Introduction

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The development of atherosclerotic diseases is associated with the accumulation of cholesterol ester in arteries. Large numbers of foam cells derived from macrophages and smooth muscle cells and containing a large amount of cholesterol ester are found in atherosclerotic lesions (Rosenfeld et al., 1987; Lewis et al., 1985; Stary et al., 1990). These foam cells have a high activity of ACAT, which catalyzes the esterification of free cholesterol (Suckling et al., 1985). The increase of lipid deposition in foam cells is due to this excessive cholesterol esterification by the ACAT action (Goldstein et al., 1980). Thus, it might be expected that atherosclerosis is reduced by the inhibition of ACAT activity.

It is known that intracellular cholesterol metabolism is regulated by some enzymes such as acid cholesterol esterase and neutral cholesterol esterase besides ACAT (Brown et al., 1979, 1980; Khoo et al., 1981, 1984; Goldberg et al., 1990). It is reported that an imbalance between cholesterol esterase and ACAT activities might induce either foam cell formation (Ishii et al., 1992, 1994, 1995b) or cell toxicity (Warner et al., 1995). For example, when ACAT activity was induced and neutral cholesterol esterase activity was low, cells accumulated excess cholesterol ester and were converted into foam cells (Ishii et al., 1992, 1994, 1995a). There are several reports on the effects of ACAT inhibitors on intracellular cholesterol content and metabolism. An ACAT inhibitor, Sandoz 58-035, reduced intracellular cholesterol ester content in J774 cells, mouse peritoneal macrophages, Fu5AH hepatoma cells (Bernard et al., 1990) and monkey arterial smooth muscle cells under conditions of hyperlipidemia (Ross et al., 1984). Other ACAT inhibitors, CL 277,082 and CI-976, also inhibited ACAT activity and cholesterol absorption (Krause et al., 1993; Largis et al., 1989). On the other hand, when free cholesterol accumulated by the inhibition of ACAT activity with Sandoz 58-035 and Pfizer 113,818, cell toxicity was induced (Warner et al., 1995). For the safe use of ACAT inhibitors, this problem must be solved. We hypothesized that an ideal condition for reducing cholesterol in foam cells by the use of ACAT inhibitors is to increase the release of intracellular free cholesterol produced by acid and neutral cholesterol esterase. Some kinds of ACAT inhibitors might indirectly stimulate this process. In this study, we investigated this possibility using a new ACAT inhibitor, HL-004 [N-(2, 6-diisopropylphenyl)tetradecylthioacetamide], compared with CI-976 (2,2-dimethyl-N1-(2,4,6-trimethylphenyl)-dodecanamide).

	Materials and Methods

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Materials. HL-004 was synthesized by Taisho Pharmaceutical (Ohmiya, Japan). HL-004 was synthesized as reported (Japanese patent No. JP-316129, 1995), and the purity was >99% by HPLC. CI-976 was obtained from Warner-Lambert (Ann Arbor, MI). HL-004 and CI-976 were first dissolved in DMSO, and the final concentration was 10⁴ M. [³H]Cholesterol oleate (3.0 GBq/mmol) and [¹⁴C]oleic acid (1.5 GBq/mmol) were purchased from New England Nuclear Corporation (Boston, MA). [8-³H]Adenine (666 GBq/mmol) was purchased from Amersham Corp. (Buckinghamshire, UK). TPA was obtained from Sigma (St. Louis, MO). THP-1 cells (human monocyte leukemia cell line) were obtained from Japan Cell Culture System (Tokyo, Japan). DMEM and RPMI 1640 were obtained from Nissui Pharmaceutical (Tokyo, Japan).

Preparation of cells. THP-1 cells (5 × 10⁶ cells/ml) were treated with 1 × 10⁸ M TPA overnight in RPMI 1640 containing 10% FBS (10% FBS/RPMI), and the adherent cells were used as macrophages (Hayashi et al., 1991).

