THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 35, Issue of August 30, pp. 31893–31899, 2002
Printed in U.S.A.

Elimination of Cholesterol Ester from Macrophage Foam Cells byAdenovirus-mediated Gene Transfer of Hormone-sensitive Lipase*
Received for publication, April 25, 2002, and in revised form, May 23, 2002
Published, JBC Papers in Press, May 29, 2002, DOI 10.1074/jbc.M204016200

Hiroaki Okazaki‡, Jun-ichi Osuga‡, Kazuhisa Tsukamoto‡, Naoyuki Isoo‡, Tetsuya Kitamine‡,
Yoshiaki Tamura‡, Sachiko Tomita‡, Motohiro Sekiya‡, Naoya Yahagi‡, Yoko Iizuka‡,
Ken Ohashi‡, Kenji Harada‡, Takanari Gotoda‡, Hitoshi Shimano§, Satoshi Kimura‡,
Ryozo Nagai¶, Nobuhiro Yamada§, and Shun Ishibashi‡储**
From the ‡Department of Metabolic Diseases and the ¶Department of Cardiovascular Medicine, Faculty of Medicine,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, §Metabolism, Endocrinology, and Atherosclerosis, Instituteof Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, and 储Division of Endocrinology
and Metabolism, the Department of Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi,
Kawachi-gun, Tochigi 329-0498, Japan

Cholesterol ester (CE)-laden foam cells are a hallmark
of atherosclerosis. To determine whether stimulation of
the hydrolysis of cytosolic CE can be used as a novel
therapeutic modality of atherosclerosis, we overexpressed hormone-sensitive lipase (HSL) in THP-1 macrophage-like cells by adenovirus-mediated gene delivery, and we examined its effects on the cellularcholesterol trafficking. We show here that the overexpression of HSL robustly increased neutral CE hydrolase activity and completely eliminated CE in the cells
that had been preloaded with CE by incubation withacetylated low density lipoprotein. In these cells, cholesterol efflux was stimulated in the absence or presenceof high density lipoproteins, which might be at least
partially explained by the increase in the expression of
ABCA1. Importantly, these effects were achieved without the addition of acyl-CoA:cholesterol acyltransferase
inhibitor, cAMP, or even high density lipoproteins. Furthermore, the uptake and degradation of acetylated low
density lipoprotein was significantly reduced probably
by decreased expression of scavenger receptor A and
CD36. Notably, the cells with stimulated CE hydrolysis
did not exhibit either buildup of free cholesterol or cytotoxicity. In conclusion, increased hydrolysis of CE by
the overexpression of HSL leads to complete eliminationof CE from THP-1 foam cells not only by increasing
efflux but also by decreasing influx of cholesterol.

Cholesterol ester (CE)1-laden macrophage foam cells are ahallmark of fatty streak lesions in atherosclerotic plaques.
* This work was supported by a grant-in-aid for Scientific Researchfrom the Ministry of Education, Science and Culture, the Promotion of
Fundamental Studies in Health Science from the Organization for
Pharmaceutical Safety and Research, and Health Sciences Research
grants (Research on Human Genome and Gene Therapy) from the
Ministry of Health and Welfare. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Division of Endocrinology and Metabolism, Dept. of Medicine, Jichi Medical School,
3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 3290498, Japan. Tel.: 81-285-58-7355; Fax: 81-285-40-6035; E-mail:ishibash@jichi.ac.jp.
1
The abbreviations used are: CE, cholesterol ester; TG, triacylglycerol; FC, free cholesterol; PBS, phosphate-buffered saline; SR, scavenger receptor; ACAT, acyl-CoA:cholesterol acyltransferase; NCEH,
neutral cholesterol ester hydrolase; ABCA1, ATP-binding cassetteThis paper is available on line at http://www.jbc.org

Besides cleaning up extracellularly deposited atherogenic lipoproteins, macrophage foam cells secrete a wide variety of
substances, which include inflammatory cytokines, chemokines, growth factors, and proteases (1). Furthermore, lipidrich lesions that are characterized by a plethora of macrophage
foam cells are associated with massive infiltration with activated T lymphocytes and prone to rupture, thereby leading tothrombotic coronary occlusion (2). Thus, elimination of CE fromfoam cells is potentially a promising therapeutic strategy tostabilize rupture-prone atherosclerotic plaques.
Foam cells are generated by the uptake of modified lipoproteins through scavenger receptors, such as scavenger receptor
A (SR-A) and CD36 (3). Hydrolysis of the lipoprotein-associated
CE by lysosomal acid lipase liberates free cholesterol (FC),which is subsequently re-esterified by acyl-CoA:cholesterolacyltransferase 1 (ACAT1) to form CE for storage in the cytoplasmic lipid droplets (4, 5). The cytoplasmic CE is in turnhydrolyzed by neutral CE hydrolase (NCEH) to generate FC,
which is transported to a compartment for re-esterification by
ACAT1. The rest of the FC is released out of the cells primarily
through ATP-binding cassette transporter A1 (ABCA1) (6, 7).
Thus, the balance between synthesis and hydrolysis of CE
conceivably governs the level of CE in macrophages.
Inhibition of ACAT activity is shown to suppress CE accumulation with concomitant promotion of the net hydrolysis of
CE in mouse peritoneal macrophages (8). Supporting these invitro results, ACAT inhibitors have been reported to suppress
atherosclerosis in many animal models (9). Consistently, we
have reported that atherosclerotic lesion areas as well as aortic
CE contents are moderately reduced in mice that are deficientin ACAT1 in the setting of apoE or the LDL receptor deficiency(10). Paradoxically, however, Fazio et al. (11) reported thatmacrophage-specific ablation of ACAT1 aggravated atherosclerotic lesions. It was speculated that inhibition of ACAT was
detrimental because of cytotoxicity of excess FC in macrophages, as has been reported in cells treated with ACAT inhibitors (12, 13). Together, it remains unknown whether selective

