Increased Metabolite Levels of Glycolysis and Pentose
Phosphate Pathway in Rabbit Atherosclerotic Arteries
and Hypoxic Macrophage
2

Atsushi Yamashita1., Yan Zhao2., Yunosuke Matsuura1,3, Kazuaki Yamasaki , Sayaka Moriguchi-Goto1,
Chihiro Sugita1, Takashi Iwakiri1,3, Nozomi Okuyama1, Chihiro Koshimoto4, Keiichi Kawai5,6,
Nagara Tamaki7, Songji Zhao2, Yuji Kuge8", Yujiro Asada1" *1 Department of Pathology, University of Miyazaki, Miyazaki, Japan, 2 Department of Tracer Kinetics & Bioanalysis, Hokkaido University, Sapporo, Japan, 3 Department ofInternal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, 4 Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan, 5 Faculty of
Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan, 6 Biomedical Imaging Research Center, University of
Fukui, Fukui, Japan, 7 Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Japan, 8 Central Institute of Isotope Science,
Hokkaido University, Sapporo, Japan

Abstract
Aims: Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many
metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims toidentify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured
macrophages.
Methods: Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral
arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated withlipopolysaccharides (LPS) and interferon-c (INFc) were cultured under normoxic and hypoxic conditions. We evaluatedcomprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresistime of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using 18Ffluorodeoxyglucose (18F-FDG) and pimonidazole, a marker of hypoxia.
Results: The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, comparedwith those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6;
tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of 18F-FDG in arterial walls measured by autoradiography
positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69
for both; p,0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia
inducible factor-1a and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose
phosphate pathway (4 of 6) metabolites increased in LPS and INFc stimulated macrophages under hypoxic but notnormoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated withmetabolic pathways in the macrophages.
Conclusion: Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but notclassical activation might augment glycolytic and pentose phosphate pathways in macrophages.
Citation: Yamashita A, Zhao Y, Matsuura Y, Yamasaki K, Moriguchi-Goto S, Sugita C, et al. (2014) Increased Metabolite Levels of Glycolysis and Pentose Phosphate
Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage. PLoS ONE 9(1): e86426. doi:10.1371/journal.pone.0086426
Editor: Toshiyuki Miyata, National Cerebral and Cardiovascular Center, Japan
Received June 13, 2013; Accepted December 10, 2013; Published January 23, 2014
Copyright: ß 2014 Yamashita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported in part by Grants-in-Aid for Scientific Research in Japan (Nos.23790410, 25460440 AY, 23659573 YK, 23390084 YA), Mitsubishi
Pharma Research Foundation (AY), and Integrated Research Project for Human and Veterinary Medicine, University of Miyazaki (AY, YA), and Project forDeveloping Innovation Systems from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (NT). The funders had no rolein study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: yasada@med.miyazaki-u.ac.jp. These authors contributed equally to this work.
" These authors also contributed equally to this work.

emission tomography (PET) imaging using [18F]-fluorodeoxyglucose (18F-FDG) is as a non-invasive diagnostic modality that candirectly measure arterial inflammation in patients at high risk foratherosclerosis [2]. The cellular accumulation of 18F-FDG reflects

Introduction
Inflammation is considered a feature of atherosclerosis, andinflammation in atherosclerotic plaques is associated with plaqueinstability and future thrombotic complications [1]. Positron
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metabolic activity because 18F-FDG is a direct surrogate of glucose
uptake. In addition, glucose utilization and O2 uptake/mg DNA
are increased in rabbit and monkey atherosclerotic aortae [3]. On
the other hand, adenosine triphosphate (ATP) depletion in the
cores of advanced rabbit atherosclerotic plaques is associated withhypoxic areas [4]. Because hypoxia can affect cellular metabolism
and inflammation [5], metabolism might differ between atherosclerotic and normal arteries. Hypoxia is a potent and sufficient
stimulus for increased glucose uptake in macrophages [6].
However, little comprehensive knowledge is available about the
metabolic status of atherosclerotic arteries.
Metabolome analysis has been widely applied to understandbiological traits and identify novel biomarkers and several clinicalstudies of metabolomes have revealed plasma metabolites that are
related to cardiovascular risk factors, myocardial injury and thedevelopment of diabetes. [7–9]. Capillary electrophoresis/mass
spectrometry (CE-MS) can separate metabolites with highresolution and quantify virtually all charged low-molecular weightcompounds [10]. We analyzed the metabolomes of rabbit
atherosclerotic arteries using capillary electrophoresis-time offlight mass spectrometry (CE-TOFMS) to identify comprehensive
metabolic changes in atherosclerosis. We also assessed therelationship between 18F-FDG uptake and vascular hypoxia in
rabbits and cultured macrophages.

Cell culture
Human THP-1 cells (Dainippon Sumitomo Pharm., Suita,
Japan) were cultured in RPMI1640 (Dainippon Sumitomo
Pharm.) supplemented with 10% heat-inactivated fetal bovine
serum, penicillin (100 U/ml), and streptomycin (100 mg/ml).
THP-1 cells (1.06105 cells/cm2) were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA, 320 nM,
Sigma, Saint Louis, MO, USA), and polarized as previously
reported [12]. For M1 polarization, cells were treated with PMA
for 6 hours and then cultured with PMA plus lipopolysaccharide(LPS, O111: B4, 10 ng/ml, Sigma-Aldrich, Saint Louis) andinterferon-c (INFc, 20 ng/ml, R & D Systems, Minneapolis, MN,
USA) for another 42 hours. For control macrophages, THP-1 cells
were cultured with PMA for 48 hours. To evaluate the appropriate induction of polarization, we measured expression of M1
marker genes, interleukin (IL)-6, tumor necrosis factor (TNF)-a,and IL-1b. After replacement of culture medium, PMA-treatedcontrol macrophages or M1 polarized macrophages were incubated under normoxic (21% O2) or hypoxic (1% O2) conditionsfor 6 hours for metabolome analysis. We measured tissue factor(TF) and plasminogen activator inhibitor-1 (PAI-1) levels in the
supernatant with Elisa kits (Quantikine ELISA for human TF and
PAI-1, R & D Systems, Inc., Minneapolis, MN, USA), andanalyzed correlation between these levels and metabolite levels inmacrophages.

