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Ahigher level of serum low-density lipoprotein choles-terol (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 aretreated aggressively with statins to reduce LDL-C levels


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Shimizu et al Rosuvastatin Promotes RCT 2247

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.9–11 Although the administration of

rosuvastatin and atorvastatin resulted in a significant regres-

sion of coronary atherosclerosis12 and significantly increased

HDL-C levels,12–16 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 atorv-

astatin on RCT. Therefore, the aim of the present study was to

clarify the effects of rosuvastatin and atorvastatin in an in vivomacrophage RCT system and their underlying mechanisms.

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

Results

Rosuvastatin and Atorvastatin Prevented theIncrease in Triglyceride Without Changesin HDL-C, LDL-C, or non–HDL-C

Changes in the lipid profile before and after 6 weeks of statin

administration are shown in the Table. The control group

did not show significant changes in plasma total cholesterol,

LDL-C, or non–HDL-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 treat-

ment. 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.

Rosuvastatin, but Not Atorvastatin, IncreasedPre-β HDL Without Decreasing LCAT ActivityNext, we analyzed HDL subfractions as assessed by capil-

lary isotachophoresis on a Beckman P/ACE MDQ system

(Beckman-Coulter, Tokyo, Japan) as described previously,17

as shown in Figure 1A and 1B. Capillary isotachophoresis is

a technique for analyzing charge-based lipoprotein subfrac-tions 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-β 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

(Figure 1C). There were no differences in the activity of LCAT

between the groups, indicating that rosuvastatin increased pre-

β HDL without decreasing LCAT activity.

Rosuvastatin, but Not Atorvastatin, DecreasedmRNA Levels of Apolipoprotein A-I in the liverWe isolated the livers from mice in the rosuvastatin, atorv-

astatin, and control groups at 6 weeks and analyzed mRNA

expression levels of various factors (Figure 2). Rosuvastatin and

atorvastatin had no significant effects on mRNA levels of SR-BI

Nonstandard Abbreviations and Acronyms

ABC ATP-binding cassette transporter

ApoA-I apolipoprotein A-I

CE cholesteryl ester

HDL high-density lipoprotein

HDL-C high-density lipoprotein cholesterolLCAT lecithin-cholesterol acyltransferase

LDL-C low-density lipoprotein cholesterol

RCT reverse cholesterol transport

SR-BI scavenger receptor class B type I

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

Group Weeks TC, mg/dL HDL-C, mg/dL LDL-C, mg/dL Triglyceride, mg/dL Non–HDL-C, mg/dL

Rosuvastatin (n=8) 0 95±12 51±16 41±10 21±8 45±9

6 86±20 44±10 46±14 21±10* 42±22

Atorvastatin (n=8) 0 95±12 51±14 40±11 23±7 45±12

6 93±11 43±9 45±7 30±8* 50±8

Control (n=7) 0 99±14 51±15 43±11 24±11 48±8

6 101±14 50±15 42±10 45±11† 51±10

HDL-C indicates high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; and TC, total cholesterol.

*P


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2248 Arterioscler Thromb Vasc Biol October 2014

and ABCA1 (Figure 2A 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 signifi-

cantly lower than those in the control group (Figure 2C–2F).

The atorvastatin group also showed a significant reduction in

liver X receptor mRNA expression compared with the control

group (Figure 2C). Furthermore, ApoA-I mRNA expression

in the rosuvastatin group was significantly less than that in the

atorvastatin group (Figure 2E). On the contrary, there were no

significant differences in ABCG5 or sterol regulatory element-

binding protein 2 among the 3 groups (Figure 2G and 2H).

Rosuvastatin, but Not Atorvastatin, IncreasedCholesterol Efflux via ABCA1 Ex Vivo

We examined whether cholesterol efflux via the ABCA1pathway was enhanced by rosuvastatin, because rosuvastatin

increased pre-β 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 macro-

phages. Before the experiments, apolipoprotein B–containing

protein was excluded from pooled plasma. For the preparation

of apolipoprotein B–depleted plasma, plasma was mixed with

phosphotungstic acid and magnesium chloride. As shown

in Figure 3A to 3C, we measured plasma HDL-C–mediated

efflux in bone marrow macrophage with or without probucol,

which specifically inhibits ABCA1. Plasma from the rosuvas-

tatin group had significantly greater cellular cholesterol effluxcapacity than plasma from the atorvastatin and control groups

(Figure 3A). However, no significant difference was seen with

the use of probucol (Figure 3B). In addition, plasma from the

rosuvastatin group had a significantly greater capacity to pro-

mote ABCA1-specific efflux than plasma from the atorvastatin

and control groups (Figure 3C). Similar results were observed

regarding cholesterol efflux using J774 cells. The rosuvastatin

group had significantly greater total efflux than the atorvas-

tatin and control groups (Figure 3D). 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 (Figure 3E).

