British Journal of Pharmacology (2001) 133, 1005 ± 1012

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Direct eect of an acyl-CoA:cholesterol acyltransferase inhibitor,
F-1394, on atherosclerosis in apolipoprotein E and low density
lipoprotein receptor double knockout mice
Tsuyoshi Chiwata, 1Katsumi Aragane, *,1Koji Fujinami, 1Kazuhiro Kojima, 2Shun Ishibashi,
Nobuhiro Yamada & 1Jun Kusunoki

1
3

1

Central Research Laboratories, Fujirebio Incorporation, 51 Komiya-cho, Hachioji 192-0031, Tokyo, Japan; 2Department ofMetabolic Disease, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113-8655, Japan and 3Metabolism, Endocrinology
and Atherosclerosis, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
1 The acyl-CoA:cholesterol acyltransferase (ACAT) enzyme is thought to be responsible for foamcell formation and the subsequent progression of atherosclerosis. The apolipoprotein E and low
density lipoprotein receptor double knockout (apoE/LDLr-DKO) mouse is an animal model thatdevelops severe hyperlipidaemia and atherosclerosis.
2 Here we have examined the eect of oral administration of an ACAT inhibitor, F-1394, on
atherosclerosis in apoE/LDLr-DKO mice fed a regular chow diet.
3 In en face analysis, a dose of 10, 30, or 100 mg kg71 day71 F-1394 for 10 weeks reduced theextent of lesions visible in the aorta by 24, 28 and 38%, respectively, as detected by staining with oil
red O, without aecting serum cholesterol level in these mice. At the highest dose
100 mg kg71 day71 of F-1394, the reduction was statistically signi®cant.
4 For quantitative analysis of the cellular and non-cellular components comprising the lesions at
the aortic sinus, the eects of an oral dose of 100 mg kg71 day71 F-1394 for 15 weeks were studied.
There was a signi®cant reduction (31.9%) in the oil-red O-stained area in cross-sections of the aortic
sinus. In addition, the neointimal area, as well as levels of ACAT-1 protein tended to be decreased(15.2 and 25.8%, respectively, not signi®cant). However, the areas containing macrophages, smoothmuscle cells, and collagen were not aected by F-1394.
5 In vitro, F-1394 attenuated foam cell formation in mouse peritoneal macrophages.
6 These results indicate that ACAT may be primarily responsible for lipid accumulation inatherosclerotic lesions, and that its inhibition diminishes the lipid deposition via a direct eect onmacrophages in the arterial wall.
British Journal of Pharmacology (2001) 133, 1005 ± 1012
Keywords: ACAT; ACAT-inhibitor; F-1394; atherosclerosis; apolipoprotein E; low density lipoprotein receptor; knockout
mouse; macrophages; foam cells; smooth muscle cells

Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; apoE, apolipoprotein E; EC, esteri®ed cholesterol; FC, freecholesterol; HDL -C, high density lipoprotein cholesterol; LDL, low density lipoprotein; SMC, smooth muscle
cell; TC, total cholesterol; TG, triglyceride

Introduction
Coronary heart disease (CHD) is a major cause of morbidity
and mortality in much of the world (Breslow, 1997). In thepast 10 years, a great deal of information has accumulated on
the molecular mechanisms underlying hypercholesterolaemiainitiated atherosclerosis. In these conditions, low density
lipoprotein (LDL) is thought to be modi®ed in the
subendothelial space, and this modi®ed lipoprotein activates
endothelial cells, which then attract circulating monocytes(Cybulsky & Gimbrone, 1991; Kume et al., 1992; Witztum &Steinberg, 1991). The monocytes enter the vessel wall,dierentiate into macrophages, and then endocytose themodi®ed lipoproteins via a scavenger receptor pathway. This
unrestricted uptake, which is not regulated by the intracellular cholesterol level, eventually leads to the formation of*Author for correspondence; E-mail: kj-fujinami@fujirebio.co.jp

lipid-®lled foam cells ± the initial step in premature atherosclerosis (Brown & Goldstein, 1983; Ross, 1999). In thisprocess, cholesterol esteri®cation via the acyl-CoA:cholesterolacyltransferase (ACAT)-1 enzyme in macrophages is thought
to be a major step in foam cell formation (Brown &Goldstein, 1983; Miyazaki et al., 1998). Thus, inhibition of
ACAT would be expected to improve or limit thedevelopment of atherosclerosis (Matsuda, 1994; Sliskovic &White, 1991). Many types of ACAT inhibitor have therefore
been tested for their direct eect on atherosclerosis
development in dierent atherosclerotic models using various
protocols (Bocan et al., 1991; 1993; Murakami et al., 1995;
Nicolosi et al., 1998). However, these models do not seem
accurately to re¯ect human hyperlipidaemia.
Recently, genetically altered hypercholesterolaemic animalmodels such as apolipoprotein E (apoE) -de®cient mice havebeen produced, which develop a full range of atherosclerotic

