Rosuvastatin Activates ATP-Binding Cassette Transporter

A1Dependent Efflux Ex Vivo and Promotes Reverse

Cholesterol Transport in Macrophage Cells in Mice Fed
a High-Fat Diet
Tomohiko Shimizu, Shin-ichiro Miura, Hiroyuki Tanigawa, Takashi Kuwano, Bo Zhang,
Yoshinari Uehara, Keijiro Saku
ObjectiveIt is controversial whether statins improve high-density lipoprotein (HDL) function, which plays an important
role in reverse cholesterol transport in vivo. The aim of the present study was to clarify the effects of rosuvastatin and
atorvastatin on reverse cholesterol transport in macrophage cells in vivo and their underlying mechanisms.
Approach and ResultsMale C57BL mice were divided into 3 groups (rosuvastatin, atorvastatin, and control groups)
and orally administered rosuvastatin, atorvastatin, or placebo for 6 weeks under feeding with a 0.5% cholesterol+10%
coconut oil diet. After administration, although there were no changes in plasma HDL cholesterol levels among the
groups, plasma from the rosuvastatin group showed an increased ability to promote ATP-binding cassette transporter A1
mediated cholesterol efflux ex vivo. In addition, capillary electrophoresis revealed a shift in HDL toward the pre- HDL
fraction only in the rosuvastatin group. Mice in all 3 groups were intraperitoneally injected with 3H-cholesterollabeled
and cholesterol-loaded macrophages and then were monitored for the appearance of 3H-tracer in plasma and feces. The
amount of 3H-tracer excreted into feces during 48 hours in the rosuvastatin group was greater than that in the control
group. Finally, 3H-cholesteryl oleate-HDL was intravenously injected into all groups, blood samples were taken, and
the count of 3H-cholesterol was analyzed. Plasma 3H-cholesteryl oleate-HDL changed similarly, and no differences in
fractional catabolic rates were observed.
ConclusionsRosuvastatin enhanced the ATP-binding cassette transporter A1dependent HDL efflux function
of reverse cholesterol transport, and this finding highlights the potential of rosuvastatin for the regression of
atherosclerosis.(Arterioscler Thromb Vasc Biol. 2014;34:2246-2253.)
Key Words: ABC transporters lipoproteins, HDL macrophages statins

higher level of serum low-density lipoprotein cholesterol (LDL-C) is a major risk factor for atherosclerosis,
which is a cause of cardiovascular disease. Several clinical
trials have demonstrated that cholesterol-lowering therapy
with statin reduces the risk of cardiovascular events.1,2 On
the contrary, a lower level of serum high-density lipoprotein
cholesterol (HDL-C) is considered to be independently and
inversely associated with the risk of cardiovascular disease,3
and in addition to a lower LDL-C level, an increase in HDL-C
has been suggested to be a secondary lipid target for reducing
the risk of cardiovascular disease.4 Even in patients who are
treated aggressively with statins to reduce LDL-C levels <70
mg/dL, low levels of HDL-C remain a significant predictor of
major cardiovascular events.5
Statins are becoming increasingly recognized for their pleiotropic effects, which include anti-inflammation, antioxidant,

vasodilation, improved endothelial function, and stabilization

of atherosclerotic plaque. Statins promote the regression of
atherosclerotic plaque in the carotid artery, coronary artery,
and thoracic aorta6,7 through their ability to reduce LDL-C
in addition to pleiotropic effects. Little is known about their
effects on reverse cholesterol transport (RCT) in macrophage
cells, which is the main function of HDL. Thus, it is controversial whether they improve HDL function, which plays an
important role in macrophage RCT, whereas statins significantly decrease LDL-C.
RCT is the physiological process by which excess cholesterol in peripheral tissues is excreted to outside of the body.8
The process of RCT can be divided into 3 stages. First, cellular cholesterol is effluxed from peripheral cells to HDL,
and macrophage ATP-binding cassette transporter (ABC) A1,
ABCG1, and scavenger receptor class B type I (SR-BI) play

Received on: August 14, 2013; final version accepted on: July 23, 2014.
From the Departments of Cardiology (T.S., S.M., H.T., T.K., Y.U., K.S.), Molecular Cardiovascular Therapeutics (S.M., Y.U., K.S.), Biochemistry (B.Z.),
and Advanced Therapeutics for Cardiovascular Disease (H.T., K.S.), Fukuoka University School of Medicine, Fukuoka, Japan.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.303715/-/DC1.
Correspondence to Shin-ichiro Miura, MD, Department of Cardiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka
814-0180, Japan. E-mail miuras@cis.fukuoka-u.ac.jp
2014 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org

DOI: 10.1161/ATVBAHA.114.303715

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at BIREME / UNIFESP on February 26, 2016
2246

Shimizu et al Rosuvastatin Promotes RCT 2247

did not show significant changes in plasma total cholesterol,
LDL-C, or nonHDL-C levels before and after 6 weeks. Both
the rosuvastatin and atorvastatin groups also did not show
changes of the levels before and after treatment. In addition,
there were no significant changes in the levels of HDL-C in
all 3 groups before and after 6 weeks. However, there were
significant changes in plasma triglyceride levels after treatment. There was a significant increase in the triglyceride level
at 6 weeks in the control group, whereas neither rosuvastatin
nor atorvastatin changed the triglyceride level, indicating that
statins prevented the increase in the triglyceride level at 6
weeks.

Nonstandard Abbreviations and Acronyms

ABC
ApoA-I
CE
HDL
HDL-C
LCAT
LDL-C
RCT
SR-BI

ATP-binding cassette transporter

apolipoprotein A-I
cholesteryl ester
high-density lipoprotein
high-density lipoprotein cholesterol
lecithin-cholesterol acyltransferase
low-density lipoprotein cholesterol
reverse cholesterol transport
scavenger receptor class B type I

important roles. Second, HDL-C is transported in blood to the

liver, and the cholesterol in HDL is converted to cholesteryl
ester (CE) by the enzyme lecithin-cholesterol acyltransferase
(LCAT) and carried as CE in the core of the HDL particle to
the liver. Finally, CE is excreted from the liver into bile, either
directly or after conversion to bile acids.
Atorvastatin and rosuvastatin are well known to be the most
effective statins for lowering LDL-C levels. Several clinical
trials have reported that aggressive lipid-lowering therapy not
only suppresses the progression of atherosclerosis, but also
contributes to its regression.911 Although the administration of
rosuvastatin and atorvastatin resulted in a significant regression of coronary atherosclerosis12 and significantly increased
HDL-C levels,1216 the effects of statins on the 3 stages of RCT
in vivo have not yet been reported. We hypothesized that these
strong statins might promote the expression of several factors
involved in RCT independent of lowering LDL-C, and there
might be differences in the effects of rosuvastatin and atorvastatin on RCT. Therefore, the aim of the present study was to
clarify the effects of rosuvastatin and atorvastatin in an in vivo
macrophage RCT system and their underlying mechanisms.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Rosuvastatin, but Not Atorvastatin, Increased

Pre- HDL Without Decreasing LCAT Activity
Next, we analyzed HDL subfractions as assessed by capillary isotachophoresis on a Beckman P/ACE MDQ system
(Beckman-Coulter, Tokyo, Japan) as described previously,17
as shown in Figure1A and 1B. Capillary isotachophoresis is
a technique for analyzing charge-based lipoprotein subfractions directly in plasma.18,19 Capillary isotachophoresis can
separate plasma lipoproteins into 3 HDL subfractions (fast-,
intermediate-, and slow-migrating HDL) according to their
electrophoretic mobilities. The rosuvastatin group, but not the
atorvastatin and control groups, showed a significant increase
in pre- HDL, which indicates peaks in slow-migrating HDL,
after administration. The absolute amounts of slow-migrating
HDL in each group were analyzed at weeks 0 and 6. There
were no significant differences among the 3 groups at week
0. At 6 weeks, the rosuvastatin group showed a significant
increase in the percentage of the slow-migrating HDL fraction
compared with the atorvastatin and control groups. Because
plasma LCAT is critical for HDL maturation, we analyzed
whether these changes were influenced by LCAT activity
(Figure1C). There were no differences in the activity of LCAT
between the groups, indicating that rosuvastatin increased pre HDL without decreasing LCAT activity.