Rosuvastatin, but Not Atorvastatin,Promoted Macrophage RCT In VivoWe investigated the differences in the effects of rosuvastatin

and atorvastatin on RCT in macrophage cells. As shown in

Figure 4A, after 3H-cholesterol–labeled J774 cells were injected

into the peritoneal cavity of mice, the 3H-cholesterol counts in

plasma (3H-plasma) were determined and expressed as a per-

centage 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, whereas3H-plasma in the rosuvastatin group was significantly greater at

all time points compared with those in the atorvastatin and controlgroups. However, there was no difference in 3H-Liver between

the rosuvastatin and atorvastatin groups (Figure 4B). Although

there was no difference in 3H-Liver between the rosuvastatin

and control groups, 3H-Liver in the atorvastatin group was sig-

nificantly 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 (Figure 4C).

Rosuvastatin, but Not Atorvastatin,Increased Cholesterol Excretion From theLiver Without Increasing Turnover

Finally, we investigated the effects of statins on the turn-over and fecal excretion of HDL-derived cholesterol. Plasma

LCAT

rosuvastatin atorvastatin control

0

100

200

300

400

A c t i v i t y ( n m o l / m

l / h r )

C

B

A

Figure 1. A , Lipoprotein subfractions as assessed by capillaryisotachophoresis (cITP) in plasma taken from wild-type (WT)mice before and after the administration of statins. The cITPtechnique separates plasma lipoproteins into 3 high-densitylipoprotein (HDL) subfractions (fast-, intermediate-, and slow-migrating HDL [fHDL, iHDL, and sHDL]) according to their elec-trophoretic mobilities. Dotted squares indicate pre-β HDL whichshows peak in sHDL. B, Absolute amount of sHDL as pre-β HDL

in plasma taken from WT mice before (week 0) and after (week6) the administration of statins. The levels of cITP HDL subfrac-tions can be determined as peak areas relative to that of aninternal marker. *P


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3H-cholesteryl oleate-loaded HDL was decreased equally in

all 3 groups (Figure 5A), consistent with the results regarding

fractional catabolic rate (Figure 5B). On the contrary, there

were differences in fecal excretion. The rosuvastatin group

showed a significant increase in 3H-cholesterol in feces com-

pared with the atorvastatin and control groups (Figure 5C).

Interestingly, the rosuvastatin group showed a significant

increase in the fecal excretion of 3H-free-cholesterol, but not3H-bile acids (Figure 5D and 5E). These results indicate that

rosuvastatin, but not atorvastatin, enhanced the excretion of

HDL-derived cholesterol into feces and support the findingthat rosuvastatin promoted RCT in macrophage cells. This

is consistent with the notion that rosuvastatin increases the

excretion of cholesterol from the liver.

DiscussionIn the present study, rosuvastatin, but not atorvastatin,

increased macrophage-to-HDL RCT by activating cholesterol

efflux via ABCA1. Furthermore, rosuvastatin increased liver-

to-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 treat-ment 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 choles-

terol 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 understand-ing 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 cho-

lesterol into feces. As the same conditions as the occasion where

cholesterol efflux from a peripheral macrophage is equivalent

(turnover study; Figure 5), we observed cholesterol excretion

into feces after the intravenous injection of HDL containing an

equivalent amount of cholesterol. As a result, cholesterol excre-

tion into feces was increased as in the RCT study (Figure 4). 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

rosuvastatin atorvastatin control0.0

0.5

1.0

1.5

SR-B1 mRNA expression

(AU)


0.5

1.0

1.5

2.0

LXR mRNA expression

* *

(AU)


0.5

1.0

1.5

ABCA1 mRNA expression

(AU)

A B C


0.5

1.0

1.5 FXR mRNA expression

*(AU)

D


0.5

1.0

1.5

ABCG8 mRNA expression

*(AU)


0.5

1.0

1.5Apo A-1 mRNA expression

**(AU)

E F


0.5

1.0

1.5

ABCG5 mRNA expression

(AU)


0.5

1.0

1.5

2.0

SREBP-2 mRNA expression

(AU)

G H

Figure 2. Effects of statins on mRNA expression levels of various factors in mouse hepatocytes (n=7 in each group). Scavenger receptorclass B type I (SR-BI; A ), ATP-binding cassette transporter A1 (ABCA1; B ), liver X receptor (LXR; C ), farnesoid X receptor (FXR; D ), apo-lipoprotein A-I (ApoA-I; E ) ABCG8 ( F ), ABCG5 ( G ), and sterol regulatory element-binding protein 2 (SREBP-2; H ). *P


