Cell Metabolism

ArticleAblation of Neutral Cholesterol
Ester Hydrolase 1 Accelerates Atherosclerosis
Motohiro Sekiya,1 Jun-ichi Osuga,1,3 Shuichi Nagashima,3 Taichi Ohshiro,3 Masaki Igarashi,1 Hiroaki Okazaki,1Manabu Takahashi,3 Fumiko Tazoe,3 Taeko Wada,4 Keisuke Ohta,1 Mikio Takanashi,1 Masayoshi Kumagai,1Makiko Nishi,1 Satoru Takase,1 Naoya Yahagi,1 Hiroaki Yagyu,3 Ken Ohashi,1 Ryozo Nagai,2 Takashi Kadowaki,1
Yusuke Furukawa,4 and Shun Ishibashi3,*
1Departments of Metabolic Diseases
2Cardiovascular Medicine
Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan
3Divison of Endocrinology and Metabolism
4Division of Stem Cell Regulation
Department of Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan*Correspondence: ishibash@jichi.ac.jp
DOI 10.1016/j.cmet.2009.08.004

SUMMARY

Cholesterol ester (CE)-laden macrophage foam cells
are the hallmark of atherosclerosis, and the hydrolysis
of intracellular CE is one of the key steps in foam cell
formation. Although hormone-sensitive lipase (LIPE)
and cholesterol ester hydrolase (CEH), which is
identical to carboxylsterase 1 (CES1, hCE1), were
proposed to mediate the neutral CE hydrolase(nCEH) activity in macrophages, recent evidences
have suggested the involvement of other enzymes.
We have recently reported the identification of acandidate, neutral cholesterol ester hydrolase 1(Nceh1). Here we demonstrate that genetic ablationof Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable
degree of nCEH activity in macrophages and together
account for most of the activity. Mice lacking both
Nceh1 and Lipe aggravated atherosclerosis in an
additive manner. Thus, Nceh1 is a promising target
for the treatment of atherosclerosis.
INTRODUCTION
Atherosclerotic cardiovascular diseases are the leading causesof mortality in industrialized countries, despite advances in the
management of coronary risk factors. Heart attacks arise fromthe thrombotic occlusion of coronary arteries following the
rupture of plaques. Lipid-rich plaques, which are characterized
by a plethora of cholesterol ester (CE)-laden macrophage foamcells, are prone to rupture (Libby, 2002). The hydrolysis of intracellular CE is the initial step of reverse cholesterol transport(RCT) (Brown et al., 1979). As the hydrolysis of CE preceding
RCT takes place at neutral pH, the enzymes catalyzing it havebeen collectively called neutral CE hydrolases (nCEHs) to distinguish them from lysosomal acid lipase, the CE hydrolase in lysosomes whose optimal pH is acidic. Since this step is rate-limiting
particularly in macrophage foam cells (Ishii et al., 1992; Krithar-ides et al., 1998), it is important to clarify the mechanisms thatmediate the hydrolysis of CE in foam cells.
To date, three enzymes have been proposed to serve as nCEHs
in macrophages: hormone-sensitive lipase (LIPE) (Yeaman,
2004); cholesteryl ester hydrolase (CEH)(Ghosh, 2000), which is
identical to human liver carboxylesterase1 (CES1, hCE-1) (Longet al., 1991) or macrophage serine esterase1 (HMSE1) (Munger
et al., 1991), also known as a human ortholog of triacylglycerolhydrolase (TGH) (Lehner and Vance, 1999); and neutral cholesterol ester hydrolase 1(Nceh1, MGI ID 2443191) (Okazaki et al.,2008), which is also known as KIAA1363 or arylacetamide deacetylase-like 1 (Aadacl1) (Nomura et al., 2005). Lipe is expressed inmacrophages, and its overexpression inhibits the accumulationof CE in THP-1 macrophages (Escary et al., 1998; Okazaki
et al., 2002). However, peritoneal macrophages (MPMs) of
Lipe-deficient (Lipe/) mice in a mixed genetic background stillretain substantial nCEH activity (Osuga et al., 2000), indicating
the presence of additional nCEH(s). Ghosh reported CES1 as
a promising candidate for an nCEH, because its macrophagespecific overexpression driven by the promoter of macrophagescavenger receptor-1 protected against diet-induced atherosclerosis in low-density-lipoprotein receptor (Ldlr)-deficient(Ldlr/) mice (Zhao et al., 2007). However, the effects of loss offunction of CES1 on nCEH activity in macrophages have notbeen reported. Furthermore, a mouse ortholog of CES1, TGH,
was expressed at a low level in MPMs (Okazaki et al., 2008) andpossessed negligible nCEH activity (Okazaki et al., 2006).
In contrast, Nceh1 is robustly expressed in MPMs as well as inatherosclerotic lesions. Its overexpression inhibits the accumulation of CE in THP-1 macrophages and the knockdown of Nceh1
by short interference RNA significantly reduced nCEH activity in
MPMs (Okazaki et al., 2008). Therefore, Nceh1 is more likely to
be involved in the hydrolysis of CE in MPMs. In the present study,
we took advantage of gene targeting to underscore the importance of Nceh1 as well as Lipe in the atherogenesis.
RESULTS
Nceh1 Mediates nCEH Activity in Macrophages
The targeting strategy was designed so that exon 4, which
encodes the catalytic domain, of the endogenous Nceh1 gene

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Absence of Nceh1 Accelerates Atherosclerosis

