Reduced ABCA1-Mediated Cholesterol Efflux and
Accelerated Atherosclerosis in Apolipoprotein E–Deficient
Mice Lacking Macrophage-Derived ACAT1
Yan Ru Su, MD; Dwayne E. Dove, PhD; Amy S. Major, PhD; Alyssa H. Hasty, PhD;
Branden Boone, MS; MacRae F. Linton, MD; Sergio Fazio, MD, PhD
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Background—Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have
been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation.
Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a
consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on
atherogenesis in apoE-deficient (apoE⫺/⫺) mice with or without the restoration of macrophage apoE.
Methods and Results—We used bone marrow transplantation to generate apoE⫺/⫺ mice with macrophages of 4 genotypes:
apoE⫹/⫹/ACAT1⫹/⫹ (wild type), apoE⫹/⫹/ACAT1⫺/⫺ (ACAT⫺/⫺), apoE⫺/⫺/ACAT1⫹/⫹ (apoE⫺/⫺), and apoE⫺/⫺/
ACAT1⫺/⫺ (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency
did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency
increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced
(30% to 40%; P⬍0.001) in ACAT1⫺/⫺ peritoneal macrophages compared with ACAT1⫹/⫹ controls regardless of apoE
expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein
levels relative to ACAT1⫹/⫹ macrophages. Microarray analyses of ACAT1⫺/⫺ macrophages showed increases in
proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and
apoptosis.
Conclusions—Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE⫺/⫺ mice but
has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion
impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane
stability, susceptibility to apoptosis, and inflammatory response may also be important in this process. (Circulation.
2005;111:-.)
Key Words: acyltransferases 䡲 apolipoproteins 䡲 apoptosis 䡲 atherosclerosis 䡲 cholesterol
E
fficient cellular cholesterol homeostasis is critical for
maintaining normal cell function. It is particularly important for arterial macrophages to protect against atherosclerosis. The homeostatic intracellular cholesterol content is
regulated by cholesterol synthesis, influx, and efflux. Cholesterol is stored either as free cholesterol (FC) in the
membrane or as cholesterol ester in cytoplasmic vesicles. The
dynamic equilibrium between FC and cholesterol ester in the
cell is tightly controlled by acyl-coenzyme A:cholesterol
acyltransferase (ACAT).1– 4 Two different isoforms of ACAT
have been cloned in mammals.5–10 In humans, ACAT1 is
expressed in most tissues, including hepatocytes, skin cells,
adrenal cells, Kupffer cells, intestinal enterocytes, and macrophages; ACAT2 is expressed in the intestinal mucosa cells
and hepatocytes. In mice, the tissue distribution pattern is
similar to that in humans, except the major isoform in
hepatocytes is ACAT2.5
ACAT1 promotes accumulation of cholesterol ester in
vascular macrophages, thereby contributing to foam cell
formation, a hallmark of early atherosclerosis. Because of the
potential role of ACAT in atherosclerosis, numerous ACAT
inhibitors have been developed and tested in animals as
potential agents for the treatment of atherosclerosis, but so
far, the results have been inconsistent.11,12 Nevertheless,
previous work in our laboratory has shown that the deletion
of ACAT1 in macrophages confers increased susceptibility to
atherosclerosis in LDLR⫺/⫺ mice fed a high-fat diet, raising
the concern that selective disruption or complete inhibition of
Received July 12, 2004; revision received November 18, 2004; accepted December 29, 2004.
From the Atherosclerosis Research Unit, Department of Medicine, Division of Cardiovascular Medicine (Y.R.S., D.E.D., A.S.M., M.F.L., S.F.),
Department of Molecular Biology and Molecular Biophisics (A.H.H.), Vanderbilt Microarray Shared Resource (B.B.), Department of Pharmacology
(M.F.L.), and Department of Pathology (S.F.), Vanderbilt University Medical Center, Nashville, Tenn.
Reprint requests to Yan Ru Su, MacRae Linton, or Sergio Fazio, Vanderbilt University Medical Center, Division of Cardiovascular Medicine, 317
Preston Research Bldg, 2220 Pierce Ave, Nashville TN 37232-6300. E-mail [email protected], [email protected], or
[email protected]
© 2005 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000164236.19860.13
1
2
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May 10, 2005
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ACAT1 in macrophages can promote, rather than protect
against, atherosclerotic lesion formation.13 We therefore hypothesized that the negative effects of macrophage ACAT1
deficiency on plaque formation are accentuated by hypercholesterolemic conditions that lead to increased cellular cholesterol burden but may not be as important under normocholesterolemic conditions. Apolipoprotein (apo)E is a strong
driver of cholesterol efflux,14 and the increased atherogenicity
of apoE-deficient macrophages may be due to defective
elimination of cholesterol.15,16 We and others have shown that
macrophage apoE influences atherosclerosis both systemically, by controlling plasma cholesterol levels, and locally, by
regulating cholesterol homeostasis in the macrophage.15,17
Thus, in the present study, we used bone marrow transplantation (BMT) to generate apoE⫺/⫺ mice with or without
macrophage ACAT1 and apoE to address the effects of
ACAT1 deletion on the development of atherosclerosis under
both hypercholesterolemic and normocholesterolemic conditions, and we investigated the underlying molecular mechanisms of the accelerated atherosclerosis resulting from
ACAT1 deficiency.