Materials and Methods

Metabolomic analysis

Ethical Statement

Regarding rabbit arterial tissue, surrounding soft tissue wasremoved and then the arteries were dissected out, frozen in liquid
nitrogen and stored at 280uC. Metabolites were extracted fromarterial samples (42–70 mg) as follows. Samples were immersed in
500 mL of methanol containing 50 mM Internal Standard Solution
1 (Solution ID: H3304-1002, Human Metabolome Technologies,
Tsuruoka, Japan) at 0uC and then homogenized three times at
1,500 rpm for 120 seconds to inactivate enzymes. The homogenate was then mixed with 200 mL of Milli-Q water and 500 mL ofchloroform and centrifuged at 2,3006g for 4 minutes at 4uC. The
upper aqueous layer (400 mL) was centrifugally filtered through a
5-kDa cutoff filter (Millipore) to remove proteins and then thefiltrate was lyophilized and suspended in 50 mL of Milli-Q water.
Regarding the cell culture experiment, adherent cells on thedishes were washed with 5% mannitol aqueous solution at room
temperature. The cells were immersed in 400 mL of methanol for
30 seconds, and 275 mL of the Internal Standard Solution (10 mM,
Solution ID: H3304-1002, Human Metabolome Technologies) for
30 seconds. The extraction liquid was centrifuged at 2,3006g for 5
minutes at 4uC. The supernatant (400 mL) was centrifugally
filtered at 9,1006g for 4 hours at 4uC through a 5-kDa cutoff filter(Millipore) to remove proteins, and then the filtrate was lyophilized
and suspended in 25 mL of Milli-Q water.
The metabolite suspension was analyzed by CE-TOFMS using
an Agilent capillary electrophoresis (CE) system equipped with an
Agilent 6210 TOFMS, an 1100 isocratic high-performance liquid
chromatography pump, a G1603A CE-MS adapter kit and a
G1607A CE-electrospray ionization-mass spectrometry (ESI-MS)
sprayer kit (Agilent Technologies, Waldbronn, Germany). The
system was controlled using G2201AA ChemStation software
version B.03.01 for CE (Agilent).
Cationic metabolites were analyzed using a fused silica capillary(50 mm i.d.680 cm total length), with commercial cation electrophoresis buffer (Solution ID: H3301-1001, Human Metabolome
Technologies) as the electrolyte. The sample was injected at apressure of 50 mbar for 10 seconds (approximately 10 nL). The
applied voltage was set at 27 kV. ESI-MS proceeded in the

The Animal Care Committee of Miyazaki University and
Hokkaido University approved the animal research protocols(permit number 2010-511), which also conformed to the Guide for
the Care and Use of Laboratory Animals published by the US
National Institutes of Health. All efforts were made to minimize
suffering.

Experimental protocol
Twenty-two male Japanese white rabbits weighing 2.5 to 3.0 kg
were fed with a conventional or 0.5% cholesterol diet and thenatherosclerotic lesions were induced in the right iliac-femoral
arteries of all of them using a balloon catheter. Three weeks later,
the rabbits were assigned to three groups for histological andmetabolome (n = 6 each) analyses and an 18F-FDG uptake study(n = 10).

Atherosclerosis model
Surgery proceeded under aseptic conditions and generalanesthesia was achieved via an intravenous injection of pentobarbital (25 mg/kg). An angioplasty balloon catheter (diameter,
2.5 mm; length, 9 mm; QUANTUM, Boston Scientific, Galway,
Ireland) was inserted via the carotid artery into the right femoral
artery under fluoroscopic guidance one week after feeding with aconventional or a 0.5% cholesterol diet to induce atherosclerotic
lesions in the right iliac-femoral artery. The catheter was inflated
to 1.5 atm and retracted three times to denude the endothelium[11]. Three weeks later, the rabbits were fasted for six hours, andinjected with heparin (500 U/kg, i.v.) and sacrificed with an
overdose of pentobarbital (60 mg/kg, i.v.). The animals were
perfused with 50 mL of saline for the metabolome analysis, andwith 0.01 mol/L of phosphate buffered saline and 50 mL of 4%paraformaldehyde for the histological analysis. The animals
involved in the 18F-FDG uptake study were not perfused, because
a preliminary study showed that perfusion reduces arterialradioactivity levels.
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positive ion mode and the capillary was set at 4,000 V. The
spectrometer scanned samples from 50 to 1,000 m/z. Other
conditions were as described [13].
Anionic metabolites were analyzed using a fused silica capillary(50 mm i.d.680 cm length), with a commercial anionic electrophoresis buffer (Solution ID: H3302-1021, Human Metabolome
Technologies) as the electrolyte. The sample was injected at apressure of 50 mbar for 25 sec (approximately 25 nL). The applied
voltage was 30 kV. ESI-MS was conducted in the negative ion
mode, and the capillary was set at 3,500 V. The spectrometerscanned samples from 50 to 1,000 m/z. Other conditions were as
described [14].
Raw data obtained by CE-TOFMS were processed using
MasterHands [15]. Signal peaks corresponding to isotopomers,
adduct ions and other product ions of known metabolites were
excluded, all signal peaks potentially corresponding to authentic
compounds were extracted, and then their migration times (MT)were normalized against those of the internal standards. Thereafter, peaks were aligned according to the m/z and normalized
MT values. Finally, peak areas were normalized against those of
the internal standards, methionine sulfone and D-Camphor-10sulfonic acid, for cations and anions, respectively. The resultant
relative area values were further normalized by sample weight.
Annotation tables were produced from CE-ESI-TOFMS values
for standard compounds, and aligned with datasets according tosimilar m/z and normalized MT values.
18

