Upregulation of aldose reductase during foam cell formation as possible link among diabetes, hyperlipidemia, and atherosclerosis.

Abstract

Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway. In diabetes, it is related to microvascular complications. We discovered AR expression in foam cells by gene chip screening and hypothesized that it may be relevant in atherosclerosis.
AR gene expression and activity were found to be increased in human blood monocyte-derived macrophages during foam cell formation induced by oxidized LDL (oxLDL, 100 microg/mL). AR activity as photometrically determined by NADPH consumption was effectively inhibited by the AR inhibitor epalrestat. oxLDL-dependent AR upregulation was further increased under hyperglycemic conditions (30 mmol/L D-glucose) as compared to osmotic control, suggesting a synergistic effect of hyperlipidemia and hyperglycemia. AR was also upregulated by 4-hydroxynonenal, a constituent of oxLDL. Upregulation was blocked by an antibody to CD36. AR inhibition resulted in reduction of oxLDL-induced intracellular oxidative stress as determined by 2'7'-dichlorofluoresceine diacetate (H2DCFDA) fluorescence, indicating that proinflammatory effects of oxLDL are partly mediated by AR. Immunohistochemistry showed AR expression in CD68+ human atherosclerotic plaque macrophages.
These data show that oxLDL-induced upregulation of AR in human macrophages is proinflammatory in foam cells and may represent a potential link among hyperlipidemia, atherosclerosis, and diabetes mellitus.