Measurment of ACAT inhibitor-induced cell toxicity. ACAT inhibitor-induced cell toxicity was measured by the modified (Warner et al., 1995; Reid et al., 1992) method of Shirhatti and Krishna (Shirhatti et al., 1985). Briefly, after 18-hr incubation of cells with DMEM containing 0.2% BSA, 37 kBq of [³H]adenine was added to each well and cells were incubated for 2 hr. Then, wells were washed 3 times with 1 ml of DMEM containing 0.2% BSA (0.2% BSA/DMEM) and incubated for an additional 10 min with 0.2% BSA/DMEM. Media were removed, and fresh 0.2% BSA/DMEM was added with ACAT inhibitor. At indicated times, 200 µl of the media was removed from triplicate cultures and their radioactivities were counted. [³H]Adenine release from macrophages was expressed as a percentage of release compared with control treatment (0.2% BSA): (cpm in medium of treatment  cpm in medium of control)/([³H]adenine content at time zero) × 100, or as a percent release of total cell [³H]adenine content at time zero.

Preparation of lipoproteins. HDL (d = 1.063 ~ 1.21 g/ml) and LPDS (d > 1.25 g/ml) were isolated from human plasma by sequential ultracentrifugation and were dialyzed against 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 0.15 M NaCl and 0.02% NaN₃ for 24 hr (Havel et al., 1955).

Preparation of reconstituted [³H]cholesterol oleate into VLDL. VLDL (density < 1.006) was isolated from cholesterol-fed rabbit serum by ultracentrifugation for 16 hr (Brown et al., 1975). Incorporation of [³H]cholesterol oleate into VLDL was done essentially by the method of Brown et al (1975). Then, 37 MBq of [³H]cholesterol oleate was added with 1 ml of DMSO. The mixture was sonicated for 30 sec. Then 2 ml of plasma density buffer (0.154 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 0.01% NaN₃) was added, and the mixture was resonicated for 30 sec. It was then added dropwise to 6 ml of VLDL (10 mg total cholesterol/ml) in 3 min. The solution was incubated for 8 hr at 37°C and then was dialyzed against 3 liters of plasma density buffer for 10 hr. After dialysis, the solution was centrifuged for 16 hr at 105,000 × g. The top layer was used as [³H]cholesterol oleate-incorporated VLDL. The specific activity was about 3.4 × 10⁷ dpm/mg total cholesterol and 5.4 × 10⁷ dpm/mg cholesterol ester.

[³H]Cholesterol oleate-VLDL incorporation into cells. TPA-treated THP-1 cells (1 × 10⁶ cells) and atherosclerotic lesion cells (5 × 10⁵ cells) were plated in 12-well plates and incubated for certain time periods in 0.75 ml of DMEM containing 5% LPDS (5% LPDS/DMEM) and 200 µg cholesterol of [³H]cholesterol oleate-VLDL (1.6 × 10⁶ dpm). Then, after 8-hr incubation, the cells were washed 3 times with 10% FBS/DMEM, and their radioactivity was measured with a scintillation counter. Furthermore, to determine the free [³H]cholesterol released from the cells during incubation, organic solvent (chloroform:methanol = 2:1) was added to the medium, and lipids were extracted from the chloroform layer. The lipids were applied to thin layer chromatography (Gillies et al., 1986). The radioactivity in the free cholesterol fraction was then counted. The total uptake amounts were the sums of intracellular radioactivity and free [³H]cholesterol radioactivity in the medium.

Release of radioactivity from [³H]cholesterol oleate-VLDL-loaded cells. TPA-treated THP-1 cells (2 × 10⁶ cells) and atherosclerotic lesion cells (2 × 10⁶ cells) were plated in 12-well plates and incubated for 24 hr in 1 ml of 5% LPDS/DMEM containing 200 µg of [³H]cholesterol oleate-VLDL. Then the cells were washed 3 times with 5% LPDS/DMEM. These [³H]cholesterol oleate-VLDL-loaded cells were incubated further in 2 ml of 5% LPDS/DMEM containing HDL (0.3 mg protein/ml). At the times indicated in figure 3, 0.4 ml of the medium was removed, and its radioactivity was measured.

Synthesis of cholesterol [¹⁴C]oleate from [¹⁴C]oleate in intact cells. Cells (2 × 10⁵ cells/well) were treated with VLDL (100 µg cholesterol/ml) and albumin-[¹⁴C]oleic acid complex (0.1 µCi/ml) (Goldstein et al., 1974). After incubation for 8 hr, the cells were washed with medium three times and with PBS twice. The intracellular lipids were extracted by chloroform/methanol (2:1) and separated by thin-layer chromatography (Macherery-Nagel Duren, Merk) using a solvent, hexane/diethyl ether/acetic acid (146:50:4). Nonlabeled free cholesterol and cholesterol ester were spotted as marker in the plate, and the spot was detected by iodide. The radioactivity of the cholesterol ester fraction was estimated as the ACAT activity in intact cells.