Methods
Animals
Six-week-old male apoE-deficient (apoE⫺/⫺) mice were purchased
from the Jackson Laboratory (Bar Harbor, Maine). ACAT-knockout
(KO) and 2KO (ACAT⫺/⫺, apoE⫺/⫺) mice were bred in our laboratory from founders generously provided by Dr Robert V. Farese
(Gladstone Institute of Cardiovascular Disease, San Francisco,
Calif). C57BL/6 mice were originally obtained from Jackson Laboratory and reproduced in our mouse colony. All mice used in this
study were on C57BL/6 background and were maintained in microisolator cages on a rodent chow diet (4.5% fat; Purina Mills Inc)
and autoclaved acidified water ad libitum. All experimental protocols were performed according to the guidelines of Vanderbilt
University’s Institutional Animal Care and Usage Committee.
BMT Experiments
We used BMT to generate apoE⫺/⫺ mice either wild type (WT) or
null for macrophage ACAT1 and apoE expression. The recipients
were 6-week-old male apoE⫺/⫺ mice. We used 4 types of agematched male donor mice: (1) WT mice (ACAT⫹/⫹, apoE⫹/⫹), (2)
ACAT⫺/⫺ mice (ACAT⫺/⫺, apoE⫹/⫹), (3) apoE⫺/⫺ mice (apoE⫺/⫺,
ACAT⫹/⫹), and (4) ACAT, apoE double-KO mice (ACAT⫺/⫺,
apoE⫺/⫺). BMT experiments were done according to the method
described previously.17 After reconstitution, the macrophages from
the recipient mice are expected to have the same genotype as the
donor mice and were identified accordingly: WT ACAT1, WT
(ACAT1⫹/⫹, apoE⫹/⫹), ACAT1 KO, ACAT⫺/⫺ (ACAT1⫺/⫺, apoE⫹/⫹),
apoE KO, apoE⫺/⫺ (ACAT1⫹/⫹, apoE⫺/⫺), and 2KO (ACAT1⫺/⫺,
apoE⫺/⫺). After BMT, mice were fed a regular chow diet (4.5% fat;
Purina Mills Inc) for 12 weeks.
and snap-frozen in liquid nitrogen for further analysis. Frozen
sections (5 and 10 ␮m) were processed according to the procedures
commonly used in our laboratory and described previously.17
Lipoprotein Isolation and Modification
Human plasma was isolated from healthy volunteers. Lipoproteins
were isolated by sequential ultracentrifugation with plasma density
adjusted to 1.019 to 1.063 g/mL for LDL. Lipoproteins were
dialyzed overnight in 0.15 mol/L NaCl and 0.3 mmol/L EDTA at pH
7.4. Acetylated LDL (acLDL) was prepared by repeatedly adding
acetic anhydride (Sigma) to LDL in a sodium acetate solution and
dialyzed in 0.15 mol/L NaCl and 0.3 mmol/L EDTA at 4°C for 2
days.19
3
H-Cholesterol Efflux in Peritoneal Macrophages
Cholesterol efflux from peritoneal macrophages was determined by
a modified procedure from Lin et al.20 Twelve weeks after BMT,
peritoneal macrophages were collected from recipient mice 3 days
after intraperitoneal injection with 3% thioglycollate. Cells were
harvested by peritoneal lavage and cultured in DMEM/10% FBS at
37°C and 5% CO2 overnight. The culture media was changed to
DMEM/4% FBS containing 2 ␮Ci/mL of 3H-cholesterol (NEN) and
50 ␮g/mL acLDL, and cells were loaded for 24 hours. Monolayers
were washed 3 times in serum-free DMEM/0.2% BSA and equilibrated in serum-free DMEM/0.5% BSA for 4 hours at 37°C and 5%
CO2. Efflux media with serum-free DMEM/human apoA-I (10
␮g/mL) was used to measure efflux mediated by the ABC-A1
transporter. Cells were incubated at 37°C and 5% CO2, with 100-␮L
samples removed from each well at 0, 3, 6, and 18 hours. 3H-cholesterol counts were detected with a scintillation counter. Total
cellular 3H-cholesterol counts and total cellular protein mass were
determined by lysing labeled cells with 0.1N NaOH. Protein concentration in the cell lysate was measured by the method of Lowry.