Rosuvastatin, but Not Atorvastatin, Decreased

mRNA Levels of Apolipoprotein A-I in the liver

Rosuvastatin and Atorvastatin Prevented the

Increase in Triglyceride Without Changes
in HDL-C, LDL-C, or nonHDL-C
Changes in the lipid profile before and after 6 weeks of statin
administration are shown in the Table. The control group

We isolated the livers from mice in the rosuvastatin, atorvastatin, and control groups at 6 weeks and analyzed mRNA
expression levels of various factors (Figure2). Rosuvastatin and
atorvastatin had no significant effects on mRNA levels of SR-BI

Table 1. Lipid Profile in Rosuvastatin, Atorvastatin, and Control Groups at 0 and 6 Weeks
Group

Weeks

Rosuvastatin (n=8)

TC, mg/dL

HDL-C, mg/dL

LDL-C, mg/dL

Triglyceride, mg/dL

NonHDL-C, mg/dL

9512

5116

4110

218

459

8620

4410

4614

2110*

4222

Atorvastatin (n=8)

9512

5114

4011

237

4512

9311

439

457

308*

508

Control (n=7)

9914

5115

4311

2411

488

10114

5015

4210

4511

5110

HDL-C indicates high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; and TC, total cholesterol.
*P<0.05 vs control group at weeks 6.
P<0.05 vs control group at weeks 0.

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2248 Arterioscler Thromb Vasc Biol October 2014

atorvastatin group (Figure2E). On the contrary, there were no
significant differences in ABCG5 or sterol regulatory elementbinding protein 2 among the 3 groups (Figure2G and 2H).

Rosuvastatin, but Not Atorvastatin, Increased

Cholesterol Efflux via ABCA1 Ex Vivo
We examined whether cholesterol efflux via the ABCA1
pathway was enhanced by rosuvastatin, because rosuvastatin
increased pre- HDL after treatment. Pooled plasma from mice
that had been treated with rosuvastatin, atorvastatin, or placebo
for 6 weeks was used for ex vivo cholesterol efflux studies
with bone marrow macrophage from mice and J774 macrophages. Before the experiments, apolipoprotein Bcontaining
protein was excluded from pooled plasma. For the preparation
of apolipoprotein Bdepleted plasma, plasma was mixed with
phosphotungstic acid and magnesium chloride. As shown
in Figure3A to 3C, we measured plasma HDL-Cmediated
efflux in bone marrow macrophage with or without probucol,
which specifically inhibits ABCA1. Plasma from the rosuvastatin group had significantly greater cellular cholesterol efflux
capacity than plasma from the atorvastatin and control groups
(Figure3A). However, no significant difference was seen with
the use of probucol (Figure3B). In addition, plasma from the
rosuvastatin group had a significantly greater capacity to promote ABCA1-specific efflux than plasma from the atorvastatin
and control groups (Figure3C). Similar results were observed
regarding cholesterol efflux using J774 cells. The rosuvastatin
group had significantly greater total efflux than the atorvastatin and control groups (Figure3D). In addition, with J774
cells in which ABCA1 was stimulated by 3-5-cAMP, total
efflux in the rosuvastatin group was significantly greater than
those in the atorvastatin and control groups (Figure3E).

C
Activity (nmol/ml/hr)

400

LCAT

300
200

Rosuvastatin, but Not Atorvastatin,

Promoted Macrophage RCT In Vivo

100
0

rosuvastatin atorvastatin

control

Figure 1. A, Lipoprotein subfractions as assessed by capillary

isotachophoresis (cITP) in plasma taken from wild-type (WT)
mice before and after the administration of statins. The cITP
technique separates plasma lipoproteins into 3 high-density
lipoprotein (HDL) subfractions (fast-, intermediate-, and slowmigrating HDL [fHDL, iHDL, and sHDL]) according to their electrophoretic mobilities. Dotted squares indicate pre- HDL which
shows peak in sHDL. B, Absolute amount of sHDL as pre- HDL
in plasma taken from WT mice before (week 0) and after (week
6) the administration of statins. The levels of cITP HDL subfractions can be determined as peak areas relative to that of an
internal marker. *P<0.05 vs atorvastatin and control groups at 6
weeks. C, Activity of lecithin-cholesterol acyltransferase (LCAT)
in plasma at week 6.

and ABCA1 (Figure2A and 2B). The mRNA levels of liver

X receptor, farnesoid X receptor, apolipoprotein A-I (ApoA-I),
and ABCG8 in the liver in the rosuvastatin group were significantly lower than those in the control group (Figure2C2F).
The atorvastatin group also showed a significant reduction in
liver X receptor mRNA expression compared with the control
group (Figure2C). Furthermore, ApoA-I mRNA expression
in the rosuvastatin group was significantly less than that in the

We investigated the differences in the effects of rosuvastatin

and atorvastatin on RCT in macrophage cells. As shown in
Figure4A, after 3H-cholesterollabeled J774 cells were injected
into the peritoneal cavity of mice, the 3H-cholesterol counts in
plasma (3H-plasma) were determined and expressed as a percentage of the total label injected (percentage of injection).
There was no difference in the percentage of injection at each
time point between the atorvastatin and control groups, whereas
3
H-plasma in the rosuvastatin group was significantly greater at
all time points compared with those in the atorvastatin and control
groups. However, there was no difference in 3H-Liver between
the rosuvastatin and atorvastatin groups (Figure4B). Although
there was no difference in 3H-Liver between the rosuvastatin
and control groups, 3H-Liver in the atorvastatin group was significantly lower than that in the control group. Furthermore, the
rosuvastatin group had significantly higher 3H-cholesterol levels
in 3H-Feces than the atorvastatin and control groups (Figure4C).

Rosuvastatin, but Not Atorvastatin,

Increased Cholesterol Excretion From the
Liver Without Increasing Turnover
Finally, we investigated the effects of statins on the turnover and fecal excretion of HDL-derived cholesterol. Plasma

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Shimizu et al Rosuvastatin Promotes RCT 2249

A
1.5

SR-B1 mRNA expression

ABCA1 mRNA expression

1.5

1.5

1.0

1.0

LXR mRNA expression

2.0

(AU)

(AU)

(AU)

1.0
0.5

0.5

0.0

1.5

(AU)

1.0

rosuvastatin atorvastatin

control

FXR mRNA expression

0.0

rosuvastatin

E
1.5

atorvastatin

control

Apo A-1 mRNA expression

(AU)

G
1.5

control

0.0

rosuvastatin atorvastatin

control

ABCG8 mRNA expression

1.5

1.0

0.5

rosuvastatin atorvastatin

0.0

(AU)

1.0

0.5

0.0

0.5

0.5

rosuvastatin atorvastatin

ABCG5 mRNA expression

H
2.0

control

0.0

rosuvastatin atorvastatin

control

SREBP-2 mRNA expression

(AU)

(AU)

1.5

1.0

1.0

0.5

0.0

0.5

rosuvastatin atorvastatin

control

0.0

rosuvastatin atorvastatin

control

Figure 2. Effects of statins on mRNA expression levels of various factors in mouse hepatocytes (n=7 in each group). Scavenger receptor
class B type I (SR-BI; A), ATP-binding cassette transporter A1 (ABCA1; B), liver X receptor (LXR; C), farnesoid X receptor (FXR; D), apolipoprotein A-I (ApoA-I; E) ABCG8 (F), ABCG5 (G), and sterol regulatory element-binding protein 2 (SREBP-2; H). *P<0.05. AU indicates
arbitrary units. All values represent the meanSD of 3 replicates from each liver.