Measurement of acid and neutral cholesterol esterase activity. Macrophages (2 × 10⁷ cells) were washed 3 times with PBS and suspended in 1 ml of 10 mM Tris-HCl (pH 7.4) containing 0.25 M sucrose. Then the cells were sonicated twice for 15 sec and used as the enzyme solution. The reaction mixture (Ishii et al., 1992; Ishii et al., 1994, 1995) contained 0.5 mM cholesterol oleate, 0.37 MBq of cholesterol [¹⁴C]oleate, 0.5 mM phosphatidic acid and 100 mM Tris-HCl (pH 7.4) for neutral cholesterol esterase, or 0.5 mM phosphatidylcholine and sodium acetate buffer (pH 4.0) for acid cholesterol esterase, certain concentration of ACAT inhibitors, and the enzyme solution, in a total volume of 200 µl. The incubation was carried out at 37°C for 1 hr. The [¹⁴C]oleate released was extracted by a modification of the method of Belfrage et al (1969). Briefly, the reaction was stopped with 3.25 ml of chloroform/methanol/heptane (1.42:1.25:1.00), and then 1 ml of 0.1 N NaOH was added. The radioactivity in the water phase was measured.

Determination of cholesterol ester content. The cholesterol ester content was measured as the difference between the total and free cholesterol contents (Ishii et al., 1995). The lipids of the washed cells in each well were extracted with 1 ml of hexane/isopropanol (2:1). The organic solvent was evaporated and the pellet was dissolved in 100 µl of methanol, and the total and free cholesterol contents in the methanol solution were assayed enzymatically with Determiner TC 555 and Determiner FC 555 kits (Kyowa Medics, Tokyo, Japan).

Determination protein concentration. Protein concentration was determined with a kit using Bradford's method (BioRad, Protein Assay, Richmond, CA).

Statistics treatment. The significance of differences in a treatment series was determined by a one-way analysis of variance. Individual treatments were compared with the control by Dunnett's test.

	Results

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Effect of HL-004 on synthesis of cholesterol [¹⁴C]oleate in TPA-treated THP-1 cells. To determine the effect of the ACAT inhibitor, HL-004 on cholesterol esterification in macrophages compared with the effect of CI-976, we examined the inhibition of cholesterol [¹⁴C]oleate synthesis in TPA-treated THP-1 cells. Figure 1A shows that HL-004 decreased the synthesis of cholesterol [¹⁴C]oleate in a concentration-dependent manner and the effect of HL-004 was stronger than that of CI-976, the inhibition being ~90% at 10⁷ M and ~100% at 10⁶ M. Then, 50% inhibition (IC₅₀) of HL-004 and CI-976 were 1.1 × 10⁹ M and 1.2 × 10⁷ M, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effect of concentration of HL-004 and CI-976 on cholesterol [14C]oleate synthesis in TPA-treated THP-1 cells and release of [3H]adenine. A, Cholesterol [14C]oleate synthesis; TPA-treated THP-1 cells (5 × 106 cells/well) were preincubated with HL-004 at indicated concentrations for 24 hr. The medium was changed and the cells were treated with VLDL (100 µg cholesterol/ml), [14C]oleate (37 MBq/well) and HL-004 for 8 hr. Incorporation of [14C]oleate into cellular cholesterol [14C]oleate was measured as described in Materials and Methods. 100% Value was 28.3 nmol/mg cell protein. B, Release of [3H]adenine. Release of [3H]adenine from TPA-treated THP-1 cells (5 × 106 cells/well) treated with HL-004 was measured as described in Materials and Methods. , HL-004-treated cells; , CI-976-treated cells. Triplicate determination was measured at each experiment. Values are mean ± S.D. for three experiments. *P < .05, **P < .01. (Asterisks beside circles, compared with nontreated groups.)