Figure 1. nCEH Activity and Cholesterol Trafficking in Nceh1/ Macrophages(A) Peritoneal macrophages (n = 7–8) were incubated withDMEM containing 10% FCS. Neutral cholesterol esterhydrolase (nCEH) activity or triglyceride lipase (TGL)
activity of whole-cell lysates were measured.
(B) The nCEH activity after subcellular fractionation (n = 8).
(C)Intracellular levels of cholesterol ester (CE) or freecholesterol (FC) in macrophages. Peritoneal macrophages(n = 7–8) were incubated with increasing concentrationsof acLDL for 24 hr (symbol definitions given in the inset,
n = 7–8).
(D) CE formation. Peritoneal macrophages (n = 7) wereincubated with acLDL and [1-14C]oleate-albumin complex
for 24 hr, and the incorporation of [1-14C]oleate into CE
fraction was measured.
(E) Cholesterol efflux. After labeling of cells (n = 5) withradiolabeled acLDL, the medium was changed to
DMEM/FCS containing acLDL with or without HDL3. After
24 hr, the radioactivity of an aliquot of the medium and of
the cell lysate was measured. The percent efflux was
calculated as (media dpm)/(cell + media dpm) 3 100.
(F) Microsomal ACAT activity (n = 8). ACAT activity inmicrosomes was determined by the rate of incorporationof [1-14C]oleoyl-CoA into the CE fraction. Data areexpressed as the mean ± SEM. * and ** denote p < 0.05
and p < 0.01, respectively, as determined by ANOVA
followed by the Tukey-Kramer posthoc test.

could be replaced with a gene encoding neo resistance (Figures
S1A and S1B). Analysis of progeny showed that mice heterozygous (+/) and homozygous (/) with respect to the disrupted
Nceh1 allele were born in numbers consistent with Mendelian
inheritance of the mutant allele, survived normally, and werefertile. Nceh1-deficient (Nceh1/) mice maintained on normalchow exhibit a normal metabolic phenotype (Table S1).
To elucidate the contribution of Nceh1 to the nCEH activity
in macrophages, MPMs were examined in vitro. Successful
deletion of Nceh1 was confirmed at both the protein andmRNA levels (Figures S1C and S1D). Nceh1 deficiency reducednCEH activity by 50%, and triacylglycerol lipase (TGL) activity by
20%, respectively (Figure 1A).
Consistent with our previous report that Nceh1 is a microsomal
enzyme (Igarashi et al., 2009), Nceh1 deficiency selectively
reduced the microsomal nCEH activity but did not affect thecytosolic nCEH activity (Figure 1B).
Nceh1 Is Involved in Foam Cell Formation
Foam cells were produced in vitro by incubation of MPMs withacetylated LDL (acLDL). The incubation with an increasingly
higher concentration of acLDL resulted in an increase in intracellular CE content by 50% in Nceh1/ macrophages comparedwith wild-type macrophages, whereas the intracellular free cholesterol (FC) content was notaffected by genotype (Figure 1C). The formationof CE was also monitored by the incorporationof [14C]oleate into cholesterol oleate, and theintracellular [14C]cholesterol oleate was accumulated in Nceh1/ macrophages approximately 2.5-fold compared with wild-type
macrophages (Figure 1D). Cholesterol efflux was reduced by
35% in Nceh1/ macrophages compared with wild-type
macrophages in the presence or absence of high-density lipoproteins (HDL)3 (250 mg/ml) as an exogenous lipid acceptor(Figure 1E). Upon exposure to acLDL, excess cholesterol in
the endoplasmic reticulum (ER) becomes esterified by acylCoA:cholesterol acyltransferase (ACAT) and stored in cytoplasmic lipid droplets (LDs) (Chang et al., 2001). The ACAT
activity did not differ between wild-type and Nceh1/ macrophages, and the ACAT activity did not explain the excess accumulation of CEs in Nceh1/ macrophages (Figure 1F).
We also investigated the expression profiles of genes involvedin the metabolism of cholesterol and formation of foam cells.
Nceh1 deficiency did not alter the expression profile, while
acLDL loading altered the expression of several genes suchas the Ldlr, ATP-binding cassette transporter A1(Abca1),3-hydroxy-3-methyl-glutaryl-CoA synthase (Hmgcs1), and class
B scavenger receptor (Scarb1) (Figure S2A). The expression of
Lipe was not affected by Nceh1 deficiency at the mRNA orprotein level (Figures S2B–S2D).
To clarify the importance of Nceh1 in multiple receptor pathways, we investigated the effects of two other kinds of modified
lipoproteins: beta very low-density lipoproteins (VLDL) and220 Cell Metabolism 10, 219–228, September 2, 2009 ª2009 Elsevier Inc.

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Absence of Nceh1 Accelerates Atherosclerosis

Figure 2. Foam Cell formation Induced by betaVLDL or Oxidized LDL(A) Peritoneal macrophages (n = 5–6 for each genotype)were incubated with beta-VLDL (10 or 20 mg/ml) for
24 hr, and intracellular CE content and FC content were
determined. As a control, the same cells were also incubated with acLDL (100 mg/ml) simultaneously.
(B) Peritoneal macrophages (n = 7 for each genotype) wereincubated with oxLDL (50 mg/ml) or acLDL (50 mg/ml) for
24 hr, and intracellular CE content and FC content were
determined.
(C and D) CE formation. Peritoneal macrophages (n = 6–7
for each genotype) were incubated with beta-VLDL (10 or
20 mg/ml, C) or oxidized LDL (oxLDL) (50 mg/ml, D) in thepresence of [1-14C]oleate-albumin complex for 24 hr.
Intracellular lipids were separated by thin layer chromatograpy (TLC), spots corresponding to the CE fraction
were scraped off, and radioactivity was measured. As
a control, the same cells were incubated with acLDL.
Data are expressed as the mean ± SEM. * and ** denotep < 0.05 and p < 0.01, respectively, as determined by
ANOVA followed by the Tukey-Kramer posthoc test.

