Advanced Glycation End Product Receptor-1 Transgenic Mice Are Resistant to Inflammation, Oxidative Stress, and Post-Injury Intimal Hyperplasia

Division of Experimental Diabetes and Aging, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
American Journal Of Pathology (Impact Factor: 4.59). 09/2009; 175(4):1722-32. DOI: 10.2353/ajpath.2009.090138
Source: PubMed
The high levels of oxidative stress (OS) and inflammation associated with cardiovascular disease are linked to pro-oxidants such as advanced glycation end products (AGEs). AGEs interact with multiple receptors, including receptor 1 (AGER1), which promotes AGE removal and blocks OS and inflammation, and RAGE, which enhances inflammation. In this study, we evaluated metabolic and vascular changes in AGER1 transgenic mice (AGER1-tg) subjected to an atherogenic diet and arterial wire-injury. Both baseline and postatherogenic diet serum and tissue AGEs as well as plasma 8-isoprostane levels were lower in AGER1-tg mice than in wild-type mice. The levels of injected (125)I-AGE in tissues were decreased as well in AGER1-tg mice. After ingesting a high-fat diet, AGER1-tg mice had a normal glucose tolerance and only 7% were hyperglycemic, whereas 53% of wild-type mice had stable hyperglycemia. After wire-injury, intimal lesions in AGER1-tg mice were small, whereas wild-type mice had diffuse intimal hyperplasia, a high intima/media ratio, and inflammatory cell infiltrates. In addition, AGER1 staining, prominent in AGER1-tg mice, was attenuated in 30 to 40% of wild-type cells, although all cells were strongly positive for AGEs. Thus, AGER1 overexpression in mice reduces basal levels of AGEs and OS, enhances resistance to diet-induced hyperglycemia and OS, and protects against injury-induced arterial intimal hyperplasia and inflammation, providing protection against OS and inflammation induced by AGEs and high-fat diets in vivo.


Available from: Massimo Torreggiani
Vascular Biology, Atherosclerosis and Endothelium Biology
Advanced Glycation End Product Receptor-1
Transgenic Mice Are Resistant to Inflammation,
Oxidative Stress, and Post-Injury Intimal Hyperplasia
Massimo Torreggiani,* Huixian Liu,* Jin Wu,*
Feng Zheng,* Weijing Cai,* Gary Striker,*
and Helen Vlassara*
From the Division of Experimental Diabetes and Aging,*
Department of Geriatrics, and the Division of Nephrology,
Department of Medicine, Mount Sinai School of Medicine,
New York, New York
The high levels of oxidative stress (OS) and inflam-
mation associated with cardiovascular disease are
linked to pro-oxidants such as advanced glycation
end products (AGEs). AGEs interact with multiple re-
ceptors, including receptor 1 (AGER1), which pro-
motes AGE removal and blocks OS and inflammation,
and RAGE, which enhances inflammation. In this
study, we evaluated metabolic and vascular changes
in AGER1 transgenic mice (AGER1-tg) subjected to an
atherogenic diet and arterial wire-injury. Both base-
line and postatherogenic diet serum and tissue AGEs
as well as plasma 8-isoprostane levels were lower in
AGER1-tg mice than in wild-type mice. The levels of
I-AGE in tissues were decreased as well in
AGER1-tg mice. After ingesting a high-fat diet,
AGER1-tg mice had a normal glucose tolerance and
only 7% were hyperglycemic, whereas 53% of wild-
type mice had stable hyperglycemia. After wire-in-
jury, intimal lesions in AGER1-tg mice were small,
whereas wild-type mice had diffuse intimal hyper-
plasia, a high intima/media ratio, and inflamma-
tory cell infiltrates. In addition, AGER1 staining,
prominent in AGER1-tg mice, was attenuated in 30
to 40% of wild-type cells, although all cells were
strongly positive for AGEs. Thus, AGER1 overex-
pression in mice reduces basal levels of AGEs and
OS, enhances resistance to diet-induced hypergly-
cemia and OS, and protects against injury-induced
arterial intimal hyperplasia and inflammation, pro-
viding protection against OS and inflammation in-
duced by AGEs and high-fat diets in vivo.
(Am J Pathol
2009, 175:1722–1732; DOI: 10.2353/ajpath.2009.090138)
Atherosclerosis, one of the principal underlying lesions in
cardiovascular disease (CVD), is a multistep process in
which high oxidative stress (OS) and inflammation play a
crucial role.
OS may play a role in vascular progenitor
function and contributes to alterations in vascular injury
repair and insulin resistance.
A characteristic feature
of OS is an imbalance between reactive oxygen species
(ROS) generation and antioxidant status. While ROS are
necessarily generated during normal metabolism, abnor-
mally increased levels may arise from excess metabo-
lites, ie, glucose or fatty acids, but also from exogenous
oxidants consumed with the diet.
Advanced glyca-
tion end products (AGEs), known contributors to CVD
and chronic kidney disease, are derived either from
metabolism or from the diet.
6,8 –10
AGEs, resulting from nonenzymatic additions of reduc-
ing sugars to Lys or Arg of proteins (AGEs), lipids (ad-
vanced lipoxidation end products) and nucleic acids,
can generate increased levels of intracellular ROS, fur-
ther promoting AGE formation in a “vicious cycle.”