Cholesterol efflux, calculated from the total supernatant counts, is
expressed as a percentage of the total lysate counts.
Real-Time Reverse-Transcriptase Polymerase
Chain Reaction for Quantification of ABCA1,
ABCG1, and SR-BI gene Expression
The relative quantities of ABCA1, ABCG1, and SR-BI message
were quantified by real-time reverse-transcriptase polymerase chain
reaction (RT-PCR) using a previously described method developed
in our laboratory.21–23 The primers and probes were synthesized by
Invitrogen and ABI, respectively. TaqMan 1-step RT-PCR master
mix reagent kit (ABI, P/N 4309169) was used for RT-PCR. Relative
quantification of ABCA1, ABCG1, and SR-BI was normalized with
18S ribosomal RNA as an endogenous control. Data were analyzed
by use of the comparative CT method and were conformed by
standard curve method. The mRNA quantity for the calibrator (WT
untreated macrophages) is expressed as 1⫻ sample; all other
quantities are expressed as the number of fold changes relative to the
calibrator.
Western Blot Analysis for ABCA1, ABCG1,
and SR-BI
Serum samples were collected after an overnight fast at several time
points: 2 weeks before BMT and 4, 8, and 12 weeks after BMT.
Serum cholesterol and triglyceride levels were determined by methods described previously.18 Serum obtained at 12 weeks after BMT
was fractionated by fast-performance liquid chromatography through
a Pharmacia Superose-6 column (Pharmacia Biotech Inc), and
fractions were measured for total cholesterol concentration.
Western blotting was performed as previously described with modifications.22 Briefly, peritoneal macrophages were isolated from WT,
ACAT⫺/⫺, apoE⫺/⫺, and 2KO mice. Cells were cultured in either
DMEM with 2% FBS (untreated) or DMEM with 2% FBS plus 50
␮g /mL acLDL for 24 hours (treated). Cell proteins (40 ␮g) were
separated by 4% to 12% Bis-Tris gel (Invitrogen). Polyclonal
antibodies to ABCA1 and SR-BI were purchased from Novus
Biologicals. Rabbit anti-mouse ABCG1 antibody was purchased
from Alpha Diagnostic International. Mouse anti–␤-actin antibody
was obtained from Sigma.
Quantification of Atherosclerotic Lesions in the
Proximal Aorta
cDNA Microarray Analysis of
Peritoneal Macrophages
Twelve weeks after BMT, all recipient mice were euthanized, and
the hearts with proximal aorta were harvested, embedded in OCT,
Total RNA was extracted from peritoneal macrophages with the
Trizol reagent (Invitrogen). The isolated RNA samples were treated
Serum Lipid Analysis
Su et al
with RNAse-free DNAse I (Qiagen) and cleaned with RNeasy
column (Qiagen). All samples were run on an Agilent 2100 Bioanalyzer to assess RNA integrity. cDNA microarray was performed in
the Vanderbilt Microarray Shared Resource according to standard
protocol (http://array.mc.vanderbilt.edu/support). All arrays were
scanned with an Axon 4000B scanner. Raw data, generated by the
accompanying software GenePix 4.0, were analyzed with GeneTraffic 2.6 (Lobion Informatics, LLC). All 20 773 spots were first
validated by the quality of signal intensity and were removed from
analysis if the following criteria were not met: Cy5 or Cy3
signal-to-background-intensity ratio ⬍1.2 and Cy5 or Cy3 signal
intensity ⬍200. Data were normalized with the Lowess subgrid method.
Lists of genes with a ⬎2-fold change were generated for each experiment. The molecular functions of each gene were based on the databases
of SOURCE (http://genome-www5.stanford.edu/cgi-bin/source), NIA
Mouse cDNA Database (http://lgsun.grc.nia.nih.gov), and Celera Discovery System (http://www.celeradiscoverysystem.com).
TUNEL Staining of Peritoneal Macrophages
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Terminal deoxynucleotidyl transferase–mediated dUTP nick-end
labeling (TUNEL) and MOMA-2 staining were performed as previously described.13 To visualize macrophages, we incubated sections
with the primary antibody directed against rat MOMA-2 (Accurate
Chemical & Scientific Corp) and used a TRITC-conjugated anti-rat
secondary antibody (Pharmingen). For TUNEL staining, sections
were incubated with deoxynucleotidyl transferase and fluoresceinlabeled nucleotide mixture according to manufacturer’s specifications (In Situ Cell Death Detection Kit, Roche Diagnostics GmbH).
Stained sections were analyzed with fluorescent microscopy (Olympus AX70 microscope) and an Optronics digital camera. Controls for
each experiment included serial sections treated with DNase (positive control) or incubated with the labeling solution without terminal
transferase (negative control).