oxidized low-density lipoproteins (oxLDL). When Nceh1/macrophages were exposed to beta-VLDL or oxLDL, the intracellular CE content was increased by 194%–231% and 150%,
respectively, compared with wild-type macrophages (Figure 2A
for beta-VLDL and Figure 2B for oxLDL). As predicted,[14C]oleate’s incorporation into intracellular CE was also
increased in Nceh1/ macrophages by 195% and 190%,
respectively, upon exposure to beta-VLDL and oxLDL (Figure 2C
for beta-VLDL and Figure 2D for oxLDL).
Nceh1 Deficiency Aggravates Atherosclerosis
To evaluate the impact of a deficiency of Nceh1 on the development of atherosclerotic lesions, Nceh1/ mice were backcrossed to the C57BL6/J background for at least five generations
and cross-bred with Apoe-deficient (Apoe/) mice. The offspringwere fed a western-type diet containing 0.15% (w/w) cholesteroland 20% (w/w) fat starting at 5 weeks of age for 12 or 16 weeks,
and the degree of atherosclerosis was determined either by quantifying sudanophilic en face lesions in pinned out aortas (Figures
3A and 3B) or by quantifying Oil Red O-stained lesions in cross-sections from the aortic root (Figures 3C and
3D). The areas of the aortic surface coveredby atherosclerotic lesions were increased inNceh1/;Apoe/ mice compared to Nceh1+/+;
Apoe/ mice (2.2-fold for males and 2.3-fold forfemales at 17 weeks of age, and 1.7-fold formales and 2.4-fold for females at 21 weeks ofage). The results of the en face analysis wereconfirmed by quantification of Oil Red O-stained
aortic root sections (1.8-fold for male and 1.2fold for female at 17 weeks of age, and 1.8-foldfor male and 1.6-fold for female at 21 weeks ofage). It is known that the correlation between
the morphometric assessment of lesions andthe content of FC and CE is high (Veniant et al.,2000); therefore aortic lipids were extracted
and measured (Figure 3E). Consistent with theincreased lesion area in Nceh1/:Apoe/ mice, FC as well as
CE content was significantly higher in Nceh1/;Apoe/ aortas
than Nceh1+/+;Apoe/ aortas (1.5-fold and 1.3-fold increase
for male and female FC content, respectively, and 2.0-fold and1.3-fold increase for male and female CE content, respectively).
Next, we determined whether the deficiency of Nceh1 in Apoe/mice influenced whole-body metabolism. None of the metabolic
parameters (plasma total cholesterol and triglycerides concentrations, blood glucose level, and body weight) was affected by the
deficiency (Table S2).
Furthermore, it was confirmed that the deficiency of Nceh1
in Apoe/ mice had no effect on plasma lipoprotein profiles
evaluated by high performance liquid chromatography (HPLC)(Table S3). Taken together, Nceh1 deficiency accelerated thedevelopment of atherosclerosis without affecting whole-bodymetabolism. Immunohistochemistry using the macrophage
marker F4/80 was carried out, and the percentage of macrophage-infiltrated area per unit lesion area was not significantly
different between Nceh1+/+;Apoe/ and Nceh1/;Apoe/mice (Figures 3F and 3G).

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Figure 3. Atherosclerosis in Nceh1/;Apoe/Mice(A) Nceh1-deficient mice were crossed with Apoe-deficient mice, and fed a Western-type diet starting at 5 weeks
of age for 12 weeks. Representative images of pinned
Sudan-IV stained aortas from mice (en face analysis,
Nceh1 genotype, and quantification values indicated).
(B) Quantification of the surface area occupied by thelesions (% area occupied by the lesions, n = 10–11). The
duration of the dietary load (12 weeks or 16 weeks) andNceh1 genotype are indicated below the graphs in (B)and (D).
(C) Representative photomicrographs of aortic root
cross-sections stained with Oil-Red O. Nceh1 genotype
is indicated above the images.
(D) Quantification of the area occupied by lesions using
cross-sections of aortic roots (n = 10–15). For analyzing
the area occupied by atherosclerotic lesions, both theMann-Whitney U test and two-tailed Student’s t test
were used. The P values generated by the two analyses
were consistent.
(E) Extraction of aortic wall lipids. After 12 weeks ona western-type diet, mice (n = 11–12 for males and n =14–16 for females) were euthanized and the aortas wereprepared.
(F) Representative photomicrographs of F4/80-stained
aortic root cross-sections from male mice fed a westerntype diet for 12 weeks.
(G) The quantification of the F4/80-stained area (n = 10 foreach genotype). The macrophage lesion area is indicatedas a percentage of the total lesion area. Data areexpressed as the mean ± SEM. * denotes p < 0.05 asdetermined with the two-tailed Student’s t test for (E)and (G) (NS denotes nonsignificant difference).

Both Nceh1 and Lipe Mediate nCEH Activity
To characterize the remaining nCEH activity in Nceh1/ macrophages, we examined whether the expression of Lipe was
induced in Nceh1/ macrophages. The expression of Lipe was
not altered at either the mRNA or protein level irrespective of
the incubation with acLDL (Figures S2A–S2C). We also performed a western blot analysis of individual samples. There
was no significant difference in the expression of Lipe protein
between wild-type and Nceh1/ macrophages (data notshown). Next, we separated microsomal and cytosolic subcellular fractions by ultracentrifugation. Nceh1 deficiency did notaffect the expression of Lipe protein in the cytosolic or microsomal fraction (Figure S2D). To test whether Lipe is responsible
for the remaining nCEH activity in Nceh1/ macrophages,
Nceh1/ mice were cross-bred with Lipe-deficient (Lipe/)mice to produce mice deficient in both Nceh1 and Lipe
(Nceh1/;Lipe/). The nCEH activity in Nceh1/;Lipe/macrophages was dramatically reduced, approximately to 10%of that in wild-type macrophages, and Nceh1 and Lipe were
responsible for approximately 50% of the nCEH activity, respec-tively (Figure 4A). Thus, these two lipases were
responsible for the majority of the nCEH activity
in MPMs. Upon subcellular fractionation, Nceh1
contributed solely to the microsomal nCEH
activity, while Lipe contributed to both cytosolicand microsomal nCEH activity (Figure 4C).
Meanwhile, substantial TGL activity remained even in theNceh1/;Lipe/ macrophages (Figure 4B) (72% without acLDL
and 83% with acLDL, of that in wild-type macrophages). To
investigate the roles of Nceh1 and Lipe in foam cell formation,
the macrophages of each genotype were incubated with acLDL.
Upon exposure to acLDL, Nceh1 and Lipe deficiency resulted in
an increase of intracellular CE content by 29% and 51%
compared with the wild-type, respectively (Figure 4D). Interestingly, Nceh1 and Lipe contributed in an additive manner to theintracellular CE content of Nceh1/;Lipe/ macrophages (anincrease of 70% compared with the wild-type, Figure 4D). The
extent of foam cell formation for each genotype was also
confirmed by the incorporation of [14C]oleate into the cellular
CE (Figure 4E).
Although the nCEH activity of Nceh1/ macrophages wasalmost identical to that of Lipe/ macrophages, Lipe/ macrophages were less prone to form foam cells than Nceh1/ macrophages. Nceh1 and Lipe also contributed in an additive manner tocholesterol efflux (Figure 5A, reduction by 11%–5%, 23%–
21%, and 43%–45% in Nceh1+/+;Lipe/, Nceh1/;Lipe+/+,222 Cell Metabolism 10, 219–228, September 2, 2009 ª2009 Elsevier Inc.