Abnormal accrual of AGEs, previously thought to be
restricted to diabetes, is now known to occur in persons
considered to be in good health.
A role for AGE-en-
riched Western diets in the causation of increased sys-
temic AGE, OS, and CVD, first studied in animals, has
been documented in clinical studies that include subjects
with diabetes and/or kidney disease.
This asso-
ciation is also present in healthy individuals.
Several cell surface AGE receptors have been identified,
each of which mediates different responses. Among these,
AGE receptor 1 (AGER1) promotes the uptake and removal
of AGEs and blocks cellular AGE-mediated ROS generation
and inflammation,
16 –18
whereas receptor for AGEs (RAGE)
promotes ROS generation and inflammatory responses.
Because AGER1 and RAGE compete for AGEs, the bind-
ing of AGEs to RAGE may increase when AGER1 levels are
decreased, resulting in the induction of increased OS. In
this instance RAGE signaling is unopposed by AGER1.
Supported by AG00943 and HL73417 to H.V.
Accepted for publication June 19, 2009.
Current address of H.L.: Cornell University Medical Center, New York, NY.
Address reprint requests to Helen Vlassara, Division of Experimental Dia-
betes and Aging, Mount Sinai School of Medicine, Box 1640, One Gustave
Levy Place, New York, NY 10029. E-mail: helen.vlassara@
The American Journal of Pathology, Vol. 175, No. 4, October 2009
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.090138
Page 1
AGER1 is a type I transmembrane receptor found on
the plasma membrane
and in the endoplasmic reticu
The cell surface membrane-associated AGER1
blocks responses to AGEs by blocking the induction of
ROS-mediated activation of mitogen-activated protein ki-
nases/Ras and inflammatory molecules, induced in part
via RAGE.
In addition, AGER1 suppresses AGE-induced
ROS by inhibiting phosphorylation of epidermal growth fac-
tor receptors, Grb-2/Shc and extracellular signal-regulated
Downstream actions of AGER1 include the inhi
bition of ser-36 phosphorylation of p66
a highly OS-
responsive isoform of Shc, involved in the AGE-mediated
inactivation of forkhead box transcription factors and man-
ganese superoxide dismutase suppression in vitro, as well
as in vascular disease and lifespan in vivo.
sion of these pathways by AGER1 is thought to support
intracellular anti-oxidant systems, to enhance tissue resis-
tance to ROS, and to prolong lifespan.
Circulating monocyte AGER1 levels correlate strongly
with systemic AGEs and OS, independent of age in
healthy adults.
However, AGER1 was found to be
down-regulated in states of chronic high OS, such as
diabetes and kidney disease.
The reasons for this
paradoxical decrease are unknown. However, studies in
mice and humans suggest that dietary AGEs may be
involved, because dietary restriction of AGE intake pre-
serves AGER1 expression, reduces circulating AGEs and
OS levels, and decreases age-related metabolic, vascular,
and renal changes in aging mice.
This is also associ
ated with an extended lifespan in mice. These data com-
plement clinical studies in which reduced dietary AGE in-
take normalized abnormally low AGER1 levels and reduced
OS and inflammation in patients with high AGEs and OS
due to chronic kidney disease.
The findings underscore
the potential significance of AGER1 expression levels and
the critical role it may play in maintaining systemic anti-
oxidant balance. However, this evidence is inferential, and
direct in vivo links between AGER1 expression, AGE levels,
and OS have not been established.
The present studies, using mice transgenic for murine
AGER1, show that the overexpression of AGER1 in mice is
associated with (a) decreased basal levels of circulating and
tissue AGEs and OS, (b) decreased serum AGE levels and
enhanced anti-oxidant reserves after exposure to a high-fat
diet, as evidenced by resistance to hyperglycemia, and (c)
significant protection against wire injury-induced femoral artery
intimal hyperplasia and inflammation.
Materials and Methods
Generation of AGER1 Transgenic Mice
Full-length murine AGER1 cDNA was generated by PCR
by using mouse embryonic stem cells with the following
CTTCTCCTTCTCCTTCATG-3. PCR products were puri-
fied, subcloned into a pcDNA3.1 vector (Invitrogen;
Carlsbad, CA) and confirmed by sequence analysis. A
CMV promoter was used. Because we introduced murine
AGER1 cDNA into mouse ova, a V5-tag was included in
the 3-end of construct for the purpose of identifying the
transgene (Figure 1A). The resultant 2.236kb hybrid gene
was linearized with SalI and SphI sites between the CMV
promoter and the bGH polyA sequence and microin-
jected into fertilized eggs. The founder transgenic mice
were identified by PCR of DNA isolated from tail snips
with a 5 primer of AGER1 (5-TTGTCCGCATTGATC-
CTTTTGT-3)anda3 primer of V5-tag (5-AGAGGGT-
TAGGGATAGGCTTAC-3). Nine out of 43 newborn off-
spring carried the murine AGER1-V5 tag cDNA. Founder
mice expressing high levels of AGER1 were used for
propagation. AGER1 mice were born in the expected
Mendelian ratio and have not shown a survival advantage
or disadvantage during more than 3 years of study. F1– 4
mice had normal fecundity, litter size, and birth weight.