transporter A1; LDL, low density lipoproteins; HDL, high density lipoproteins; HSL, hormone-sensitive lipase; acLDL, acetylated LDL;
BSA, bovine serum albumin; Ad-LacZ, recombinant adenovirus carrying ␤-galactosidase expression cassette; Ad-HSL, recombinant adenovirus carrying HSL expression cassette; m.o.i., multiplicity of infection;
RT, reverse transcriptase; FBS, fetal bovine serum; RPMI, Roswell
Park Memorial Institute; apo, apolipoprotein; CYP27, cholesterol 27hydroxylase; PPAR, peroxisome proliferator-activated receptor; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

31893

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Elimination of CE from Macrophage Foam Cells

inhibition of ACAT1 in macrophages is useful as a therapeutictool.
The role of CE hydrolysis in the development of atherosclerosis is less clear, because molecular identity of NCEH inmacrophages has yet to be determined. Several investigators(14 –16) have attributed the NCEH activity in macrophages tohormone-sensitive lipase (HSL), which is a multifunctional
enzyme that catalyzes both triacylglycerol (TG) and CE invarious organs including adipose tissue and testis (17). This isapparently consistent with the results that treatment withcAMP, which is known to activate HSL, stimulated the CEhydrolysis in macrophages (18, 19). However, cAMP stimulates
the expression of ABCA1 (20, 21), another rate-limiting step forcholesterol efflux, making it difficult to conclude that CE hydrolysis and cholesterol efflux are primarily mediated by endogenous HSL. Furthermore, we showed that NCEH activity
was not reduced in peritoneal macrophages obtained from HSL
knockout mice (22), indicating that another enzyme that is
distinct from HSL mediates NCEH activity in macrophages. In
this context, it is noteworthy that a new NCEH has been cloned
from the human macrophages library (23). Thus, neither molecular identity nor characteristics of NCEHs in macrophages
are completely elucidated.
Previously, Escary et al. attempted to achieve increased hydrolysis of CE by overexpression of HSL using macrophagespecific enhancer/promoter of SR-A in RAW 264.7 cells (24) andtransgenic mice (25). However, they were not successful inobtaining sufficient increases in CE hydrolysis. In the transfected cells, without the addition of ACAT inhibitor, increasedhydrolysis of CE was not observed (24). In macrophages from
HSL transgenic mice, CE was paradoxically increased whenincubated with acetylated LDL (acLDL) in vitro, which was
ascribed to the compensatory activation of ACAT1 with lowlevel of increase in NCEH activity (25). Thus, it still remains
unclear whether stimulation of CE hydrolysis suppresses foamcell formation.
To achieve higher expression of HSL in macrophages, wehave used adenovirus-mediated gene delivery. We show herethat the overexpression of HSL caused a robust increase in
NCEH activity, which accompanied complete elimination of CE
from the cells loaded with CE without affecting cell viability in
THP-1 macrophages. These results implicate that adenovirusmediated overexpression of HSL can be used as a therapeuticstrategy to regress and stabilize rupture-prone foam celllesions.
EXPERIMENTAL PROCEDURES