H-cholesteryl oleate-loaded HDL was decreased equally in

all 3 groups (Figure5A), consistent with the results regarding
fractional catabolic rate (Figure5B). On the contrary, there
were differences in fecal excretion. The rosuvastatin group
showed a significant increase in 3H-cholesterol in feces compared with the atorvastatin and control groups (Figure5C).
Interestingly, the rosuvastatin group showed a significant
increase in the fecal excretion of 3H-free-cholesterol, but not
3
H-bile acids (Figure5D and 5E). These results indicate that
rosuvastatin, but not atorvastatin, enhanced the excretion of
HDL-derived cholesterol into feces and support the finding
that rosuvastatin promoted RCT in macrophage cells. This
is consistent with the notion that rosuvastatin increases the
excretion of cholesterol from the liver.
3

Discussion
In the present study, rosuvastatin, but not atorvastatin,
increased macrophage-to-HDL RCT by activating cholesterol
efflux via ABCA1. Furthermore, rosuvastatin increased liverto-feces RCT by promoting the fecal excretion of HDL-derived
cholesterol. This difference in the effects of rosuvastatin and
atorvastatin may promote the regression of atherosclerosis.
We clarified the differences in RCT in vivo between treatment with rosuvastatin and atorvastatin in mice. There are 3

key steps in RCT: cholesterol efflux from macrophage cells

to plasma HDL acceptors, uptake from plasma to the liver
by HDL-C, and HDL-derived cholesterol excretion from the
liver to bile. In the overall mechanism of RCT, rosuvastatin
activates both the extraction of cholesterol from peripheral
macrophage cells and the excretion of cholesterol from the
liver into feces or bile. Rosuvastatin increased both cholesterol efflux via ABCA1 ex vivo and cholesterol excretion from
the liver without increasing turnover. These results suggest a
mechanism for the regression of atherosclerosis independent
of lowering LDL-C and have implications for our understanding of the effects of rosuvastatin on RCT.
With regard to the mechanism by which rosuvastatin activates
RCT, not only it can activate cholesterol efflux from peripheral
macrophage cells, but it can also increase the excretion of cholesterol into feces. As the same conditions as the occasion where
cholesterol efflux from a peripheral macrophage is equivalent
(turnover study; Figure5), we observed cholesterol excretion
into feces after the intravenous injection of HDL containing an
equivalent amount of cholesterol. As a result, cholesterol excretion into feces was increased as in the RCT study (Figure4). If
cholesterol transfer to the liver by HDL is increased, cholesterol
excretion into feces would also be increased. Unexpectedly, there
were no differences in cholesterol transfer to the liver by HDL

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2250 Arterioscler Thromb Vasc Biol October 2014

15
10
5
0

rosuvastatin atorvastatin

Cholesterol Efflux, %

10

6
4
2
0

control

rosuvastatin atorvastatin

Total efflux cAMP(-)

*
4

rosuvastatin atorvastatin

ABCA1 dependent efflux

10

ABCA1 independent efflux

Cholesterol Efflux, %

Cholesterol Efflux, %

20

Cholesterol Efflux, %

Total efflux

6
4
2
0

control

rosuvastatin atorvastatin

control

ABCA1 stimulated efflux cAMP(+)

Cholesterol Efflux, %

10

6
4
2
0

control

rosuvastatin atorvastatin

control

Figure 3. Effects of statins on cholesterol efflux ex vivo. Cultured bone marrow macrophages or J774 macrophages were incubated for
20 hours with/without probucol or cAMP. Cellular cholesterol efflux was determined for 4 hours in the presence or absence of 2% pooled
plasma that excluded apolipoprotein Bcontaining protein taken from wild-type mice after 6 weeks of statin administration. Total (without
probucol; A), ATP-binding cassette transporter A1 (ABCA1)-independent (with probucol; B), and ABCA1-dependent efflux (C) in bone
marrow macrophage cells with acetylated low-density lipoprotein (ac-LDL). Total (without cAMP; D) and ABCA1-stimulated (with cAMP;
E) efflux in J774 macrophages with ac-LDL. *P<0.05.

between the groups after the intravenous injection of 3H-labeled

cholesteryl-loaded HDL, which indicated that rosuvastatin did
not influence the transfer of HDL from peripheral tissue to the
liver (Figure5). Although cholesterol in feces was increased
(Figure4), there were no differences in the attenuation of cholesterol in blood after intravenous injection. This observation was
important. Specifically, rosuvastatin did not alter blood cholesterol levels. Cholesterol that is transported to the liver might be
excreted into bile as free cholesterol, without being metabolized
to bile acid. Thus, it is not possible to obtain a final conclusion
from these results.
Recently, Le May et al20 reported that transintestinal cholesterol excretion is an important pathway for hepatobiliary secretion by statin. Little is known about the effects of statins on the
intestine. Transporters, ABCG5/8 and ABCB1a, in addition
to Niemann-Pick C1-Like 1 and LDL receptor, significantly

atorvastatin

% of injection/ml

H-Plasma

rosuvastatin

control

4
2
0

10

20

30

Time(hrs)

40

50

2
1

rosuvastatin atorvastatin

H-Feces

H-Liver

% CPM injected

% CPM injected

contribute to transintestinal cholesterol excretion. Although

we analyzed mRNA levels of ABCG5, ABCB1a, NiemannPick C1-Like 1, and LDL receptor in the intestine after statin
treatment (Figure I in the online-only Data Supplement), there
were no significant changes in the levels of these mRNAs. In
addition, we also examined whether statins effect an intestinal
absorption of cholesterol. Although we performed an additional experiment, there was no difference in the intestinal
absorption of cholesterol between rosuvastatin and atorvastatin (Figure II in the online-only Data Supplement). We could
not explain transintestinal cholesterol excretion with respect
to the mechanism of the differential effects of RCT between
rosuvastatin and atorvastatin.
There are several clinical reports that the administration of
statins increases HDL-C.1316 Unlike these clinical results, a
significant increase in plasma HDL-C was not seen after the

control

rosuvastatin atorvastatin

control

Figure 4. Effects of statins on reverse cholesterol transport (RCT) in mice. C57BL6 (wild type) mice were fed a high-cholesterol diet for 2
weeks and then divided into rosuvastatin, atorvastatin, and placebo control groups (n=7 each). The respective drugs were administered
orally for 6 weeks, and RCT was studied as described in the Materials and Methods in the online-only Data Supplement. 3H-cholesterol
in plasma after macrophage injection (A), 3H-cholesterol in the liver (B), and 3H-cholesterol in feces (C). CPM indicates counts per minute.
*P<0.05 atorvastatin and control groups in each time point (A). *P<0.05 (B and C).