Transgenic mice were backcrossed with C57B6 mice for
more than six generations, having free access to water
and a regular chow diet (5% fat/g, PicoLab Rodent Diet
20, #5053, Purina Mills; St. Louis, MO). To evaluate the
anti-AGE and anti-OS properties of AGER1 during sus-
tained OS burden, AGER1 transgenic (AGER1-tg; 12
months, n 8 male and n 9 female) and age- and
sex-matched C57B6 mice were placed on a high-fat diet
(21.2% fat/g, TD.88137, Harlan Teklad; Madison, WI) (Ta-
ble 1), which is shown to raise AGEs and OS in addition
to lipids or glucose,
for 8 weeks.
PCR and Real-Time PCR
Total RNA was extracted from tissues and purified with the
SV Total RNA Isolation System (Promega; Madison, WI)
Figure 1. A: Schematic representation of the AGER1 construct. B: Relative
AGER1 mRNA levels in various tissues. Real time-PCR data are expressed as the
mean SEM gene copies in AGER1-tg above those in wild-type (WT) mice.
Table 1. Characteristics of Dietary Formulas
Protein, % by weight, g 20 17.3
Fat, % by weight, g 4.5 21.2
Carbohydrate, % by weight, g 54.8 48.5
Total calories/day, kcal 4.02 4.5
Lipid-CML, U/g 225 2.7 10
Lipid-MG, nmol/g 5.9 21.2
AGER1 Reduces OS and Vascular Inflammation 1723
AJP October 2009, Vol. 175, No. 4
Page 2
according to the manufacturer’s instructions. One
total RNA was reverse transcribed by using the Transcriptor
First Strand cDNA Synthesis Kit (Roche; Indianapolis, IN).
PCR was performed with the genotyping AGER1 primers or
primers internal to the AGER1 gene (forward 5-CTGCT-
GCCACAGCTAGTTC-3), by using the TaqPCR Master Mix
Kit (Qiagen; Valencia, CA) under the following conditions:
initial denaturation 94°C 3; denaturation 94°C 45,
annealing 55°C 45, elongation 72°C 1 for 36 cycles, and
final elongation 72°C 10. Real-Time PCR was performed by
using the Sybr Green PCR Master Mix (Applied Biosystems;
Warrington, UK) under standard conditions.
Western Analysis
Cellular or tissue samples (20 mg) were manually macer-
ated in CelLytic MT Mammalian Tissue Lysis/Extraction Re-
agent (Sigma; St. Louis, MO) after the addition of a Protease
Inhibitor Cocktail (Pierce; Rockford, IL), EDTA (Pierce) and
Phosphatase Inhibitor (Pierce). Samples were centrifuged at
10,000 rpm for 10 minutes and supernatant was recovered.
The protein concentration was measured by a colorimetric
assay (DC Protein Assay, Bio-Rad; Hercules, CA) according to
the manufacturer’s instruction. Fifty
g of protein for each
sample was heated at 100°C with Laemmli sample buffer
containing 2%
-mercaptoethanol for 5 minutes and electro-
phoresed on a 10% SDS-polyacrylamide electrophoresis gel.
Separated proteins were transferred onto polyvinylidene fluo-
ride membranes, which were blocked with PBS-Tween20 with
5% nonfat dry milk for 1 hour and then incubated with the
primary antibody (rabbit polyclonal anti-OST48, Santa Cruz
Biotechnologies, Santa Cruz, CA, dilution 1:1000; Mouse
monoclonal anti-
-actin, Sigma, dilution 1:4000), followed by
a 1-hour incubation with a 1:2000 dilution of the appro-
priate secondary antibody (goat anti-mouse IgG HRP-
conjugate or Goat anti-rabbit HRP-conjugate from Bio-
Rad). Bands were detected by using an enhanced
chemiluminescence method (Roche).
Serum and Tissue AGE
AGE concentrations in mouse sera and tissues were deter-
mined by enzyme-linked immunosorbent assay, by using
monoclonal antibodies reacting with N
(CML) (4G9; Alteon; Northvale, NJ) or methylglyoxal (MG)-
derived AGEs (3D11).
The CML-bovine serum albu
min (BSA) standard contained 23 modified lysines/mol,
whereas the MG-BSA standard contained 22 modified
arginines/mol, based on high pressure liquid chroma-
tography/gas chromatography-mass spectrometry.
Plasma 8-isoprotane, Blood, and Tissue GSH/
GSSG Levels
Blood was collected at sacrifice by cardiac puncture and
endogenous lipid peroxidation products (8-isoprostanes)
were determined in fresh plasma samples by using an
enzyme immunoassay kit, which correlates with values de-
termined by GC-MS (Cayman Chemical; Ann Arbor, MI).
Reduced glutathione and oxidized gluthathione were ana-
lyzed with a colorimetric reaction kit (OxisResearch; Port-
land, OR) according to the manufacturer’s instructions.
Preparation of
AGE-BSA were iodinated as previously described
by incubating AGE-BSA with
I in the presence of
IODO-Beads (Pierce) at room temperature for 1 hour.