Materials—Phorbol 12-myristate 13-acetate, triolein, lecithin, bovine
serum albumin fraction V (BSA), and leupeptin were purchased from
Sigma. Cholesterol esterase from Pseudomonas sp., horseradish peroxidase, p-hydroxyphenylacetic acid, and sodium taurocholate were purchased from Wako Pure Chemicals (Osaka, Japan). Cholesterol oxidase
was purchased from Roche Molecular Biochemicals. Tri[3H]oleoylglycerol and cholesterol [1-14C]oleate, Na125I, [cholesteryl-1,2,6,
7-3H]cholesteryl linoleate, [cholesteryl-1,2,6,7-3H]cholesteryl oleate,[1-14C]oleic acid, and [1-14C]oleoyl-CoA were purchased from Applied
Biosystems (Foster City, CA). MTT assay kit was purchased from
Chemicon International, Inc. (Temecula, CA). Adenovirus plasmidcarrying ␤-galactosidase (pAd-LacZ) was described previously (26).
Construction of Recombinant Adenoviruses—Recombinant adenovirus that carried murine HSL cDNA under the control of cytomegalovirus promoter, designated as Ad-HSL, was constructed using the cDNA
cloned by reverse transcriptase-PCR (RT-PCR) from mouse white adipose tissue (22) as described previously (27). The recombinant adenoviruses were expanded in HEK293 cells and purified by cesium chloride
ultracentrifugation. The purified viruses were stored in 10% (v/v) glycerol/phosphate-buffered saline (PBS) at ⫺80 °C. In our preparations, 1
multiplicity of infection (m.o.i.) corresponded to 25 particles of adenovirus per cell, and cells were infected at 100 m.o.i., 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 high density lipoproteins (HDL) (d 1.063–1.21 g/ml)
were isolated from the plasma by sequential density ultracentrifugation(28). LDL was acetylated by repetitive additions of acetic anhydride(29). acLDL was radioiodinated with Na125I by the iodine monochloride
method (30). CE in acLDL was reconstituted with [cholesteryl-1,2,6,73
H]cholesteryl linoleate as described previously (31).
Cell Culture—THP-1 cells were cultured in RPMI-1640 containing
10% (v/v) FBS and differentiated to THP-1 macrophages by the treatment with 100 nmol/liter phorbol 12-myristate 13-acetate for 48 h.
P388D1 cells were cultured in RPMI 1640 containing 10% (v/v) FBS.
J774, Raw 264.7, and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) FBS. Mouse peritoneal
macrophages were harvested as described previously (22). Mononuclear
cells, which were isolated from peripheral blood of healthy donors using
Lymphoprep (NYCOMED, Roskilde, Denmark) and adhered to plastic
dishes, were used as human monocytes.
Cells were loaded with cholesterol by incubation with RPMI 1640
containing 100 ␮g/ml acLDL and 5 mg/ml BSA for 24 h at 37 °C. Aftertransduction with Ad-HSL or Ad-LacZ for 72 h at 37 °C in RPMI
containing 5 mg/ml BSA and 100 ␮g/ml acLDL, cells were harvested.
Adenovirus Transduction Efficiency—Cells were plated at 0.5 ⫻ 106
cells per well in 12-well tissue culture plates and infected with Ad-LacZ
at 100 m.o.i. On day 3, ␤-galactosidase activity in the cellular proteins
was measured by standard kits (Promega, Madison, WI). Cellular protein was determined by BCA protein assay (Pierce).
Western Blot Analysis—Cells were sonicated in buffer A (50 mmol/
liter Tris-HCl, pH 7.0, 250 mmol/liter sucrose, 1 mmol/liter EDTA, 2
␮g/ml leupeptin) and centrifuged at 100,000 ⫻ g for 45 min at 4 °C. The
supernatant was used for Western blot analysis as described previously(22) using an anti-HSL antibody that was raised according to thedescribed method (32).
NCEH and TG Lipase Activities—Cells were sonicated in buffer A
and centrifuged at 100,000 ⫻ g for 45 min at 4 °C. The supernatant wasused for the enzyme assay. NCEH and TG lipase activities were measured as described previously (22).
Cholesterol Determination—Cellular lipids were extracted by hexane/isopropyl alcohol, and cholesterol contents were determined byenzymatic fluorometric microassay according to the method of Heider
and Boyett (33), with minor modifications (10).
Oil Red O Staining—THP-1 macrophages were plated in 4-chamber
plates at 0.5 ⫻ 106 cells per well and treated with acLDL and recombinant adenovirus, as described above. Cells were washed twice with
PBS, fixed with 3% (w/v) paraformaldehyde in PBS, and stained with
Oil Red O in 60% (v/v) isopropyl alcohol, and hematoxylin.
MTT Assay—Colorimetric MTT assay for cell survival and proliferation was performed following manufacturer’s protocol.
CE Formation—CE formation from [1-14C]oleic acid was determined
as described previously (34) with minor modifications.
Microsomal ACAT Activity—Cells were sonicated in buffer A andcentrifuged at 100,000 ⫻ g for 45 min at 4 °C. The precipitates were
resuspended and used for the assay. ACAT activity in microsomes wasdetermined by the rate of incorporation of [1-14C]oleoyl-CoA into the CE
fraction according to Yagyu et al. (10).
Cholesterol Efflux—Cholesterol efflux assays were performed as described previously (31). Cells were incubated for 24 h at 37 °C in culture
media containing 5 mg/ml BSA and 100 ␮g/ml acLDL whose CE wasreconstituted with [cholesteryl-1,2,6,7-3H]cholesteryl linoleate. Cells
were then infected with recombinant adenoviruses at indicated m.o.i. at
37 °C for additional 72 h in RPMI 1640 containing 5 mg/ml BSA and 100
␮g/ml acLDL in the presence or absence of 250 ␮g/ml HDL. An aliquotof the medium was removed and centrifuged at 15,000 ⫻ g for 2 min,and the radioactivity in the supernatant was determined by a liquid
scintillation counter. Total cell-associated radioactivity was determined
after dissolving the cells at time 0 in 0.1 N NaOH. Cholesterol efflux ofradioactive cholesterol from the cells into the medium was determined
as a percentage to the total radioactivity in the cells at time 0.
Uptake and Degradation of Lipoproteins—Cells were incubated in
RPMI containing 5 mg/ml BSA and 125I-labeled acLDL at the indicatedconcentrations at 37 °C for 6 h. The amounts of 125I-labeled acLDL
associated with or degraded by the cells were determined as described(30). Nonspecific values were determined by adding a 50-fold excess of
unlabeled acLDL. Specific values were calculated by subtracting thenonspecific value from the total value.
Northern Blot Analyses—Five ␮g of total RNA, which was isolated
from the cells by TRIzol reagent (Invitrogen), was used for Northernblot analysis as described (22). Probes for human adipophilin, CD36,

Elimination of CE from Macrophage Foam Cells

31895

SRBI, SR-A, ABCA1, human ABCG1, human apoE, and cholesterol
27-hydroxylase (CYP27) were constructed from cDNA fragments amplified by RT-PCR using cDNA obtained from THP-1 monocyte/macrophages as a template. Probes for murine adipocyte lipid-binding protein(aP2) and HSL (exon 8 probe) were constructed from cDNA fragments
amplified by RT-PCR using cDNA obtained from mouse adipose tissue
as a template (22).
Statistical Analyses—Results are presented as means ⫾ S.D. Student’s t test was employed to compare the means. All calculations were
performed with STAT view version 5.0 for Macintosh (SAS InstituteInc.).
RESULTS

