Hypaque gradient centrifugation. The mononuclear cells thus obtained were resuspended in RPMI 1640 (Nissui Seiyaku Co) supplemented with 20% autologous serum, 0.1 mg/mL streptomycin, and 100 U/mL penicillin, plated on serum-treated 10-cm dishes (Falcon), and incubated for 2 hours. The nonadherent cells were removed by washing three times with PBS, and the adherent cells were then detached by incubation in PBS/5% autologous serum containing 0.02% EDTA at 4°C for 30 minutes. The cells were then washed extensively and resuspended in RPMI 1640 supplemented with 5% autologous serum, 0.1 mg/mL streptomycin, and 100 U/mL penicillin (medium A). The cells were plated on 10-cm dishes and incubated for 9 days to differentiate into macrophages. The medium was aspirated and replaced every 3 days with fresh medium A.

After 9 days' incubation, differentiation of monocytes into macrophages was judged by three categories: adherence to the culture plates, morphological features, such as mononuclear cells after Giemsa staining, and the capacity to take up carbon particles. The cells contained more than 95% macrophages and were more than 98% viable as determined by trypan blue staining. All cellular experiments were performed at 37°C in a humidified atmosphere of 5% CO₂ in air.

MTT Assay and Tritiated Thymidine Incorporation Assay
Human monocyte–derived macrophages were adjusted to 5x10⁴ cells/mL for the MTT assay and 4x10⁵ cells/mL for the tritiated thymidine ([³H]thymidine) incorporation assay. Cell suspensions (0.1 mL) were dispersed in each well of 96-well tissue-culture plates (6.4-mm diameter, Falcon) and incubated for 90 minutes at 37°C. The nonadherent cells were removed by washing three times with 0.1 mL prewarmed medium A. Adherent cells were cultured at 37°C with 0.1 mL medium A in the presence or absence of the lipoproteins to be tested without a medium change. After the incubation, macrophage growth was determined by using the MTT method⁶ and the [³H]thymidine incorporation assay.⁶ The MTT assay is based on the cellular reduction of MTT to MTT formazan by mitochondrial dehydrogenase. During the last 4 hours of incubation, cells were treated with 50 ng/mL MTT, and then 1.5% SDS for 16 hours to dissolve the MTT formazan, which was measured spectrophotometrically at 570 nm.

Cell-Counting Assay
For the cell-counting assay, macrophages were adjusted to 1.5x10⁴ cells/mL, and 1 mL cell suspension was dispersed in each well of 24-well tissue-culture plates (16-mm diameter, Falcon). Nonadherent cells were removed by washing three times with 1 mL prewarmed medium A, and adherent cells were cultured with 1 mL medium A in the presence or absence of the lipoproteins being tested without a medium change. After the incubation the medium was discarded, and the adherent cells in triplicate wells were lysed in 1% (wt/vol) Triton X-100, and the number of naphthol blue black–stained nuclei were counted in a hemocytometer.⁵

Morphological Observations
Cells were cultured with 1 mL medium A in the presence or absence of lipoproteins. After the incubation the cells were washed three times with PBS, fixed with 10% paraformaldehyde for 30 minutes, and stained with oil red O. The cells were counterstained with hematoxylin for 10 minutes and then examined by using inverted phase-contrast microscopy.²⁶ To examine the uptake of carbon particles, 1 µL black drawing ink (Rotring, Art. 591 017) was added to 1 mL of each well of 24-well plates; the cells were incubated for 2 hours, washed with PBS, and examined by using inverted phase-contrast microscopy.⁵

Chemical Analysis
Phospholipid contents of lipoproteins were determined on a Hitachi 7450 automatic analyzer by using a standard enzymatic method,²⁷ and the content of lysoPC in each lipoprotein was determined.⁶

	Results

Top Abstract Introduction Methods Results Discussion References

 
Ox-LDL Promotes Growth of Human Peripheral Blood Monocyte-Derived Macrophages
The mitogenic activity of Ox-LDL for human macrophages was first examined by MTT assay. When human macrophages were incubated with medium alone, MTT values were not altered (Fig 1). In contrast, when macrophages were incubated with Ox-LDL, MTT values were significantly increased in a dose- and time-dependent manner (Fig 1), suggesting that the growth of human monocyte–derived macrophages was induced by Ox-LDL. This observation was further characterized by the following experiments.