Samples were then passed through a Sephadex G-25
PD-10 column (GE Health Care; Piscataway, NJ), 10
1-ml fractions were collected, and the radioactivity was
measured (Top Count NXT scintillation counter, Packard;
Meriden, CT). Protein fractions with the highest radioactivity
were extensively dialyzed against PBS with a Slide-A-Lyzer
Cassette (Pierce) and precipitated with trichloroacetic acid.
Samples with a specific activity 94% were used.
AGE Kinetics in Vivo
AGER1-tg and age- and sex-matched C57B6 mice
(3 months old, n 5/group) were anesthetized by Halo-
thane and placed in a supine position with the lower
extremities extended. After making a groin incision and
exposing the femoral vein,
I-AGE-BSA (150
g of pro
tein in PBS) was infused into the vein. Two hours after
injection, the mice were perfused with PBS until the per-
fusate from the inferior vena cava was clear. Tissues were
isolated and aliquots (20 mg) were used for total protein
extraction and assessment of tissue-bound radioactivity,
as described with minor modifications.
Briefly, the
degraded AGE-BSA (30 kDa) was separated by using a
Microcon 30 column (Amicon; Beverly, MA). The protein
concentration and radioactivity were determined in each
fraction (Top Count NXT scintillation counter, Packard) and
normalized to the weight of the initial tissue sample.
Glucose Tolerance Test
After 2 months on a high-fat diet and after an overnight
fast, mice were injected i.p. with a 20% glucose solution
(2 g glucose/kg body weight). Blood samples were col-
lected before, and at 15, 30, 60, and 120 minutes after
the injection. Blood glucose levels were measured with
an Elite glucometer (Bayer; Mishawaka, IN).
Insulin Tolerance Test
After a 3-hour fast, mice were injected with insulin (No-
volin R 100 U/ml, i.p.) at a concentration of 5 U/kg BW in
sterile 0.9% NaCl solution. Blood samples were collected
before, and at 15, 30, 60, and 120 minutes after the
injection. The blood glucose concentration was mea-
sured with an Elite glucometer (Bayer).
Femoral Artery Injury
After 4 weeks on a high-fat diet (21.2% fat/g), the se-
lected AGER1-tg (8 males, 9 females) and age- and
sex-matched C57B6 mice (n 13) were subjected to a
1724 Torreggiani et al
AJP October 2009, Vol. 175, No. 4
Page 3
femoral artery injury. The injury was performed as previ-
ously described.
In brief, mice were anesthetized
with Halothane and placed in the supine position with the
lower extremities extended, bilateral groin incisions were
made, and the segment of femoral artery between the epi-
gastric and saphenous arteries was separated from the
vein. A left femoral artery arteriotomy was performed inferior
to the epigastric branch, through which a 0.25-mm-diame-
ter angioplasty guide wire (Advanced Cardiovascular Sys-
tems; Temecula, CA) was introduced into the lumen. The
wire was advanced and pulled back three times, each time
reaching beyond the aortic bifurcation. The wire was then
removed, and the arteriotomy site was ligated. The con-
tralateral artery was sham-operated, by exposing the blood
vessels, and used as an uninjured control. In this model,
flow is maintained through the injured segment of femoral
artery, because the epigastric artery and muscular
branches superior to the ligation are preserved.
Evaluation of Femoral Artery Injury Repair
Mice were sacrificed 1 month after the femoral artery
injury. The hind limbs and pelvis were excised en bloc,
postfixed in 4% paraformaldehyde in PBS overnight, and
decalcified in 10% formic acid for an additional 12 hours.
The specimens, containing the femoral vessels, were cut
transversely, dividing the common femoral artery into
multiple 5-mm segments. Specimens underwent stan-
dard dehydration, paraffin embedding, sequential sec-
tioning, and staining by Masson’s trichrome for light mi-
croscopy and for immunohistochemistry. Sequential
sections were cut from each segment. Sections at the
same distance from the site of the arteriectomy were
used for morphometry. Adjacent sections were used for
light microscopy and immunohistochemistry. Captured
images (Leica DM 5000 B microscope, digitalized with
a Leica DFC 300 FX camera and the Leica Application
Suite software version 2.4.0R1, Leica Microsystems;
Heerbrugg, Switzerland) were evaluated with the Image
Pro Plus software (version, Media Cybernetics, Be-
thesda, MD). The intima/media ratio was calculated as the
area defined by the internal elastic lamina (IEL) of the ves-
sel, minus the lumen area, divided by the area defined by
the external elastic lamina (EEL) minus the area defined by
the IEL.
IEL Lumen
Paraffin sections from AGER1-tg and control mice were
deparaffinized before staining for AGEs (MG-derived
AGEs and CML), AGER1, macrophages (F4/80) and
-smooth muscle actin (
-SMA), as previously de-
34 –36
In brief, sections were heated for 2 hours at
58°C, soaked three times in xylene, serially passed
through decreasing concentrations of alcohol, and
washed three times with PBS. Antigen unmasking, when
needed, was performed by heating the slides in a citrate
buffer (Sodium Citrate 0.0008M, Citric Acid 0.002M, pH
6.1) for 15 minutes. Samples were then washed three
times with PBS and incubated 20 minutes in methanol
with 2% H
. Peroxidase in the sections was blocked by
using a Vector Avidin/Biotin Blocking Kit and either a Vector
VECTASTAIN Elite ABC Kit (Rat IgG) or a Vector M.O.M.