Full text

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ISSN: 1524-4636
Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 1079-5642. Online
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Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.
DOI: 10.1161/ATVBAHA.107.158295
published online May 1, 2008;
Arterioscler Thromb Vasc Biol
Christian A. Gleissner, John M. Sanders, Jerry Nadler and Klaus Ley
Among Diabetes, Hyperlipidemia, and Atherosclerosis
Upregulation of Aldose Reductase During Foam Cell Formation as Possible Link
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Upregulation of Aldose Reductase During Foam Cell
Formation as Possible Link Among Diabetes,
Hyperlipidemia, and Atherosclerosis
Christian A. Gleissner, John M. Sanders, Jerry Nadler, Klaus Ley
Objective—Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway. In diabetes, it is related to
microvascular complications. We discovered AR expression in foam cells by gene chip screening and hypothesized that
it may be relevant in atherosclerosis.
Methods and Results—AR gene expression and activity were found to be increased in human blood monocyte-derived
macrophages during foam cell formation induced by oxidized LDL (oxLDL, 100 ?g/mL). AR activity as photometri-
cally determined by NADPH consumption was effectively inhibited by the AR inhibitor epalrestat. oxLDL-dependent
AR upregulation was further increased under hyperglycemic conditions (30 mmol/L D-glucose) as compared to osmotic
control, suggesting a synergistic effect of hyperlipidemia and hyperglycemia. AR was also upregulated by
4-hydroxynonenal, a constituent of oxLDL. Upregulation was blocked by an antibody to CD36. AR inhibition resulted
in reduction of oxLDL-induced intracellular oxidative stress as determined by 2?7?-dichlorofluoresceine diacetate
(H2DCFDA) fluorescence, indicating that proinflammatory effects of oxLDL are partly mediated by AR. Immunohis-
tochemistry showed AR expression in CD68? human atherosclerotic plaque macrophages.
Conclusions—These data show that oxLDL-induced upregulation of AR in human macrophages is proinflammatory in
foam cells and may represent a potential link among hyperlipidemia, atherosclerosis, and diabetes mellitus. (Arterioscler
Thromb Vasc Biol. 2008;28:1137-1143)
Key Words: atherosclerosis ? diabetes mellitus ? lipoproteins ? macrophage ? plaque
D
perturbs cellular integrity include formation of advanced
glycation end products (AGE), activation of protein kinase C
(PKC), upregulation of 12/15-lipoxygenase, and increased
flux through the hexosamine or the polyol pathway.2,3,3aAGE
formation may result in cross-linking and subsequent confor-
mational changes of many proteins, increased inflammatory
signaling, and oxidative stress in various cell types including
macrophages and endothelial cells.2Similarly, activation of
PKC isoforms leads to endothelial dysfunction by activation
of NADPH oxidase, enhanced vascular permeability through
upregulation of vascular endothelial growth factor, and
thrombosis by increasing plasminogen activator inhibitor
(PAI)-1 levels.4The contribution of the polyol pathway to
diabetes-induced atherogenesis is currently not completely
understood.
The polyol pathway includes aldose reductase (AR,
AKR1B1), which is the rate-limiting enzyme, and sorbitol
dehydrogenase (SDH, SORD).3Its physiological role is con-
troversial. AR activity has been proposed to be protective
iabetes mellitus represents one of the major risk factors
for atherosclerosis.1Mechanisms by which diabetes
against oxidative stress by detoxifying lipid peroxidation
products, eg, in vasculitis,5,6but inhibiting AR does not
increase lipid peroxidation products.7By contrast, AR activ-
ity has also been related to greater oxidative stress and
increased damage during brain ischemia in mice.8In keeping
with a proinflammatory role of AR, AR null mice exhibit
reduced oxidative stress and are protected from ischemic
injury.8
The main physiological substrates of the polyol pathway
are aldehydes, but sugars like glucose or galactose have also
been shown to be metabolized by AR.9With rising intracel-
lular glucose levels as seen in diabetics, glucose can be
shunted into the polyol pathway resulting in sorbitol produc-
tion, which is subsequently metabolized by sorbitol dehydro-
genase to fructose.3This leads to increased AGE formation
attributable to increased fructose levels and increased oxida-
tive stress attributable to consumption of NADPH, which is
needed to regenerate glutathione.3Thus, the polyol pathway
has been implicated in microvascular diabetic complications
like cataract,10nephropathy,11and neuropathy.12Pharmaco-
logical inhibition of AR has been shown to improve nerve
Original received October 23, 2007; final version accepted March 15, 2008.
From the Division of Inflammation Biology (C.A.G., K.L.), La Jolla Institute for Allergy & Immunology, Calif; Robert M. Berne Cardiovascular
Research Center (J.M.S.) and the Department of Endocrinology and Metabolism (J.N.), University of Virginia, Charlottesville.
Correspondence to Christian A. Gleissner, MD, Division of Inflammation Biology, La Jolla Institute for Allergy & Immunology, 9420 Athena Circle
Drive, La Jolla, CA 92037. E-mail christian@liai.org
© 2008 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.orgDOI: 10.1161/ATVBAHA.107.158295
1137
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conduction velocity and symptoms of polyneuropathy.13Re-
cently, the polyol pathway has been linked to atherosclerosis,
as overexpression of human AR in LDL receptor–deficient
diabetic mice (which physiologically display only low AR
expression) increased atherosclerotic lesions.14However, the
mechanisms by which AR promotes atherosclerosis remain
still to be elucidated.
In a study of gene expression during foam cell formation
by gene chip analysis, we found a significant increase of AR
mRNA expression after treatment of primary human blood
monocyte-derived macrophages with oxidized low density
lipoprotein (oxLDL).15We therefore hypothesized that in-
creased AR expression in human foam cells may represent a
novel proinflammatory mechanism by which oxLDL in-
creases oxidative stress and which may link 2 of the major
risk factors for atherosclerosis, diabetes and hyperlipidemia.
Materials and Methods
Macrophages and Foam Cells
Macrophages were derived from monocytes isolated from either
peripheral blood or buffy coats provided by the Virginia Blood
Services (Richmond, Va) as described previously.15The study was
approved by the institutional review committee. All subjects gave