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Absence of Nceh1 Accelerates Atherosclerosis

CS-505 or HDL. Under such conditions, cholesterol efflux wasvery slow in the absence of HDL and strikingly facilitated by
HDL, and the FC was effluxed from macrophages of each genotype in a quite reproducible fashion (Figure 5C). The contributionsof Nceh1 and Lipe to the cholesterol efflux were further verified inthese additional experiments.
ACAT1 is an allosteric enzyme regulated by FC (Chang et al.,2001), and the hydrolysis of cellular CE would supply substrate
for ACAT. Consistent with this assumption, microsomal ACAT
activity was reduced in Nceh1/;Lipe/ macrophages when
they were loaded with acLDL (45% reduction compared withwild-type macrophages) (Figure 5D).
We performed a northern blot analysis of the genes involvedin the metabolism of cholesterol during the formation of foamcells. Cholesterol loading by incubation with acLDL increased
the mRNA expression of Cd36, Abca1, and Abcg1, while itdecreased the mRNA expression of Ldlr. However, there wasno obvious difference in the mRNA expression of the genes
examined among four genotypes under conditions of cholesterolloading (Figure 5E). We also compared the endocytic uptake anddegradation of acLDL by macrophages of each genotype(Figure 5F). Supporting the results of the northern blot analysis,
there was no significant difference in the internalization anddegradation of acLDL among the 4 genotypes.

Figure 4. nCEH Activity and Foam Cell Formation in Lipe/, Nceh1/,or Nceh1/;Lipe/ Macrophages(A and B) Peritoneal macrophages (n = 6–7 for each genotype) were incubatedwith or without acLDL (100 mg/ml) for 24 hr, and nCEH (A) and TGL (B) activities
were determined.
(C) The nCEH activity after subcellular fractionation (n = 6–7).
(D) Peritoneal macrophages (n = 8) were incubated with or without acLDL for
24 hr, and intracellular CE content and FC content were measured.
(E) CE formation. Peritoneal macrophages (n = 7–8) were incubated with
acLDL in the presence of [1-14C]oleate-albumin complex for 24 hr, and theincorporation of [1-14C]oleate into CE fraction was measured. Data areexpressed as the mean ± SEM. * and ** denote p < 0.05 and p < 0.01, respectively, as determined by ANOVA followed by the Tukey-Kramer posthoc test.

and Nceh1/;Lipe/ macrophages compared with wild-type
macrophages, respectively). The basal efflux to bovine serum
albumin (BSA) alone without an extracellular lipoprotein acceptor
or serum was also examined (Figure 5B). To eliminate the possibility that the constant cycle of hydrolysis and resynthesis of
CE modulates cholesterol efflux, the experiment was conductedin the presence or absence of an ACAT inhibitor, CS-505. Since
it was reported that the simultaneous cholesterol-loading and
ACAT inhibition led to cytotoxicity in macrophages (Tabas,
2005), macrophages were loaded with acLDL reconstituted
with [3H]cholesteryl linoleate for 24 hr, and the medium waschanged to that without acLDL in the presence or absence ofAtheroprotective Role of Nceh1 and Lipe
Deficiency of Lipe or Nceh1 in whole body may affect atherosclerosis by the systemic phenotypes irrelevant to the local
phenotypes in macrophages. Indeed, Lipe deficiency changed
glucose tolerance as well as plasma lipoprotein profiles (Table
S4). To specifically assess the biological roles of macrophagenCEHs in atherogenesis in vivo, we took advantage of bonemarrow transplantation to selectively disrupt the Nceh1 gene
and/or Lipe gene in hematopoietic cells. Female Ldlr-deficient(Ldlr/) mice were exposed to total-body irradiation to eliminate the endogenous bone marrow-derived cells, and subsequently transplanted with bone marrow cells isolated frommale donor mice of each genotype. Successful reconstitutionof recipients with cells of donor origin after bone marrow transplantation was established by PCR-assisted amplification of the
Sry gene (a male marker) and Nceh1 or Lipe mutant gene
(Figure 6A).
During the course of the experiment, a deficiency of Nceh1
and/or Lipe in hematologic cells did not alter serum lipid levels(Table S5). An analysis using HPLC did not detect any differencein the cholesterol and triglycerides levels of each lipoprotein fraction (data not shown). The atherosclerotic surface area wereincreased by 1.7-fold in Lipe/(BM)/Ldlr/ mice and 3.1-foldin Nceh1/(BM)/Ldlr/ mice compared to wild-type(BM)/Ldlr/ mice. The effect was most pronounced in Nceh1/;Lipe/(BM)/Ldlr/ mice (a 4.3-fold increase compared towild-type(BM)/Ldlr/ mice) and was approximately additive
with respect to the contribution (Figures 6B and 6C). Although
the difference between wild-type(BM)/Ldlr/ mice and Lipe/(BM)/Ldlr/ mice was not statistically significant when analyzed
by an one-way ANOVA, it was statistically different whenanalyzed by a two-tailed Student’s t test (p = 0.016). Quantification of cross-sectional lesion area in aortic root sections further
supported the significance of Nceh1 and Lipe in atherogenesis