Peroxidase Kit (Vector Laboratories; Burlingame, CA). Sec-
tions were incubated overnight at 4°C with the primary
antibody (anti- methylglyoxal-derived AGEs 1:20 [3D11];
-CML epitopes 1:100 [4G9; Alteon]; anti-OST48 1:4
[Santa Cruz Biotechnologies]; anti-F4/80 1:25 [Caltag Lab-
oratories, Burlingame, CA]; and anti-
-SMA 1:60 [Sigma]).
After washing with PBS, slides were incubated with the
appropriate secondary antibody using a Vector M.O.M. Per-
oxidase Kit or a Vector VECTASTAIN Elite ABC Kit (Rat IgG)
according to the manufacturer’s instructions and stained
with a Vector DAB Substrate Kit or an Alkaline Phosphatase
Substrate Kit III (Vector Laboratories). Nuclei were stained
with hematoxylin (Millipore, Billerica, MA) and mounted by
using Permount (Fisher Scientific, Pittsburgh, PA).
Triglycerides and Cholesterol Levels
Blood triglyceride and total cholesterol concentrations
were measured, at baseline and after exposure to the
high-fat diet, with two different kits (BioVision, Mountain
View, CA) according to the manufacturer’s instructions.
All data are expressed as mean SEM. Differences of
means between groups were analyzed by the unpaired,
two-tailed Student’s t-test. Statistical significance was de-
fined as a P value of 0.05. All data analyses were
performed by using the GraphPad Prism statistical pro-
gram (GraphPad Software, Inc., San Diego, CA).
Characterization of AGER1-tg Mice: Transgene
AGER1 transgene expression was found significantly in-
creased in multiple tissues of AGER1-tg mice including
aorta and heart, as compared with wild-type mice (Figure
1B). AGER1 protein levels were also significantly increased
in several highly vascular tissues of AGER1-tg mice, ie,
heart, liver, spleen, and kidney (Figure 2, A and B).
AGER1-tg Mice Have Increased Serum and
Tissue AGE Turnover and Decreased OS
Because AGER1 mediates AGE uptake in vitro,
tested the effect of overexpression of the AGER1 trans-
gene on the levels of native AGEs in vivo. Serum CML
levels were significantly decreased in serum of AGER1-tg
mice at baseline (3 months of age, P 0.05) compared
with wild-type mice (C57B6) (Figure 2C). Basal tissue levels
for two AGEs, CML and MG, did not differ between
AGER1-tg and wild-type mice, and although without reach-
ing significance, liver AGE levels were lower in the
AGER1-tg mice than in wild-type mice (Table 2). A lower
basal oxidant burden in the AGER1-tg mice was also re-
AGER1 Reduces OS and Vascular Inflammation 1725
AJP October 2009, Vol. 175, No. 4
Page 4
flected in the lower levels of 8-isoprostanes, endogenous
lipid peroxidation products (Figure 2D). As a test of the
ability of AGER1 transgene product to remove exogenous
AGEs, tissue-associated
I-AGE-BSA was measured in
AGER1-tg mice within 2 hours of injection. The tissue levels of
labeled peptides in AGER1-tg mice were decreased to 10 to
20% of that in wild-type controls (P 0.05) (Figure 2E).
Tissue levels of both CML and MG were also assessed
after the high-fat diet. While in wild-type mice they were
found increased above the baseline after the high-fat
diet, they were not increased in AGER1-tg mice (Table 2).
The CML levels in kidney and liver tissue of AGER1-tg
mice remained below those in wild-type mice after the
high-fat diet (Figure 3A). Although MG levels were ele-
vated in the kidneys of wild-type mice after the high-fat
diet, the levels in AGER1-tg mice were not different from
baseline values (Table 2). In addition, immunostaining
for MG and CML in the vascular wall of AGER1-tg mice
was lower than in wild-type controls, by immunohisto-
chemical staining (Figure 3B). Antioxidant GSH status
in different tissues was assessed at baseline and after
the high-fat diet to assess intracellular OS. A higher
basal GSH/GSSG ratio was found in AGER1-tg mice in both
kidney and liver tissues, and was maintained at higher
levels in the AGER1-tg kidneys after the fatty diet overload
(Table 2). These results were consistent with a higher anti-
oxidant capacity in the AGER1-tg mice.
AGER1-tg Mice Are Resistant to Diet-Induced
Glucose Intolerance
Because increased AGEs and OS after a high-fat diet
are known to contribute to impaired glucose tolerance
and diabetes,
we examined whether AGER1 overex
pression would reduce these changes in AGER1-tg
mice. Both AGER1-tg and wild-type mice had similar
glucose and lipid levels at baseline. However, after 8
weeks of a high-fat diet, both groups of mice had
significantly increased fasting circulating lipids, but
only wild-type mice had increased blood glucose (Ta-
ble 2, Figure 4D). Before the diet, there were no differ-
ences between the groups with respect to glucose
tolerance (Figure 4A). However after 8 weeks on the
high fat diet, fasting glucose was higher in wild-type
than in AGER1-tg (Table 2). In addition, a glucose
tolerance test after this period revealed that blood
glucose levels returned more rapidly to baseline in
AGER1-tg mice than in wild-type mice (Figure 4B).