Statistical Analysis
All results are expressed as mean⫾SE unless indicated elsewhere.
Differences between groups were assessed by use of 1-way ANOVA
for comparisons among groups, followed by the Bonferroni posttest
when statistical significance was detected. Atherosclerotic lesion
assessment was also analyzed by 2-way ANOVA for apoE and
ACAT status. Values of P⬍0.05 were considered statistically
significant.
Results
Serum Lipid Profile in Recipient Mice
The average serum cholesterol at baseline was between 320
and 380 mg/dL, with no significant differences between
groups. As expected, the ACAT1⫹/⫹/apoE⫹/⫹3apoE⫺/⫺ and
ACAT1⫺/⫺/apoE⫹/⫹3apoE⫺/⫺ mice had a dramatic decrease
in total serum cholesterol as a result of the presence of
macrophage apoE in plasma.17 However, there were no
significant differences in plasma total cholesterol (Figure 1A)
and triglycerides (Figure 1B) between mice receiving
ACAT1⫺/⫺ and those receiving ACAT1⫹/⫹ marrow. Similarly,
fast-performance liquid chromatography analysis of the serum lipoproteins revealed patterns similar to previously
published effects resulting from the presence or absence of
macrophage apoE,17 but there was no apparent influence from
the presence or absence of ACAT1 (Figure 1C).
Development of Atherosclerosis
There was a significant increase in the average lesion area
(2-fold) in the proximal aorta of ACAT1⫺/⫺/apoE⫺/⫺
3apoE⫺/⫺ compared with ACAT1⫹/⫹/apoE⫺/⫺3apoE⫺/⫺
mice. Consistent with our previous results,17 atherosclerosis
Reduced Cholesterol Efflux in apoE-Deficient Mice
3
was dramatically reduced in ACAT1⫹/⫹/apoE⫹/⫹3apoE⫺/⫺
and ACAT1⫺/⫺/apoE⫹/⫹3apoE⫺/⫺ mice as a result of the
restoration of circulating apoE and the consequent reduction
in serum cholesterol (Figure 2A). The presence or absence of
macrophage ACAT1 did not affect the extent of atherosclerosis in mice receiving apoE⫹/⫹ marrow but increased lesion
size in mice receiving apoE⫺/⫺ marrow, suggesting that
ACAT1 deficiency has no influence on atherosclerosis under
normocholesterolemic conditions but has a prominent effect
under hypercholesterolemic conditions (Figure 2B).
ABCA1-Mediated Cholesterol Efflux in ACAT1ⴚ/ⴚ
Peritoneal Macrophages
In the presence of human apoA-I as an acceptor, cholesterol
efflux was reduced ⬇30% to 40% in ACAT1⫺/⫺ macrophages
compared with ACAT1⫹/⫹ macrophages in either the presence or absence of apoE (Figure 3). Cholesterol efflux
experiments were performed under mild acLDL loading
conditions; under these conditions, there was no difference
between the total cholesterol mass of WT and ACAT1⫺/⫺
macrophages (P⫽0.291; n⫽3).
Real-Time Quantitative RT-PCR and Western
Blot Analysis of ABCA1, ABCG1, and SR-BI
There were no significant changes in ABCG1 and SR-BI
mRNA expression levels (data not shown). No significant
changes were seen in ABCG1 and SR-BI protein levels as
detected by Western blot and normalized to ␤-actin (Figure
4C and 4D). However, ABCA1 mRNA level was significantly increased by 3- to 4-fold in the 2KO and 2KO with
mild acLDL loading compared with WT macrophages.
ACAT⫺/⫺ macrophages had a 2-fold increase in ABCA1
mRNA compared with the WT macrophages (Figure 4A).
Nevertheless, ABCA1 protein levels were decreased in both
ACAT1⫺/⫺-acLDL and 2KO macrophages as detected by
Western blot (Figure 4B), indicating abnormal posttranscriptional regulation of ABCA1 in ACAT1⫺/⫺ macrophages.