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Figure 5. Cholesterol trafficking in Lipe/,Nceh1/, or Nceh1/;Lipe/ Macrophages(A and B) Cholesterol efflux. Peritoneal macrophages (n =6, A, n = 6–7, B) were incubated for 24 hr in DMEM containing 10% FCS (A) or 5% BSA (B) with acLDL (100 mg/ml)
whose CE was reconstituted with cholesteryl[1,2-3H]linoleate. After labeling of the cells, the medium was changed
to DMEM containing 10% FCS (A) or 5% BSA (B) with
acLDL (100 mg/ml)in the presence or absence of HDL3
(250 mg/ml).
(C) Peritoneal macrophages (n = 5–6) were incubatedfor 24 hr in DMEM containing 5% BSA with 100 mg/ml
acLDL whose CE was reconstituted with cholesteryl[1,2-3H]linoleate. After labeling of the cells, the medium
was changed to DMEM containing 5% BSA in the presence or absence of an ACAT inhibitor, CS-505 (10 mM)
and/or HDL3 (250 mg/ml). After 24 hr, the radioactivity ofan aliquot of the medium and cell lysate was measured.
The percent efflux was calculated as (media dpm)/(cell +media dpm) 3 100.
(D) Microsomal ACAT activity. Peritoneal macrophages(n = 6–7) were incubated with or without acLDL (100 mg/ml)
for 24 hr. ACAT activity in microsomes was determined by
the rate of incorporation of [1-14C]oleoyl-CoA into the CE
fraction.
(E) Gene expression profile. Peritoneal macrophages (n =4) were incubated with or without acLDL (100 mg/ml) for
24 hr. Nceh1, neutral cholesterol ester hydrolase 1; Lipe,
hormone-sensitive lipase; Msr1, type A scavenger
receptor; Scarb1, scavenger receptor class B type-I;
Ldlr, the low-density lipoprotein receptor; Soat1, acylCoA:cholesterol acetyltransferase 1; Abca1, ATP-binding
cassette transporter A1; Abcg1, ATP-binding cassettetransporter G1. Loading was normalized by the expressionof Rplp0 (acidic ribosomal phosphoprotein P0).
(F) Cellular uptake and degradation of acLDL. Peritoneal
macrophages (n = 5) were incubated with a medium containing varying concentrations of 125I-acLDL with orwithout a 40-fold excess of unlabeled acLDL for 5 hr at
37 C. The amounts of 125I-acLDL either degraded by or
associated with the cells were measured. Data areexpressed as the mean ± SEM. * and ** denote p < 0.05
and p < 0.01, respectively, as determined by ANOVA(AD) or repeated-measures ANOVA (F) followed by the
Tukey-Kramer posthoc test.

(a 1.2-fold increase in Lipe/ (BM)/Ldlr/ mice, a 1.6-foldincrease in Nceh1/ (BM)/Ldlr/ mice, and a 2.0-fold increasein Nceh1/;Lipe/ (BM)/Ldlr/ mice compared to wildtype(BM)/Ldlr/ mice, respectively (Figures 6D and 6E).
DISCUSSION
The hydrolysis of cellular CEs is the first committed step in theefflux of cholesterol. Subsequently, cholesterol is transferred
to plasma membrane where ABCA1 and/or ABCG1 mediate final
transfer to apoA-I and/or HDL, respectively. Under normal
circumstances, the efflux of cholesterol mediated by ABC transporters is rate-limiting in the overall process, since the combined
deficiency severely impaired cholesterol efflux (Out et al., 2008;
Wang et al., 2007; Yvan-Charvet et al., 2007), and since cholesterol efflux was stimulated when the expression of these ABCtransporters were induced by the activation of LXR (Venkateswaran et al., 2000; Tazoe et al., 2008). Under conditions where

an excess of cholesterol is stored as CE in LDs of macrophage
foam cells, the hydolysis of cellular CE may become rate-limiting
as was observed in the present study (Figures 1E and 5A–5C). It
is of note that the deficiency of Nceh1 caused a more profounddecrease in cholesterol efflux than the deficiency of Lipe,even though the reduction in nCEH activity was comparable(Figure 4A). These results suggest that the roles of Lipe andNceh1 in the mobilization of cholesterol from LDs to the medium
are somewhat different: Nceh1 is more efficient than Lipe in thisprocess.
Based on our current results, the contribution of the orthologof CEH, TGH1, and Ces1, to the nCEH activity of MPM would
be marginal. In contrast to MPMs, human monocyte-derived
macrophages express CEH, a macrophage serine-esterase.
Even in human macrophages, CES1 is not a major enzyme mediating the hydrolysis of CE, and NCEH1 (NM_020792) also playsa central role in the CE hydrolysis in human macrophages(M.I., unpublished data).

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Figure 6. Aherosclerosis in Ldlr/ Mice with
Nceh1/, Lipe/, or Nceh1/;Lipe/ Bone
Marrow(A)Irradiated female Ldlr/ mice (age 8 weeks, n = 12–17)
were transplanted with bone marrow cells isolated frommale wild-type, Lipe/, Nceh1/, Nceh1/;Lipe/
mice (age 8 weeks). Mice were maintained on a chow dietfor the first 4 weeks, then switched to a high-cholesterol
diet. After 12 weeks on the high-cholesterol diet, the mice
were euthanized to assess their lesions. (A) shows verification of successful reconstitution with donor hematopoietic
cells by PCR amplification of specific fragments of several
genes. Genomic DNA from bone marrow of chimeric mice(the genotype of donor mice is indicated below) or tail wasused as a template. The following templates were used as
positive and negative controls (lane A and B): Tail DNA froma Nceh1+/+ and Nceh1/ mouse for Nceh1 gene amplification, tail DNA from a Lipe+/+ and Lipe/ mouse for Lipe
gene amplification, tail DNA from a Ldlr+/+ and Ldlr/
mouse for Ldlr gene amplification, and tail DNA froma female and male C57BL6/J mouse for Sry gene amplification in lane A and B, respectively.
(B–E) Quantification by en face analysis (B) (quantification
values indicated below the images) (C), and crosssectional analysis (D and E). The genotypes of donor cells
are indicated below the graphs. * and ** denote p < 0.05
and p < 0.01, respectively, as determined by ANOVA
followed by the Tukey-Kramer posthoc test.