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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 (Figure 5). Although cholesterol in feces was increased

(Figure 4), there were no differences in the attenuation of choles-

terol in blood after intravenous injection. This observation was

important. Specifically, rosuvastatin did not alter blood choles-terol 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 choles-

terol excretion is an important pathway for hepatobiliary secre-

tion 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

contribute to transintestinal cholesterol excretion. Although

we analyzed mRNA levels of ABCG5, ABCB1a, Niemann-

Pick 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 addi-tional experiment, there was no difference in the intestinal

absorption of cholesterol between rosuvastatin and atorvas-

tatin (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.13–16 Unlike these clinical results, a

significant increase in plasma HDL-C was not seen after the

3H-Plasma

0 10 20 30 40 50

0

2

4

6

8

A B C

rosuvastatin

atorvastatin

control

*

* *

Time(hrs)

% o

f i n j e c t i o n / m l


0

1

2

3

4

3H-Liver

% C

P M i

n j e c t e d

*

3H-Feces


0

2

4

6

**

% C

P M

i n j e c t e d

Figure 4. Effects of statins on reverse cholesterol transport (RCT) in mice. C57BL6 (wild type) mice were fed a high-cholesterol diet for 2weeks and then divided into rosuvastatin, atorvastatin, and placebo control groups (n=7 each). The respective drugs were administeredorally 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 ),3

H-cholesterol in the liver ( B ), and3

H-cholesterol in feces ( C ). CPM indicates counts per minute.*P


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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 extra-

hepatic ABCA1 expression. The increase in HDL-C levels

by statins in humans may be partly attributable to increasedliver-specific transcripts as a result of the activation of a liver-

type 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 inde-

pendent 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-β HDL fraction,which has higher cholesterol efflux capacity. Recently, an

exciting new therapeutic strategy that uses cholesterol ester

transfer protein inhibitors has gained attention.22–24 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 trans-

fer protein inhibitors with rosuvastatin may promote RCT via

the induction of pre-β HDL.

LCAT is critical for the maturation and maintenance of nor-

mal HDL metabolism. Hydrophobic CE by LCAT moves to the

core of HDL particles, where it contributes to the generationof 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 help-

ing 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 adminis-

tration 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 rosuvas-

tatin nor atorvastatin altered LCAT activity. In this study, rosu-

vastatin shifted the composition of HDL from mature HDL

to pre-β HDL without any changes in LCAT activity. In addi-

tion to LCAT, both hepatic lipase and endothelial lipase affect

the composition of HDL particles. Endothelial lipase expres-

sion resulted in the generation of small pre-β HDL particles

in wild-type mice.29 Hepatic lipase induced the formation of

pre-β1 HDL from triacylglycerol-rich HDL

2.30 If rosuvastatin

activates hepatic lipase and endothelial lipase, this activation

may change the composition of HDL and increase pre-β 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 lipo-

protein lipase activity, atorvastatin improved diabetic dyslip-

idemia and increased lipoprotein lipase activity in vivo while

reducing LDL-C, triglyceride, and very-low-density lipopro-

tein cholesterol.31

We also determined the mRNA expression of variousfactors in the liver. SR-BI receptor expressing a hepatocyte

3H-Plasma

0 20 40 60

1

10

100

rosuvastatin

atorvastatin

control

Time(hrs)

% C

P M i n j e c t e d


0.0

0.1

0.2

0.3 FCR

F C R ( p o o l s / h r )


0

10

20

30

Feces

% c

p m i

n j e c t e d

**


0

5

10

15

20

25

3H-free cholesterol

% c

p m i

n j e c t e d

**


0.0

0.5

1.0

1.5

2.0

2.5

3H-bile acids

% c p m i

n j e c t e d

A B

C D E

Figure 5. Effects of statins on the clearance of 3H-cholesteryl oleate–loaded 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 inthe placebo control group), which were orally administered their respective drugs for 6 weeks. HDL turnover studies were then performedas described in the Materials and Methods in the online-only Data Supplement. Decay curve of 3H-cholesteryl oleate-loaded HDL inplasma ( A ) and the plasma fractional catabolic rate (FCR; B ), 3H-cholesterol in feces ( C ), 3H-free cholesterol in feces ( D ), and 3H-bile acidin feces ( E ). CPM indicates counts per minute. *P


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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, rosuvas-

tatin decreased ApoA-I mRNA, although clinical studies

have shown an increase in ApoA-I levels.34–37 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 choles-

terol depletion attributable to the inhibition of statin, which

may reduce hepatic HDL production.

Study LimitationsThere 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-derived3H-cholesterol in Figure 5 because we used 3H-cholesteryl

oleate in the experiment, which is excreted into the fecesthrough 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.