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Shimizu et al Rosuvastatin Promotes RCT 2251

3

20

Time(hrs)

40

20

10

rosuvastatin atorvastatin

0.1

0.0

rosuvastatin atorvastatin

control

20

10
5
rosuvastatin atorvastatin

control

H-bile acids

2.5

15

control

H-free cholesterol

25

% cpm injected

0.2

60

Feces
30

FCR

0.3

FCR(pools/hr)

% CPM injected

rosuvastatin
atorvastatin
control

10

% cpm injected

H-Plasma

100

%cpm injected

2.0
1.5
1.0
0.5
0.0

rosuvastatin atorvastatin

control

Figure 5. Effects of statins on the clearance of 3H-cholesteryl oleateloaded high-density lipoprotein (HDL) in mice. C57BL6 (wild type)
mice were fed a high-cholesterol diet for 2 weeks and then divided into 3 groups (5 each in the rosuvastatin and atorvastatin groups, 4 in
the placebo control group), which were orally administered their respective drugs for 6 weeks. HDL turnover studies were then performed
as described in the Materials and Methods in the online-only Data Supplement. Decay curve of 3H-cholesteryl oleate-loaded HDL in
plasma (A) and the plasma fractional catabolic rate (FCR; B), 3H-cholesterol in feces (C), 3H-free cholesterol in feces (D), and 3H-bile acid
in feces (E). CPM indicates counts per minute. *P<0.05.

administration of statins. A lack of cholesterol ester transfer

protein in mice might be one of the reasons for this result.
In addition, Tamehiro et al21 reported that the dual promoter
system driven by sterol regulatory element-binding protein 2
and liver X receptor, respectively, regulates hepatic and extrahepatic ABCA1 expression. The increase in HDL-C levels
by statins in humans may be partly attributable to increased
liver-specific transcripts as a result of the activation of a livertype promoter. In the present study, statins did not increase
the mRNA levels of sterol regulatory element-binding protein
2 or liver X receptor in the liver. Fortunately, there were no
significant differences in HDL-C concentrations among the 3
groups, and we could perform further experiments to analyze
the mechanisms by which rosuvastatin enhanced RCT independent of plasma HDL-C levels. Although rosuvastatin did
not change HDL-C levels compared with those in the other
groups, cholesterol efflux was enhanced via ABCA1. We
considered that rosuvastatin altered the HDL composition
and analyzed this composition by capillary isotachophoresis.
Rosuvastatin significantly increased the pre- HDL fraction,
which has higher cholesterol efflux capacity. Recently, an
exciting new therapeutic strategy that uses cholesterol ester
transfer protein inhibitors has gained attention.2224 Cholesterol
ester transfer protein inhibitors markedly increase HDL-C and
decrease LDL-C when administered as monotherapy or when
administered in combination with statins. Based on the results
of the present study, the combination of cholesterol ester transfer protein inhibitors with rosuvastatin may promote RCT via
the induction of pre- HDL.
LCAT is critical for the maturation and maintenance of normal HDL metabolism. Hydrophobic CE by LCAT moves to the
core of HDL particles, where it contributes to the generation
of mature HDL particles and their progressive enlargement.

Glomset25 proposed that LCAT plays a central role in RCT by

removing free cholesterol from the surface of HDL, thus helping to maintain a gradient of free cholesterol from cells to HDL.
We considered that rosuvastatin reduces LCAT activity and
increases the nascent HDL component. The effects of statins
on LCAT activity are controversial. For example, the administration of rosuvastatin in rats did not change LCAT activity.26
Conversely, when mice were administered simvastatin or when
humans were administered atorvastatin or pravastatin, LCAT
activities increased.14,27,28 In our experiment, neither rosuvastatin nor atorvastatin altered LCAT activity. In this study, rosuvastatin shifted the composition of HDL from mature HDL
to pre- HDL without any changes in LCAT activity. In addition to LCAT, both hepatic lipase and endothelial lipase affect
the composition of HDL particles. Endothelial lipase expression resulted in the generation of small pre- HDL particles
in wild-type mice.29 Hepatic lipase induced the formation of
pre-1 HDL from triacylglycerol-rich HDL2.30 If rosuvastatin
activates hepatic lipase and endothelial lipase, this activation
may change the composition of HDL and increase pre- HDL.
With regard to the lipid profile, such as the plasma levels
of LDL-C, HDL-C, and triglyceride, both rosuvastatin and
atorvastatin suppressed the increase in triglyceride levels after
a high-cholesterol diet, whereas a control group showed a
significant increase in triglyceride levels. In clinical studies,
statins have been shown to reduce not only LDL-C levels but
also triglyceride levels.13 Although we did not analyze lipoprotein lipase activity, atorvastatin improved diabetic dyslipidemia and increased lipoprotein lipase activity in vivo while
reducing LDL-C, triglyceride, and very-low-density lipoprotein cholesterol.31
We also determined the mRNA expression of various
factors in the liver. SR-BI receptor expressing a hepatocyte

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2252 Arterioscler Thromb Vasc Biol October 2014

cell surface is important for cholesterol transfer from HDL
to the liver.32 However, there was no significant difference
in SR-BI mRNA expression in this study. Statins did not
change macrophage SR-BI expression in mice or hepatic
SR-BI expression in dogs.27,33 Based on our HDL turnover
study, rosuvastatin may activate RCT without increasing
transport from HDL to the liver. Unexpectedly, rosuvastatin decreased ApoA-I mRNA, although clinical studies
have shown an increase in ApoA-I levels.3437 Marchesi
et al38 reported that rosuvastatin did not increase ApoA-I
transcription or hepatic secretion. The unexpected result
obtained in our study, that is, rosuvastatin reduced hepatic
ApoA-I mRNA, could be a consequence of hepatic cholesterol depletion attributable to the inhibition of statin, which
may reduce hepatic HDL production.

Study Limitations
There were several study limitations in this study. First,
there was a problem about optimal doses of rosuvastatin
and atorvastatin. We analyzed the mRNA levels of Cyp7A1,
3-hydroxy-3-methylglutaryl coenzyme A reductase, and LDL
receptor (Figure III in the online-only Data Supplement). The
mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A
reductase in the atorvastatin group, but not the rosuvastatin
group, were significantly higher than those in the control
group. Although the dose of rosuvastatin in the present study
may not be optimal, rosuvastatin enhanced RCT in vivo. On
the contrary, although the dose of atorvastatin was optimal,
atorvastatin did not enhance RCT. We think that rosuvastatin
could enhance RCT, even though the dose was low. Second,
we did not analyze the hepatic levels of HDL-derived
3
H-cholesterol in Figure5 because we used 3H-cholesteryl
oleate in the experiment, which is excreted into the feces
through bile without accumulating in the liver. Hence, we
used 3H-cholesterol ester which accumulates at a higher
rate in the liver 39 in an additional experiment. Although the
hepatic level of HDL-derived 3H-cholesteryl oleate in the
atorvastatin group was similar to that in the control group,
the level in the rosuvastatin group was significantly lower
than that in the atorvastatin group (data not shown). Thus,
the amount of cholesterol trapped in the liver decreased after
the administration of rosuvastatin. These results suggest that,
in the rosuvastatin group, cholesterol may pass through some
tissue or organ other than the liver. Further studies will be
needed to resolve this issue.

Conclusions
Our results indicate that rosuvastatin, but not atorvastatin,
promotes RCT in macrophage cells in vivo by activating
ABCA1-dependent efflux from peripheral macrophage cells
and increasing the excretion of cholesterol from the liver to
bile. The regression of atherosclerosis by rosuvastatin may
be more powerful than the effect of atorvastatin, and rosuvastatin might be able to further reduce the incidence of cardiovascular events.

Acknowledgments
We thank Y. Matsuo and S. Abe for providing excellent technical
assistance.