Based on the current findings, it is expected that inductionof the expression of Nceh1 in macrophages can be used
for the treatment or prevention of atherosclerosis. In vitro, we
demonstrated that adenovirus-mediated overexpression ofNceh1 decreased foam cell formation (Okazaki et al., 2008). To
substantiate these findings, we need to prove the hypothesis
in vivo either by transgenic overexpression of Nceh1 or by its
pharmacological activation. In this context, it is noteworthy
that this hypothesis has been tested by overexpressing
LIPE or CEH to hydrolyze CE in a macrophage-specific manner.
The results for LIPE are somewhat complicated. Overexpression of LIPE paradoxically increases the development of
atherosclerosis (Escary et al., 1998) and the concomitant cooverexpression of a cholesterol acceptor, apoA-IV, is anti-atherogenic(Choy et al., 2003). Moreover, overexpression of CES1
was reported to reduce the atherosclerosis of Ldlr/ mice(Zhaoet al., 2007).
The human ortholog of Nceh1, KIAA1363, is identical tohuman AADACL1 (Probst et al., 1994). Recently, Tiwari et al.
reported that steryl deacetylase 1 in S. cerevisiae, which has
homology to mammalian AADAC and AADACLs, controls theexport of sterols by regulating the deacetylation/acetylation ofcholesterol (Tiwari et al., 2007). In this report, it was shown thathuman AADAC, but not AADACL1, catalyzed the deacetylationof acetylated cholesterol. Thus, it is not
surprising that the substrate of AADACL1(NCEH1) is CE.
In foam cells, CE is the major lipid component
in LDs with triglyceride typically comprising 10%
or less of the total lipid. While Nceh1 and Lipe
were responsible for the majority of the nCEH
activity in macrophages, substantial amount of TGL activity inmacrophages was remained even in the Nceh1/;Lipe/macrophages. In adipose tissue, adipose triglyceride lipase(PNPLA2) was identified as a non-Lipe triglyceride lipase, andPnpla2 is also expressed in macrophages (Zimmermann et al.,2008). Pnpla2 might be a promising candidate responsible forthe remaining TGL activity in Nceh1/;Lipe/ macrophages.
It was also reported that KIAA1363 hydrolyzes 2-acetyl monoalkylglycerol and the chemical inhibition of KIAA1363 modulates
the metabolism phospholipids such as lysophosphatidinic acidand platelet-activating factor (Chiang et al., 2006). Lysophosphatidinic acid and platelet-activating factor are implicated inmonocyte adhesion and transmigration, and promote atherosclerosis through their inflammatory activities (Ninio, 2005).
Thus, it remains possible that the Nceh1 deficiency aggravates
atherosclerosis at least in part by altering the composition ofphospholipids, which have pro-atherogenic or anti-atherogenic
potentials.
In conclusion, Nceh1 plays a crucial role in the development ofboth foam cells and atherosclerotic lesions. Nceh1 and Lipe are
two major enzymes with nCEH activity in murine macrophages.
These findings should provide the basis for understanding thepathophysiology of atherosclerosis and can be exploited todevelop an attractive therapeutic approach.

Cell Metabolism 10, 219–228, September 2, 2009 ª2009 Elsevier Inc. 225

Cell Metabolism
Absence of Nceh1 Accelerates Atherosclerosis

EXPERIMENTAL PROCEDURES
Generation of the Nceh1/ MiceA replacement-type targeting vector was constructed; the 1 kb short arm
spanning intron 3, and the 10 kb long-arm fragment encompassing intron
4–5 were generated by PCR using genomic DNA from the 129/Sv mouse asa template. Primers used were as follows; 50 -ctcgagatcagctcaaggctggagt
gtg-30 and 50 -ctcgagctctttgtaaagtacatgtggccc-30 for the short arm and 50 -acctttctcacggggttttctgaagtgggt-30 and 50 -gcatcctgcctgaatctgcaagtgctgag-30 for
the long arm. After subcloning into pGEM-T easy vector (Promega, Madison,
WI), the fragments were cut out and inserted into the Xho I and Not I sites,
respectively, of pPol II -short-neo-bpA-HSVTK as described previously (Ishibashi et al., 1993). Thus, a 1.2 kb region spanning exon 4 of the Nceh1 gene
was replaced by a neomycin-resistant cassette, which was expected toabolish translation of the catalytic motif. After linearization by digestion with
SalI, the vector was electroporated into JH1 embryonic stem cells (a giftfrom Dr. J Herz). Targeted clones, which had been selected in the presenceof G418 and 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodouracil, wereidentified by PCR using the following primers; sense (50 -gacttgacctcacagtgtgt
gaacc-30 ) and antisense (50 - gattgggaagacaatagcaggcatgc-30 ).
Homologous recombination was verified by Southern blot analysis afterdigestion with BglII using a 50 -flanking probe. Targeted embryonic stem cellclones were injected into the C57BL/6J blastocysts yielding two lines ofchimeric mice, which transmitted the disrupted allele through the germ line.
Genotyping was performed with PCR using specific primers: sense (50 -tccagta
gaagagggcacagaggtcc-30 ), antisense (50 -ccatcccgtttaccaataaagcttaaggc-30 )and neo (50 -aggattgggaagacaatagcaggcat-30 ).
The genetic background used in this study was as follows: (1) experiments
in vitro using Nceh1/ mice were performed on matched littermates from 129/
Sv-C57BL/6J hybrid descendants (F1 and subsequent generations) unlessotherwise stated. (2) Nceh1/ mice were crossed onto the C57BL/6J background for more than five generations and then crossed with Apoe/ miceon a C57BL/6 background (the Jackson Laboratory). (3) Nceh1/ mice
were crossed onto the C57BL/6J background for more than five generations
and then crossed with Lipe/ mice (crossed onto the C57BL6/J backgroundfor more than ten generations) (Osuga et al., 2000) for experiments in vitro
using Nceh1/;Lipe/ macrophages. (4) For donor mice in the bone marrowtransplantation experiments, mice were crossed onto the C57BL6/J background for more than ten generations. Mice were maintained on a 12 h dark/light cycle and were fed a normal chow diet (Lab Diet 5053, PMI Nutrition International) unless otherwise stated. All experimental procedures involving
animals were conducted according to our institutional guidelines.