To identify macrophages or macrophage-like cell lines thatwere effectively transduced with recombinant adenovirus, we
compared the expression of LacZ in macrophage-like cell lines(THP-1, J774, RAW 264.7, and P388D1), primary cultures ofmacrophages such as murine peritoneal macrophages andhuman monocyte-derived macrophages, and HEK293 cells,
after infection with Ad-LacZ. Compared with HEK293 cells,
macrophage-like cells expressed extremely low activity of LacZ
as follows: THP-1, 0.71%; J774, 0.01%; RAW 264.7, ⬍0.01%.
LacZ activity was undetectable in the following cells: P388D1,
murine peritoneal macrophages, and human monocyte-derived
macrophages. Among these cells, THP-1 macrophages weretransduced with adenovirus most effectively. Thus, we used 30,
100, and 300 m.o.i., which correspond to 0.21, 0.71, and 2.13
copies of the virus infected into a THP-1 cell, respectively.
To verify the expression of exogenous HSL after the infection
with Ad-HSL, we performed Western blot analysis and measured activities for NCEH and TG lipase in the cells (Fig. 1).
HSL protein was expressed in THP-1 macrophages infectedwith Ad-HSL in a dose-dependent manner but not in cells
infected with Ad-LacZ (Fig. 1A). In parallel, NCEH activity wasrobustly stimulated after infection with Ad-HSL in cells
treated with acLDL in a dose-dependent manner; 120-fold increase was observed at 300 m.o.i. (Fig. 1B). Similarly, TG lipase
activity was stimulated after infection with Ad-HSL in cells
treated with acLDL in a dose-dependent manner; 4-fold increase was observed at 300 m.o.i. (Fig. 1C). Fold increase wasapparently more prominent in NCEH activity than in TG
lipase activity, because basal level of endogenous NCEH activity was much lower than that of endogenous TG lipase activity.
To examine whether the increased NCEH activity hydrolyzes
cellular CE, we measured CE and FC contents in THP-1 macrophages infected with various doses of Ad-LacZ or Ad-HSL (Fig.
2). Incubation with acLDL increased both CE and FC in thecells. The net amount of these lipids was not decreased by theaddition of HDL, which is a physiological acceptor of FC that isreleased from cells and conceivably stimulates the efflux of FC
from the cells to the medium (Fig. 2, compare A with C and B
with D). Infection with Ad-LacZ did not change the contents ofeither CE or FC. In contrast, infection with Ad-HSL significantly decreased the CE contents in a dose-dependent manner;
CE was almost undetectable at 300 m.o.i. Importantly, theelimination of CE did not accompany an increase in FC. HDL
had no significant effect on the CE contents (Fig. 2, compare A
with C).
In agreement with these results, Oil Red O staining revealed that the number and size of intracellular lipid droplets, which were increased by incubation with acLDL, wereremarkably reduced by the infection with Ad-HSL but not by
the infection with Ad-LacZ (Fig. 3A). To rule out the possibility that the overexpression of HSL is cytotoxic and thereby
decreases the lipid accumulation, we performed MTT assay(Fig. 3B). Incubation with acLDL increased MTT activity by
51%. Infection with either Ad-LacZ or Ad-HSL decreased
MTT activity by 41 and 33%, respectively; there was no

FIG. 1. Expression of HSL protein (A), NCEH (B), and TG lipase
activities (C) in THP-1 macrophages infected with recombinantadenoviruses. THP-1 macrophages were infected with Ad-HSL or
Ad-LacZ at the indicated m.o.i. On day 3 after the infection, 10 ␮g ofcytosolic proteins were subjected to Western blot analysis (A). THP-1
macrophages were infected with Ad-HSL or Ad-LacZ at the indicatedm.o.i. 24 h after incubation with acLDL. On day 3 after the infection,
cytosolic proteins were extracted for the measurements of NCEH activity (B) and TG lipase activity (C). Data are expressed as means ofduplicate measurements.

significant difference between the cells infected with twoviruses.
To determine how Ad-HSL decreased cellular CE contents,
we compared CE formation from [14C]oleic acid between AdLacZ and Ad-HSL (Fig. 4A). The CE formation, which wasstimulated by acLDL, was substantially inhibited by the infection with Ad-HSL but not by Ad-LacZ. It is of note that theseresults were nearly identical to those on CE contents. To determine whether the decreased CE formation resulted fromdecreased ACAT activity, we measured microsomal ACAT activities (Fig. 4B). Incubation with acLDL stimulated microsomal ACAT activity. Neither Ad-HSL nor Ad-LacZ reduced theactivities.
Because FC contents were not increased despite the increased hydrolysis of CE, we hypothesized that FC, which is
generated by the breakdown of CE, was effectively transportedout of the cells to the media. To verify the possibility, wemeasured the amounts of FC released from the cells to themedia (cholesterol efflux) (Fig. 5, A and B). Ad-HSL stimulated
the cholesterol efflux in a dose-dependent manner. HDL increased the cholesterol efflux by 50% (compare Fig. 5, A and B).
Suppression of the uptake of acLDL may explain the de-31896