informed consent. Blood was drawn from 15 healthy individuals (8
female, 7 male, age 20 to 40 years). Blood monocytes were
differentiated to macrophages by macrophage colony stimulating
factor (M-CSF) or platelet factor-4 (CXCL4) for 6 days and exposed
to vehicle, oxLDL (Biomedical Technologies), or 4-hydroxynonenal
(4-HNE, Sigma) as indicated. For receptor blocking experiments,
cells were preincubated with mAb to CD36 antibody (clone CB38)
or IgM control (0.5 ?g/1?106cells, both BD Biosciences) for 30
minutes before addition of oxLDL16(see supplemental materials,
available online at http://atvb.ahajournals.org).
Gene Chip Experiments
RNA was extracted from cells using the RNEasy kit (Qiagen)
according to the manufacturer’s instructions. Gene expression was
measured in duplicates at the University of Virginia Gene Expression
Core Facility using Affymetrix equipment and H133A chips as
described. Gene expression data are available at the NCBI gene
expression and hybridization array data repository (http://
www.ncbi.nlm.nih.gov/geo/, series GSE7138).
Real-Time Polymerase Chain Reaction
Total RNA was isolated from cultured macrophages and foam cells
using the RNEasy Mini Kit with DNase treatment. Reverse tran-
scription was performed with the Omniscript RT Kit (all Qiagen).
Gene expression was measured using iQ SYBR green supermix in an
iCycler iQ Real-Time Detection System (both BioRad). Product
specificity was ascertained by melting curve analysis and initially by
electrophoresis on agarose gels. Primers were purchased from
Integrated DNA Technologies. Primer sequences were as follows:
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5?-
GGCTCATGACCACAGTCCAT-3?, reverse 5?-GCCTGCTTCA-
CCACCTTCT-3?,17aldose reductase (AKR1B1) forward 5?-
AGTCGGGCAATGTGGTTCCC-3?, reverse 5?-GGATTAACTT-
CTCCTGAGTG-3?.14All samples were run in duplicates. Values
were determined using iCycler iQ Real-Time Detection System
software (version 3.1; Bio-Rad). Relative gene expression was
calculated as described previously using GAPDH as housekeeping
gene.18
Aldose Reductase Activity Assay and
Inhibition Experiments
AR activity was measured photometrically as described previous-
ly5,19(see supplemental materials). For AR inhibition experiments,
epalrestat (Sequioa Research Products) was used. The dosage was
chosen based on the IC50(ranging from 0.01220to 0.021 ?mol/L21)
and effective inhibition of AR activity in monocyte-derived macro-
phages. For AR inhibition experiments, epalrestat was added to the
culture medium at a concentration of 0.2 ?mol/L 24 hours before
addition of oxLDL, which completely inhibited AR activity.
Oxidative Stress Assay
Generation of reactive oxygen species was measured as described
previously using 2?7?-dichlorofluoresceine diacetate (H2DCFDA), a
dye which converts to fluorescent 2?7?-dichlorfluorescein (DCF) in
the presence of reactive oxygen species22(see supplemental data).
Immunohistochemistry
Human coronary arteries were obtained from the University of
Virginia Department of Pathology/Tissue bank and were stained for
AR, CD68, or smooth muscle ?-actin. Immunofluorescence was
performed to determine colocalization of AR with CD68 or smooth
muscle ?-actin (for details of staining procedure see supplemental
materials). Coronary arteries from 8 donors were studied, 2 of whom
were diabetic.
Statistics
Differences between the groups were evaluated by 2-tailed t test or
1-way ANOVA followed by a posthoc Tukey test. The exact details
of gene array data analysis have been described previously15(see
supplemental materials). Generally, P?0.05 was considered statis-
tically significant. Data are presented as mean?SEM. Statistical
analysis was performed using PRISM (GraphPad).
The authors had full access to the data and take responsibility for
its integrity. All authors have read and agreed to the manuscript as
written.
Results
AR Expression in Human Macrophages Is
Increased During oxLDL-Dependent
Foam Cell Formation
Cell culture of human blood–derived monocytes with M-CSF
(100 ng/mL) or CXCL4 (100 ng/mL) for 6 days resulted in
macrophage formation. Large scale analysis of gene expres-
sion using Affymetrix gene HU133A arrays revealed a
significant increase of AR mRNA in macrophages gener-
ated with either M-CSF or CXCL4 as compared to periph-
eral blood mononuclear cells (PBMCs) or monocytes
(P?0.0001). Incubation of monocyte-derived macrophages
with 100 ?g/mL mmLDL or oxLDL for 48 hours reproduc-
ibly resulted in foam cell formation as assessed by oil red O
staining (supplemental Figure IB). AR gene expression was
further increased during foam cell formation caused by
oxLDL but not by mmLDL (P?0.10 for M-CSF, P?0.0001
for CXCL4) (supplemental Figure IIA). A full set of other
genes significantly regulated during foam cell formation by
Affymetrix chip analysis is given in supplemental Table I.
The time course of oxLDL-dependent upregulation of AR
mRNA was investigated by real-time PCR revealing an early
peak 4 to 6 hours after addition of oxLDL. The peak reached
about 60% and 80% above baseline in macrophages gener-
ated with M-CSF or CXCL4, respectively (P?0.01 in both
cases, Figure 1A and supplemental Figure IIB). A second
increase was seen between 24 and 48 hours.
To investigate whether increased AR gene expression was
accompanied by functional changes, AR activity was deter-
mined in macrophages generated with M-CSF or CXCL4
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before and 48 hours after addition of oxLDL. AR activity was
significantly increased in oxLDL-treated macrophages irre-
spective of the growth factor used for differentiation
(P?0.01). Treatment of macrophages with epalrestat
(0.2 ?mol/L) resulted in complete inhibition of AR activity in
both cases (Figure 1B and supplemental Figure IIC).
Molecular Mechanism of oxLDL-Induced
AR Upregulation
To investigate the mechanisms by which oxLDL upregulates
AR gene expression, different constituents of oxLDL were
tested for their ability to upregulate AR in macrophages.
Previous studies have reported increased AR expression in rat
vascular smooth muscle cells or human lymphocytes after
stimulation with 4-hydroxynonenal (4-HNE), a constituent of
oxLDL, displaying a peak at a 4-HNE concentration of
5 ?mol/L.5,6We confirm this finding in macrophages (Figure
2A). To identify the receptor involved, we used a blocking
mAb against the scavenger receptor CD36, which accounts
for up to two thirds of oxLDL uptake by macrophages.23
Anti-CD36 antibody significantly reduced oxLDL-induced
AR upregulation (Figure 2B).
oxLDL-Dependent Upregulation of AR Depends