Gene Expression Analysis of
ACAT1ⴚ/ⴚ Macrophages
ACAT1⫺/⫺ macrophages showed a 2- to 3-fold decrease in
mRNA levels of genes involved in membrane integrity (zinc
finger DHHC-containing domain 5), phospholipid binding
(milk fat globule–EGF factor 8 protein), functional and
dynamic regulation of mitotic spindles (sperm-associated
antigen 5), and a critical enzyme involved in collagen
maturation (procollagen-proline, 2-oxoglutarate 4-dioxygenase) compared with WT ACAT1 macrophages. Conversely, a
2- to 3-fold increase in procollagen mRNA was observed in
ACAT1⫺/⫺ macrophages. The 2KO macrophages showed a
gene expression pattern similar to the ACAT1⫺/⫺ macrophages except for the involvement (decreased expression) of
a number of genes that protect cells from oxidative damage
(heme oxygenase, kynurenine 3-hydroxylase, cytochrome c
oxidase) and apoptosis such as cystein proteinases (cathepsin
H) (Figure 5). Gene expression profiling under acLDL
loading conditions (70 ␮g acLDL for 16 hours) is summarized in the Table. Of note, ACAT1-deficient macrophages
showed a downregulation of translation initiation factor-3 and
4
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May 10, 2005
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Figure 1. Serum lipid profile before and after BMT. Total serum cholesterol (A) and triglycerides (B) were measured before and at 4, 8,
and 12 weeks after BMT. C, Two representative samples (12 weeks after BMT) from each recipient group.
protein tyrosine phosphatase receptor type D, both involved
in posttranscriptional gene expression control, and upregulation of ABCA1 and a number of protein kinases and serine/
threonine kinases.
macrophages as determined by MOMA staining (Figure 6).
The collagen content in the proximal aortic lesion area, as
assessed by trichrome staining, was increased ⬇3-fold in
ACAT1⫺/⫺/apoE⫺/⫺3apoE⫺/⫺ mice compared with ACAT1⫹/⫹/
apoE⫺/⫺3apoE⫺/⫺ mice (data not show).
Analyses of Lesion Composition
A significant increase in apoptotic cells within the lesion area
was observed in ACAT1⫺/⫺/apoE⫺/⫺3apoE⫺/⫺ mice relative
to the other recipients. The apoptotic cells appeared to be
Discussion
We used BMT to generate apoE⫺/⫺ mice with or without
macrophage ACAT1 to address the effects of ACAT1 dele-
Su et al
Reduced Cholesterol Efflux in apoE-Deficient Mice
5
Figure 2. Proximal aortic lesions 12
weeks after BMT. A, Representative
10-␮mol/L cross sections of aortic root of
apoE⫺/⫺ recipient mice 12 weeks after
BMT. Sections were stained with oil red O
and hematoxylin. A, WT ACAT1 (apoE⫹/⫹,
ACAT1⫹/⫹3apoE⫺/⫺); B, ACAT1 KO
(apoE⫹/⫹, ACAT1⫺/⫺3apoE⫺/⫺); C, apoE
KO (apoE⫺/⫺, ACAT1⫹/⫹3apoE⫺/⫺); D,
2KO (apoE⫺/⫺, ACAT1⫺/⫺3apoE⫺/⫺). B,
Quantification of lesion area. Data represent mean⫾SEM (n⫽9 per group).
*P⬍0.05, ***P⬍0.001 as determined by
1-way ANOVA to compare difference in
lesion size among groups. When analyzed
by 2-way ANOVA, atherosclerotic lesion
as function of apoE and ACAT status,
there is significant difference between
apoE⫺/⫺ and 2KO (P⬍0.01) and no significant difference between WT and ACAT⫺/⫺
groups (P⬎0.05).
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tion on the development of atherosclerosis under both hypercholesterolemic conditions, when systemic apoE is absent,
and normocholesterolemic conditions, when macrophage
apoE is restored on the background of systemic apoE deficiency. We showed that ACAT1 deficiency aggravates lesion
formation in the absence of macrophage apoE but has no
effects when the hypercholesterolemia is corrected by macrophage apoE synthesis. To further investigate the effect of
ACAT1 deficiency on macrophage cholesterol homeostasis,
we performed cholesterol efflux experiments and demonstrated that ABCA1-mediated efflux to apoA-I is reduced
30% to 40% in ACAT1⫺/⫺ macrophages. The reduction in
cholesterol efflux was found to be independent of the presence of macrophage apoE and probably was due to the
decrease in ABCA1 protein (Figure 4A). The reduction in
ABCA1-mediated cholesterol efflux observed in ACAT1⫺/⫺
Figure 3. Macrophage cholesterol efflux to apoA-I. Twelve
weeks after BMT, peritoneal macrophages from WT, ACAT1⫺/⫺,
apoE⫺/⫺, and 2KO mice (n⫽3 for each group) were collected.