Elimination of CE from Macrophage Foam Cells

FIG. 2. Cellular contents of CE (A and C) and FC (B and D) in THP-1 macrophages infected with recombinant adenoviruses in theabsence (A and B) and presence (C and D) of HDL in the medium. Twenty four h after the incubation with acLDL, THP-1 macrophages were
infected with recombinant adenoviruses, with (C and D) or without (A and B) the addition of 250 ␮g/ml HDL. On day 3 after infection, intracellular
contents of CE (A and C) and FC (B and D) were measured by an enzymatic fluorometric microassay. Data are expressed as means ⫾ S.D. of
triplicate wells and are representative of four independent experiments. *, p ⬍ 0.001, Ad-HSL versus Ad-LacZ.

crease in CE content of Ad-HSL-infected cells. To test thispossibility, we measured the amounts of 125I-labeled acLDL
associated with or degraded by the cells infected with theviruses (Fig. 5, C and D). Even the infection with Ad-LacZ
reduced the amounts of 125I-labeled acLDL degraded by or
associated with the cells by 2-fold. The infection with Ad-HSL
further decreased the uptake and degradation by 30.2 and50.8%, respectively.
To investigate the changes in the expression of genes thatgovern cholesterol trafficking in the cells, we have performed
Northern blot analyses (Fig. 6). The overexpression of HSL
accompanied an ⬃2-fold increase in the mRNA expression of
ABCA1 but not the expression of other genes involved in cholesterol efflux, such as ABCG1, apoE, and CYP27. Apparently
consistent with the reduced uptake and degradation of acLDL,
the mRNA expression of the members of scavenger receptor
family such as SR-A, CD36, and SRBI was decreased by ⬃2fold in the cells infected with Ad-HSL. Finally, we found nochanges in the mRNA expression of the molecules whose expression was reported to be increased in response to oxidized
LDL, such as adipophilin (35) and adipocyte lipid-binding
protein (36).
DISCUSSION

Foam cell formation, accumulation of CE in monocyte/macrophages, is an initial step in the development of atherosclerotic
lesions. Thus, elimination of CE from foam cells would be of
considerable therapeutic benefit. This study was designed todetermine whether the hydrolysis of CE is the rate-limiting

step in the removal of CE from foam cells. We show here thatoverexpression of HSL leads to the complete elimination of CE
from THP-1 macrophage foam cells without affecting cellularviability. These changes are associated not only with an increase in cholesterol efflux but also with a decrease in lipoprotein uptake, which may effectively protect foam cells fromexcessive accumulation of FC.
Previously, Escary et al. (24) attempted to overexpress HSL
in macrophages. They took advantage of plasmid transfection
to RAW 264.7 macrophage-like cells (24) as well as transgenic
mice using SR-A enhancer/promoter (25). Unfortunately, however, the levels of the expression were relatively low (5-fold inin vitro study, and 7-fold in in vivo study), and did not sufficiently overcome the compensatory activation of endogenous
ACAT activity (2-fold increase in both studies). Indeed, themacrophages isolated from the transgenic mice showed paradoxical accumulation of CE when incubated with acLDL. Similar paradoxical worsening of atherosclerotic lesions was observed in the transgenic mice. In this regard, it is noteworthy
that our study was the first to achieve a significant increase in
the hydrolysis of CE in macrophages by virtue of adenovirusmediated gene delivery (Fig. 1), which allowed us to investigate
the role of CE hydrolysis in foam cell formation without theneed for concomitant addition of ACAT inhibitors and/or cAMPanalogues, which were required to demonstrate the increased
CE hydrolysis in the studies of Escary et al. (24, 25).
The resultant increased activity of NCEH led to complete
elimination of CE accumulated in THP-1 macrophages that

Elimination of CE from Macrophage Foam Cells

FIG. 3. Accumulation of lipid droplets (A) and cell viability (B)of THP-1 macrophages infected with recombinant adenoviruses. Twenty four h after the incubation with acLDL, THP-1 macrophages were infected with recombinant adenoviruses and were incubated for the following 72 h. A, cells were fixed and stained with Oil Red
O and hematoxylin (⫻100 magnification). The number of cells was⬃100 for each group. B, MTT assay was performed to assess cell
viability. Data are expressed as percentages to control cells. Data areexpressed as means of triplicate wells and representative of threeexperiments. *, p ⬍ 0.005, compared with other groups.

had been preloaded with CE by incubation with acLDL (Fig. 2).
The elimination of CE was not caused by inhibition of cholesterol esterification, because microsomal ACAT activity was notdecreased (Fig. 4). The suppression of CE formation from
[14C]oleic acid (Fig. 4) could be explained not only by the increased efflux (Fig. 5, A and B) but also by the decreased influxof cholesterol (Fig. 5, C and D).
It is noteworthy that FC contents were not increased in thesecells overexpressing HSL (Fig. 2). FC that was generated by thehydrolysis of CE might be effectively removed out of the cells by
the increase in cholesterol efflux (Fig. 5), indicating that CEhydrolysis is the rate-limiting step in foam cell formation of
THP-1 macrophages. In comparison with murine macrophages
such as mouse peritoneal macrophages and J774.2 cells, humanmacrophages including THP-1 macrophages are reportedly resistant to cholesterol efflux; neither cAMP nor the addition of
HDL enhanced the cholesterol efflux (37, 38). This particularnature of THP-1 macrophages has been ascribed to the low activity of endogenous NCEH, which may in part account for our
success in obtaining the significant increase in NCEH activity
above the endogenous level after the transduction with Ad-HSL.
The increased cholesterol efflux was at least partly in agreementwith the up-regulation of the expression of ABCA1, which is a key
transporter facilitating cellular cholesterol efflux (7) (Fig. 6).
Hydrolysis of CE generates cholesterol and fatty acids, both ofwhich conceivably stimulate the expression of ABCA1, because
fatty acids, their oxidized forms, and oxysterols might activate
PPAR␣, PPAR␥, and liver X receptor ␣, respectively, which increase the expression of ABCA1 in turn (7).