1006

T. Chiwata et al

Anti-atherosclerotic effects in hyperlipidemic micelesions (Plump et al., 1992; Zhang et al., 1992), from fatty
streaks to raised ®brous plaques which are similar to thoseseen in humans (Nakashima et al., 1994). Thus, these miceare considered to represent a suitable model to study
atherogenesis, as well as the eectiveness of drugs on disease(Breslow, 1996). To date, however, there have been no studies
investigating the in¯uence of ACAT-1 disruption bypharmacological inhibitors on the development of atherosclerosis in these mouse models.
More recently, apoE and LDL-receptor (LDLr) doubleknockout (apoE/LDLr-DKO) mice have been created(Ishibashi et al., 1994), representing a new mouse model thatdevelops severe hyperlipidaemia and atherosclerosis (Bonthu
et al., 1997). It has been reported that, even on a regularchow diet, the progression of atherosclerosis is usually moremarked in apoE/LDLr-DKO mice than in mice de®cient for
apoE alone (Witting et al., 1999). Thus, the apoE/LDLrDKO mouse is a suitable model in which to study the antiatherosclerotic eect of compounds without having to feed
the animals an atherogenic diet.
Here we have tested the direct eect of (1S,2S)-2-[3-(2,2dimethylpropyl)-2-nonylureido]cyclohexane-1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate(F-1394), which potently inhibits both ACAT-1 and ACAT-2(Kusunoki et al., 1995b), on the development of atherosclerosis in apoE/LDLr-DKO mice fed a regular chow diet.

Tissue preparation and histochemistry

Methods

Images from a cross-section or from the aorta were collected
in one of two ways: either using a microscope (Nikon, Japan)®tted with a 3-CCD Color Camera (KY-F55MP; Victor) or
using a video-microscope (PV-10; Olympus). Quanti®cationof the images was carried out using Mac Scope software(Mitani Corporation) after carefully outlining a speci®c areaof the image.

Animals and diet
ApoE/LDLr-DKO mice were created by mating apoEde®cient mice and LDLr-knockout mice, as described
previously (Ishibashi et al., 1994). The genetic backgroundof these mice was derived from the 129/Ola and C57BL/6J
strains. ApoE/LDLr-DKO mice were maintained in atemperature- and humidity-regulated animal facility(22+28C, 55+15%) with controlled lighting (12-h light/darkcycle). They had free access to both tap water and acommercial regular chow diet (CRF-1; Oriental Yeast)
throughout the study. All experiments were performed on
both male and female mice, and approved by the Animal
Ethics Committee of Fujirebio Incorporated.

Study protocol
To determine how the ACAT enzyme aects atherosclerosis
formation, an orally active ACAT inhibitor, compound F1394 (Fujirebio Inc., Tokyo, Japan), was used to inhibit
ACAT in vivo (Aragane et al., 1998; Kusunoki et al., 1995a).
For oral administration, F-1394 was suspended in 0.5%
sodium carboxymethylcellulose solution.
For en face analysis, the apoE/LDLr-DKO mice weredivided into four groups at 5 weeks of age, and each group
was given F-1394 at a dose of 0, 10, 30, or
100 mg kg71 day71 for 10 weeks. For detailed histologicalanalysis of the aortic sinus, the DKO mice were divided into
two groups at 5 weeks of age, and each group was given F1394 at a dose of 0 or 100 mg kg71 day71 for 10 ± 15 weeks.
At the end of each experiment, blood was collected from theanimals after overnight fasting.
British Journal of Pharmacology vol 133 (7)After the mice were sacri®ced under ether anaesthesia, theheart and the whole aorta were perfused with phosphatebuered formaldehyde (10%, pH 7.4) containing 5% sucrose,
before being removed and ®xed in the same solution.
The whole aorta was used for en face lipid staining(Tangirala et al., 1995). After the adventitia had been
removed, the aorta was cut open longitudinally and stainedwith oil red O to visualize the extent of the lipid deposition.
For the analysis of the aortic sinus, the heart wasembedded in OTC compound (Milles Inc.), and serial crosssections 8 mm thick were cut by cryostat from any lesions
seen in the aortic sinus. The serial sections were stained withoil red O and with Mayer's hematoxylin, Elastica van Gieson,or Sirius red (Junqueira et al., 1979; Kratky et al., 1996) tovisualize lipid, ®brous extracellular matrixes, or collagen(type I, II, and III), respectively. Macrophages, smoothmuscle cells (SMC), and ACAT-1 protein in a given section
were visualized by anti-mouse macrophage monoclonal
antibody (BM8; BMA Biochemicals), anti-human SMC
monoclonal antibody (1A4; Dako), and anti-mouse ACAT1 monoclonal antibody (KF-620; Fujirebio Inc.), respectively.
These immunostained sections were then counterstained withmethyl green.