ConclusionsOur 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 rosuvas-

tatin might be able to further reduce the incidence of cardio-

vascular events.

Acknowledgments

We thank Y. Matsuo and S. Abe for providing excellent technicalassistance.

DisclosuresK. Saku received research and education grants, consulting fees, andpromotional speaking fees from Pfizer Co Ltd. K. Saku is a ChiefDirector and S. Miura is a Director of NPO Clinical and AppliedScience, Fukuoka, Japan. K. Saku has an Endowed Department ofMolecular Cardiovascular Therapeutics supported by MSD, CoLtd and “Department of Advanced Therapeutics for CardiovascularDisease” supported by Boston Scientific Japan, Japan MedtronicInc, Japan Lifeline Co Ltd and St. Jude Medical Japan Co, Ltd.S. Miura and Y. Uehara belong to the Department of MolecularCardiovascular Therapeutics which is supported by MSD, Co Ltd.H. Tanigawa belongs to the Department of Advanced Therapeuticsfor Cardiovascular Disease supported by Boston Scientific Japan,Japan Medtronic Inc, Japan Lifeline Co Ltd and St. Jude MedicalJapan Co, Ltd. The other authors report no conflicts.

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idemia and low high-density lipoprotein cholesterol levels. Am J Cardiol.

2003;91:1304–1310.

37. Hunninghake DB, Stein EA, Bays HE, Rader DJ, Chitra RR, SimonsonSG, Schneck DW. Rosuvastatin improves the atherogenic and atheropro-

tective lipid profiles in patients with hypertriglyceridemia. Coron Artery

Dis. 2004;15:115–123.

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P, Camera M, Brambilla M, Sirtori CR, Chiesa G. Rosuvastatin does not affect

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39. Glass C, Pittman RC, Weinstein DB, Steinberg D. Dissociation of tissue

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and gonad. Proc Natl Acad Sci U S A. 1983;80:5435–5439.

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-β

HDL even if cholesterol ester transfer protein activity is blocked.

Significance


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Yoshinari Uehara and Keijiro SakuTomohiko Shimizu, Shin-ichiro Miura, Hiroyuki Tanigawa, Takashi Kuwano, Bo Zhang,

Diet High-Fatand Promotes Reverse Cholesterol Transport in Macrophage Cells in Mice Fed a

Dependent Efflux Ex Vivo−Rosuvastatin Activates ATP-Binding Cassette Transporter A1

Print ISSN: 1079-5642. Online ISSN: 1524-4636Copyright © 2014 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.114.3037152014;34:2246-2253; originally published online August 7, 2014; Arterioscler Thromb Vasc Biol.

http://atvb.ahajournals.org/content/34/10/2246

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1

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


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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


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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 manufacturer’s

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


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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 4°C 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 (0.25×106 /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 μCi/mL [1,2-3H]cholesterol and 50

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


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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 37°C 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 (0.25×106 /mL) in DMEM

with 10 % FBS plus 0.5 % penicillin G and streptomycin at 37°C. After 4 days,

macrophages were labeled for 24 h at 37°C with 2 μCi/mL [1, 2-3H]cholesterol and 25

μg/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 μM) in serum-free medium. For cholesterol efflux, BMM were treated with or

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


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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 μCi/mL 3H-cholesterol and cholesterol enriched with 25 μg/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.


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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


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mean ± standard deviation (SD). A probability value


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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. J 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


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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


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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.

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

atorvastatin and control groups.

rosuvastatin atorvastatin control0

10

20

30

Feces

% c

p m i n j e c t e d

**

Methods for measurement of the intestinal absorption of cholesterol

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


0.5

1.0

1.5

NPC1L1 mRNA express ion

(AU)


0.5

1.0

1.5

ABCB1a mRNA expr ess ion

(AU)


0.5

1.0

1.5

2.0

2.5

LDL-R mRNA expressi on

(AU)


0.5

1.0

1.5

ABCG5 mRNA exp ressio n

(AU)


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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 μl of safflower oil (195-15372, Wako Pure

Chemical Industries, Ltd. Japan) that contained a mixture of 2 μCi [5,6-3H] sitostanol

(ART 0361, American Radiolabeled Chemicals Inc., St. Louis, MO, USA) and 1 μCi

[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 calculatedas 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.


0.5

1.0

1.5

Cyp7A1 mRNA expression

(AU)


0.5

1.0

1.5

2.0

HMG-CoA reductase mRNA expression

(AU)

* *


0.5

1.0

1.5

LDL-R mRNA express ion

(AU)

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Arterioscler Thromb Vasc Biol 2014 Shimizu 2246 53