Disclosures
K. Saku received research and education grants, consulting fees, and
promotional speaking fees from Pfizer Co Ltd. K. Saku is a Chief
Director and S. Miura is a Director of NPO Clinical and Applied
Science, Fukuoka, Japan. K. Saku has an Endowed Department of
Molecular Cardiovascular Therapeutics supported by MSD, Co
Ltd and Department of Advanced Therapeutics for Cardiovascular
Disease supported by Boston Scientific Japan, Japan Medtronic
Inc, Japan Lifeline Co Ltd and St. Jude Medical Japan Co, Ltd.
S. Miura and Y. Uehara belong to the Department of Molecular
Cardiovascular Therapeutics which is supported by MSD, Co Ltd.
H. Tanigawa belongs to the Department of Advanced Therapeutics
for Cardiovascular Disease supported by Boston Scientific Japan,
Japan Medtronic Inc, Japan Lifeline Co Ltd and St. Jude Medical
Japan Co, Ltd. The other authors report no conflicts.

References
1. Cannon CP, Braunwald E, McCabe CH, Rader DJ, Rouleau JL, Belder R,
Joyal SV, Hill KA, Pfeffer MA, Skene AM; Pravastatin or Atorvastatin
Evaluation and Infection Therapy-Thrombolysis in Myocardial
Infarction 22 Investigators. Intensive versus moderate lipid lowering
with statins after acute coronary syndromes. N Engl J Med. 2004;350:
14951504.
2. Kasai T, Miyauchi K, Kajimoto K, Kubota N, Kurata T, Amano A, Daida
H. The impact of pravastatin therapy on long-term outcome in patients
with metabolic syndrome undergoing complete coronary revascularization. Circ J. 2009;73:21042109.
3. Toth PP. High-density lipoprotein as a therapeutic target: clinical evidence
and treatment strategies. Am J Cardiol. 2005;96(9A):50K58K; discussion 34K.
4. Third report of the national cholesterol education program (NCEP)
expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III) final report. Circulation.
2002;106:31433421
5. Barter P, Gotto AM, LaRosa JC, Maroni J, Szarek M, Grundy SM,
Kastelein JJ, Bittner V, Fruchart JC; Treating to New Targets Investigators.
HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular
events. N Engl J Med. 2007;357:13011310.
6. Corti R, Fuster V, Fayad ZA, Worthley SG, Helft G, Smith D, Weinberger
J, Wentzel J, Mizsei G, Mercuri M, Badimon JJ. Lipid lowering by simvastatin induces regression of human atherosclerotic lesions: two years
follow-up by high-resolution noninvasive magnetic resonance imaging.
Circulation. 2002;106:28842887.
7. Corti R, Fuster V, Fayad ZA, Worthley SG, Helft G, Chaplin WF,
Muntwyler J, Viles-Gonzalez JF, Weinberger J, Smith DA, Mizsei G,
Badimon JJ. Effects of aggressive versus conventional lipid-lowering
therapy by simvastatin on human atherosclerotic lesions: a prospective,
randomized, double-blind trial with high-resolution magnetic resonance
imaging. J Am Coll Cardiol. 2005;46:106112.
8. Moreno PR, Sanz J, Fuster V. Promoting mechanisms of vascular health:
circulating progenitor cells, angiogenesis, and reverse cholesterol transport. J Am Coll Cardiol. 2009;53:23152323.
9. Hiro T, Kimura T, Morimoto T, Miyauchi K, Nakagawa Y, Yamagishi
M, Ozaki Y, Kimura K, Saito S, Yamaguchi T, Daida H, Matsuzaki M;
JAPAN-ACS Investigators. Effect of intensive statin therapy on regression
of coronary atherosclerosis in patients with acute coronary syndrome: a
multicenter randomized trial evaluated by volumetric intravascular ultrasound using pitavastatin versus atorvastatin (JAPAN-ACS [Japan assessment of pitavastatin and atorvastatin in acute coronary syndrome] study).
J Am Coll Cardiol. 2009;54:293302.
10. Schoenhagen P, Tuzcu EM, Apperson-Hansen C, Wang C, Wolski K,
Lin S, Sipahi I, Nicholls SJ, Magyar WA, Loyd A, Churchill T, Crowe T,
Nissen SE. Determinants of arterial wall remodeling during lipid-lowering
therapy: serial intravascular ultrasound observations from the Reversal of
Atherosclerosis with Aggressive Lipid Lowering Therapy (REVERSAL)
trial. Circulation. 2006;113:28262834.
11. Nozue T, Yamamoto S, Tohyama S, et al.; TRUTH Investigators.

Comparison of arterial remodeling and changes in plaque composition
between patients with progression versus regression of coronary atherosclerosis during statin therapy (from the TRUTH study). Am J Cardiol.
2012;109:12471253.
12. Nicholls SJ, Ballantyne CM, Barter PJ, Chapman MJ, Erbel RM, Libby
P, Raichlen JS, Uno K, Borgman M, Wolski K, Nissen SE. Effect of two

Downloaded from http://atvb.ahajournals.org/ at BIREME / UNIFESP on February 26, 2016

Shimizu et al Rosuvastatin Promotes RCT 2253

intensive statin regimens on progression of coronary disease. N Engl J
Med. 2011;365:20782087.
13. Saku K, Zhang B, Noda K; PATROL Trial Investigators. Randomized
head-to-head comparison of pitavastatin, atorvastatin, and rosuvastatin for
safety and efficacy (quantity and quality of LDL): the PATROL trial. Circ
J. 2011;75:14931505.
14. Kawano M, Nagasaka S, Yagyu H, Ishibashi S. Pitavastatin decreases
plasma prebeta1-HDL concentration and might promote its disappearance rate in hypercholesterolemic patients. J Atheroscler Thromb.
2008;15:4146.
15. Schaefer JR, Schweer H, Ikewaki K, Stracke H, Seyberth HJ, Kaffarnik H,
Maisch B, Steinmetz A. Metabolic basis of high density lipoproteins and
apolipoprotein A-I increase by HMG-CoA reductase inhibition in healthy
subjects and a patient with coronary artery disease. Atherosclerosis.
1999;144:177184.
16. Asztalos BF, Horvath KV, McNamara JR, Roheim PS, Rubinstein JJ,
Schaefer EJ. Comparing the effects of five different statins on the HDL
subpopulation profiles of coronary heart disease patients. Atherosclerosis.
2002;164:361369.
17. Zhang B, Kaneshi T, Ohta T, Saku K. Relation between insulin resistance
and fast-migrating LDL subfraction as characterized by capillary isotachophoresis. J Lipid Res. 2005;46:22652277.
18. Zhang B, Miura S, Fan P, Kumagai K, Takeuchi K, Uehara Y, McMahon
M, Rye KA, Saku K. ApoA-I/phosphatidylcholine discs remodels fastmigrating HDL into slow-migrating HDL as characterized by capillary
isotachophoresis. Atherosclerosis. 2006;188:95101.
19. Schmitz G, Mllers C, Richter V. Analytical capillary isotachophoresis of
human serum lipoproteins. Electrophoresis. 1997;18:18071813.
20. Le May C, Berger JM, Lespine A, Pillot B, Prieur X, Letessier E, Hussain
MM, Collet X, Cariou B, Costet P. Transintestinal cholesterol excretion
is an active metabolic process modulated by PCSK9 and statin involving
ABCB1. Arterioscler Thromb Vasc Biol. 2013;33:14841493.
21. Tamehiro N, Shigemoto-Mogami Y, Kakeya T, Okuhira K, Suzuki K,
Sato R, Nagao T, Nishimaki-Mogami T. Sterol regulatory elementbinding protein-2- and liver X receptor-driven dual promoter regulation of hepatic ABC transporter A1 gene expression: mechanism
underlying the unique response to cellular cholesterol status. J Biol Chem.
2007;282:2109021099.
22. de Grooth GJ, Kuivenhoven JA, Stalenhoef AF, de Graaf J, Zwinderman
AH, Posma JL, van Tol A, Kastelein JJ. Efficacy and safety of a novel cholesteryl ester transfer protein inhibitor, JTT-705, in humans: a randomized
phase II dose-response study. Circulation. 2002;105:21592165.
23. Clark RW, Sutfin TA, Ruggeri RB, Willauer AT, Sugarman ED, MagnusAryitey G, Cosgrove PG, Sand TM, Wester RT, Williams JA, Perlman ME,
Bamberger MJ. Raising high-density lipoprotein in humans through inhibition of cholesteryl ester transfer protein: an initial multidose study of
torcetrapib. Arterioscler Thromb Vasc Biol. 2004;24:490497.
24. Brousseau ME, Schaefer EJ, Wolfe ML, Bloedon LT, Digenio AG,
Clark RW, Mancuso JP, Rader DJ. Effects of an inhibitor of cholesteryl
ester transfer protein on HDL cholesterol. N Engl J Med. 2004;350:
15051515.
25. Glomset JA. The plasma lecithins:cholesterol acyltransferase reaction. J
Lipid Res. 1968;9:155167.