Evaluation of atherosclerotic lesions

Serum lipid measurement
Serum total cholesterol (TC) and triglyceride (TG) levels were
measured by enzymatic/colorimetric methods (Cholesterol EHA Test Wako; Triglyceride E-HA Test Wako; Wako Pure
Chemical Industries, Osaka, Japan). The high density
lipoprotein cholesterol (HDL-C) level was also determined
enzymatically after selective precipitation of all lipoproteinsexcept HDL (HDL cholesterol Precipitation Reagent Set;
Wako Pure Chemical Industries).

Peritoneal macrophage assay
Human plasma was obtained from healthy volunteers, andLDL (d=1.019 ± 1.063 g ml71) and HDL3 (d=1.125 ±
1.210 g ml71) were isolated by ultracentrifugation as described previously (Havel et al., 1955). Acetylated LDL(acLDL) was prepared according to the method of Basu et al.
(1976). Peritoneal macrophages were collected from male
ddY mice (Sankyo Labo Service), and cells that adhered tothe culture dish were used in this study. Brie¯y, macrophages(26106 cells) were plated and preincubated for 2 h in
Dulbecco's modi®ed Eagle's medium (DMEM) containing
10% foetal bovine serum (FBS, Gibco) and antibiotics. Then,the following 24-h incubations were carried out in DMEM
containing 0.1% bovine serum albumin (BSA): (i) to induce

T. Chiwata et alfoam cells, 25 mg ml71 acLDL was added to the media; and(ii) to determine the eect of ACAT inhibition on foam cell
formation, 25 mg ml71 acLDL and 5 mg ml71 HDL3 with orwithout F-1394 dissolved in dimethylsulfoxide (®nal concentration, 0.1%) was added to the media. After incubation,
intracellular lipids were extracted with n-hexane/2-isopropanol (3 : 2, v v71), and TC and free cholesterol (FC) were
determined enzymatically. Esteri®ed cholesterol (EC) was
calculated by subtraction of FC from TC.

Statistical analysis
The results are expressed as mean+standard error (s.e.mean).
Statistical dierences among the groups were assessed using
Williams' multiple range test, and dierences between groups
were assessed using Student's t-test or Aspin ± Welch's test

Anti-atherosclerotic effects in hyperlipidemic mice

1007

(SAS software package). Values of P50.05 were considered
to be signi®cant.

Results
En face analysis of the aorta
In the en face lipid staining analysis, oil red O stained
8.01+1.41% of the luminal surface of the whole aorta in thecontrol group. In contrast, administration of F-1394
diminished lipid deposition in the aorta in a dose-dependent
manner; at the highest dose of F-1394, lipid deposition was
signi®cantly reduced by 38.1% (Figure 1). The mean serum
TC level in the control group was 732+44 mg dl71, which
was not aected by the administration of F-1394.

Atherosclerosis at the aortic sinus

Figure 1 Quantitative analysis of en face lipid-stained area in whole
aorta from apoE/LDLr DKO-mice at 15 weeks of age. Each column
is the mean+s.e.mean of results from eight mice. Statistical analysis
was performed by Williams' multiple range testing. #P50.10;*P50.05 vs control.