View larger version (21K): [in this window] [in a new window]   Figure 1. Monocyte-derived macrophages (5x103) were incubated with 10 µg/mL (), 20 µg/mL (), or 50 µg/mL () Ox-LDL or medium alone (). After the incubation, cells in each well were treated with MTT for 4 hours and sodium dodecyl sulfate for 16 hours, and MTT formazan was measured as described in "Methods." Data represent mean of three separate experiments; error bars indicate SD.

Effects of PLA₂-Treated Acetyl-LDL on the Growth of Human Monocyte–Derived Macrophages
Because lysoPC is an active part of Ox-LDL that is responsible for its mitogenic activity,⁶ we tested whether lysoPC could stimulate the growth of human monocyte–derived macrophages. Acetyl-LDL was treated with PLA₂, and the effect on cell growth was examined. Treatment of acetyl-LDL with PLA₂ converted 75% of the total phosphatidylcholine into lysoPC (Table 1). Whereas untreated acetyl-LDL had no growth-stimulating activity, the significant cell growth–promoting activity of PLA₂-treated acetyl-LDL was equivalent to that of Ox-LDL (Fig 2). In contrast, the mitogenic activity of PLA₂-treated LDL was negligible (Fig 2), although its lysoPC content was indistinguishable from that of PLA₂-treated acetyl-LDL (Table 1).

View this table: [in this window] [in a new window]   Table 1. LysoPC Contents of LDL and Acetyl-LDL After Treatment With PLA2

View larger version (23K): [in this window] [in a new window]   Figure 2. Monocyte-derived macrophages (5x103) were incubated with the indicated concentrations of native LDL (), Ox-LDL (), acetyl-LDL (), PLA2-treated LDL (), or PLA2-treated acetyl-LDL (). On day 4, cells in each well were treated with MTT for 4 hours and sodium dodecyl sulfate for 16 hours, and MTT formazan was measured as described in "Methods." Data represent mean of three separate experiments; error bars indicate SD.

Combined Effects of LysoPC and Acetyl-LDL on the Growth of Human Monocyte–Derived Macrophages
We next determined the cell growth–promoting activity of three types of lysoPC: oleoyl-lysoPC, palmitoyl-lysoPC, and stearoyl-lysoPC. None of these liposomes significantly induced cell growth (Tables 2 and 3 and Fig 3). Instead, lysoPC at concentrations >100 µmol/L induced cell death rather than cell growth (Fig 3). However, when lysoPC was added in combination with acetyl-LDL, the [³H]thymidine incorporation into cells was increased to an extent that was indistinguishable from that induced by Ox-LDL (Table 2). When cell growth was assayed by determination of cell number, Ox-LDL increased cell number by 38% (Table 3). The increase in cell number by acetyl-LDL alone was not significant, but it became significant by coincubation with lysoPC (Table 3). Taken together with the results shown in Fig 2, it is likely that lysoPC behaves as a potent mitogen for human monocyte–derived macrophages only when it is presented by a modified LDL that is readily endocytosed through the scavenger receptor pathway. To further test this notion, we next determined amounts of lysoPC transferred to macrophages when cells were incubated with [¹⁴C]lysoPC liposomes or [¹⁴C]lysoPC-labeled Ox-LDL. When macrophages were incubated with 26 µmol/L [¹⁴C]lysoPC liposomes, the cell-associated lysoPC reached a plateau level almost instantaneously (Fig 4). Since this level did not increase, but rather decreased slightly on further incubation, it may reflect lysoPC absorbed nonspecifically to the surface membranes of macrophages rather than that endocytosed by these cells. In sharp contrast, when cells were incubated with [¹⁴C]lysoPC-labeled Ox-LDL, the cell-associated [¹⁴C]lysoPC was much lower than that of [¹⁴C]lysoPC liposomes but increased gradually with time (Fig 4), indicating that the lysoPC of Ox-LDL particles was transferred to these cells by a mechanism different from that of lysoPC liposomes (probably by endocytic uptake through the scavenger receptor). These results likely support the notion that the endocytic uptake of lysoPC through the scavenger receptor is crucial for Ox-LDL–induced macrophage growth.