Macrophages of same genotype were pooled and plated on
24-well plate. Six wells of macrophages from each genotype
were loaded with 50 ␮g/mL acLDL and 3H-cholesterol for 24
hours and then cultured in efflux media with 10 ␮g/mL human
apoA-I. 3H-cholesterol efflux measured as a percentage of cellular 3H-cholesterol counts showed that efflux is reduced 30% to
40% in ACAT1-KO and double-KO macrophages vs WT and
apoE-KO macrophages.
macrophages may contribute to the increase in atherosclerosis. ABCA1 is a key player in reverse cholesterol transport
and is critical in regulating cellular cholesterol homeostasis.25–28 ABCA1 is regulated both at the transcriptional level
via liver X receptor and retinoid X receptor and at the
posttranscriptional level via changes in trafficking and the
turnover rate of ABCA1 protein.26,28 Zinc finger protein 202
(ZNF202) has been reported to suppress ABCA1 expression.29 Downregulation of ZNF202 could result in a loss of
repression, leading to an increase in ABCA1 expression as
one of the regulatory mechanisms at the transcriptional
level.29 Our microarray data in the ACAT1-deficient macrophages show a downregulation of zinc finger DHHC containing protein 5 (Zdhhc5), a member of a recently identified zinc
finger superfamily that has been implicated in transcriptional
regulation of nuclear export signal and in protein-protein,
protein-DNA, and protein-lipid interaction in the nucleus and
cytoplasm.30 It is tempting to speculate that Zddhc5 may also
play a role in regulating ABCA1 expression.
The increase in ABCA1 message could also be a compensatory response to FC accumulation via liver X receptor/
retinoid X receptor activation. Our results show a discordant
expression between ABCA1 message and its protein, suggesting a defect in posttranscriptional regulation of ABCA1. The
decrease in ABCA1 protein may be due to either an increase
in protein degradation or a reduction in translation. Interestingly, reduced expression of translation initiation factor-3
was found in ACAT1⫺/⫺ macrophages loaded with acLDL
(the Table). In agreement with our results, Feng and Tabas31
have shown that ABCA1-mediated cholesterol and phospholipid efflux was inhibited by ⬇80% in pretoxic FC-loaded
macrophages and was due mainly to an increase in ABCA1
degradation. Moreover, it is possible that the downregulation
of Zddhc5 and other membrane proteins could affect ABCA1
trafficking and localization to the cell membrane, thereby
leading to a reduction in ABCA1-mediated cellular cholesterol efflux. The effects of ABCA1 in macrophages resulting
from ACAT1 deficiency in the presences of apoE (ACAT⫺/⫺)
are not as dramatic as in 2KO macrophages. However, even
under mild loading conditions, ACAT⫺/⫺ macrophages have
6
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May 10, 2005
Figure 4. Quantification of ABCA1,
ABCG1, and SR-BI in peritoneal macrophages. A, Relative quantity of ABCA1
mRNA is significantly increased in 2KO
macrophages and 2KO⫹acLDL vs WT
(P⬍0.001). B, C, D, Western blots of
ABCA1, ABCG1, and SR-BI, respectively. Total cell lysates (40 ␮g) were
loaded in each lane. Top, ABCA1,
ABCG1, and SR-BI protein; bottom,
␤-actin. Relative ratios of intensitynormalized ␤-actin are shown on bar
graph. B, ABCA1 protein in ACAT⫺/⫺acLDL and 2KO macrophages is significantly reduced. No significant differences
in ABCG1 and SR-BI protein levels are
seen among groups.
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significantly decreased ABCA1 protein, suggesting that
ACAT deficiency alone could affect the ABCA1-mediated
cholesterol efflux, as indicated in Figure 3. ABCG1 and
SR-BI are also important mediators of cholesterol efflux and
play critical roles in maintaining cellular cholesterol homeostasis. However, ACAT1 deficiency has no significant
effect on either the mRNA or protein levels of both ABCG1
and SR-BI measured by real-time RT-PCR and Western blot,
respectively, suggesting that the primary effect on cholesterol
homeostasis resulting from ACAT1 deficiency is likely due
to the downregulation of the ABCA1-mediated pathway.
The importance of macrophage apoE in the pathogenesis of
atherosclerosis has been demonstrated by our previous studies and others.15,17,32,33 However, the interaction between
ACAT1 and apoE in the effect of cellular cholesterol homeostasis has not been studied. Our data suggest that even
though both ACAT1 and apoE have significant influence on
cholesterol efflux, it is likely that such an effect is through
independent mechanisms. Nevertheless, the combined deletion of apoE and ACAT1 had a greater effect on macrophage
cholesterol homeostasis and resulted in a significant increase
in atherogenesis compared with the deletion of apoE alone.
However, the all-or-none effects of macrophage apoE on
serum cholesterol make it difficult to identify possible physiological connections between these proteins. We have previously reported that the absence of either macrophage apoE
or ACAT1 increases atherosclerosis in LDLR⫺/⫺ mice in the
presence of a more severe hypercholesterolemia than that
Figure 5. A, cDNA microarray analysis of
ACAT1⫺/⫺ macrophages. Red shows fold
increase in gene expression compared
with ACAT1⫹/⫹ macrophages; green, fold
decrease. B, Gene expression changes
in double-KO macrophages vs apoE⫺/⫺
macrophages.