on Hyperglycemic Conditions
As mentioned previously, AR expression has been shown to
be of clinical importance in diabetic individuals. To assess the
effects of oxLDL on AR expression and activity not only
under standard cell culture conditions with a glucose concen-
tration of 17.5 mmol/L,15but also under normoglycemic and
hyperglycemic conditions, AR expression and activity were
measured with 5 mmol/L glucose or 30 mmol/L glucose in
the culture media.
Real-time PCR revealed a significant increase of AR gene
expression under hyperglycemic as compared to normogly-
cemic conditions (P?0.05). Under hyperglycemic conditions,
stimulation of macrophages with oxLDL resulted in a signif-
icant increase of AR gene expression (P?0.05; Figure 3A).
Exposure of macrophages to oxLDL resulted in a 10-fold
increase of AR activity under hyperglycemic, but not normo-
glycemic conditions (P?0.01). The activity increase ex-
ceeded the increase in mRNA expression levels (Figure 3B).
oxLDL-Dependent AR Upregulation Is Not
Related to Increased Gene Expression of
Proinflammatory Mediators but to Oxidative
Stress in Macrophages
AR expression has been related to expression of proinflam-
matory cytokines induced by lipopolysaccharide (LPS) in
mouse peritoneal macrophages.24However, exposure of mac-
rophages to oxLDL under the conditions applied in this study
did not result in increased gene expression of inflammatory
mediators like tumor necrosis factor (TNF)-?, interleukin
(IL)-6, or IL-1? (supplemental Figure III).
oxLDL has been described to increase production of
reactive oxygen species in macrophages.25AR activity in
A
hours of oxLDL stimulation (100 µg/ml)
0
0 6 12 18 24 30 36 42 48
AR gene expression
(n-fold change)
***
**
**
1.0
1.2
1.4
1.6
1.8
2.0
2.2
(glc 17.5 mM)
B
unstimulated oxLDL (100 µg/ml)
Epalrestat -
(200 nM)
+ - +
(glc 17.5 mM)
AR activity
(mU/µg protein)
0
2
4
6
8
**
**
Figure 1. AR gene expression and activity during foam cell for-
mation. A, Gene expression in macrophages exposed to oxLDL
(100 ?g/mL) for indicated periods. B, AR activity in macro-
phages after exposure to oxLDL (48 hours) with or without epal-
restat treatment (means?SEM, **P?0.01, ***P?0.001).
B
unstimulated oxLDL (100 µg/ml)
(glc 17.5 mM)
*
0
1
2
AR gene expression
(fold increase)
-
anti-
CD36IgM
-
anti-
CD36IgM
*
A
(glc 17.5 mM)
*
**
0
1
2
AR gene expression
(fold increase)
unst.
oxLDL
(100 µg/ml)
4-HNE
(5 µM)
Figure 2. 4-hydroxynonenal upregulates AR mRNA expression
to the same extent as oxLDL. A, Gene expression in macro-
phages treated with vehicle (unst.), oxLDL, or 4-hydroxynonenal
for 6 hours. B, Gene expression in macrophages treated with
vehicle or oxLDL in the presence of IgM to CD36 (anti-CD36)
or irrelevant IgM (means?SEM, *P?0.05, **P?0.01).
Gleissner et alAldose Reductase in Foam Cells
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foam cells may represent a proinflammatory mechanism, by
which oxLDL increases oxidative stress in the macrophage.
To test this hypothesis, intracellular oxidative stress was
measured in macrophages under different conditions using
2,7-dichlorfluorescein, a dye which displays increased fluo-
rescence intensity in the presence of reactive oxygen species.
Exposure of macrophages to oxLDL (100 ?g/mL) for 48
hours resulted in a significant increase of DCF fluorescence
under normoglycemic (5 mmol/L) as well as under hypergly-
cemic (30 mmol/L) conditions (P?0.001). Under hypergly-
cemia, oxLDL induced an 8-fold increase of DCF fluores-
cence, compared to 5-fold under normoglycemic conditions
(Figure 4). Pretreatment of macrophages with epalrestat
(0.2 ?mol/L) for 24 hours before and throughout exposure to
oxLDL resulted in a statistically significant (P?0.01) reduc-
tion of ROS formation under hyperglycemic conditions.
Treatment of macrophages with epalrestat alone did not have
any effect. These findings suggest that under hyperglycemic
conditions ?20% to 30% of the oxidative stress in macro-
phage foam cells is attributable to AR activity.
AR Is Expressed in Macrophages in Human
Atherosclerotic Plaque
To assess the relevance of our findings for the pathogenesis of
atherosclerosis, AR expression in human atherosclerotic
plaque was investigated. Immunohistochemistry revealed that
AR was abundantly expressed in a wide range of cells in the
vascular wall, mostly vascular smooth muscle cells, including
cells in atherosclerotic plaques (Figure 5A). AR expression in
macrophage foam cells was verified by immunofluorescence
demonstrating colocalization of CD68 and AR in atheroscle-
rotic lesions (Figure 5B). AR was also colocalized with CD68
in macrophages in the vascular adventitia and perivascular
adipose tissue (Figure 5B). Colocalization with smooth mus-
cle ?-actin confirmed AR expression in vascular smooth
muscle cells, which had been described previously26(Figure
5C). The proportion of AR-expressing macrophages ranged
from 75% to 100% and was similar in the adventitia,
perivascular adipose tissue, and within the atherosclerotic
lesions (supplemental Figure IV).
Discussion
This article describes the discovery of increased aldose
reductase (AR) gene expression and activity in macrophages
during foam cell formation induced by oxLDL in the pres-
ence of high glucose. AR was similarly upregulated by
4-hydroxynonenal, a constituent of oxLDL. Most of the
oxLDL-induced upregulation of AR expression was blocked
by mAb to CD36. Inhibition of AR using the pharmacological
inhibitor epalrestat indicates that AR upregulation ac-
counts for about 20% of oxLDL-induced oxidative stress.
AR gene expression
(fold increase)
AR activity
(mU/µg protein)
5 mM glucose30 mM glucose
5 mM glucose30 mM glucose
0
2
4
6
8
10
12
14
16
*
0
1
2
3
4
5
6
*
unstimulated
oxLDL (100 µg/ml
48 hours)
A
B
Figure 3. High glucose levels and oxLDL have synergistic
effects on AR expression and activity. A, Gene expression in
macrophages treated with oxLDL for 48 hours under normogly-
cemic (glucose 5 mmol/L) or hyperglycemic (glucose 30 mmol/L)
conditions. B, AR activity in macrophages treated with oxLDL
under normoglycemic or hyperglycemic conditions (means?SEM,
*P?0.05).
A
0
DCF fluorescence
(fold change over
untreated cells)
unstimulatedoxLDL (100 µg/ml,
48 hours)
Epalrestat -
(200 nM)
+ -+
***
2
4
6
8
10
(glc 5 mM)
***
B
unstimulatedoxLDL (100 µg/ml,
48 hours)
Epalrestat -
(200 nM)
+ -+
(glc 30 mM)
**
0
2
4
6
8
10
DCF fluorescence
(fold change over
untreated cells)
Figure 4. oxLDL-induced AR upregulation causes increased for-
mation of reactive oxygen species. Macrophages generated
under (A) normoglycemic or (B) hyperglycemic conditions were