View this table: [in this window] [in a new window]   Table 2. Combined Effects of LysoPC and Acetyl-LDL on Human Macrophages as Determined by [3H]Thymidine Incorporation Assay

View this table: [in this window] [in a new window]   Table 3. Combined Effects of LysoPC and Acetyl-LDL on Human Macrophages as Determined by Counting of Solubilized Nuclei

View larger version (22K): [in this window] [in a new window]   Figure 3. Monocyte-derived macrophages (5x103) were incubated with the indicated concentrations of oleoyl-lysoPC () or stearoyl-lysoPC (). As a control experiment, macrophages were incubated with the indicated lysoPC concentrations of the Ox-LDL preparation (), which contained 634 nmol lysoPC/mg protein (Table 1). On day 4, cells in each well were treated with MTT for 4 hours and sodium dodecyl sulfate for 16 hours, and MTT formazan was measured as described in "Methods." Data represent mean of three separate experiments; error bars indicate SD.

View larger version (18K): [in this window] [in a new window]   Figure 4. Monocyte-derived macrophages (1x105) were incubated for the indicated times with 26 µmol/L [14C]palmitoyl-lysoPC liposome () or 40 µg protein/mL (26 µmol/L on the basis of lysoPC) [14C]lysoPC-labeled Ox-LDL (). After the incubation, the cellular radioactivity from [14C]lysoPC was counted in a liquid scintillation spectrophotometer. Data represent mean of three separate experiments; error bars indicate SD.

A parallel experiment with mouse resident peritoneal macrophages showed that the cell number was increased 2.3-fold by Ox-LDL (3.2x10⁴/well) compared with that of nonloaded cells (1.4x10⁴/well), indicating that the increase in cell number was more prominent in mouse than in human macrophages.

Morphological Observation
Morphological observation showed that significant intracellular accumulation of lipids did not occur in human monocyte–derived macrophages when incubated with medium alone (Fig 5A) or LDL (Fig 5B). In contrast, when human macrophages were incubated with acetyl-LDL (Fig 5C), cells were strongly stained with oil red O. When cells were incubated with Ox-LDL, intracellular lipid accumulation virtually occurred (Fig 5D). However, the lipid droplets induced by Ox-LDL appeared smaller and much finer than those induced by acetyl-LDL. Moreover, the cells exposed to Ox-LDL also showed rounded and/or enlarged shapes (Fig 5D). These morphological features of macrophages after incubation with Ox-LDL have been reported with mouse peritoneal macrophages.²⁸ More than 95% of the oil red O–positive foam cells obtained after incubation with Ox-LDL were mononuclear cells that were able to take up carbon particles (data not shown). Thus, it is likely that the cells that proliferate in response to Ox-LDL are macrophages but not other contaminated cells.

View larger version (144K): [in this window] [in a new window]   Figure 5. Photomicrographs showing monocyte-derived macrophages that were incubated with medium alone (A) or 20 µg/mL LDL (B), acetyl-LDL (C), or Ox-LDL (D) for 7 days (oil red O, x100).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Ox-LDL has a growth-stimulating activity for mouse resident peritoneal macrophages,⁵ and lysoPC plays an essential role in this mitogenic activity.⁶ These findings were extended in the present study to human monocyte–derived macrophages. The results demonstrate that the growth of human monocyte–derived macrophages was virtually induced by Ox-LDL in which lysoPC had a key role in the mitogenic activity. This conclusion was supported by the following two observations: first, while untreated acetyl-LDL did not show a significant mitogenic effect, PLA₂ treatment of acetyl-LDL resulted in a marked increase in lysoPC content (75% of total phospholipids) (Table 1) with a concomitant increase in the mitogenic effect on human monocyte–derived macrophages (Fig 2); second, cell growth was significantly induced when lysoPC was coincubated with acetyl-LDL (Tables 2 and 3).

Various atherogenic functions of lysoPC have been reported: chemotactic activity for monocytes,²⁹ induction in endothelial cells of cell adhesion molecules³⁰ and growth factors such as platelet-derived growth factor and heparin-binding epidermal growth factor–like protein,³¹ and impairment of endothelium-dependent arterial relaxation.³² The growth-stimulating activity for human monocyte–derived macrophages is a novel aspect of lysoPC.