31897

FIG. 4. CE formation from [14C]oleic acid (A) and microsomal
ACAT activity (B) in THP-1 macrophages infected with recombinant adenoviruses. A, 24 h after the incubation with acLDL and 1%(w/v) [14C]oleate-BSA complex, THP-1 macrophages were infected withrecombinant adenovirus at the indicated m.o.i. and incubated for thefollowing 72 h. Radioactivity of intracellular CE formed from [14C]oleate
was determined. Data are expressed as means ⫾ S.D. of triplicate wells
and are representative of four independent experiments. *, p ⬍ 0.005,
Ad-HSL versus Ad-LacZ. B, microsomal protein was extracted from thecells and used for measurement of microsomal ACAT activity. Data areexpressed as means ⫾ S.D. of triplicate assays.

It is important to note that cholesterol efflux was increased
even in the absence of HDL in the medium (Fig. 5). Thus, theincreased hydrolysis of CE stimulated cholesterol efflux via apathway that is distinct from ABCA1, which requires HDL, in
addition to cholesterol efflux via the ABCA1-mediated pathway. Thus far, it has been reported that the HDL-independent
efflux of cholesterol is mediated either by apoE (39) or by
CYP27, an enzyme that catalyzes hydroxylation of cholesterol
to form 27-hydroxycholesterol for elimination as bile acids fromthe liver (40, 41). Recently, PPAR␦ has been shown to regulate
the expression of CYP27 at the transcriptional level (42).
Therefore, it is tempting to speculate that FC released by the
CE hydrolysis might be directed to a distinct subcellular compartment where FC is preferentially metabolized by this enzyme, thus facilitating cholesterol efflux. In this context, it isinteresting to note that inhibition of ACAT results in accumulation of FC in endoplasmic reticulum, not in cytosol (43).
Increased cholesterol efflux should have a therapeutic benefit, because accumulation of excess FC might be toxic to thecells. For example, many investigators (12, 13) have reported
the cytotoxicity of ACAT inhibition in macrophages, whereas
there are some who disagree (37). In this context, it is noteworthy that the LDL receptor knockout mice whose bone marrow31898Elimination of CE from Macrophage Foam Cells

FIG. 5. Stimulation of cholesterol efflux in the absence (A) and presence (B) of HDL in the media, and specific uptake (C) anddegradation (D) of 125I-labeled acLDL in THP-1 macrophages infected with recombinant adenoviruses. THP-1 macrophages wereincubated with 100 ␮g/ml acLDL whose CE was reconstituted with [cholesteryl-1,2,6,7-3H]cholesteryl linoleate. After 24 h, the cells were washed,infected with recombinant adenoviruses at the indicated m.o.i., and incubated for the following 72 h, in the absence (A) or presence of HDL (B).
Radioactivities in the medium and cells were measured. Cholesterol efflux to the medium was determined as a percentage to the total radioactivity
in the cells at time 0. The values for the non-infected cells were subtracted from those for the infected cells to obtain cholesterol efflux that was
specifically stimulated by the infection with adenoviruses. Data are expressed as means ⫾ S.D. of triplicate wells. *, p ⬍ 0.005, Ad-HSL versus
Ad-LacZ. Twenty four h after the incubation with acLDL, THP-1 macrophages were infected with recombinant adenoviruses and incubated for thefollowing 72 h. On day 3 after the infection, cells were washed extensively and incubated with 125I-labeled acLDL at the indicated concentrations
at 37 °C for 6 h. The amounts of 125I-labeled acLDL associated with (C) and degraded by (D) the cells were determined. Nonspecific values wereobtained by adding a 50-fold excess of unlabeled acLDL. Specific values were calculated by subtracting the nonspecific value from the total value.
Data are expressed as means ⫾ S.D. of triplicate wells. *, p ⬍ 0.005, and **, p ⬍ 0.05, Ad-HSL versus Ad-LacZ.

FIG. 6. Northern blot analyses for genes involved in cholesterol trafficking in THP-1 macrophages infected with recombinant adenoviruses. Twenty four h after the incubation with acLDL,
THP-1 macrophages were infected with recombinant adenoviruses. On
day 3 after the infection, 5 ␮g of total RNA isolated from the cells was
subjected to Northern blot analyses.

was repopulated with that from ACAT1-deficient mice developed more severe atherosclerosis than the LDL receptor knockout mice (11), which was ascribed to potential cytotoxic effects
of FC accumulated in macrophages due to ACAT inhibition. We
did not observe either FC accumulation or cytotoxicity in
THP-1 cells overexpressing HSL (Figs. 2 and 3).
Unexpectedly, we found that THP-1 macrophages overexpressing HSL showed reduced ability to take up and degrade
acLDL (Fig. 5). In agreement with this, the expression of SR-A,
CD36, and SRBI was decreased (Fig. 6). SR-A (44) and CD36