treated with vehicle (unstimulated) or oxLDL with or without
24-hour pretreatment with epalrestat. Cells were stained with
2?7?-dichlorofluoresceine diacetate (10 ?mol/L) for 30 min-
utes, and fluorescence was determined by flow cytometry
(means?SEM of fold change over untreated cells, **P?0.01,
***P?0.001).
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Figure 5. AR expression in atherosclerotic lesions. A, Immunohistochemistry demonstrating expression of CD68 (macrophages),
smooth muscle ?-actin (smooth muscle cells), and AR in atherosclerotic lesions of human coronary artery. 100? and 400? magnifica-
tion. B, Immunofluorescence demonstrating colocalization of CD68 (green) and AR (red) in cells in atherosclerotic lesions, adventitia,
and perivascular adipose tissue of human coronary artery. DAPI (blue) is shown as nuclear stain. *Indicates the lumen of the vessel,
400? magnification. C, Immunofluorescence demonstrating colocalization of smooth muscle ?-actin (red) and AR (green) in cells in athero-
sclerotic lesions in human coronary artery. *Indicates the lumen
of the vessel, 400? magnification.
Gleissner et alAldose Reductase in Foam Cells
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AR expression in macrophages in atherosclerotic lesions
and the vascular adventitia suggests that macrophage AR
may represent a link among diabetes, hyperlipidemia and
atherosclerosis.
In diabetics, glucose is shunted into the polyol pathway and
thus converted by AR into sorbitol and subsequently to
fructose by sorbitol dehydrogenase.9For several reasons, this
is considered harmful: First, activation of AR results in
decreased NADPH levels leading to reduced regeneration of
glutathione which is a key protective factor against oxidative
stress.3Reduced NADPH levels may also lead to increased
levels of 12-hydroperoxyeicosatetraonic acid (12-HPETE),
which cannot be reduced to less toxic 12-hydroxyei-
cosatetraenonic (12-HETE) by glutathione peroxidase.27In-
creased fructose levels cause increased advanced glycation
end products, which interfere with many cellular processes.3
AR expression and activity have been described in various
tissues including the lens of the eye, kidney, and nerve in
several animal models including rat, rabbit, and pig.28A
causal relation between metabolization of glucose by AR and
diabetic complications was shown in several animal models
of cataract29and renal dysfunction30or neuropathy.31Simi-
larly, AR activity has been associated with cataract,10ne-
phropathy,11and polyneuropathy12in diabetic patients. Clin-
ical studies have demonstrated that AR inhibition reduces
symptoms of diabetic neuropathy.13In the vasculature, AR is
expressed in rat smooth muscle cells as demonstrated in rats14
and human umbilical vein endothelial cells.32Furthermore,
AR expression was found in smooth muscle cells and
mononuclear cells in vivo in human temporal artery speci-
mens from patients with giant cell arteriitis.6
A potential role for AR in atherosclerosis was demonstrated
in LDL receptor knock-out mice overexpressing human AR.14
Normoglycemic AR overexpressing Ldlr?/?mice fed a Western
diet did not display increased atherosclerotic lesions as com-
pared to Ldlr?/?mice not expressing human AR. However, a
significant increase in lesion size was demonstrated in the
AR-overexpressing mice after streptozotocin (STZ) injection,
which induces diabetes by selectively killing pancreatic ? cells,
supporting a pathogenetic role of the polyol pathway for athero-
genesis in diabetes.14The mechanism described here may at
least partially account for increased atherogenesis in this mouse
model. Macrophages harvested from human atherosclerotic
plaques by laser capture microdissection showed 75% increased
AR mRNA expression as measured by gene chip analysis
(Volger OL et al, http://www.ncbi.nlm.nih.gov/geo/, series
GSE7074), supporting a role for the polyol pathway in human
atherosclerosis.
Our findings that oxLDL-induced AR upregulation in-
creases oxidative stress suggest that the harmful effects
clearly outweigh any potentially beneficial role of the polyol
pathway. The protective effects of AR inhibition on ROS
formation seem to be more pronounced under hyperglycemic
conditions, suggesting that harmful effects of AR require
oxLDL as well as elevated glucose levels. AR accounts for
20% to 30% of oxidative stress as demonstrated by the
significant reduction of oxidative stress after inhibition of AR
with epalrestat. Other prooxidant pathways like lipoxygen-
ases,33cyclooxygenases,33and NADPH oxidase34are likely
to contribute the remaining oxidative stress.
AR gene regulation involves 3 osmotic response elements
and 1 androgen response element; there may also be 1
glucose response element.35Accordingly, increased AR ex-
pression under hyperglycemic conditions has been shown in
different cell types.36,37Here, we extend this finding to human
monocyte-derived macrophages. According to our results,
4-HNE is a key constituent of oxLDL responsible for AR
upregulation. This is consistent with previous data demon-
strating 4-HNE–dependent AR upregulation in PBL.6The
fact that blocking experiments using antibody against
CD36—a scavenger receptor responsible for about two thirds
of oxLDL uptake into macrophages23—resulted in a signifi-
cant inhibition of oxLDL-induced AR upregulation suggests
that uptake of oxLDL mediated by CD36 is necessary for AR
upregulation.
In conclusion, we demonstrate hyperglycemia-related up-
regulation of AR gene expression and activity as a new
proinflammatory mechanism of oxLDL in human macro-
phages with potential relevance for atherogenesis, thereby
identifying a potential link between 2 of the most impo-
rtant atherosclerotic risk factors, namely diabetes and
hyperlipidemia.
Acknowledgments
We thank Dr Nancy Harthun, University of Virginia, Charlottesville
for providing carotid endatherectomy specimens, Dr Maria-Beatriz
Lopes, University of Virginia, Charlottesville for providing postmor-
tem coronary artery specimens, and Dr Aruni Bhatnagar, University
of Kentucky, Louisville for providing an antibody against human
aldose reductase.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft
(grant GL599/1-1 to C.A.G.), by NIH grant HL58108 (to K.L.), and
by NIH grant HL55798 (to K.L. and J.L.N.).
Disclosures
None.
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Gleissner et al Aldose Reductase in Foam Cells
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Supplementary data Gleissner et al., Aldose reductase in foam cells
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Materials and Methods