26. Vaziri ND, Liang K. Effects of HMG-CoA reductase inhibition on

hepatic expression of key cholesterol-regulatory enzymes and receptors in
nephrotic syndrome. Am J Nephrol. 2004;24:606613.
27. Song G, Liu J, Zhao Z, Yu Y, Tian H, Yao S, Li G, Qin S. Simvastatin
reduces atherogenesis and promotes the expression of hepatic genes associated with reverse cholesterol transport in apoE-knockout mice fed highfat diet. Lipids Health Dis. 2011;10:8.
28. Bouzas L, San Jos E, Tutor JC. Chitotriosidase activity in pleural effusions. Clin Lab. 2007;53:449452.
29. Nijstad N, Wiersma H, Gautier T, van der Giet M, Maugeais C, Tietge
UJ. Scavenger receptor BI-mediated selective uptake is required for the
remodeling of high density lipoprotein by endothelial lipase. J Biol Chem.
2009;284:60936100.
30. Barrans A, Collet X, Barbaras R, Jaspard B, Manent J, Vieu C, Chap H,
Perret B. Hepatic lipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases. J Biol Chem.
1994;269:1157211577.
31. Schneider JG, von Eynatten M, Parhofer KG, Volkmer JE, Schiekofer S,
Hamann A, Nawroth PP, Dugi KA. Atorvastatin improves diabetic dyslipidemia and increases lipoprotein lipase activity in vivo. Atherosclerosis.
2004;175:325331.
32. Fidge NH. High density lipoprotein receptors, binding proteins, and

ligands. J Lipid Res. 1999;40:187201.
33. Briand F, Magot T, Krempf M, Nguyen P, Ouguerram K. Effects of atorvastatin on high-density lipoprotein apolipoprotein A-I metabolism in dogs.
Eur J Clin Invest. 2006;36:224230.
34. Brown WV, Bays HE, Hassman DR, McKenney J, Chitra R, Hutchinson
H, Miller E; Rosuvastatin Study Group. Efficacy and safety of rosuvastatin compared with pravastatin and simvastatin in patients with hypercholesterolemia: a randomized, double-blind, 52-week trial. Am Heart J.
2002;144:10361043.
35. Davidson MH, McGarry T, Bettis R, Melani L, Lipka LJ, LeBeaut AP,
Suresh R, Sun S, Veltri EP. Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. J Am Coll Cardiol.
2002;40:21252134.
36. Capuzzi DM, Morgan JM, Weiss RJ, Chitra RR, Hutchinson HG,

Cressman MD. Beneficial effects of rosuvastatin alone and in combination with extended-release niacin in patients with a combined hyperlipidemia and low high-density lipoprotein cholesterol levels. Am J Cardiol.
2003;91:13041310.
37. Hunninghake DB, Stein EA, Bays HE, Rader DJ, Chitra RR, Simonson
SG, Schneck DW. Rosuvastatin improves the atherogenic and atheroprotective lipid profiles in patients with hypertriglyceridemia. Coron Artery
Dis. 2004;15:115123.
38. Marchesi M, Parolini C, Caligari S, Gilio D, Manzini S, Busnelli M, Cinquanta
P, Camera M, Brambilla M, Sirtori CR, Chiesa G. Rosuvastatin does not affect
human apolipoprotein A-I expression in genetically modified mice: a clue to
the disputed effect of statins on HDL. Br J Pharmacol. 2011;164:14601468.
39. Glass C, Pittman RC, Weinstein DB, Steinberg D. Dissociation of tissue
uptake of cholesterol ester from that of apoprotein A-I of rat plasma high
density lipoprotein: selective delivery of cholesterol ester to liver, adrenal,
and gonad. Proc Natl Acad Sci U S A. 1983;80:54355439.

Significance
Statin therapy reduces the incidence of cardiovascular events. It is controversial whether statins improve high-density lipoprotein (HDL)
function, which plays an important role in reverse cholesterol transport (RCT). We clarified the effects of rosuvastatin and atorvastatin on in
vivo macrophage RCT and the underlying mechanisms. Rosuvastatin, but not atorvastatin, increased macrophage-to-HDL RCT by activating
cholesterol efflux via ATP-binding cassette transporter A1. Furthermore, rosuvastatin increased liver-to-feces RCT by promoting the fecal
excretion of HDL-derived cholesterol. The differential effects of rosuvastatin and atorvastatin may be associated with the regression of
atherosclerosis. The regression induced by rosuvastatin may be more powerful than that induced by atorvastatin. Furthermore, cholesterol
ester transfer protein inhibitors markedly increase HDL cholesterol and decrease low-density lipoprotein cholesterol when administered in
combination with statins. Cholesterol ester transfer protein inhibitors combined with rosuvastatin may promote RCT via the induction of pre-
HDL even if cholesterol ester transfer protein activity is blocked.

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Rosuvastatin Activates ATP-Binding Cassette Transporter A1Dependent Efflux Ex Vivo

and Promotes Reverse Cholesterol Transport in Macrophage Cells in Mice Fed a High-Fat
Diet
Tomohiko Shimizu, Shin-ichiro Miura, Hiroyuki Tanigawa, Takashi Kuwano, Bo Zhang,
Yoshinari Uehara and Keijiro Saku
Arterioscler Thromb Vasc Biol. 2014;34:2246-2253; originally published online August 7, 2014;
doi: 10.1161/ATVBAHA.114.303715
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636

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Supplement
Materials and Methods
Materials
The following antibodies and reagents were purchased or provided: Dulbecco modified
Eagle medium (DMEM) and phosphate buffer saline (PBS) (Wako Pure Chemical
Industries Ltd., Osaka, Japan); [1,2-3H]cholesterol, [1,2-3H]cholesteryl oleate (Perkin
Elmer Life & Analytical Sciences Inc.); rosuvastatin and atorvastatin (Toronto Research
Chemicals, Inc., Canada); 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate
(8-CTP-cAMP) (Enzo Life Sciences, Inc., New York, NY); human HDL (Calbiochem,
Darmstadt, Germany); and the liver X receptor (LXR) agonist T0-901317 and the
retinoid X receptor (RXR) agonist 9-cis-retinoic acid (Sigma-Aldrich, St. Louis, MO).

Mice, Diets and Experimental Design

Wild-type (WT) C57BL/6J mice were purchased from Japan SLC Inc. (Shizuoka,
Japan). Mice were housed in specific pathogen-free barrier facilities at Fukuoka
University. Mice were maintained under a 12-h light/dark cycle and fed a 0.5 %
high-cholesterol diet + 10 % coconut oil (purchased from Oriental Yeast Co. Ltd.,
Tokyo, Japan) (Content of diet were shown in Supplementary Table I), which was

provided ad libitum for 2 weeks before and during the study. In all cases, fasting
plasma was obtained from the retro-orbital plexus while the mice were under isoflurane
anesthesia.