Su et al
Reduced Cholesterol Efflux in apoE-Deficient Mice
7
Deferential Gene Expression in ACAT1ⴚ/ⴚ Macrophages Loaded With acLDL
UNIQID
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Fold Change
Name
H3133B01
⫺3.1
Eukaryotic translation initiation factor-3 subunit 7 (eif-3
zeta) (eIf-3p66) (eif3 d)
Translation initiation factor
Function*
596629
⫺2.5
Transcribed sequence with strong similarity to protein
pir: S12207 (Mus), S12207 hypothetical protein (B2
element)–Mus
Mitochondrial gene, unknown
481955
⫺2.1
Protein tyrosine phosphatase, receptor type, D
Cell adhesion receptor, protein tyrosine phosphatase
activity, regulation of cell growth, differentiation, mitotic
cycle
598396
⫺2.0
WD repeat domain 26
Structural protein, unknown
605155
2.8
Glutamyl-prolyl-tRNA synthetase
ATP binding, regulate protein translation, apoptosis
suppressor
336726
2.7
LIM domain binding 3
Intracellular signal, protein interaction
425885
2.6
Hypothetical protein A430031N04
536663
2.5
Muskelin 1, intracellular mediator containing kelch
motifs
Mediate cell spreading and cytoskeletal response
464892
2.2
3-Monooxgenase/tryptophan 5-monooxgenase
activation protein, ␥ polypeptide
Multifunctional regulator of the cell signaling processes
mediated by protein kinase C
420509
2.2
PCTAIRE-motif protein kinase 1
Serine/threonine kinase
644906
2.2
DNA segment, Chr 1, ERATO Doi 161, expressed
Transcription factor, DNA binding
524155
2.2
Ubiquilin 1
Involved in spindle pole body duplication
439354
2.1
Vacuolar protein sorting 4a (yeast)
Postendosomal sorting of cholesterol
457503
2.1
Adducin 3 (␥)
Membrane cytoskeleton-associated protein
516417
2.1
TGF-␤–regulated gene 4
Unknown
350167
2.1
Insulin-induced gene 2
Cell growth and differentiation
575050
2.1
RIKEN cDNA 2410004N11 gene
Protein binding, voltage-gated K⫹ channel activity
579422
2.1
ATP-binding cassette, sub-amily A (ABC1), member 1
Cellular cholesterol transporter
582683
2.1
RIKEN cDNA 5031400M07 gene
Integral to membrane
478504
2.0
Calpain 6
Unknown
482370
2.0
Intersectin (SH3 domain protein 1A)
Protein binding, Ca2⫹ binding
The gene function is based largely on the information obtained from SOURCE at http://genome-www5.stanford.edu/cgi-bin/source, MGI at http://www.informatics.
jax.org, and Celera Discovery System at http://www.celeradiscoverysystem.com.
observed in the present study.13,16 We interpret this finding to
mean that the increased effect on atherogenesis resulting from
absence of both macrophage ACAT1 and apoE in the apoE⫺/⫺
model is most likely not mediated by the degree of plasma
cholesterol changes.
The microarray analysis of ACAT1-deficient macrophages
also revealed a decreased gene expression for proteins associated with the mitotic spindle apparatus regulating mitosis
(sperm-associated antigen 5), proteins required for posttranscriptional processing and metabolism of histone mRNA
Figure 6. TUNEL staining of proximal
aortic lesions. Red represents MOMA
staining specific for macrophages; green,
apoptotic cells. Compared with apoE⫺/⫺
group, 2KO group has more positive
staining for apoptosis. With higher magnification (⫻400), TUNEL staining is seen
to localize in the macrophages.
8
Circulation
May 10, 2005
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(stem-loop binging protein),34,35 and proteins involved in
protecting cells from oxidative damage (heme oxygenase,
kynurenine 3-hydroxylase, cytochrome c oxidase). Because
of these changes in gene expression, the ACAT1⫺/⫺ macrophages may be more susceptible to cell damage and apoptosis, which is consistent with our findings of increased
apoptotic cells in the lesion area. In addition, the increase in
apoptotic cells in the lesion area may also be due to
downregulation of milk fat globule–EGF factor 8 (Figure
4A), a protein that binds apoptotic cells and facilitates their
elimination via phagocytosis.36 Feng et al37 recently elucidated the mechanisms of FC loading–induced apoptosis in
macrophages. FC loading activates the unfolded protein
response in the endoplasmic reticulum, resulting in the
activation of signaling pathways that promote programmed
cell death.37
ACAT1⫺/⫺ macrophages may also be exposed to proinflammatory factors such as interleukin-1 (IL-1) receptor.38
We observed a 3-fold increase in gene expression of IL-1
receptor type I in the double-KO macrophages compared with
apoE-KO macrophages (the Table). The IL-1 family comprises 4 members: IL-1␣, IL-1␤, IL-1 receptor antagonist
(IL-1ra), and IL-18. IL-1 binds to its receptor and triggers
intracelluar signal transduction through a p38 mitogen-activated protein kinase–activated phosphorylation cascade.