The other important aspect regarding the growth-stimulating activity of lysoPC is the notion that the scavenger receptor might play an important role in the mitogenic effect of Ox-LDL. The lysoPC content of PLA₂-treated LDL was virtually the same as that of PLA₂-treated acetyl-LDL (Table 1). However, its mitogenic activity for human macrophages was less than one fourth that of PLA₂-treated acetyl-LDL (Fig 2). Moreover, lysoPC showed significant cell growth–promoting activity when coincubated with acetyl-LDL, but coincubation with LDL failed to induce cell growth (Table 3). The Ox-LDL–induced growth of starch-induced mouse macrophages is efficiently suppressed by dextran sulfate, a polyanionic compound that could compete with Ox-LDL for its binding to the scavenger receptor.⁵ Furthermore, the amount of lysoPC transfer from lysoPC liposomes to macrophages was higher than that from Ox-LDL (Fig 4), while macrophage growth was induced by Ox-LDL but not by lysoPC liposomes (Tables 2 and 3). It is likely, therefore, that the scavenger receptor–mediated endocytosis of Ox-LDL might provide an effective route for the lysoPC supply to cells, which would lead to macrophage growth.

The growth response of human monocyte–derived macrophages to Ox-LDL (Table 3) seems weaker than that of mouse macrophages. The cell numbers of mouse macrophages (resident or starch-induced) were increased 2.3- to 2.8-fold by Ox-LDL,^{5 6} whereas the corresponding increase by Ox-LDL in human macrophages was less than 1.4-fold (Table 3). The reasons for the difference are unclear, but it could be explained, in part, by the following notions. First, the rate of cellular growth induced by mitogens might differ between mouse and human cells. Van Corven et al^{33 34} have shown that the growth of human fibroblasts induced by lysophosphatidic acid or fetal calf serum is fourfold weaker than that of rat fibroblasts.³³ Moreover, the response of rat fibroblasts to lysophosphatidic acid was less than one third that of mouse fibroblasts.³⁴ Second, there might be a species difference in the scavenger receptor activity between mouse and human macrophages. The data provided by Keider et al³⁵ and Ylä-Herttuala et al⁴ show that the absolute amount of Ox-LDL degraded by mouse macrophages was threefold and subsequent intracellular accumulation of cholesteryl esters was sixfold higher than those in human monocyte–derived macrophages.

The present study shows that the growth response of human monocyte–derived macrophages to Ox-LDL in vitro is relatively weak. However, this cannot be simply extended to the in vivo situation because macrophages in atherosclerotic lesions might be stimulated by various cytokines secreted from their own and/or other cells, such as endothelial cells and smooth muscle cells. Compared with mouse resident peritoneal macrophages, the growth response of mouse peritoneal exudate macrophages was much more sensitive to macrophage colony-stimulating factor³⁶ or phorbol esters,³⁷ indicating that an inflammatory stimulation per se increases the basal level of the responsiveness of monocyte-derived macrophages to mitogens. Since atherosclerosis is thought to be a chronic inflammatory reaction, it is possible to speculate that monocyte-derived macrophages in human atherosclerotic lesions in situ would be more potent for growth induction than in vitro.

In the present study, macrophage growth was assayed by using MTT, [³H]thymidine incorporation, and cell-counting assays. The results obtained by these methods were essentially consistent. However, an increase in MTT values by exposure to Ox-LDL seemed more prominent than a corresponding increase in cell numbers; MTT values increased twofold or more by Ox-LDL (Figs 2 and 3), whereas the corresponding increase in cell number was 1.4-fold (Table 3). This difference might be explained as follows. In addition to an increase in cell number, Ox-LDL induced an increase in cellular size (Fig 5). Moreover, the electron microscopic examination showed that the number of mitochondria per cell increased after incubation with Ox-LDL (data not shown). Therefore, it is likely that the increase in numbers of mitochondria per cell that was induced by Ox-LDL may account for the much higher increase in MTT values compared with that in cell numbers.

In conclusion, the present study demonstrates that the growth of human monocyte–derived macrophages can be induced by Ox-LDL in which lysoPC plays an essential role in growth induction. Since macrophage-derived foam cells in atherosclerotic lesions are known to originate from monocyte-derived rather than resident macrophages, the present results strengthen the possibility that the growth of macrophages in human atherosclerotic lesions could be induced by Ox-LDL.

	Selected Abbreviations and Acronyms

 

acetyl-LDL = acetylated LDL lysoPC = lysophosphatidylcholine MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Ox-LDL = oxidized LDL PLA2 = phospholipase A2

	Acknowledgments

 
This work was supported in part by a grant-in-aid for scientific research (No. 07770108) from the Ministry of Education, Science and Culture of Japan, and a grant from the HMG-CoA Reductase Research Foundation. We are grateful to Drs Toshinori Sasaki, Satoru Yui, and Masatoshi Yamazaki for helpful discussion.

Received August 31, 1995; accepted November 20, 1995.

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