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View larger version (21K): [in this window] [in a new window]   Fig. 2.   Time-dependent effect of HL-004 and CI-976 on cholesterol [14C]oleate synthesis in TPA-treated THP-1 cells. TPA-treated THP-1 cells (5 × 106 cells/well) were incubated with VLDL (100 µg cholesterol/ml) and [14C]oleate (37 MBq/well) in the presence of 107 M ACAT inhibitors for indicated times. Incorporation of [14C]oleate into cellular cholesterol [14C]oleate was measured as described in Materials and Methods. , control; , HL-004-treated cells; , CI-976-treated cells. Triplicate determination was measured at each experiment. Values are mean ± S.D. for three experiments. *P < .05, **P < .01.

Effect of HL-004 on cholesterol metabolism in TPA-treated THP-1 cells. Cholesterol metabolism of macrophages is as follows; macrophages take up lipoproteins such as VLDL and denatured LDL. First, cholesterol ester in lipoproteins is hydrolyzed by acid cholesterol esterase in lysosomes (Brown et al., 1979; Brown, et al., 1980). Then the product, free cholesterol, is reesterified by ACAT and stored as cholesterol ester in intracellular lipid droplets. After that, the reesterified cholesterol is hydrolyzed by neutral cholesterol esterase (Khoo et al., 1981, 1984; Goldberg et al., 1990), and finally, free cholesterol is released from the cells. Thus, the effects of HL-004 on the incorporation of VLDL, release of cholesterol, acid cholesterol esterase activity and neutral cholesterol esterase activity were measured compared with CI-976.

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View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of concentration of HL-004 and CI-976 on incorporation of [3H]cholesterol oleate-VLDL and release of radioactivity from TPA-treated THP-1 cells loaded with [3H]cholesterol oleate-VLDL. A, Incorporation of [3H]cholesterol oleate-VLDL into TPA-treated THP-1 cells. TPA-treated THP-1 cells (1 × 106 cells/well) were incubated with ACAT inhibitors at indicated concentrations for 12 hr. The medium was changed and the cells were treated with [3H]cholesterol oleate-VLDL, and again with ACAT inhibitors for 8 hr. Incorporation of [3H]cholesterol oleate-VLDL into the cells was measured as described in Materials and Methods. B, Release of radioactivity from TPA-treated THP-1 cells loaded with [3H]cholesterol oleate-VLDL. TPA-treated THP-1 cells (1 × 106 cells/well) were incubated with [3H]cholesterol oleate-VLDL for 24 hr. After that, cells were incubated with 5% LPDS/DMEM containing HDL (0.3 mg protein/ml) and ACAT inhibitors for 12 hr. Total incorporated and released radioactivity were measured as described in Materials and Methods. , HL-004-treated cells; , CI-976-treated cells. Triplicate determination was measured at each experiment. Values are mean ± S.D. for three experiments. *P < .05, **P < .01.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of concentration of HL-004 and CI-976 on acid and neutral cholesterol esterase activities. Acid and neutral cholesterol esterase activities were measured as described in Materials and Methods. A, Acid cholesterol esterase. B, Neutral cholesterol esterase. , HL-004-treated cells; , CI-976-treated cells. Triplicate determination was measured at each experiment. Values are mean ± S.D. for three experiments. *P < .05, **P < .01. (Asterisks beside circles, compared with nontreated groups.)

Effect of HL-004 on cholesterol metabolism of foam cells. Next, we examined the effects of HL-004 on the cholesterol metabolism of foam cells. Figure 5A shows the effect of HL-004 and CI-976 on the synthesis of cholesterol [¹⁴C]oleate in foam cells. Synthesis of cholesterol [¹⁴C]oleate of control cells was in the range of 3.7 ~ 5.7 nmol/mg protein. HL-004 decreased the synthesis in a concentration-dependent manner, the inhibition being 83% at 10⁷ M. Although CI-976 decreased the synthesis in a concentration-dependent manner, the inhibition was much lower than that of HL-004, being 21% at 10⁷ M. Figure 5B shows the effect of HL-004 and CI-976 on the release of [³H]adenine. The release of HL-004-treated groups was almost the same as CI-976-treated groups and was not different from that of nontreated group. These data on HL-004 were quite similar to those in THP-1 cell-derived macrophages (fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of concentration of HL-004 and CI-976 on cholesterol [14C]oleate synthesis in atherosclerotic lesion cells and release of [3H]adenine. A, Atherosclerotic lesion cells (5 × 105 cells/well) were preincubated with HL-004 or CI-976 at indicated concentrations for 24 hr. The medium was changed and the cells were treated with VLDL (100 µg cholesterol/ml) and [14C]oleate (37 MBq/well), and again with HL-004 or CI-976 for 8 hr. Incorporation of [14C]oleate into cellular cholesterol [14C]oleate was measured as described in Materials and Methods. 100% Value was 118.2 nmol/mg cell protein. B, Release of [3H]adenine; Release of [3H]adenine from TPA-treated THP-1 cells (5 × 106 cells/well) treated with HL-004 or CI-976 was measured as described in Materials and Methods. , HL-004-treated cells; , CI-976-treated cells. Values are mean ± S.D. for three experiments. Triplicate determination was measured at each experiment. Data are expressed as mean ± SD values obtained from 3 animals. **P < .01. (Asterisks beside circles, compared with nontreated groups.)