For each experiment, female 3- to 4-month-old C57BL/6 mice were

divided into 3 groups; 4 mg/kg/day rosuvastatin (rosuvastatin group), 8 mg/kg/day

atorvastatin (atorvastatin group) and 0.5 % methylcellulose (control group) dissolved in
freely available drinking water for 6 weeks.

In a study by de Haan et al., mice

received diet with or without 0.01% (w/w) atorvastatin for 6 weeks (i.e., approximately
10 mg/kg/day, which corresponds to a dose of 70 mg/day for an average 70 kg person)
[1]. Since the maximum doses of rosuvastatin and atorvastatin in humans are 40 mg/day
and 80 mg/day, respectively, the doses of rosuvastatin and atorvastatin in mice were
determined to be 4 mg/kg/day and 8 mg/kg/day, respectively. Animal experiments
were approved by the Fukuoka University Animal Center Committee.

Plasma Lipid, Lipoprotein and lecithin-cholesterol acyltransferase (LCAT)

Activity Analyses
Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density
lipoprotein cholesterol (LDL-C), triglycerides (TG) and LCAT activities were measured
by a commercial kit (Sekisui Medical Co. Ltd., Japan). Lipoprotein subfractions in

serum and serum fractions were analyzed by capillary isotachophoresis (cITP) on a

Beckman P/ACE MDQ system (Beckman-Coulter, Tokyo, Japan) as described
previously [2].

cITP is a technique for analyzing charge-based lipoprotein subfractions

directly in plasma [3, 4].

cITP can separate plasma lipoproteins into three HDL

subfractions [fast- (f), intermediate- (i), and slow- (s) migrating HDL] according to their
electrophoretic mobilities. The levels of cITP HDL subfractions can be determined as
peak areas relative to that of an internal marker.

RNA Quantification
Total RNA was isolated from mouse primary hepatocytes with an RNeasy Mini Kit
(QIAGEN, Hilden, Germany). cDNA was produced from total RNA via reverse
transcription with a SuperScript First-Stand Synthesis System for RT-PCR (Life
Technologies, Grand Island, NY). mRNA expression with Taqman assay systems was
quantified by an ABI 7500 Real Time PCR System according to the manufacturers
instructions (Life Technologies, Grand Island, NY) for scavenger receptor class B type I
(SR-BI), ATP-binding cassette transporter (ABC) A1, liver X receptor (LXR), farnesoid
X receptor (FXR), apolipoprotein (Apo)A-1, ABCG8, ABCG5, sterol regulatory
element-binding protein 2 (SREBP-2) and -actin RNA. The expression data were

normalized for -actin levels.

Ex Vivo Cholesterol Efflux from Bone Marrow Macrophage and J774 Macrophage
Cells
Plasma samples were collected from mice after 6 weeks of treatment. Plasma HDL
(apoB-depleted plasma)-mediated cholesterol efflux was measured in cAMP-treated
J774 macrophage cells as described above. For the preparation of apoB-depleted
plasma, plasma was mixed in a tube containing the precipitation reagent (0.55 mmol/l
phosphotungstic acid and 25 mmol/l magnesium chloride).

After 10 min at room

temperature, this mixture was centrifuged for 10 min at 3,000g. The clear supernatant
was separated and stored in capped glass tubes for a maximum of 2 days at 4C before
the cholesterol content was determined by the cholesterol oxidase/p-aminophenazone
method. We measured plasma HDL-C-mediated efflux in bone marrow macrophage
(BMM) with or without probucol, which specifically inhibits ABCA1.

J774

macrophages were plated in 12-well plates (0.25106 /mL) in DMEM with 10 % fetal
bovine serum (FBS) and 0.5 % penicillin G and streptomycin at 37 C.

After 1 day,

the cells were labeled for 24 h at 37 C with 3 Ci/mL [1,2-3H]cholesterol and 50

g/mL acetylated low-density cholesterol (ac-LDL) in the presence of 1.0 % FBS and

0.5 % penicillin G and streptomycin in DMEM. Cells were washed extensively with
PBS with 1.0 % bovine serum albumin (BSA), and J774 macrophages were then
equilibrated with or without 0.3 mmol/L cAMP, which activates mainly ABCA1, in 0.2
% BSA-containing medium for 20 h at 37 C [5].

At the end of the equilibration

period, cell monolayers were washed twice with PBS containing 1 % BSA.
Subsequently, cholesterol efflux was induced by incubation for 4 h at 37C with each
pooled plasma (excluding apo-B-containing protein) diluted to 2 % in DMEM. Free
cholesterol efflux was obtained by measuring the release of radiolabeled cholesterol into
the medium as described previously [6].
Next, BMM were isolated from mice by standard procedures [7].

Isolated

macrophages were suspended and cultured in 12-well plates (0.25106 /mL) in DMEM
with 10 % FBS plus 0.5 % penicillin G and streptomycin at 37C. After 4 days,
macrophages were labeled for 24 h at 37C with 2 Ci/mL [1, 2-3H]cholesterol and 25
g/mL ac-LDL in the presence of 1.0 % FBS and 0.5 % penicillin G and streptomycin
in DMEM. BMM were washed extensively with PBS with 1 % BSA and equilibrated
overnight in the presence of the LXR agonist T0-901317 and the RXR agonist 9-cis
(each 5 M) in serum-free medium.

For cholesterol efflux, BMM were treated with or

without 20 M probucol for 2 h at 37C [8]. After 4 h, aliquots of the medium were

removed, and the 3H-cholesterol released was measured by liquid scintillation counting.
The 3H-cholesterol present in the cells was determined by extracting cell lipids in 0.2 M
NaOH and 0.15 M NaCl.

Macrophage reverse cholesterol transport (RCT) Study

The RCT study was performed as described previously [9-11]. J774 macrophages
were grown in DMEM supplemented with 10 % FBS. The cells were radiolabeled
with 5 Ci/mL 3H-cholesterol and cholesterol enriched with 25 g/mL ac-LDL for 40 h.
The labeled foam cells were washed, equilibrated in medium with 0.2 % bovine serum
albumin for 4 hours, spun down and resuspended in DMEM immediately before use.
All mice were fed a 0.5 % high-cholesterol diet for 2 weeks before experiments as
prefeeding.

For this experiment, 21 female 3- to 4-month-old mice (n=7/each group)

were divided into 3 groups (rosuvastatin, atorvastatin and control groups).

After 6

weeks, 3H-cholesterol-labeled and acetylated LDL-C-loaded J774 cells were injected

intraperitoneally. Blood was collected at 6, 24 and 48 h, and plasma samples were
used for liquid scintillation counting.

Feces were collected continuously from 0 to 48

h and stored at 4 C before the extraction of cholesterol and bile acid.

HDL Turnover Study

Human HDL was labeled with 3H-cholesteryl oleate as described previously with slight
modifications [11, 12].

For the experiment, 15 female 3- to 4-month-old mice

(n=5/each group) were divided into 3 groups (rosuvastatin, atorvastatin and control
groups). After 6 weeks, labeled HDL was injected intravenously. Blood was
collected at 2 min and 1, 3, 6, 9, 24 and 48 h, and liver and feces were collected at 48 h.
The fractional catabolic rate (FCR) was calculated using the SAS (Statistical Analysis
System) software package (Ver. 9.2; SAS Institute Inc., Cary, NC) as described
previously [12, 13].

Statistical Analysis
The results before and after drug administration were compared with the Student t test
(2-tailed) and analysis of variance (ANOVA) using GraphPad Prism version 5.0
(GraphPad Software Inc., La Jolla, CA). For the macrophage RCT studies, the
appearance of tracer in plasma was analyzed by repeated measures ANOVA, and a
Newman-Keuls multiple comparison test was applied to correct for multiple
comparisons.