Bone Marrow Transplantation
To eliminate the endogenous bone marrow-derived cells, female Ldlr/ mice(age 8 weeks) were exposed to a single dose of 9 Gy total body irradiation.
Irradiated recipients were transplanted with 5 3 106 bone marrow cells, isolated from male wild-type, Lipe/, Nceh1/, and Nceh1/;Lipe/ mice(age 8 weeks). Mice were maintained on a chow diet for the first 4 weeks,
and then switched to a high-cholesterol diet containing 1.25% (w/w) cholesterol and 15% (w/w) cocoa butter (Oriental Yeast Company; Tokyo, Japan).
After 12 weeks on the high-cholesterol diet, the chimeras were euthanized
for the assessment of the lesions. The hematologic chimerism of the Ldlr/
mice was determined in genomic DNA from bone marrow by PCR at 12 weeks
posttransplant. Sry-s (50 -gtggtgagaggcacaagt-30 ) and Sry-as (50 -gagtacaggtgtgcagct-30 ) were used to detect the Sry gene specific to the mouse
Y chromosome(Mignon et al., 1998). The primers used to detect both thewild-type and the null mutant Nceh1 or Lipe gene were described above or
previously (Osuga et al., 2000).

Peritoneal Macrophages
Thioglycorate-elicited macrophages were obtained from 8 week-old mice as
described previously (Osuga et al., 2000). The cells were resuspended in Dulbecco’s Modified Eagle Medium (DMEM) to give a concentration of 106 cells
per ml, and 10 ml per dish was plated in 10 cm dishes. Cells were incubatedin DMEM containing 10% fetal calf serum (FCS) in an atmosphere of 5%
CO2 and 95% air unless otherwise stated.

Preparation of Lipoproteins
After an overnight fast, blood was collected from normolipidemic volunteers
to isolate plasma. LDL (d =1.019–1.063 g/ml) and HDL (d = 1.063–1.21 g/ml)
were isolated from the plasma by sequential density ultracentrifugation(Havel et al., 1955). LDL was acetylated by repetitive additions of acetic
anhydride (Goldstein et al., 1979). acLDL was radioiodinated with Na125I by
the iodine monochloride method (Goldstein et al., 1979). CE in acLDL wasreconstituted with [1,2-3H]cholesteryl linoleate as described previously(Roberts et al., 1985). To prepare oxLDL, LDL was incubated for 17 hr at
37 C with 5 mM CuSO4, followed by the addition of 2.5 mM EDTA (Sakai
et al., 1996). Beta-VLDL (d = 1.006–1.019 g/ml) was isolated by sequential
ultracentrifugation of plasma obtained from overnight fasted male JapaneseWhite rabbits maintained on a cholesterol-enriched diet containing 1% (w/w)
cholesterol.
Determination of Cholesterol Content
Cellular lipids were extracted by hexane/isopropyl alcohol, and cholesterol
content was determined by an enzymatic fluorometric microassay according
to the method of Heider and Boyett, with minor modifications (Heider andBoyett, 1978; Yagyu et al., 2000).
CE Formation
The amounts of CE formed from [1-14C]oleic acid was determined as
described previously with minor modifications (Perrey et al., 2001).
TGL and nCEH Activity Assay
Enzymatic activity was assayed as described previously (Osuga et al., 2000;
Sekiya et al., 2008). The samples were incubated at 37 C for 30–60 min in a final
volume of 200 ml of a reaction mixture containing 105 mmol/l tri[3H]oleoylglycerol (99.4 mCi/mmol), 23.7 mmol/l lecithin, 12.5 mmol/l sodium taurocholate,
1 mol/l NaCl, and 85 mmol/l potassium phosphate (pH 7.0). The high concentration of NaCl was included to inactivate lipoprotein lipase. The nCEH activity
was measured using a reaction mixture containing 6.14 mmol/l cholesterol[1-14C]oleate (48.8 mCi/mmol).
Cholesterol Efflux
Cholesterol efflux assays were performed as described previously (Roberts
et al., 1985). Cells were incubated for 24 hr at 37 C in DMEM containing
bovine serum albumin (BSA, 5 mg/ml) or 10% FCS and acLDL (100 mg/ml)
whose CE was reconstituted with [1,2-3H]cholesteryl linoleate. After labelingof the cells, the medium was changed to DMEM containing BSA (5 mg/ml) or
10% FCS with acLDL (100 mg/ml) and with/without HDL3 (250 mg/ml). After
24 hr, an aliquot of the medium was removed and centrifuged at 15,000 3
g for 2 min to remove cellular debris, and the radioactivity in the supernatant
was measured with a liquid scintillation counter. The cells were lysed in 0.1 N
NaOH, and the radioactivity in an aliquot of the cell lysate was measured.
The percent efflux was calculated as (media dpm)/(cell + media dpm) 3
100. To precisely evaluate the contribution of nCEH activity to cholesterol
efflux, additional assays were conducted in the presence of the ACAT inhibitor CS-505 (provided by Daiichi Sankyo and Kyoto pharmaceutical industries) (Takahashi et al., 2008). After cells were loaded with cholesterol for
24 hr by incubation with acLDL, whose CE was reconstituted with
[1,2-3H]cholesteryl linoleate, the medium was changed to DMEM containing
BSA (5 mg/ml) in the presence or absence of CS-505 (10 mM) or HDL3
(250 mg/ml). After incubation for 24 hr, the radioactivity in media and cells
was measured.
Microsomal ACAT Activity
Cells were sonicated in buffer A (20 mM Tris-HCl pH 7.0, 250 mM sucrose,
5 mg/ml leupeptin, and 2.8 mg/ml aprotinin), ultracentrifuged at 100,000 3 g
for 45 min at 4 C, and re-ultracentrifuged at 100,000 3 g for 45 min at 4 C.
The precipitates were resuspended and used for the assay. ACAT activity inmicrosomes was determined by the rate of incorporation of [1-14C]oleoylCoA into the CE fraction according to Yagyu et al. (Yagyu et al., 2000).
Cellular Uptake and Degradation of 125I-AcLDL
Peritoneal macrophages were plated in 12-well plates at a density of 1 3 106/
well and incubated with a medium containing varying concentrations of