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View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of HL-004 on incorporation of [3H]cholesterol oleate-VLDL into atherosclerotic lesion cells. Atherosclerotic lesion cells (5 × 105 cells/well) were incubated with HL-004 at indicated concentrations for 12 hr. The medium was changed and the cells were treated with [3H]cholesterol oleate-VLDL, and again with HL-004. Incorporation of [3H]cholesterol oleate-VLDL into the cells was measured as described in Materials and Methods. Triplicate determination was measured at each experiment. Data are expressed as mean ± S.D. values obtained from 3 animals. *P < .05.

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View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effect of HL-004 pretreatment on release of radioactivity from atherosclerotic lesion cells loaded with [3H]cholesterol oleate-VLDL. Percentage of released radioactivity to total incorporated radioactivity. Atherosclerotic lesion cells (2 × 106 cells/well) were preincubated with 5% LPDS/DMEM containing HL-004 at indicated concentrations for 12 hr. After that, cells were incubated with 5% LPDS/DMEM containing [3H]cholesterol oleate-VLDL (100 µg total cholesterol/ml) and HL-004 for 24 hr. The cells were washed with 5% LPDS/DMEM and then incubated with 5% LPDS/DMEM containing HDL (0.3 mg protein/ml) and HL-004. , control; , 109 M; , 108 M; , 107 M. Total incorporated and released radioactivity were measured as described in Materials and Methods. Triplicate determination was measured at each experiment. Data are expressed as mean ± S.D. values obtained from 3 animals. *P < .05.

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View larger version (28K): [in this window] [in a new window]   Fig. 8.   Effect of HL-004 treatment on release of radioactivity from [3H]cholesterol oleate-VLDL-loaded atherosclerotic lesion cells. Percentage of released radioactivity to total incorporated radioactivity. Atherosclerotic lesion cells (2 × 106 cells/well) were incubated with 5% LPDS/DMEM containing [3H]cholesterol oleate-VLDL (100 µg total cholesterol/ml) for 24 hr. The cells were washed with 5% LPDS/DMEM and then incubated with 5% LPDS/DMEM containing HDL (0.3 mg protein/ml) and HL-004 at indicated concentrations. , control; , 109 M; , 108 M; , 107 M. Total incorporated and released radioactivity were measured as described in Materials and Methods. Triplicate determination was measured at each experiment. Data are expressed as mean ± S.D. values obtained from 3 animals. *P < .05, **P < .01.

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View larger version (53K): [in this window] [in a new window]   Fig. 9.   Effect of HL-004 on cholesterol ester content in atherosclerotic lesion cells. Atherosclerotic lesion cells (5 × 105 cells/well) were preincubated with HL-004 for 12 hr. The cells were then incubated with VLDL (1 mg cholesterol/ml) for 12 hr. Cholesterol ester content was measured as described in Materials and Methods. Triplicate determination was measured at each experiment. Data are expressed as mean ± S.D. values obtained from 3 animals. **P < .01.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The concentration-dependent effect of HL-004 on rabbit foam cells was almost the same as that on THP-1 derived macrophages (figs. 1 and 3). It was also similar to that on rat macrophages (Murakami et al., 1995b) and smooth muscle cells (Murakami et al., 1995a). These data indicate that the inhibitory effect on ACAT by HL-004 is universal regardless of cell type and origin. From our results, the difference of HL-004 and other ACAT inhibitor (CI-976 in this report) is as follows: (1) the effective concentration of HL-004 was low (10⁹ ~ 10⁷ M), (2) the cell toxicity of HL-004 was low and (3) the effect of HL-004 on incorporation of VLDL, acid cholesterol esterase and neutral cholesterol esterase was very little, whereas CI-976 affected these metabolisms. These data suggested that HL-004 was a unique ACAT inhibitor compared with other ACAT inhibitors.