Histological analysis
Excised iliac-femoral arteries were fixed in 4% paraformaldehyde for 12 hours at 4uC and embedded in paraffin. Sections
3 mm thick were stained with hematoxylin and eosin andimmunohistochemically assessed using antibodies against muscle
actin (HHF35; DAKO, Glostrup, Denmark), rabbit macrophages(RAM11; DAKO), CD163 (AM-3K, Trans Genic Inc. Kobe,
Japan), Ki-67 (MIB-1; DAKO) and hypoxia inducible factor(HIF)-1a (H1alpha67; Abcam, Cambridge, MA, USA).
Consecutive 5-mm slices used in the 18F-FDG uptake study were
stained with hematoxylin and eosin. Immunohistochemicalassessment using antibodies against muscle actin (HHF35;
DAKO), rabbit macrophages (RAM11; DAKO), pimonidazole(Natural Pharmacia International Inc.), or hexokinase-II (Abcam)
was followed by staining with Envision (DAKO). Horseradish
peroxidase activity was visualized using 3, 39-diaminobenzidine
tetrahydrochloride. Immunostaining controls included non-immune mouse IgG instead of primary antibodies. Areas of positive
immunostaining in vessels were analyzed using the WinRoof color
imaging morphometry system (Mitani, Fukui, Japan) [18]. Data
are expressed as immunopositive areas (mm2), ratios of vascularareas (%) or numbers of immunopositive nuclei/mm2.
For double immunofluorescence, antibodies for rabbit macrophage (RbM2, TransGenic Inc. Kobe, Japan) and smooth muscle
actin (1A4, Novus biologicals, Littleton, CA, USA) were labeledwith Mix-n-Stain CF594 antibody labeling kit (Biotium, Hayward,
CA, USA). The frozen sections of arteries at 3 weeks after balloon
injury in rabbits fed with 0.5% cholesterol diet were stained with
CF488A labeled secondary antibody (Biotium) for Ki-67 andCF594 labeled antibodies for rabbit macrophage or smoothmuscle actin.

F-FDG uptake and hypoxia

We investigated 18F-FDG uptake as described with minor
modifications [16]. Rabbits (n = 10) were fasted for four hours
before being infused with 18F-FDG (average, 207 MBq/rabbit)
and 60 mg/kg of the hypoxia marker, pimonidazole (Hypoxiaprobe-1, Natural Pharmacia International Inc., Burlington, MA,
USA). Two hours later, amounts of radioactivity in the iliacfemoral arteries, and blood were measured using a well-type ccounter (1480 WIZARD 3; Wallac Co. Ltd., Turku, Finland). The
results are calculated as (%ID/g)6kg.
The non-injured or injured iliac-femoral arteries were excised,and cut into 9–10 segments or 13–15 segments in both diet groups,
respectively. The arterial segments were embedded in Tissue-Tek(Sakura, Tokyo, Japan) and frozen. Consecutive 10- or 5-mm slices
were prepared for autoradiography or histological analysis,
respectively, and then 10-mm cryostat sections were exposed tophosphor imaging plates (Fuji Imaging Plate BAS-SR 2025, Fuji
Photo Film Co. Ltd., Tokyo, Japan) for 12 h together with a set ofcalibrated standards [17]. The plates were then scanned using a
Fuji Bio-imaging Analyzer BAS-5000 with an internal resolutionof 25-mm (Fuji Photo Film) and the images were assessed using
Multi Gauge Ver. 3.0 image analysis software (Fuji Photo Film).
The amount of radioactivity in each image is expressed as
photostimulated luminescence per unit area (PSL = a6D6t, wherea is a constant, D is the amount of radioactivity exposed on theimage plate, and t is exposure time). Each PSL value/mm2 fromthe arterial tissue was recorded and converted to a ratio (%) of theactivity of the standard injected dose/mm2 of lesion area (% ID/m2). The data were normalized according to the weight of eachrabbit (%ID/m2)6kg.
We measured FDG uptake in areas with or without macrophages-rich area or hypoxia to determine whether or not hypoxia
augments FDG uptake in the rabbit iliac-femoral artery withmacrophage-rich neointima. Areas that were rich in macrophages
or pimonidazole were traced on immunohistochemical images,
and then FDG uptake was measured in corresponding areas of
autoradiographic images.
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Quantitative real-time polymerase chain reaction
Cellular total RNA isolated using Trizol (Life Technologies,
Carlsbad, CA, USA) and PureLinkTM RNA mini kit (LifeTechnologies), and converted into complementary DNA (cDNA)
using the Primescript RT Mater Mix kit (Takara Bio, Otsu,
Japan). cDNA were quantified by quantitative polymerase chain
reaction (qPCR) on an LightCycler 480 apparatus (Roche Applied
Science, Penxberg, Germany) using SYBR Premix Ex TagTM kit(Takara Bio) and specific primers indicated in table S1. Messenger
RNA (mRNA) levels were subsequently normalized to those of bactin.