Macrophages and foam cells: Mixed peripheral blood mononuclear cells (PBMCs) were
isolated by Histopaque 1.077 (Sigma Diagnostics, St. Louis, MO) and washed twice with
PBS containing 0.02% EDTA (Sigma Diagnostics, St. Louis, MO). Subsequently, mono-
cytes were isolated using a negative selection monocyte isolation kit and LS columns
(both Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of monocytes was
>97% as determined by flow cytometry using anti-CD14 antibody (Figure S1A).

Monocytes were cultured in Macrophage Serum-Free Medium (Invitrogen, Carlsbad,
CA) in the presence of 1% media supplement nutridoma-SP (Roche Molecular Bio-
chemicals, Indianapolis, IN), penicillin (100 u/mL), streptomycin (1 mg/mL, both Sigma
Diagnostics, St. Louis, MO) and macrophage colony-stimulating factor (M-CSF) or
CXCL4 (100 ng/mL, both Peprotech, Rocky Hill, NJ) for 6 days. This standard macro-
phage cell culture medium has a final glucose concentration of 17.5 mM. Macrophages
were then incubated with minimally modified LDL (mmLDL, a kind gift from Joseph
Witztum, San Diego, CA) or oxidized LDL (oxLDL, Biomedical Technolgies, Stough-
ton, MA) at a concentration of 100 µg/mL for up to 48 hours to induce foam cell forma-
tion. Controls were incubated with medium only.