Quantitative analysis of the atherosclerotic lesions found in
the aortic sinus in the control group determined theneointimal area to be approximately 3206103 mm2 and4906103 mm2 at 15 and 20 weeks of age, respectively (Figure2A). In the F-1394-treated mice, the neointimal area wasslightly reduced (by 8.4 and 15.2% at 15 and 20 weeks of age,respectively), but this dierence did not reach statisticalsigni®cance. F-1394 treatment reduced the size of the area
stained with oil red O by 30.7% (P=0.217) and 31.9%(P=0.027) in mice at 15 and 20 weeks of age, respectively(Figure 2B); this dierence did reach statistical signi®cancefor the 20-week-old group. ACAT protein in the lesions was
located in an area of 94.9+10.96103 mm2 in sections fromthe control group. F-1394 treatment reduced ACAT proteinin the lesions by 26%, but this dierence did not reachsigni®cance (P=0.1120) (Table 1). The other lesion components that we examined, namely macrophages, SMC, andcollagen, were not altered by F-1394 treatment (Table 1).
Typical cross-sections of the aortic sinus from control and
F-1394-treated mice are shown in Figure 3. Lipid staining
was con®ned to the subendothelial space in sections from

Figure 2 Quantitative analysis of the extent of neointima (A) and oil red O-positive area (B) in the aortic sinus from apoE/LDLrDKO mice. Each column is the mean+s.e.mean of results from 13 ± 14 mice. Statistical analysis was performed by Student's t-test.
*P50.05 vs corresponding control.
British Journal of Pharmacology vol 133 (7)1008

T. Chiwata et al

Anti-atherosclerotic effects in hyperlipidemic mice

Figure 3 Photomicrographs showing serial cross-sections of aortic sinus from apoE/LDLr-DKO mice at 20 weeks of age. Leftside, control group (A, C, E, G and I); right side, F-1394-treated group (B, D, F, H and J). A and B, macrophage (BM8); C and D,
neutral lipid (oil red O stain); E and F, ACAT (KF-620); G and H, collagen (Sirius red); I and J, SMC (1A4). Scale is 100 mm forall photographs. Magni®cation,625.

the F-1394-treated group, whereas lipid deposition was
clearly present throughout the entire neointima in thecontrol group. Macrophages, neutral lipids and ACAT
protein were colocalized in these sections. In contrast,
collagen was not colocalized with these components in
British Journal of Pharmacology vol 133 (7)lesions from either group. SMC were just visible in thelesions from either group.
In control mice at 20 weeks of age, serum TC, non HDLC, HDL-C, and TG levels were 538+30, 525+29, 13+2 and69+7 mg dl71, respectively. The repeated administration of

T. Chiwata et al

Anti-atherosclerotic effects in hyperlipidemic mice1009

treated mice displayed no abnormalities, such as alopecia ordermal hypertrophy during the dosing period.

Foam cell formation in mouse peritoneal macrophages
Figure 4 shows the eect of F-1394 on the cholesterol content
of foam cells. In normal macrophages, the intracellularconcentrations of TC, FC, and EC were 61.5+2.6, 61.5+1.8,
and 0.0+0.6 nmol mg protein71, respectively. In acLDLinduced foam cells, the concentrations of TC and FC were2.02 fold and 1.54 fold higher, respectively, and the EC
content in particular was increased markedly (to29.7+1.0 nmol mg protein71). HDL signi®cantly decreased
the concentrations of TC and EC (by 18.0 and 37.4%,
respectively), but not the FC concentration found in acLDLinduced foam cells. Moreover, further reductions in TC andEC concentrations were observed in the HDL/F-1394-treatedfoam cells; the reduction observed was dependent on theconcentration of F-1394. At the highest concentration of F1394 used (1.0 mmol l71), the concentrations of TC and EC
were 61 and 12.3%, respectively, of the concentrations seen in
the foam cells treated only with HDL. The FC concentration
was not aected by F-1394 at 0.3 mmol l71 or less, but wassigni®cantly reduced (by 12.3%) following treatment with F1394 at 1.0 mmol l71.

Discussion

Figure 4 Eect of F-1394 on accumulation of TC (A), EC (B), andFC (C) in mouse peritoneal macrophages. Left columns: N, normal
macrophages; C, control macrophages (foam cells); H, HDL-treatedcontrol macrophages; right columns; F-1394- and HDL-treatedcontrol macrophages. Each column is the mean+s.e.mean of resultsfrom triplicate assays. Statistical analysis was performed by Student's
t-test or Aspin ± Welch's test. ##P50.01; ###P50.001 vs control
macrophages (C). *P50.05, **P50.01, ***P50.001 vs HDL-treatedcontrol macrophages (H).