Statistical analysis
All data are presented as means and standard deviation.
Differences between individual groups were tested using the one
way analysis of variance with the Bonferroni multiple comparison
test (GraphPad Prizm 4.03; GraphPad Software Inc., San Diego,
CA, USA). Biologically meaningful patterns were identified bymetabolite set enrichment analysis using MetaboAnalyst 2.0 [19].
Relationships between factors were evaluated using Pearson’s test
and P,0.05 was considered statistically significant.

Results
SMC-rich or macrophage-rich neointimal lesions in rabbit
iliac-femoral arteries
Figure 1 shows representative histological images acquired three
weeks after injuring the iliac-femoral arteries of rabbits fed with aconventional or 0.5% cholesterol diet. Balloon injury induced theformation of neointima comprising SMCs, a few macrophages andextracellular matrix in rabbits that were fed with the conventional
diet (Fig. 1A), and larger neointima comprising SMCs, more
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macrophages and more extracellular matrix in those fed with the
0.5% cholesterol diet (Fig. 1A). The ratios of macrophages and
SMC areas in the injured arteries were significantly larger andsmaller, respectively in rabbits fed with the 0.5% cholesterol dietcompared with the conventional diet (Fig. 1B). Thus, we defined
injured iliac-femoral arteries in rabbits fed with a conventional dietor a 0.5% cholesterol diet as having SMC-rich or macrophagerich neointima, respectively. CD163 positive area accounted for
0.3460.22% (n = 12) in the arteries with macrophage-richneointima. Ratio of CD163 positive area to RAM11 positive area
was 0.0260.01 (n = 12) in the arteries with macrophage-richneointima. The number of Ki-67 immunopositive nuclei wasincreased in the arteries with macrophage-rich neointima (Fig. 1B),
indicating increased proliferative activity. Double immunofluorescence showed Ki-67 immunopositive nuclei in macrophages but
not in SMCs (Fig. 1C). Macrophage accounted for 80.4614.4% of
Ki-67 positive cells. The result suggests that proliferative cells are
predominantly macrophages in the arteries.

g)6kg, p,0.001) than in the non-injured iliac-femoral arteries ofrabbits fed with either the conventional (0.08160.02 (%ID/g)6kg)
or the 0.5% cholesterol (0.08060.016% (%ID/g)6kg) diet, or
iliac-femoral arteries with SMC-rich neointima (0.08060.022(%ID/g)6kg; n = 5 each). Less radioactivity was found in the
blood than in the arteries, and did not differ between conventional
and 0.5% cholesterol diets (0.04460.012 vs. 0.04060.003 (%ID/g)6kg; Fig. 3A). Serial sections were immunohistochemicallystained with antibodies for pimonidazole, the SMC marker
HHF35 and the macrophage marker RAM11. Figure 3B shows
autoradiographic and corresponding histological images of thearteries with SMC- and macrophage-rich neointima. Significantly
more tissue radioactivity was found in the sections of arteries withmacrophage- than SMC-rich neointima (Fig. 3B and C). Areas
containing cells that were immunopositive for pimonidazole werelocalized deep in the wall and such cells were distributed inmacrophage-rich areas. However, macrophages were not always
immunopositive for pimonidazole. No cells were immunopositive
for pimonidazole in the non-injured arteries of rabbits fed withboth diets and the arteries of rabbits with SMC-rich neointima(Fig. 3B and C).
Figure 3D shows relationships between 18F-FDG uptake andvascular, SMC, macrophage and hypoxic areas in the arteries with
SMC- or macrophage-rich neointima. Vascular areas significantly
correlated with 18F-FDG uptake in the arteries with macrophagerich (r = 0.72, p,0.0001, n = 69) and SMC-rich (r = 0.76, p,
0.0001, n = 76) neointima. However, more 18F-FDG was uptakenby the arteries with macrophage- than SMC-rich neointima. The
SMC areas correlated with 18F-FDG uptake in the arteries with
SMC (r = 0.54, p,0.0001, n = 72)- but not macrophage (r = 0.20,
p = 0.10, n = 69) -rich neointima. The uptake of 18F-FDG
significantly correlated with macrophage (r = 0.76, p,0.0001,
n = 69) and hypoxic (r = 0.59, p,0.0001, n = 69) areas in thearteries with macrophage-rich neointima. A few macrophages
were evident in those with SMC-rich neointima, but areas ofmacrophages did not correlate with FDG uptake (r = 0.07,
p = 0.55, n = 72).
We measured 18F-FDG uptake in areas with or without
macrophage-rich area or hypoxia to determine whether hypoxia
augments 18F-FDG uptake in rabbit iliac-femoral arteries withmacrophage-rich neointima (Fig. 3E). More 18F-FDG was uptaken
in areas with, than without macrophages-rich area (3.861.3 vs.
2.260.8 (%ID/m2)6kg; n = 12 each; p,0.001). 18F-FDG uptake
in macrophage-rich area tended more in hypoxic area, but did not
significantly differ from non-hypoxic area (4.161.6 vs. 3.761.3(%ID/m2)6kg; n = 12 each; Fig. 3F).