Foam cell formation was verified by oil red O staining. Therefore, cells were fixed in 1 %
paraformaldehyde over night at 4°C. Cells were then washed twice with PBS and incu-
bated with 70% isopropanol for 2 minutes. Oil red O staining was performed with 0.5%
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Supplementary data Gleissner et al., Aldose reductase in foam cells
2
oil red O (Sigma Diagnostics, St. Louis, MO) in 60% isopropanol for 10 minutes fol-
lowed by counterstaining with Mayer’s hematoxylin (Sigma Diagnostics, St. Louis, MO)
for 5 minutes (Figure S1B).

For experiments under normo- and hyperglycemic conditions, cells were cultured in glu-
cose-deficient medium (glucose-free RPMI 1640 (Invitrogen, Carlsbad, CA) +10% dia-
lyzed FCS (HyClone, Logan, UT)) supplemented with either 30 mM D-glucose or 5 mM
D-glucose plus 25 mM L-glucose as osmotic control (both Sigma, St.Louis, MO).

Aldose reductase activity assay: Aldose reductase enzyme activity was measured spec-
trophotometrically by determination of the decrease in absorbance at 340 nm in absence
and presence of glyceraldehyde as substrate reflecting the consumption of NADPH 1-3 in
either cell lysates or cell culture supernatants 2. The total assay volume of 1000 µl con-
tained 730 µl sodium phosphate buffer (100 mM, pH 7.0), 50 µl NADPH (1.6 mM, both
Sigma Diagnostics, St. Louis, MO), 20 µl diphenylhydantion (10 mM), and 100 µl of en-
zyme solution. To start the reaction, 10 µl DL-glyceraldehyde (100 mM, Sigma Diagnos-
tics, St. Louis, MO) were added and the absorption at 340 nm was measured at 25°C for 3
minutes. Glyceraldehyde was dedimerized for 10 mins at 85°C before assay. Diphenylhy-
dantoin was added to inhibit unspecific metabolization of glyceraldehydes by aldehyde de-
hydrogenase. Absorbance values were corrected for blanks containing all components of the
reaction except the substrate. AR activity was normalized to protein content as measured
photometrically 4 and expressed in mU/µg (nmol NADPH oxidized per minute per µg
protein).

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Supplementary data Gleissner et al., Aldose reductase in foam cells
3
Oxidative stress assay: Macrophages were exposed to 100 µg/mL oxLDL for 48 hours,
with or without pre-treatment with 0.2 µM epalrestat for 24 hours. Cells were then
washed with PBS and incubated with 2’7’-dichlorofluoresceine diacetate (H2DCFDA, 10
µM, Invitrogen, Carlsbad, CA) at 37°C for 30 minutes. After an additional wash step
with PBS, cells were harvested by scraping and fluorescence was assessed using a FACS
Calibur instrument (BD Biosciences, Franklin Lakes, NJ). Results were expressed as fold
change of geometric means over unstimulated cells

Immunohistochemistry: Five µm paraffin sections of coronary arteries were used. Fol-
lowing heat induced antigen retrieval using antigen unmasking solution (Vector Labora-
tories, Burlingame, CA) tissue sections were either incubated with antibodies against al-
dose reductase (a kind gift from Dr. Aruni Bhatnagar, University of Kentucky, Louisville,
KY; for later experiments SC-17732, Santa Cruz Biotechnology, Santa Cruz, CA), CD 68
(clone KP-1, Santa Cruz Biotechnology, Santa Cruz, CA) or α-smooth muscle actin
(clone alpha sm-1, Novocastra Laboratories, Norwell, MA). Antibodies were detected
using the ABC method (Vector Laboratories). Antibodies were visualized using DAB
(DAKO corporation, Carpinteria, CA). Sections were then counterstained with hematox-
ylin (Richard-Allan Scientific, Kalamazoo, MI). Sections were analyzed for light micros-
copy using an Olympus BX51 microscope and Image Pro Plus 3.0 software (Media Cy-
bernectics, Silver Spring, MD).

For fluorescence microscopy was performed after heat induced antigen retrieval using
antigen unmasking solution. Sections were co-incubated with either antibodies against
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Supplementary data Gleissner et al., Aldose reductase in foam cells
4
aldose reductase (SC-17732) and CD68-Alex Fluor-488 (clone KP-1, both Santa Cruz
Biotechnology, Santa Cruz, CA) or aldose reductase and smooth muscle α-actin-Cy3
conjugated (clone IA-4, Sigma Diagnostics, St. Louis, MO). For CD68 and aldose reduc-
tase, aldose reductase was detected using donkey anti-Goat Alexa Fluor 555 (Molecular
Probes, Carlsbad, CA). For SMA and aldose reductase, AR was detected using Donkey
anti-Goat Alexa Fluor 488 (Molecular Probes, Carlsbad, CA). All slides were cover-
slipped using Vector Shield Hardset with DAPI (Vector Laboratories, Burlingname, CA).
Sections were analyzed by fluorescence microscopy (Olympus BX51 microscope) and
Image Pro Plus 3.0 software (Media Cybernectics, Silver Spring, MD).

For quantification of AR positive macrophages in atherosclerotic plaques and adventitia
AR positive and CD68 positive cells were counted in at least four fields of vision at a
magnification of 400X and the proportion of AR positive cells out of CD68 positive cells
was calculated.