HL-004 was effective on macrophages and foam cells at a very low concentration (10⁹ ~ 10⁷ M), apparently without any cell toxicity (figs. 1B and 5B). Orally administered ACAT inhibitors generally inhibit ACAT activity of various organs such as the liver and intestine, but the side effect of adrenocortical cytotoxicity has been reported (Dominick et al., 1993). Then, the question arises why HL-004 is free from toxicity. HL-004 is a noncompetitive inhibitor against oleoyl-CoA and its specificity for ACAT is very high because it does not influence the synthesis of phospholipids and triglycerides. If this ACAT inhibitor acts on only ACAT and free cholesterol is transported out of cells, cell toxicity would not occur. To find the differences between both HL-004 and CI-976, the stable structures were obtained by a semiempirical molecular orbital method with P3 approximation (Stewart, 1989a, 1989b) in figure 10. For HL-004, the structure of molecule at grand minimized potential energy was a linear conformation. Phenyl ring and peptide bond might be conjugated but area of delocalization might be smaller compared with that of CI-976. When "S" in HL-004 was changed to "C" and the minimum energy was calculated, the energy of a turned conformation was 1.9 kcal/mol low compared with the energy of a linear conformation. Thus, a presence of "S" may be important for a linear conformation. For CI-976, the structure was folded between peptide bond and alkyl chain. Conjugation of phenyl ring, methoxy group and peptide bond would raise up delocalization of electrons, and then the molecule might be stable. To clear this reason, we calculated the potential energy of grand minimum between a linear conformation and a turned conformation of CI-976. The energy of the turned conformation was 2.1 kcal/mol low. Therefore, the turned conformation was stable compared with a linear conformation. These results suggested that the unique structure of HL-004 indicates that this inhibitor specifically acts on ACAT as a noncompetitive inhibitor and does not affect other proteins related to the free cholesterol transport system.

View larger version (25K): [in this window] [in a new window]   Fig. 10.   The stable structures of ACAT inhibitors obtained by PM3 method. Computation program used was MOPAC Ver. 6 (Stewart, 1989c). Numerals are the change densities calculated by Mulliken's population analysis. , H; shaded circle, C; large dots, O; small dots, O; stipes, S.

Cholesterol efflux was very low compared with that in figures 7 and 8 without HDL (data not shown). This suggested that HL-004 enhanced the HDL-mediated cholesterol efflux from atherosclerotic lesion cells. It is reported that ACAT inhibitors enhanced HDL-mediated cholesterol efflux from macrophages (Furuchi et al., 1993; Schmitz et al., 1988, 1985). In the experiments of figures 7 and 8, a sufficient level of HDL (0.3 mg protein/ml) was added to the medium. In a similar experiment using 10% FBS/DMEM, the total release of cholesterol was low (the released cholesterol was up to ~30% of the total incorporated cholesterol) and there was no statistical difference between control and HL-004-treated foam cells (data not shown). Both cholesterol-releasing pathways from lysosome and cytosol required HDL (figs. 7 and 8). It was recently reported that free cholesterol produced by the addition of ACAT inhibitor induced cell toxicity (Warner et al., 1995). This points out the need for sufficient acceptors for the released cholesterol, such as HDL, to avoid such toxicity by ACAT inhibitors.

Clinically, it is speculated that plaque rupture caused by foam cells containing a lot of cholesterol ester is a trigger of acute coronary syndrome (Davies et al., 1985; Hackett et al., 1988). Our studies showed that HL-004 was effective at low concentrations (10⁹ ~ 10⁷) and might decrease foam cell formation without toxicity. Considering these findings, it seems reasonable to expect that acute coronary syndrome (acute coronary thrombosis, and so on) can be reduced by the use of HL-004 together with a sufficient presence of HDL.