Metabolome analysis in rabbit iliac-femoral arteries
We comprehensively evaluated metabolism in non-injured iliacfemoral arteries of rabbits fed with a conventional diet, and iniliac-femoral arteries with SMC- and macrophage-rich neointima
by metabolomic analysis using CE-TOFMS. Figure 2 shows levelsof central carbon metabolites in the arteries. Levels of 4 of 9
glycolytic, 4 of 6 pentose phosphate cycle, and 4 of 6 citric acidcycle metabolites were increased in the arteries with macrophagerich neointima compared with the arteries that were not injured
and those with SMC-rich neointima. Levels of glucose 1phosphate, an intermediate of glyconeogenesis/glycogenolysis,
and glycerol 3-phosphate, an intermediate of triacylglycerol/
glycerophospholipids, also increased in the arteries with macrophage-rich neointima. The metabolite levels shown in Figure 2 did
not significantly differ between the arteries that were not injured
and those with SMC-rich neointima from rabbits fed with aconventional diet.
Table S2 shows the levels of all metabolites analyzed in thisstudy. In addition to the above, levels of 10 of 20 nucleotides, 2oxoisovaleric acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid,and glycolic acid were increased in the arteries with macrophagerich neointima, compared with those that were not injured andthose with SMC-rich neointima. Levels of nicotinamide adenine
dinucleotide and nicotinamide adenine dinucleotide phosphate
were also increased in the arteries with macrophage-richneointima.
Levels of gluconic acid were significantly decreased in thearteries with SMC- and macrophage-rich neointima, comparedwith those that were not injured (Table S2).
Analysis of metabolite set enrichment revealed significant
changes in glycolysis (p,0.001), the pentose phosphate pathway(p,0.0001), the tricarboxylic acid (TCA) cycle (p,0.001), purine
metabolism (p,0.001), ribonucleic acid (RNA) transcription (p,0.00001) and pyrimidine metabolism (p,0.05) in the arteries withmacrophage-rich neointima, compared with those that were notinjured (conventional diet) and those with SMC-rich neointima.

Nuclear localization of HIF-1a and expression of HK-II in
iliac-femoral arteries
We examined the nuclear localization of HIF-1a and theexpression of HK-II, a glycolysis enzyme induced by HIF-1 toevaluate hypoxic responses in iliac-femoral arteries. Due to thetransitory nuclear localization of HIF-1a [20], we immunohistochemically stained HIF-1a in 4% paraformaldehyde fixed-paraffin
embedded sections. Figure 4 shows immunohistochemical imagesof RAM11, pimonidazole, HIF-1a, and HK-II staining in thearteries with macrophage-rich neointima. Areas that were positive
for nuclei containing HIF-1a and HK-II closely localized withthose that were positive for macrophages and pimonidazole. No
nuclei immunopositive for HIF-1a or HK-II immunoreactivity
were evident in the non-injured arteries from rabbits fed withconventional or 0.5% cholesterol diets or in the arteries with
SMC-rich neointima.

18

F-FDG uptake and its relationship to hypoxia in rabbit
iliac-femoral arteries

We evaluated glucose uptake and its relationship to vascularhypoxia by injecting rabbits with both 18F-FDG and the hypoxia
marker, pimonidazole. Figure 3A shows the amount of radioactivity in excised arteries two hours after 18F-FDG infusion.
Significantly more radioactivity was found in the iliac-femoral
arteries with macrophage-rich neointima (0.17160.041 (%ID/
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Figure 1. Histological findings of iliac-femoral arteries that were not injured or those at three weeks after balloon injury in rabbits

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fed with conventional or 0.5% cholesterol diets. A. Representative histological images of arterial sections stained with hematoxylin eosin (HE),and antibodies for muscle actin or rabbit macrophages. N, neointima; BI, balloon injury. B. Vascular areas, as well as areas that are immunopositive for
SMCs and macrophages, and numbers of Ki-67-immunopositive nuclei in arteries. White and black bars, conventional and 0.5% cholesterol diet,respectively; n = 12 each). BI, balloon injury. Arteries at three weeks after balloon injury shows neointimal formation rich in SMCs (conventional diet)or macrophages (0.5% cholesterol diet), compared with non-injured arteries. C. Representative double immunofluorescent images for Ki-67 andmacrophage or SMC in arteries 3 weeks after balloon injury in rabbits fed with 0.5% cholesterol diet. Images stained with CF488A labeled anti-Ki-67
antibody (green), CF495-labeled anti-macrophage antibody (red) or anti-SMC antibody (red), and merged immunofluorescent images. There are Ki-67
immunopositive nuclei in macrophages (upper row). The cells with Ki-67-immunopositive nuclei don’t have SMC-positive cytoplasm (arrows).
doi:10.1371/journal.pone.0086426.g001

levels significantly differed in M1 polarized macrophages. Figure 5
shows levels of central carbon metabolites in the macrophages.
Levels of 3 of 8 glycolytic, 2 of 4 pentose phosphate cycle
metabolites, and 5 of 6 TCA cycle metabolites were altered in M1
polarized macrophages compared with those in control macrophages. The metabolite levels of TCA cycle except for 2oxoglutalic acid decreased in M1 polarized macrophages. In M1
polarized macrophages, the hypoxic condition increased in levelsof all glycolytic-, all pentose phosphate cycle metabolites, glucose
1-phosphate, and glycerol 3-phosphate (Fig.5). In addition, thehypoxic condition affected one-half of amino acid levels and onethird of nucleotide levels (Table S3).