(45, 46) are receptors for both acLDL and oxidized LDL; SRBI
is a receptor for acLDL as well as for HDL (47). Down-regulation of these proteins may contribute to the decreased uptake ofacLDL as observed in our study. Since thiazolidinediones werereported to down-regulate the expression of SR-A (48), it ispossible that the HSL overexpression generates oxidized fatty
acids, which suppress the expression of SR-A by activating
PPAR␥. If this is the case, it is difficult to explain the suppression of the expression of CD36, which was reported to be upregulated by the activation of PPAR␥ (49). Intracellular cholesterol content may play a more central role in the regulationof CD36 than fatty acids (50).
Ghosh (23) has recently reported the cloning of a novel CEhydrolase expressed in macrophages. There may be multiple
NCEHs in macrophages: HSL, the CEH cloned by Ghosh (23)and possibly others (22). It is yet to be known which enzyme is
predominant in macrophages.
Although the successful elimination of CE from macrophage
foam cells was shown only in the artificial system at cellularlevel, adenovirus-mediated gene transfer of HSL can be applied
to the treatment of foam cell lesions in atherosclerotic plaques
after solving issues at the in vivo level, such as gene delivery,
tissue-specific expression, level of expression, side effects, etc.
In summary, we show here that the overexpression of HSL
robustly increases NCEH activity and completely eliminates
CE in foam cells. In these cells, cholesterol efflux was stimulated in the absence or presence of HDL, which might be atleast partially explained by the increase in the expression ofElimination of CE from Macrophage Foam Cells
ABCA1. Furthermore, the uptake and degradation of acLDL
were significantly reduced probably by decreased expression ofscavenger receptors such as SR-A and CD36. These changes incholesterol trafficking, which are associated with the increased
CE hydrolysis, may protect cells from cytotoxic effects of FC
buildup. Thus, increased hydrolysis of CE by the overexpression of HSL leads to complete elimination of CE from THP-1
foam cells not only by increasing efflux but also by decreasing
influx of cholesterol. Further studies are needed to clarify themechanisms underlying the changes in cholesterol trafficking
in cells with increased hydrolysis of CE.
Acknowledgments—We thank Z. Chen, S. Perrey, M.
Amemiya-Kudo, T. Yoshikawa, and A. H. Hasty for comments anddiscussion.
REFERENCES
1. Ross, R. (1999) N. Engl. J. Med. 340, 115–126
2. Libby, P. (2001) Circulation 104, 365–372
3. de Winther, M. P., and Hofker, M. H. (2000) J. Clin. Invest. 105, 1039 –1041
4. Brown, M. S., and Goldstein, J. L. (1983) Annu. Rev. Biochem. 52, 223–261
5. Chang, T. Y., Chang, C. C., and Cheng, D. (1997) Annu. Rev. Biochem. 66,
613– 638
6. Lawn, R. M., Wade, D. P., Garvin, M. R., Wang, X., Schwartz, K., Porter, J. G.,
Seilhamer, J. J., Vaughan, A. M., and Oram, J. F. (1999) J. Clin. Invest. 104,
R25–R31
7. Santamarina-Fojo, S., Remaley, A. T., Neufeld, E. B., and Brewer, H. B., Jr.
(2001) J. Lipid Res. 42, 1339 –1345
8. Brown, M. S., Ho, Y. K., and Goldstein, J. L. (1980) J. Biol. Chem. 255,
9344 –9352
9. Buhman, K. F., Accad, M., and Farese, R. V. (2000) Biochim. Biophys. Acta
1529, 142–154
10. Yagyu, H., Kitamine, T., Osuga, J., Tozawa, R., Chen, Z., Kaji, Y., Oka, T.,
Perrey, S., Tamura, Y., Ohashi, K., Okazaki, H., Yahagi, N., Shionoiri, F.,
Iizuka, Y., Harada, K., Shimano, H., Yamashita, H., Gotoda, T., Yamada,N., and Ishibashi, S. (2000) J. Biol. Chem. 275, 21324 –21330
11. Fazio, S., Major, A. S., Swift, L. L., Gleaves, L. A., Accad, M., Linton, M. F., andFarese, R. V., Jr. (2001) J. Clin. Invest. 107, 163–171
12. Warner, G. J., Stoudt, G., Bamberger, M., Johnson, W. J., and Rothblat, G. H.
(1995) J. Biol. Chem. 270, 5772–5778
13. Tabas, I., Marathe, S., Keesler, G. A., Beatini, N., and Shiratori, Y. (1996)
J. Biol. Chem. 271, 22773–22781
14. Small, C. A., Goodacre, J. A., and Yeaman, S. J. (1989) FEBS Lett. 247,
205–208
15. Khoo, J. C., Reue, K., Steinberg, D., and Schotz, M. C. (1993) J. Lipid Res. 34,
1969 –1974
16. Reue, K., Cohen, R. D., and Schotz, M. C. (1997) Arterioscler. Thromb. Vasc.
Biol. 17, 3428 –3432
17. Holm, C., Osterlund, T., Laurell, H., and Contreras, J. A. (2000) Annu. Rev.
Nutr. 20, 365–393
18. Khoo, J. C., Mahoney, E. M., and Steinberg, D. (1981) J. Biol. Chem. 256,12659 –12661
19. Bernard, D. W., Rodriguez, A., Rothblat, G. H., and Glick, J. M. (1991) J. Biol.
Chem. 266, 710 –716
20. Oram, J. F., Lawn, R. M., Garvin, M. R., and Wade, D. P. (2000) J. Biol. Chem.
275, 34508 –34511
21. Santamarina-Fojo, S., Peterson, K., Knapper, C., Qiu, Y., Freeman, L., Cheng,
J. F., Osorio, J., Remaley, A., Yang, X. P., Haudenschild, C., Prades, C.,