Furthermore, stable hyperglycemia, found in only 1/14
AGER1-tg mice (7%), was present in a significantly
greater number of wild-type mice (7/13 or 53.8%) (P
0.02) (Figure 4C). There were no significant differences
in insulin tolerance between the two groups after the
diet (Figure 4D).
(cpm x 10
CML (U/ml)
Serum AGEs
Fold of WT
8-iso (pg/ml)
Figure 2. A: AGER1 protein expression in heart, liver, spleen, and kidney tissues from AGER1-tg mice based on Western analysis. B: Densitometric data from
Western blots, shown as a ratio of AGER1 to
-actin (n ⫽⬃3/group), (C) Serum CML levels. D: Plasma 8-isoprostane levels from the same mice as in A and B,
data shown as mean SEM. E: Tissue associated radioactivity 2 hours after injection of
I-AGE-BSA in protein extracts of kidney, liver, and spleen, shown as
mean SEM. *P 0.05 versus wild-type (WT) mice; **P 0.01 versus WT mice.
1726 Torreggiani et al
AJP October 2009, Vol. 175, No. 4
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AGER1-tg Mice Have Markedly Reduced
Neointimal Formation and Inflammatory
Response after Femoral Artery Injury
Eight weeks after initiating the high-fat diet and 4 weeks
after acute vascular injury, neointima hyperplasia was
prominent and uniformly present in wild-type mice (Fig-
ure 5A). The thickened intima contained many nucle-
ated cells and a large amount of extracellular matrix.
The media was irregular in width, and where it was
focally thickened there was an increased amount of
connective tissue in association with a decreased num-
ber of nuclei. The thickened areas were bordered by
inflammatory infiltrates in both the adventitia (Figure
5A, upper arrow) and the media (Figure 5A, lower
arrow). The intima/media ratio was significantly higher
in the wild-type mice compared with AGER1-tg mice
(P 0.01) (Figure 5C). A large number of macro-
phages were irregularly scattered throughout the me-
dia of wild-type vessels (Figure 5D, lower arrow), par-
ticularly in association with small mononuclear
inflammatory cells. Similarly, macrophages and inflam-
matory cells were locally prominent in the adventitia
(Figure 5, A and D, upper arrows). Although the cells
that were most consistently stained for
-SMA were in
the media, many of the cells in the thickened intima
and some of the adventitial cells in wild-type mice were
positively stained (Figure 5F).
The femoral artery lumen in high-fat-fed AGER1-tg
mice was widely patent and there were only focal, small
areas in which neointima formation was noted (Figure 5B,
upper arrow; Figure 6F, arrow). When present, the neo-
intima was hypocellular and contained an increased
amount of connective tissue by trichrome staining. In
contrast to wild-type vessels, infiltration with inflammatory
cells or macrophages was almost undetectable in any
segment of the vessel wall in AGER1-tg mice (Figure 5, B
and E). There were focal areas in the media of AGER1-tg
mice in which there was an increased amount of connec-
tive tissue, and decreased numbers of cells (Figure 5B,
double arrow). Such fibrotic areas contained extracellular
CML deposits (Figure 6F, double arrow). Extracellular
CML was also present in the localized intimal lesions of
AGER1-tg mice (Figure 6F, single arrow). In contrast to
wild-type mice,
-SMA-positive cells in the injured femo-
ral arteries of AGER1-tg mice were limited to the media of
AGER1-tg mice (Figure 5G). Note that the areas corre-
sponding to the areas of increased connective tissue in
the media contained fewer
-SMA-positive cells, and the
cells were smaller.