Metabolome analysis in cultured macrophages
Macrophages treated with LPS and INFc showed significant
upregulation of IL-6 (control: 0.1260.03, M1: 0.8460.52, p,
0.01, n = 6), TNF-a (control: 0.3460.04, M1: 0.9060.15, p,
0.0001, n = 6), and IL-1b mRNA (control: 4.4360.57, M1:
6.2460.18, p,0.0001, n = 6) compared with PMA-treated control
macrophages.
To evaluate comprehensive metabolic status in macrophages,
we performed metabolomic analysis with CE-TOFMS in PMAtreated control and M1 polarized macrophages under normoxic
condition or M1 polirized macrophages under hypoxic (1% O2)
conditions for 6 hours. Table S3 shows levels of 110 metabolites in
the cultured macrophages. Under normoxic condition, 42
metabolite levels significantly differed between control and M1
polirized macrophages. Under hypoxic condition, 54 metabolite

Figure 2. Levels of metabolites of glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle and glyconeogenesis/
glycogenolysis in rabbit iliac-femoral arteries. Gray, blue and red bars: iliac-femoral arteries that were not injured (conventional diet), and thosewith SMC-rich (conventional diet) and macrophage-rich (0.5% cholesterol diet) neointima, respectively (n = 3 for all). Metabolite levels are expressed
as nmol/g. *p,0.05 vs. other groups, {p,0.05 vs. non-injured femoral artery. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglyceric acid; 3PG, 3phosphoglyceric acid; 2OG, 2-oxoglutaric acid; 6PG, 6-phosphogluconic acid; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F1-6P,
fructose 1,6-diphosphate; F6P, fructose 6-phosphate; FFA, free fatty acid; G1P, glucose 1-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose
6-phosphate; Glu, glucose; Gly, glycogen; PEP, phosphoenolpyruvic acid; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7phosphate; Xu5P, xylulose 5-phosphate.
doi:10.1371/journal.pone.0086426.g002

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Figure 3. Arterial 18F-FDG uptake and its relationship to hypoxia. A. Radioactivity levels in iliac-femoral arteries and blood. White and black
bars, conventional and 0.5% cholesterol diets, respectively; n = 5). BI, balloon injury. B. Representative findings of macrophages and pimonidazole in
the iliac-femoral arteries at three weeks after balloon injury in rabbits fed with conventional (upper row) or 0.5% cholesterol diet (lower row).
Autoradiographic images show increased 18F-FDG uptake in the artery with macrophage-rich neointima. Immunohistochemical staining shows
hypoxic area (pimonidazole positive area) localized in macrophage-rich area in deep portion of wall. C. Uptake of 18F-FDG and immunopositive areafor pimonidazole in arterial sections. White and black bars, conventional and 0.5% cholesterol diets, respectively. D. Correlations between 18F-FDG
uptake and vascular area, SMC-, macrophage-, and pimonidazole-immunopositive areas in sections of arteries at three weeks after BI. E.
Representative trace of macrophage-rich and/or pimonidazole immunopositive area in autoradiographic image. Areas that were rich in macrophages
or pimonidazole were traced on immunohistochemical images, and then FDG uptake was measured in corresponding areas of autoradiographic
images. F. Autoradiogram of arteries with macrophage-rich neointima shows 18F-FDG uptake in areas with or without macrophage-rich area or
pimonidazole immunopositivity.
doi:10.1371/journal.pone.0086426.g003

macrophage-rich atherosclerotic iliac-femoral arteries from rabbits. We also found that hypoxia affects the levels of glycolytic andpentose phosphate pathways in M1 polarized macrophages, andthat PAI-1 protein levels are closely associated with metabolic
pathways in macrophages.
Clinical and preclinical studies have positively associatedincreased 18F-FDG uptake in atherosclerotic arteries [21] withmacrophage density [22]. Morrison et al. [3] examined glucose
utilization in rabbit and rhesus monkey aortic atherosclerosis
induced by high-fat diets, and found increased glucose utilizationand degradation to lactic acid in atherosclerotic intima and media.
Our results support the notion that glycolysis is enhanced inmacrophage-rich atherosclerotic arteries and that it might reflect
metabolic activity in macrophage-rich lesion rather than SMCrich lesion, because levels of glycolytic metabolites, 18F-FDG
uptake and HK-II expression in iliac-femoral arteries with SMCrich neointima did not significantly increase (Table S2, Figs. 2 and
3). Hypoxia is a potent stimulus for increased glucose uptake inmacrophages and foam cells in vitro, suggesting that hypoxia
rather than pro-inflammatory cytokines augments 18F-FDG
uptake in atherosclerotic plaque [6]. In consistent with theprevious study [6], HIF-1a nuclear translocation and HK-II
expression were observed in areas that were positive forpimonidazole (Fig. 4) and 18F-FDG uptake was positively
associated with hypoxic areas (Fig. 3D). In addition, hypoxia
increased in glycolytic metabolite levels in M1 polarized macrophages (Fig.5 and Table S3). However, 18F-FDG uptake did not
significantly differ between hypoxic and non-hypoxic macrophagerich areas of the arteries with macrophage-rich neointima (Fig. 3F).
This may be due to metabolic status of SMCs, endothelial cells,
and lymphocytes in macrophage-rich area. Relative reduction ofblood flow in hypoxic area might affect 18F-FDG supply. And, thediscrepancy might be due to some difference between 18F-FDG
uptake and the levels of down stream metabolites, or special
resolution of the autoradiography. Classical but not innate
activation of cultured macrophages significantly increases 2deoxy-D-glucose uptake, accompanied by glycolytic pathway
activation [23]. Classical activation increases lactate production
with a decrease in oxygen consumption by mitochondria. In our invitro study, M1 polarization decreased in levels of TCA cycle
metabolites but not increased in levels of glycolytic metabolites(Fig. 5). The present findings support the notion that hypoxic
stimuli enhance glycolysis in macrophage-rich atheroscleroticarteries and M1 polarized macrophages. However, it remains
controversial whether non-hypoxic stimuli enhance glycolysis inmacrophages.
The TCA cycle is a series of chemical reactions that generates
energy through the oxidization of acetate derived from carbohydrates, fats and proteins to carbon dioxide. Glucose oxidationaccounts for only 10% of the total O2 uptake in normal rabbit andmonkey aortae, whereas oxygen uptake doubles in atherosclerotic
aortae, although glucose oxidation does not increase [3]. A series

Relation between TF or PAI-1 and metabolite levels in
cultured macrophages
We assessed the correlation between TF or PAI-1 levels in the
supernatant and metabolite levels in the macrophages. The TF
levels negatively correlated with macrophage creatine level (r = 2
0.495, p,0.05, n = 18). While, the PAI-1 levels correlated with
one-half of the metabolite levels in macrophages. Moreover, thecorrelation tended a similar manner within the pathways or
metabolite groups. The PAI-1 levels positively correlated with
TCA cycle metabolites and nucleotide, and negatively correlatedwith glycolysis, pentose phosphate pathway metabolites, andamino acids (Table S4).