F-1394 did not aect these values. In mice at 15 weeks of age,the levels of most of these serum lipids were much greaterthan at 20 weeks, but there were no dierences in levels
between control and F-1394-treated mice (Table 2).
At the end of the F-1394 dosing period (at 20 weeks ofage), the mean body weight of both control and F-1394treated mice was 22 g (Table 2). Moreover, the F-1394-

In preliminary studies, early small lesions comprising only offoam cells in the aortic sinus were observed in some of the
apoE/LDLr-DKO mice at 5 weeks of age, and these lesions
stained with oil red O (data not shown). Therefore we started
administration of F-1394 to the mice at 5 weeks of age.
As expected, F-1394 signi®cantly reduced the extent of en
face lipid staining in the whole aorta in a dose-dependent
manner (Figure 1). In addition, F-1394 signi®cantly decreased
lipid deposition detected by oil red O in the neointima of theaortic sinus (Figure 2B). In general, ACAT inhibitors are
predicted to have an anti-atherosclerotic eect because
ACAT-1 catalyzes both cholesterol esteri®cation in macrophages and the subsequent formation of foam cells ± adistinctive feature of premature atherosclerosis (Matsuda,1994). We previously reported that F-1394 strongly inhibited
ACAT activity in a murine macrophage cell line (Kusunoki etal., 1995b). In the present study, we found that F-1394
decreased foam cell formation in mouse peritoneal macrophages in a dose-dependent manner in vitro (Figure 4).
Furthermore, a previous pharmacokinetic study had shownthat the dose of F-1394 administered to mice resulted in asuciently high concentration in the circulation to exert theseeects in vivo (unpublished data). These ®ndings suggest thatthe bene®cial eect of F-1394 documented in this study is due
to diminished ACAT-1 activity in macrophages, decreased
foam cell formation, and a subsequent reduction in lipid
deposition on the arterial wall. Moreover, this does not seemto be due to a hypolipidaemic eect of F-1394, but to a direct
action at the arterial wall, because the drug did not aect thelevels of any serum lipids (Table 2).
Previous investigators have proposed that ACAT inhibitors
may have a direct action on atherosclerosis in a rabbit modelin which atherosclerosis is induced by an atherogenic diet
British Journal of Pharmacology vol 133 (7)1010

T. Chiwata et al

Anti-atherosclerotic effects in hyperlipidemic mice

Table 1 Quantitative analysis of lesion components in the aortic sinus of apoE/LDLr-DKO mice at 20 weeks of age
Group

n

Macrophage

SMC

Control
F-1394, 100 mg kg71
P-value14
13

30.9+3.6
30.9+6.6
0.998360.3+0.67
54.6+0.93
0.6200ACATCollagen94.9+10.9
70.4+10.0
0.112021.8+6.1
21.0+6.7
0.8067(6103 mm2)

Macrophage, SMC, and ACAT area was determined by computerized image analysis of cross-sections stained with speci®c probes. For
collagen area, Sirius Red-stained sections were used in the same system. Data are the mean+s.e.mean; statistical analysis was
performed by Student's t-test.

Table 2 Body weight and serum lipid levels in apoE/LDLr-DKO miceAge
15 weeks
20 weeks

GroupnBody weight(g)TC(mg dl71)

TG(mg dl71)

HDL-C(mg dl71)

nonHDL-C(mg dl71)

Control
F-1394, 100 mg kg71
Control
F-1394, 100 mg kg71

14
13
14
13

20+0
19+1
22+1
22+1

858+43
797+89538+30530+36

149+17155+21
69+7
80+9

12+1
14+1
13+2
17+2

846+43
783+88525+29513+35TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; nonHDL-C, non-high-density lipoproteincholesterol; The program of oral administration of F-1394 or its vehicle was started in the mice at 5 weeks of age. Data are themean+s.e.mean.