Statistical analysis of gene arrays: The exact details of gene array data analysis have
been described previously 5. Briefly, after exclusion of internal controls, gene expression
was normalized and log-transformed. The local pooled error (LPE) test for differential
expression discovery under two conditions and the heterogeneous error model (HEM) for
differential expression discovery under multiple conditions 6 were applied using the
open-source statistical software R (www.r-project.com).
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Supplementary data Gleissner et al., Aldose reductase in foam cells
5
Supplementary tables

Table S1: Genes most strongly up- or downregulated by oxLDL in monocyte-derived
macrophages. Given are Affymetrix H133A gene chip probe set ID, gene symbols, gene
titles, false discovery rate (FDR) and fold change in oxLDL versus untreated cells in
macrophages generated with either MCSF or CXCL4.
Probe set
ID
Gene
Symbol
Gene Title FDR Fold
change
MCSF
Fold
change
CXCL4

0.04
Cytokines, chemokines and their receptors
207533_at CCL1 chemokine (C-C motif)
ligand 1
chemokine (C-C motif)
ligand 4
interleukin 1 receptor an-
tagonist
interleukin 1 receptor an-
tagonist
interleukin 1 receptor an-
tagonist
lymphotoxin alpha (TNF
superfamily, member 1)
lymphotoxin beta (TNF
superfamily, member 3)
<0.001 0.34
204103_at CCL4 0.028 0.54 0.57
212659_s_at IL1RN 0.009 2.02 2.56
216243_s_at IL1RN 0.015 1.94 1.79
212657_s_at IL1RN 0.009 1.92 1.84
206975_at LTA 0.015 0.17 0.04
207339_s_at LTB <0.001 0.37 0.13
218856_at TNFRSF21 tumor necrosis factor re-
ceptor superfamily, mem-
ber 21
214228_x_at TNFRSF4
<0.001 4.22 3.22
tumor necrosis factor re-
ceptor superfamily, mem-
ber 4
tumor necrosis factor
(ligand) superfamily,
member 4 (tax-
transcriptionally activated
glycoprotein 1, 34kDa)
tumor necrosis factor
(ligand) superfamily,
member 7
chemokine (C motif)
ligand 1
0.009 0.43 0.31
207426_s_at TNFSF4 0.021 0.45 0.26
206508_at TNFSF7 0.009 0.41 0.45
206365_at XCL1 <0.001 0.41 0.36
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Supplementary data Gleissner et al., Aldose reductase in foam cells
6
206366_x_at

Genes related to lipid metabolism
203504_s_at ABCA1
XCL1 chemokine (C motif)
ligand 1
<0.001 0.37 0.17

ATP-binding cassette, sub-
family A (ABC1), member
1
ATP-binding cassette, sub-
family A (ABC1), member
1
ATP-binding cassette, sub-
family G (WHITE), mem-
ber 1
ATP-binding cassette, sub-
family G (WHITE), mem-
ber 1
adipose differentiation-
related protein
apolipoprotein C-I
fatty acid desaturase 1
fatty acid desaturase 1
fatty acid desaturase 1
fatty acid desaturase 2
3-hydroxy-3-
methylglutaryl-Coenzyme
A reductase
3-hydroxy-3-
methylglutaryl-Coenzyme
A reductase
3-hydroxy-3-
methylglutaryl-Coenzyme
A synthase 1 (soluble)
low density lipoprotein
receptor (familial hyper-
cholesterolemia)
low density lipoprotein
receptor (familial hyper-
cholesterolemia)
<0.001 2.37 1.71
203505_at ABCA1 <0.001 2.21 2.62
204567_s_at ABCG1 <0.001 2.79 2.73
211113_s_at ABCG1 <0.001 2.78 2.66
209122_at ADFP <0.001 2.28 3.60
213553_x_at APOC1
208964_s_at
208962_s_at
208963_x_at FADS1
202218_s_at
202539_s_at
<0.001
0.009
<0.001
<0.001
<0.001
<0.001
2.92
0.45
0.30
0.21
0.21
0.23
1.79
1.00
1.18
0.69
0.44
0.31
FADS1
FADS1
FADS2
HMGCR
202540_s_at HMGCR <0.001 0.17 0.29
221750_at HMGCS1 <0.001 0.27 0.31
202068_s_at LDLR <0.001 0.15 0.46
202067_s_at LDLR <0.001 0.05 0.18
Genes related to oxidative stress
212859_x_at MT1E metallothionein 1E (func-
tional)
metallothionein 1F (func-
tional)
metallothionein 1F (func-
tional)
metallothionein 1G
metallothionein 1H
<0.001 3.73 3.32
217165_x_at MT1F <0.001 4.51 4.37
213629_x_at MT1F <0.001 4.24 3.89
204745_x_at MT1G
206461_x_at MT1H
<0.001
<0.001
5.07
4.99
5.37
4.10
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Supplementary data Gleissner et al., Aldose reductase in foam cells
7
211456_x_at MT1H
217546_at
204326_x_at MT1L
208581_x_at MT1X
212185_x_at MT2A
216609_at
214205_x_at TXNL2
metallothionein 1H
metallothionein 1K
metallothionein 1L
metallothionein 1X
metallothionein 2A
thioredoxin
thioredoxin-like 2
<0.001
0.009
<0.001
<0.001
<0.001
<0.001
<0.001
3.68
21.34
5.58
3.22
2.75
2.84
2.49
3.50
11.19
6.16
3.15
2.68
2.44
2.53
MT1K
TXN

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