Discussion
The CE-TOFMS and 18F-FDG findings showed increased
levels of metabolites in the glycolytic and pentose phosphatepathways and the TCA cycle, as well as of nucleotides in

Figure 4. Immunohistochemical findings of HIF-1a and HK-II in
iliac-femoral artery with macrophage-rich neointima. Immunohistochemical staining for macrophages, pimonidazole and HIF-1a inparaffin sections (A) and for macrophages, pimonidazole and HK-II infrozen sections (B)doi:10.1371/journal.pone.0086426.g004

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Figure 5. Levels of metabolites of glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle and glyconeogenesis/
glycogenolysis in cultured macrophages. For M1 polarization, THP-1 cells were treated with PMA (320 nM) for 6 hours and then cultured with
PMA plus LPS (10 ng/ml) and INFc (20 ng/ml,) for another 42 hours. After replacement of culture medium, PMA-treated control macrophages (graybar, n = 6) and M1 macrophages (blue bar, n = 6) were incubated under normoxic (21% O2) condition for 6 hours, or the M1 macrophages wereincubated hypoxic (1% O2) conditions for 6 hours (red bar, n = 6). Metabolite levels are expressed as pmol/106 cell. *p,0.01 vs. PMA control, {p,0.05
vs. PMA control, 1p,0.01 vs. M1 normoxia, "p,0.05 vs. M1 normoxia. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglyceric acid; 3PG, 3phosphoglyceric acid; 2OG, 2-oxoglutaric acid; 6PG, 6-phosphogluconic acid; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F1-6P,
fructose 1,6-diphosphate; F6P, fructose 6-phosphate; FFA, free fatty acid; G1P, glucose 1-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose
6-phosphate; Glu, glucose; Gly, glycogen; PEP, phosphoenolpyruvic acid; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7phosphate; Xu5P, xylulose 5-phosphate.
doi:10.1371/journal.pone.0086426.g005