One-way ANOVA with a Newman-Keuls multiple comparison test was

used to compare the differences among the 3 groups. All data are presented as the

mean standard deviation (SD). A probability value <0.05 was considered statistically
significant.

References
1.

de Haan W, van der Hoogt CC, Westerterp M, Hoekstra M, Dallinga-Thie GM,

Princen HM, Romijn JA, Jukema JW, Havekes LM, Rensen PC. Atorvastatin
increases HDL cholesterol by reducing CETP expression in cholesterol-fed
APOE*3-Leiden.CETP mice. Atherosclerosis. 2008;197:57-63.

2.

Zhang B, Kaneshi T, Ohta T, Saku K. Relation between insulin resistance and

fast-migrating LDL subfraction as characterized by capillary isotachophoresis. J
Lipid Res. 2005;46:2265-2277

3.

Zhang B, Miura S, Fan P, Kumagai K, Takeuchi K, Uehara Y, McMahon M,

Rye KA, Saku K. Apoa-i/phosphatidylcholine discs remodels fast-migrating
HDL into slow-migrating HDL as characterized by capillary isotachophoresis.
Atherosclerosis. 2006;188:95-101

4.

Schmitz G, Mollers C, Richter V. Analytical capillary isotachophoresis of

human serum lipoproteins. Electrophoresis. 1997;18:1807-1813

5.

Sakr SW, Williams DL, Stoudt GW, Phillips MC, Rothblat GH. Induction of

cellular cholesterol efflux to lipid-free apolipoprotein A-I by camp. Biochim

Biophys Acta. 1999;1438:85-98
6.

Rothblat GH, de la Llera-Moya M, Favari E, Yancey PG, Kellner-Weibel G.

Cellular cholesterol flux studies: Methodological considerations. Atherosclerosis.
2002;163:1-8

7.

Yona S, Heinsbroek SE, Peiser L, Gordon S, Perretti M, Flower RJ. Impaired

phagocytic mechanism in annexin 1 null macrophages. Br J Pharmacol.
2006;148:469-477

8.

Favari E, Zanotti I, Zimetti F, Ronda N, Bernini F, Rothblat GH. Probucol

inhibits abca1-mediated cellular lipid efflux. Arterioscler Thromb Vasc Biol.
2004;24:2345-2350

9.

Zhang Y, Da Silva JR, Reilly M, Billheimer JT, Rothblat GH, Rader DJ. Hepatic
expression of scavenger receptor class b type I (SR-BI) is a positive regulator of
macrophage

reverse

cholesterol

transport

in

vivo.

Clin

Invest.

2005;115:2870-2874
10.

Tanigawa H, Billheimer JT, Tohyama J, Zhang Y, Rothblat G, Rader DJ.

Expression of cholesteryl ester transfer protein in mice promotes macrophage
reverse cholesterol transport. Circulation. 2007;116:1267-1273

11.

Alexander ET, Weibel GL, Joshi MR, Vedhachalam C, de la Llera-Moya M,

Rothblat GH, Phillips MC, Rader DJ. Macrophage reverse cholesterol transport
in

mice

expressing

apoa-i

milano.

Arterioscler

Thromb

Vasc

Biol.

2009;29:1496-1501
12.

Tietge UJ, Maugeais C, Cain W, Grass D, Glick JM, de Beer FC, Rader DJ.
Overexpression of secretory phospholipase a(2) causes rapid catabolism and
altered tissue uptake of high density lipoprotein cholesteryl ester and
apolipoprotein A-I. J Biol Chem. 2000;275:10077-10084

13.

Maugeais C, Tietge UJ, Broedl UC, Marchadier D, Cain W, McCoy MG,

Lund-Katz S, Glick JM, Rader DJ. Dose-dependent acceleration of high-density
lipoprotein catabolism by endothelial lipase. Circulation. 2003;108:2121-2126

10

Supplementary Figs and Tables.

Supplementary Table I. Content of diet.
Crude protein 20.7g/100g diet
Crude fat 15.1g/100g
Crude ash 5.2g/100g
Crude fiber 2.5g/100g
Nitrogen free extract 49.5g/100g
Water 7.1g/100g
Supplemental Figure I. mRNA levels of ABCG5, ABCB1a, NPC1L1 and LDL receptor
in the intestine in the rosuvastatin, atorvastatin and control groups.
ABCB1a mRNA expression

ABCG5 mRNA expression

1.5

1.5

(AU)

(AU)

1.0

1.0

0.5

0.5

0.0

0.0
rosuvastatin atorvastatin

rosuvastatin atorvastatin

control

LDL-R mRNA expression

control

NPC1L1 mRNA expression

2.5

1.5

(AU)

(AU)

2.0
1.0

1.5
1.0

0.5

0.5
0.0

0.0

rosuvastatin atorvastatin

control

rosuvastatin atorvastatin

control

Supplemental Figure II. Intestinal absorption of cholesterol in the rosuvastatin,

atorvastatin and control groups.
Feces

% cpm injected

30

20

10

0
rosuvastatin atorvastatin

control

Methods for measurement of the intestinal absorption of cholesterol

Wild-type mice were divided into 3 groups; 4 mg/kg/day rosuvastatin (rosuvastatin

group, n=7), 8 mg/kg/day atorvastatin (atorvastatin group, n=8) and 0.5 %

methylcellulose dissolved in water (control group, n= 8) for 2 weeks. We measured
intestinal cholesterol absorption function by a fecal dual-isotope ratio method, as
described previously [1, 2].
Briefly, mice were gavaged with 150 l of safflower oil (195-15372, Wako Pure
Chemical Industries, Ltd. Japan) that contained a mixture of 2 Ci [5,6-3H] sitostanol
(ART 0361, American Radiolabeled Chemicals Inc., St. Louis, MO, USA) and 1 Ci
[4-14C] cholesterol (NEC018250UC, PerkinElmer Life Sciences, USA), and then
returned to fresh cages. Feces were collected from individually housed mice in
wire-bottom cages after the administration of label and processed as described
previously following homogenization and neutral sterol extraction of fecal samples. The
ratio of 14C and 3H sterol in each feces sample and the dosing mixture were determined
by liquid scintillation counting and the cholesterol absorption percentage was calculated
as described previously [2, 3].
References:
1. Xie Y, Newberry EP, Young SG, Robine S, Hamilton RL, Wong JS, Luo J, Kennedy
S, Davidson NO. Compensatory increase in hepatic lipogenesis in mice with
conditional intestine-specific mttp deficiency. J Biol Chem. 2006;281:4075-4086.
2. Wang DQ, Carey MC. Measurement of intestinal cholesterol absorption by plasma
and fecal dual-isotope ratio, mass balance, and lymph fistula methods in the mouse:
an analysis of direct versus indirect methodologies. J Lipid Res.
2003;44:1042-1059.
3. Newberry EP, Xie Y, Kennedy SM, Luo J, Davidson NO. Protection against Western
diet-induced obesity and hepatic steatosis in liver fatty acid-binding protein
knockout mice. Hepatology. 2006;44:1191-1205.
Supplemental Figure III. mRNA levels of Cyp7A1, HMG-CoA reductase and LDL-R in
the liver in the rosuvastatin, atorvastatin and control groups.
Cyp7A1 mRNA expression
1.5

HMG-CoA reductase mRNA expression

(AU)

(AU)
1.5

1.0

LDL-R mRNA expression

1.5

2.0

(AU)

*
1.0

1.0

0.5

0.5
0.5

0.0

0.0

0.0

rosuvastatin atorvastatin

control

rosuvastatin atorvastatin

control

rosuvastatin atorvastatin

control