AGER1 Expression, Location, and Amount of
AGEs in AGER1-tg and Wild-Type Mice
AGER1 expression was significantly increased in the vas-
cular cells of the transgenic mouse, especially in the
TABLE 2: Epidemiologic and Biochemical Characteristics of AGER1-tg mice
Baseline (BL)
After high-fat diet
(HFD; administered for 8 weeks)
AGER1-tg Wild-type AGER1-tg Wild-type
Weight, g 22.72 0.80 19.56 1.29 25.76 0.56* 27.13 1.27
*P0.01 vs. AGER1-tg BL
P0.01 vs. wild-type BL
Fasting blood glucose,
111.3 9.86 102.6 5.25 111.9 5.26 130.7 4.69
P0.01 vs. wild-type BL
P0.05 vs. AGER1-tg HFD
Fasting insulin, ng/ml 0.287 0.06 0.272 0.03 0.363 0.03 0.365 0.04 NS
Triglycerides, mg/dl 91.70 17.08 96.48 20.06 203.6 32.35
194.0 18.15
P0.05 vs. AGER1-tg BL
P0.05 vs. wild-type BL
Cholesterol, mg/dl 106.4 16.62 111.5 7.65 190.8 20.86
170.6 16.84
P0.05 vs. AGER1-tg BL
P0.01 vs. wild-type BL
Serum CML, U/ml 15.51 1.16 20.21 0.25
48.88 3.63
51.87 4.99
P0.05 vs. AGER1-tg BL
P0.001 vs. AGER1-tg BL
P0.05 vs. wild-type BL
Serum MG, nmol/ml 0.94 0.21 1.02 0.24 2.25 0.16* 2.24 0.24
*P0.01 vs. AGER1-tg BL
P0.05 vs. wild-type BL
Kidney CML, U/mg prot 20.58 2.20 21.05 1.66 21.77 1.04 27.39 1.44**
P0.01 vs. AGER1-tg HFD
Liver CML, U/mg prot 11.99 2.51 20.02 3.51 19.22 1.55 33.47 1.48
P0.01 vs. wild-type BL
P0.001 vs. AGER1-tg HFD
Kidney MG, nmol/mg prot 1.07 0.09 1.13 0.14 0.96 0.09 1.92 0.09
P0.01 vs. wild-type BL
P0.001 vs. AGER1-tg HFD
Liver MG, nmol/mg prot 0.91 0.05 0.85 0.02 1.13 0.15 1.49 0.18 NS
Plasma 8-isoprostane,
61.18 8.68 111.16 3.53* 100.29 5.30* 127.51 8.89
*P0.01 vs. AGER1-tg BL
P0.05 vs. AGER1-tg HFD
Liver GSH/GSSG ratio 1.08 0.05 0.81 0.08
0.89 0.10 1.15 0.13
P0.05 vs. AGER1-tg BL
Kidney GSH/GSSG ratio 2.35 0.15 1.80 0.04
1.65 0.28 1.02 0.08
†‡‡ §
P0.05 vs. AGER1-tg BL
P0.05 vs. AGER1-tg HFD
P0.001 vs. wild-type BL
All values are expressed as meanSEM. NS, not significant.
AGER1 Reduces OS and Vascular Inflammation 1727
AJP October 2009, Vol. 175, No. 4
Page 6
tunica media in which the cells were larger compared
with wild-type mice (Figure 6, A and B). In addition,
endothelial cells and a variety of cells in the adventitia
stained positively for AGER1 in the transgenic mice. The
AGER1 staining appeared mostly cytoplasmic and
largely co-localized with CML and MG staining in AGER1
mice. The focal areas of the media in transgenic mice that
contained dense connective tissue contained fewer and
smaller cells with reduced amounts of CML and MG
(Figure 6D, arrow). However, some extracellular CML
was also detected in the extracellular matrix (Figure 6F,
double arrows). Interestingly, extracellular CML was also
noted in areas with a subintimal lesion of transgenic mice
(Figure 6F, arrow). The fact that increased AGER1 expres-
sion was associated with reduced or absent intracellular
MG and CML staining in the media of the transgenic mice
might be related to a higher AGE removal rate by AGER1.
The cells in the hyperplastic intima of the wild-type
mice were smaller in size, and while 30 to 40% of cells did
not stain positively for AGER1 (Figure 6A, arrow), most
contained large amounts of MG and CML (Figure 6, C
and E). This observation is consistent with the observed
decrease in AGER1 levels in the presence of persistently
high serum AGE levels in this group.
The total num
ber of cells in the vascular wall was much greater in the
wild-type mice due to the hyperplastic intima, as was the
intensity of intracellular CML and MG staining (Figure 6, C
and E). Interestingly, while an increased amount of con-
nective tissue was noted (by trichrome staining) in the
hyperplastic intima, AGE staining was mostly intracellular
in this area (Figure 6, C and E). This, coupled with the
-SMA staining in most cells of the intima,
suggests that there was a higher connective tissue turn-
over rate, such that it likely exceeded the rate of extracel-
lular AGE accumulation. However, the media did contain
extracellular CML (Figure 6E, arrow). The tunica media in
wild-type mice, as demarcated by the internal and external
elastic laminae, was irregularly thinned (Figures 5A and 6C)
compared with transgenic mice (Figures 5B and 6B). CML
deposits in the adventitia of wild-type mice were also in-
creased (Figure 6E), compared with AGER1-tg mice, and
this correlated with the large number of macrophages seen
in this area (Figure 5D).
In this report, we show that AGER1-tg mice maintained
significantly lower levels of systemic AGEs and OS and
are more resistant to high-fat diet-induced glucose
intolerance as compared with wild-type mice. Further-
more, after wire-induced femoral artery injury, the
marked neointimal hyperplasia and inflammatory cell
infiltration seen in wild-type mice were largely absent in
AGER1-tg mice.
Figure 3. Tissue AGEs are lower in AGER1-tg mice compared with wild-type
(WT) mice, after a high-fat diet. A: Kidney and liver CML immunoreactivity is
shown as mean SEM, based on enzyme-linked immunosorbent assay. B:
Arterial tissue CML- and MG-positive staining, based on specific monoclonal
antibodies (4C7 and 3D11, respectively) (Original magnification, 400).
Figure 4. AGER1-tg mice are resistant to diet-induced glucose intolerance. Intravenous glucose tolerance tests were performed before (A), or after (B) 8 weeks
of a high-fat diet in AGER1-tg mice (n 14) and wild-type (WT) mice (n 13), as described. Blood glucose levels were determined at the indicated intervals.
Data are expressed as the mean SEM percentage of peak blood glucose value after infusion. *P 0.05 versus wild-type mice; **P 0.01 versus wild-type mice.