(Bocan et al., 1991; 2000; Tanaka et al., 1994). However,apart from the work of Matsuo et al. (1995), these models do
not necessarily re¯ect the actual hypercholesterolaemic
conditions seen in patients with atherosclerosis. Matsuo etal. (1995) have reported an anti-atherosclerotic eect of an
ACAT inhibitor, FR-145237, which reduced the lesion index
at coronary arteries in rabbits with spontaneous hypercholesterolaemia that was not induced by a hypercholesterolaemic diet. Although this model closely re¯ects thehypercholesterolaemia seen in humans, these authors did
not carry out a detailed analysis of the lesions. Thus, little is
known about the changes induced by ACAT inhibitors in thecomponents of lesions in the face of persistent hypercholesterolaemia. Therefore, in the present study, we adopted a`human-like' protocol using a spontaneous hypercholesterolaemic mouse model on a regular chow diet to evaluate thedirect anti-atherosclerotic eect of the ACAT inhibitor F1394, and we also performed a detailed histochemical analysisof the lesions, which are similar to those seen in humans(Nakashima et al., 1994).
Although we found no statistically signi®cant dierences,
the neointimal area and amount of ACAT protein werereduced by 15.2 and 26%, respectively, in the F-1394-treatedgroup. However, the levels of macrophages, SMC, andcollagen ®bres (types I, II and III) were not aected. Thereis a number of possibilities to explain the reduced lesion sizein F-1394-treated mice. ACAT inhibition might cause
atrophy of the macrophages/foam cells, or might aect theextracellular matrix, which would not have been detected by
the dye used. Alternatively, ACAT inhibition might prevent
migration into the lesions of the other cellular components,
which are attracted by factors released from foam cells. In
addition, the reduced expression of ACAT-1 protein in thelesions might be attributable to attenuation of foam cell
formation by F-1394 treatment in such lesions, but theunderlying mechanisms remain unclear. It has been reported
that ACAT-1 mRNA and protein are induced duringdierentiation of monocytes into macrophages, but expresBritish Journal of Pharmacology vol 133 (7)sion of these products is not induced by foam cell formationin macrophage in vitro (Miyazaki et al., 1998; Wang et al.,1996). It could be that BM8, the anti-macrophage antibody
used here, stains not only macrophages abundantly expressing the ACAT-1 protein at the lesions, but also monocytes
which would not express this protein. Additionally, attenuation of foam cell formation by ACAT inhibition may cause adelay in the dierentiation of macrophages from monocytes
and, consequently the number of monocytes is increased at
the lesions. Therefore, it might be that ACAT protein is infact reduced, but the number of BM8-positive cells, bothmonocytes and macrophages, is not changed by F-1394treatment. Furthermore, in our preliminary experiments,
ACAT protein expression was induced in human vascularsmooth muscle cells (VSMC) by cholesterol loading but not
in rat VSMC (unpublished data). Hence, regulation of ACAT
expression may be complex and dier under various
conditions, depending on species, tissue examined, etc.
In this study, the body weights of the mice increased
normally and there were no signi®cant dierences between
the control group and the F-1394-treated group at either 15
or 20 weeks of age (Table 2). Moreover, the mice did not
develop skin diseases, such as dermal hypertrophy oralopecia. Recently, it was reported that an atherogenic diet
induced severe toxicity in both ACAT-disrupted apoE- andLDLr-knockout mice. It was proposed that a complete lackof ACAT-1 in macrophages in mice with severe hypercholesterolaemia leads to a massive accumulation of FC andsubsequent in¯ammation in tissues such as brain, skin, eyeand aortic wall (Accad et al., 2000; Yagyu et al., 2000). In
our study, the serum cholesterol level in the apoE/LDLrDKO mice would have been much lower than in apoE- or
LDLr-de®cient mice fed an atherogenic diet; this may be why
toxic changes were not observed in our double-knockout
mice. However, we have found that the ACAT inhibitor F1394 did not induce toxicity in apoE-de®cient mice fed awestern-type diet, although it did have an anti-atheroscleroticeect (unpublished data). Thus, we should emphasize that

T. Chiwata et al

partial inhibition of ACAT-1 enzyme activity by pharmacological compounds does not cause such toxic alterations as
the severe dermal xanthomas observed when there iscomplete disruption of the enzyme activity.
Esteri®ed cholesterol deposition mediated by the ACAT
pathway is thought to be one of the major factors involved inlesion stability in rupture-prone plaques in the coronary
artery in humans (Davies, 1996; Falk et al., 1995; Libby,
1995). We have shown here that excess deposition of neutral
lipids or EC is signi®cantly reduced by ACAT inhibition invivo (Figures 1, 2B and 3D) and in vitro (Figure 4). Thus, we

Anti-atherosclerotic effects in hyperlipidemic mice

1011

postulate that F-1394 might prevent the disruption of
unstable plaques as a result of a decrease in EC deposition
in foam cells at the coronary arterial wall.
In summary, ACAT inhibition by treatment with F-1394
diminished both lipid deposition and lesion size withoutaecting the serum cholesterol level, indicating that the
ACAT enzyme is involved in foam cell formation and lesion
development. These results imply that F-1394 exerts its eects
directly on the arterial wall. Thus, ACAT inhibition by F1394 treatment is a potential new therapeutic strategy foracute coronary syndromes.

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(Received December 19, 2000
Revised May 10, 2001
Accepted May 10, 2001)