226 Cell Metabolism 10, 219–228, September 2, 2009 ª2009 Elsevier Inc.

Cell Metabolism
Absence of Nceh1 Accelerates Atherosclerosis

125

I-AcLDL with or without a 40-fold excess of unlabeled acLDL at 37 C. After
5 hr, the amounts of 125I-AcLDL either degraded by or associated with the cells
were measured according to a modified method of Goldstein (Goldstein et al.,
1983; Perrey et al., 2001).
Northern Blot Analysis
Total RNA was isolated using Trizol Reagent (Life Technologies; Helgerman,
CT). Ten microgram RNA samples equally pooled from each genotype were
electrophoresed in a 1% agarose gel containing formaldehyde and transferred
to a nylon membrane. The membranes were hybridized with probes, whichwere labeled with [a-32P]dCTP using a Megaprime DNA Labeling System Kit(Amersham Biosciences; Piscataway, NJ) in Rapid-hyb Buffer (Amersham
Biosciences) or Ultra-hyb buffer (for HSL mRNA detection) (Ambion; Austin,TX) and analyzed using a BAS2000 Bioimaging Analyzer (Fuji Photo Film;
Tokyo, Japan). Loading was normalized by the expression of Rplp0 (acidic
ribosomal phosphoprotein P0) mRNA.
Quantitative Real-Time PCR
Two micrograms of total RNA was reverse-transcribed using the ThermoScript
RT-PCR System (Invitrogen). Quantitative real-time PCR was performed using
SYBR Green Dye (Applied Biosystems; Foster City, CA) in an ABI Prism 7900
PCR instrument (Applied Biosystems). The relative abundance of eachtranscript was calculated from a standard curve of cycle thresholds for serial
dilutions of a cDNA sample and normalized to Rplp0. Primer sequences areavailable upon request.
Measurement of Atherosclerotic Lesions
Nceh1/;Apoe/ mice and littermate Nceh1+/+;Apoe/ mice were fed
a western-type diet containing 0.15% (w/w) cholesterol and 20% (w/w) fat(Oriental Yeast Company; Tokyo, Japan). (2) Bone marrow chimeric Ldlr-deficient mice were fed the high-cholesterol diet. After the indicate time, they wereeuthanized and their hearts and aortas were isolated. The degree of atherosclerosis was assessed by determining lesion sizes on both pinned-open
aortas and serial cross-sections through the aortic root (Paigen et al., 1987;
Yagyu et al., 2000). The aorta was opened longitudinally along the ventral
midline from the iliac arteries to the aortic root. After branching vessels were
removed, the aorta was pinned out flat on a black wax surface. The lesions
were stained with Sudan IV for 15 min, destained with 70% ethanol, andthen fixed in 4% phosphate-buffered formalin. Aortic images were analyzed
with Adobe Photoshop 7 software (Adobe Systems, Incorporated; Mountain
View, CA). Data are reported as the percentage of the aortic surface coveredby lesions (total surface area of the atherosclerotic lesions divided by the total
surface area of the aorta). The hearts were perfused with saline containing 4%(w/v) formalin and fixed for more than 48 hr in the same solution. The basal halfof the hearts was embedded in Tissue-Tek OCT compound (Miles, Incorporated), and serial sections were captured using a Cryostat microtome (10 mmthick). Four sections were used to evaluate the lesions: two at the end of theaortic sinus and two at the junction of the sinus and ascending aorta. The
sections were stained with Oil Red O and counterstained with hematoxylin.
Immunohistochemistry
Sections were incubated with primary antibody to mouse F4/80 (1:20; Serotec,
Raleigh, NC) for 2 hr. After washes, the sections were incubated with biotinylated anti-rat antibody for 30 min at room temperature and then with avidinbiotin peroxidase complex (Vector Labs) for 30 min. Finally, the sections
were developed with DAB (Wako Pure Chemicals Co.; Osaka, Japan) andcounterstained with methylgreen.
Extraction of Aortic Wall Lipids
After 12 weeks on a western-type diet, mice were euthanized and the aortas
were prepared. After the removal of connective tissue and fat, lipids wereextracted by the chloroform-methanol method, and FC content and CE
content were measured.
Statistics
Statistical differences between groups were analyzed by one-way analysis ofvariance and the post hoc Tukey-Kramer test, unless otherwise stated.

SUPPLEMENTAL DATAThe Supplemental Data include five tables and two figures and can be foundwith this article online at http://www.cell.com/cellmetabolism/supplemental/
S1550-4131(09)00233-2.
ACKNOWLEDGMENTS
This work was supported by a grant-in-aid from the Japan Health Science
Foundation and Research Fellowships of the Japan Society for the Promotionof Science for Young Scientists, a Grant-in-Aid for Scientific Research from the
Ministry of Education and Science, the Takeda Science Foundation, theMitsubishi Pharma Research Foundation, the Program for Promotion of
Fundamental Studies in Health Sciences of the National Institute of Biomedical
Innovation (NIBIO), and JKA through its promotion funds from KEIRIN RACE.
M.S. performed the majority of the experiments, analyzed data, and wrote
the manuscript; J.O. supervised the overall project; S.N., M.T., T.O, F.T.,
T.W., and Y.F. assisted with bone marrow transplantation experiments; M.I.
provided helpful data supporting our conclusion; H.O. identified Nceh1 andcontributed to the design of targeting construct; S.I. supervised the overall
project and wrote the manuscript.
Received: May 9, 2009
Revised: July 18, 2009
Accepted: August 14, 2009
Published: September 1, 2009
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