via regenerating reduced glutathione (GSH) from its oxidative form,
GSSG. The amounts of glucose oxidized by the pentose phosphatepathway in arteries have been described as large and small [29,30].
Levels of metabolites from the pentose phosphate pathway in the
rabbit iliac-femoral artery were equivalent to or less than those ofglycolytic metabolites. These findings suggest that the pentose
phosphate pathway plays a role in atherosclerosis, and that theenhanced pentose phosphate pathway is compatible with anincrease in the numbers of proliferative cells and in 2-hydroxybutyric acid levels in the arteries with macrophage-rich neointima. When cystathionine is cleaved to cysteine that is incorporated
into glutathione, 2-hydroxybutyric acid is released as a by-productand it is associated with increased oxidative stress [31]. The role ofthe pentose phosphate pathway in atherosclerosis and its role incomplicating thrombosis are poorly understood. A reduction in
glucose 6-phosphate dehydrogenase activity decreases vascularsuperoxide and atherosclerotic lesions in mice that are deficient in
apolipoprotein E [32]. The findings of one study in vitro have raised
the notion that changes in glucose metabolism influence theinflammatory properties of M1 macrophages [33]. Stimulation withlipopolysaccharide (LPS), which drives an M1-like or classicalactivation pathway, increases glycolysis as well as metabolism via
the pentose phosphate pathway and decreases oxygen consumptionof studies found that almost all glucose is converted into lactate,very little is oxidized, and almost all oleate, (long-chain fatty acid)is converted into CO2 in peritoneal macrophages in vitro [24,25].
More citrate synthase activity is found in macrophages than inlymphocytes [26]. Therefore, the increased level of citric acid in
the arteries with macrophage-rich neointima might reflectenhanced fatty acid oxidation. On the other hand, vascularhypoxia might be associated with increased levels of malate,fumarate and succinate in femoral arteries with macrophage-richneointima, because anaerobic mitochondrial oxidation of 2oxoglutarate to succinate and reduction of fumarate to succinate
can generate ATP and maintain mitochondrial energization[27,28]. Taken together, increased levels of TCA intermediates
could reflect enhanced aerobic fatty acid oxidation and anaerobic
pathways for ATP production in macrophage-rich atheroscleroticarteries.
The pentose phosphate pathway is an alternative route of glucose
catabolism that functions in the formation of ribose 5-phosphate(R5P) for synthesis of RNA and deoxyribonucleic acid, thus
supporting cell growth and proliferation, as well as the formationof nicotinamide adenine dinucleotide phosphate (NADPH) for
biosynthetic reactions. NADPH protects directly against oxidative
stress by neutralizing reactive oxygen intermediates, and indirectly
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rates in cultured macrophages. The carbohydrate kinase-likeprotein that catalyzes the production of sedoheptulose-7-phosphate,
an intermediate of the pentose phosphate pathway, can influence
macrophage polarization and antagonize the LPS-induced production of nuclear factor-kB-regulated cytokines. In contrast to thestudy, hypoxia but not classical activation increased in metabolite
levels of pentose phosphate pathway in cultured macrophage (Fig.5
and Table S3). Because 98% of macrophages in the femoral arteries
with macrophage-rich neointima were of the non-M2 type,increased amounts of pentose phosphate pathway metabolites
might reflect their metabolic status and be affected by hypoxic
milieu.
Nucleotide levels were increased in iliac-femoral arteries withmacrophage-rich neointima. This finding is consistent with theincreased levels of metabolites of the pentose phosphate pathway
and proliferative activity in macrophage-rich atheroscleroticarteries. In contrast to our findings, one study discovered ATP
depletion in atherosclerotic plaques of the rabbit aorta [4].
Obvious depletion in the cores of plaques .500 mm thick was
associated with hypoxic areas and low glucose concentrations in
the core. Because 18F-FDG uptake was increased in hypoxic areasof deep portions of plaque (Fig. 3B), a low glucose concentration in
the core is less likely in this model. The difference might be due tothe size of the plaque and the absence of a necrotic core in thismodel.
Balloon injury induces phenotypic changes in SMCs, and theneointima mainly comprised the synthetic phenotype type of
SMCs [34]. Transcription, signalling pathways and protein
expression significantly differ between synthetic and contractile
phenotypes of SMCs [35]. Therefore, we presumed somemetabolic changes between the non-injured and injured arteriesof rabbits fed with conventional diet. However, the only analyzed
metabolite that significantly differed was gluconic acid.
Gluconic acid is a non-toxic glucose derivative that occurs
naturally in fruits and vegetables, and levels were reduced in the
injured arteries of rabbits fed with either diet. Despite many
commercial applications in pharmaceutical and food industries,
the potential roles of gluconic acid in vivo are unclear. SalukJuszczak [36] investigated the effect of sodium D-gluconic acid on
platelet activation and found that it inhibited thrombin-induced
arachidonic peroxidation, O22i production and platelet oxidation/nitration induced by peroxynitrite, suggesting that it exerts
antioxidant effects. Because oxidative stress plays a substantial rolein atherogenesis and neointimal formation after balloon angioplasty [37], a reduction of gluconic acid in injured arteries implies
an unbalanced redox state.
We found increased levels of glucose 1-phosphate, 2-oxoisovaleric acid, glycolic acid, glycerol 3-phosphate and 3-hydroxybutylicacid in arteries with macrophage-rich neointima. This indicatesthat other metabolic pathways, such as glyconeogenesis/glycogenolysis, and amino acids triglyceride and glycerophospholipid
metabolism are altered in such an environment. A clinical PET/
CT study has shown that 11C-acetate uptake, which reflects acetylCoA production, is increased in the aorta with advanced
atherosclerosis, suggesting enhanced fatty acid synthesis [38].
Further comprehensive study is required to unveil other metabolicchanges in atherosclerotic lesions.
Macrophages incubated in 1% oxygen showed an increased
triglyceride and sterol contents though HIF-1a mediated cholesterolsynthesis and reduction of ATP-binding cassette subfamily A
member1 mediated cholesterol efflux [39]. Chronic hypoxia
activated Akt pathway in human macrophages [40]. It is consideredthat Akt singling stimulates transport and metabolism of bothglucose and amino acids, with in turn support mTOR-dependent
increases in protein translation in cancer cells [41]. Our in vitro
study showed significant metabolic change in M1 polarization andhypoxic condition in macrophages (Fig.5 and Table S3). Hypoxic
and non-hypoxic stimuli may substantially modify macrophage
metabolism.
In spite of the widespread of lipid lowering therapy, atherosclerotic disease remains a leading cause of death and loss of
productive life. Metabolomic approach may discover a novel
markers and therapeutic targets of atherosclerosis. We found that
PAI-1 protein levels are closely associated with metabolic
pathways in macrophages (Table S4). Furthermore, we recently
demonstrated that 18F-FDG uptake reflect nuclear factor-kB
activation, tissue factor, an initiator of coagulation cascade,expression, and arterial thrombus formation in rabbits [42].
These findings suggest a link between vascular metabolism andthrombogenic potential of atherosclerotic artery.
There are several limitations in this study. First, the metabolites
in this study are a part of total vascular metabolites. Combined
CE-TOFMS with liquid chromatography-mass spectrometry
might unveil further comprehensive metabolic changes in atherosclerotic arteries. Second, we cannot refer to a dynamic process ofvascular metabolism, because the arterial metabolite levels
represent a metabolic point in rabbits fasted for 6 hours. Further
studies are required to reveal a metabolic rate in the pathways andmetabolic changes before and after diet in atherosclerotic artery.
Third, it is possible that neointimal SMCs contribute to themetabolic changes and 18F-FDG uptake in arteries with macrophage-rich neointima, because inflammatory stimuli mildly
enhanced glucose uptake in cultured vascular SMCs [6].
In conclusion, Infiltrative macrophages in atheroscleroticarteries might affect metabolic systems, and hypoxia but notclassical activation might augment glycolytic and pentose phosphate pathways in macrophages.

Supporting Information
Table S1 Sequences of primers used for qPCR analysis.

(PDF)
Table S2 Quantities of arterial metabolites.

(PDF)
Table S3 Quantities of macrophage metabolites.

(PDF)
Table S4 Relation between plasminogen activator
inhibitor-1 and metabolite levels.
(PDF)

Author Contributions
Conceived and designed the experiments: AY YZ YM CK KK NT SZ YK
YA. Performed the experiments: AY YZ YM KY SM TI CS NO SZ.
Analyzed the data: AY YZ KY SM CS TI NO YK YA. Contributed
reagents/materials/analysis tools: YZ YM KY. Wrote the paper: AY YZSZ YK YA.

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