31899

Chimini, G., Blackmon, E., Francois, T., Duverger, N., Rubin, E. M., Rosier,M., Denèfle, P., Fredrickson, D. S., and Brewer, H. B., Jr. (2000) Proc. Natl.
Acad. Sci. U. S. A. 97, 7987–7992
22. Osuga, J., Ishibashi, S., Oka, T., Yagyu, H., Tozawa, R., Fujimoto, A.,
Shionoiri, F., Yahagi, N., Kraemer, F. B., Tsutsumi, O., and Yamada, N.
(2000) Proc. Natl. Acad. Sci. U. S. A. 97, 787–792
23. Ghosh, S. (2000) Physiol. Genomics 2, 1– 8
24. Escary, J. L., Choy, H. A., Reue, K., and Schotz, M. C. (1998) Arterioscler.
Thromb. Vasc. Biol. 18, 991–998
25. Escary, J. L., Choy, H. A., Reue, K., Wang, X. P., Castellani, L. W., Glass, C. K.,
Lusis, A. J., and Schotz, M. C. (1999) J. Lipid Res. 40, 397– 404
26. Ye, X., Robinson, M. B., Batshaw, M. L., Furth, E. E., Smith, I., and Wilson,
J. M. (1996) J. Biol. Chem. 271, 3639 –3646
27. Tsukamoto, K., Smith, P., Glick, J. M., and Rader, D. J. (1997) J. Clin. Invest.
100, 107–114
28. Havel, R. J., Eder, H. A., and Bragdon, J. J. (1955) J. Clin. Invest. 34,
1345–1353
29. Goldstein, J. L., Ho, Y. K., Basu, S. K., and Brown, M. S. (1979) Proc. Natl.
Acad. Sci. U. S. A. 76, 333–337
30. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98,
241–260
31. Roberts, D. C., Miller, N. E., Price, S. G., Crook, D., Cortese, C., La Ville, A.,
Masana, L., and Lewis, B. (1985) Biochem. J. 226, 319 –322
32. Kraemer, F. B., Patel, S., Saedi, M. S., and Sztalryd, C. (1993) J. Lipid Res. 34,
663– 671
33. Heider, J. G., and Boyett, R. L. (1978) J. Lipid Res. 19, 514 –518
34. Perrey, S., Ishibashi, S., Kitamine, T., Osuga, J., Yagyu, H., Chen, Z.,
Shionoiri, F., Iizuka, Y., Yahagi, N., Tamura, Y., Ohashi, K., Harada, K.,
Gotoda, T., and Yamada, N. (2001) Atherosclerosis 154, 51– 60
35. Wang, X., Reape, T. J., Li, X., Rayner, K., Webb, C. L., Burnand, K. G., andLysko, P. G. (1999) FEBS Lett. 462, 145–150
36. Fu, Y., Luo, N., and Lopes-Virella, M. F. (2000) J. Lipid Res. 41, 2017–2023
37. Graham, A., Angell, A. D., Jepson, C. A., Yeaman, S. J., and Hassall, D. G.
(1996) Atherosclerosis 120, 135–145
38. Kritharides, L., Christian, A., Stoudt, G., Morel, D., and Rothblat, G. H. (1998)
Arterioscler. Thromb. Vasc. Biol. 18, 1589 –1599
39. Huang, Z. H., Lin, C. Y., Oram, J. F., and Mazzone, T. (2001) Arterioscler.
Thromb. Vasc. Biol. 21, 2019 –2025
40. Björkhem, I., Andersson, O., Diczfalusy, U., Sevastik, B., Xiu, R. J., Duan, C.,
and Lund, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8592– 8596
41. Shanahan, C. M., Carpenter, K. L., and Cary, N. R. (2001) Atherosclerosis 154,
269 –276
42. Vosper, H., Patel, L., Graham, T., Khoudoli, G. A., Hill, A., Macphee, C. H.,
Pinto, I., Smith, S. A., Suckling, K. E., Wolf, C. R., and Palmer, C. N. (2001)
J. Biol. Chem. 276, 44258 – 44265
43. Puglielli, L., Konopka, G., Pack-Chung, E., Ingano, L. A., Berezovska, O.,
Hyman, B. T., Chang, T. Y., Tanzi, R. E., and Kovacs, D. M. (2001) Nat. Cell
Biol. 3, 905–912
44. Kodama, T., Freeman, M., Rohrer, L., Zabrecky, J., Matsudaira, P., andKrieger, M. (1990) Nature 343, 531–535
45. Endemann, G., Stanton, L. W., Madden, K. S., Bryant, C. M., White, R. T., andProtter, A. A. (1993) J. Biol. Chem. 268, 11811–11816
46. Febbraio, M., Podrez, E. A., Smith, J. D., Hajjar, D. P., Hazen, S. L., Hoff, H. F.,
Sharma, K., and Silverstein, R. L. (2000) J. Clin. Invest. 105, 1049 –1056
47. Acton, S. L., Scherer, P. E., Lodish, H. F., and Krieger, M. (1994) J. Biol. Chem.
269, 21003–21009
48. Moore, K. J., Rosen, E. D., Fitzgerald, M. L., Randow, F., Andersson, L. P.,
Altshuler, D., Milstone, D. S., Mortensen, R. M., Spiegelman, B. M., andFreeman, M. W. (2001) Nat. Med. 7, 41– 47
49. Tontonoz, P., Nagy, L., Alvarez, J. G., Thomazy, V. A., and Evans, R. M. (1998)
Cell 93, 241–252
50. Han, J., Hajjar, D. P., Tauras, J. M., and Nicholson, A. C. (1999) J. Lipid Res.
40, 830 – 838