This, in turn, activates the transcription of a variety of
proinflammatory genes, including the production of IL-1
itself.39 Therefore, the IL-1 family is among the most important cytokine mediators of the inflammatory responses and is
thought to play a key role in the propagation of vessel wall
inflammation in atherosclerosis.39 IL-1ra is an endogenous
inhibitor of the IL-1 receptor, which displays affinity for
IL-1R but does not induce a cellular response. IL-1ra transgenic mice have decreased atherosclerosis,40 whereas administration of IL-1ra reduces fatty streak formation in apoE⫺/⫺
mice.41
Our microarray data showed a 2- to 3-fold upregulation of
procollagen types II and III in ACAT⫺/⫺ macrophages compared
with the ACAT⫹⫹ macrophages. Evidently, procollagen-proline,
2-oxoglutarate-4-dioxygenase, ␣-subunit (P4HA1), an enzyme
that plays a critical role in collagen maturation, is downregulated in ACAT⫺/⫺ macrophages. P4HA1 catalyzes the formation 4-hydroxyproline by hydroxylation of proline residues in
the nascent procollagen protein, and the hydroxylated proline
is known to be essential for the assembly of newly synthesized procollagen polypeptide chain into triple helical molecules.42 Therefore, one can speculate that the decrease in
P4HA1 might result in an accumulation of procollagen
peptide in the extracellular matrix of the lesion, which is
supported by an increased collagen content found in the
proximal aortic lesions by trichrome staining in the 2KO mice
(data not show). However, it has been reported that most
collagens in the lesion are made by smooth muscle cells in
later stages of the lesion,43 and the relative contribution of
macrophage produced collagens in early lesion composition
and remodeling needs to be further investigated.
The vascular consequences of systemic ACAT1 deficiency
in hyperlipidemic mice are complex and not without controversy. We reported that both LDLR⫺/⫺ and apoE⫺/⫺ mice have
reduced plasma lipid levels and increased lesion size when
ACAT1 is absent. Then, we showed that macrophage-specific
deletion of ACAT1 increases lesion size and induces apoptosis.13,44 These findings, however, are in contrast to those of a
study that reported a reduction in atherosclerosis in apoE⫺/⫺
mice.45 Because of the possible clinical utility of interfering
with ACAT function in vivo, several inhibitors have been
tested in mouse models for their effects on atherosclerosis.
The results suggest that nonselective inhibition of ACAT may
have a protective role in atherogenesis in animal models11 and
in vitro studies in macrophages.46,47 However, selective
inhibition of ACAT1 increases atherosclerosis in animal
models.12 It is reasonable to speculate that perhaps the
antiatherogenic effects are due to the inhibition of ACAT2,
with a subsequent reduction in cholesterol absorption and
plasma VLDL concentrations. This notion is also supported
by the evidence that apoE⫺/⫺ mice deficient in ACAT2 are
resistant to atherosclerosis.48 Therefore, selective inhibition
of ACAT2 has promising potential in the treatment of
atherosclerosis, whereas inhibition of ACAT1 may have too
profound an influence on macrophage cholesterol homeostasis to be a target of pharmacological inhibition.
Our study provides evidence of the detrimental biological
effects on the vasculature after macrophage ACAT1 deletion.
The underlying molecular mechanisms of disrupted cellular
cholesterol homeostasis and accelerated atherosclerosis are
likely due to the reduction in ABCA1-mediated cholesterol
efflux and predisposition to inflammation and apoptosis.
Acknowledgments
This work was supported in part by National Institutes of Health
grants HL-65709 and HL-57986 to Dr Fazio and HL–53989 to Dr
Linton. Dr Major is supported by an American Heart Association
scientist development grant. Dr Hasty was supported by the Diabetic
Research and Training Center Pilot and Feasibility Grant at Vanderbilt University (2P60DK20593-24). The study was also supported by
the Lipid, Lipoprotein and Atherosclerosis Core of the Vanderbilt
Mouse Metabolic Physiology Centre (NIH DK59637-01) and
Vanderbilt Microarray Shared Resource. We thank John Blakemore,
Tianli Zhu, Janet Matthews, and Youmin Zhang for technical
assistance.
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Reduced ABCA1-Mediated Cholesterol Efflux and Accelerated Atherosclerosis in
Apolipoprotein E-Deficient Mice Lacking Macrophage-Derived ACAT1
Yan Ru Su, Dwayne E. Dove, Amy S. Major, Alyssa H. Hasty, Branden Boone, MacRae F.
Linton and Sergio Fazio
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