C: Hyperglycemia in AGER1-tg mice (n 14) (closed bars) and wild-type mice (n 13) (shaded) after the high-fat diet (8 weeks). Data indicate the percentage
of mice with stable fasting hyperglycemia (fasting blood glucose 130 mg/dl), on at least two measurements per mouse. D: Insulin tolerance test after the high-fat
diet. Shown are blood glucose levels (*P 0.05).
1728 Torreggiani et al
AJP October 2009, Vol. 175, No. 4
Page 7
CVD is characterized by increased OS and inflamma-
tion, and progression is accelerated by conditions that
further increase OS, ie, hyperlipidemia, hyperglycemia,
and chronic kidney disease.
These conditions are
associated with elevated AGEs, whereas a lower AGE
burden correlates with reduced OS and inflammation in
patients with diabetes
and in mice with chronic vas
or kidney disease.
Cellular responses to AGEs are mediated by interac-
tions with two major classes of cell surface receptors,
which mediate distinct responses.
RAGE contributes to
the generation of ROS and enhances inflammatory re-
sponses to AGEs and other agents, primarily via NF-
In contrast, AGER1, a type I transmem
brane protein, has at least two properties that reduce OS
and inflammation. First, it mediates AGE uptake and deg-
radation, thereby reducing the extracellular levels of these
potent oxidants.
Secondly, it acts as a potent anti-
oxidant molecule, in part, via its inhibition of AGE-medi-
ated ROS-dependent signaling promoted by RAGE
as well as by epidermal growth factor receptor via Shc/
Grb2/Ras and Erk1/2 pathways.
Although RAGE and
AGER1 are up-regulated by their ligands,
responses differ in the presence of persistently high OS.
Namely, RAGE levels increase with rising OS, whereas
AGER1 levels can be decreased with long-term elevated
Because AGER1 potentially has a potent anti-
AGE and anti-inflammatory function in vivo, it is important
to understand its regulation.
The present study demonstrates that the maintenance
of high AGER1 levels provides effective protection
against AGE accumulation, OS, and inflammation in high-fat
fed mice subjected to acute vascular injury. AGER1-tg mice
had a normal phenotype. They had two- to fourfold higher
levels of AGER1 than wild-type controls, which may explain
the observation that they had decreased baseline levels of
circulating CML and of markers of OS. The tissue levels of
two distinct AGEs, CML and MG, increased in wild-type
mice as a function of exposure to the high-fat, high-AGE
However, neither AGE compound increased in tis
sues from AGER1-tg mice, despite an identical dietary bur-
den. This fact, together with lower levels of AGEs after a
Figure 5. AGER1-tg mice have suppressed intimal hy-
perplasia and inflammatory responses to a high-fat diet
and wire injury. Histological and morphometric analy-
sis of injured femoral artery sections after a high-fat diet
(8 weeks) and wire-induced injury (4 weeks) in
AGER1-tg mice (n 14) and wild-type (WT) mice
(n 13). Representative sections of injured vessels
from wild-type mice (A, D, and F, n 13) and
AGER1-tg mice (B, E, and G, n 14) after 8 weeks of
a high-fat diet. A and B: Massons-Trichrome staining
(original magnification, 200). C: Intima/Media (I/M)
ratio; AGER1-tg mice (*P 0.01) versus wild-type
mice. D and E: Anti-macrophage (F4/80) staining. E
and F:
-SMA staining. A: Inflammatory cell infiltrates;
upper arrow, adventitia; lower arrow, media. B:
Upper arrow, intimal plaque; double arrows, area of
medial fibrosis. C: I/M ratio. D: Upper arrow, macro-
phages in the adventitia; lower arrow, macrophages
in the media. E: No macrophages visualized. F:
staining is present in all three layers of the vascular
wall. G:
-SMA staining is limited to the media.
AGER1 Reduces OS and Vascular Inflammation 1729
AJP October 2009, Vol. 175, No. 4
Page 8
bolus injection of labeled AGEs, suggested that reduced
tissue levels of AGEs may be related to an internalization
function of AGE-R1, as well as to other mechanisms.
We previously found that a large proportion (70%) of
AGEs absorbed from the diet were deposited in tissues of
naive mice.
In the current study, we found that the large
influx of AGE-rich lipids from the high-fat diet led to a
parallel increase in serum AGE levels in wild-type and
AGER1-tg mice, but that serum and tissue levels of AGEs
were not increased in AGER1-tg mice. Taken together
these findings suggest that although the ingested AGEs
were partly deposited in the tissues in wild-type mice,
high AGER1 expression in AGER1-tg mice facilitated the
clearance of the dietary load AGEs from tissues. This may
explain the decreased levels of OS and inflammation in
the AGER1-tg mice.
AGEs, such as the relatively inert terminal product
CML, or the highly reactive oxidant AGE precursor, MG,
and its derivatives form intracellularly during normal me-
They are also present in the diet, conjugated
with proteins and lipids.
Prolonged consumption
of a high-fat diet, which is also AGE-enriched by way of
pre-exposure to heat,
increases the levels of circulating
oxidants, such as 8-isoprostanes, which promote further
OS and AGE formation.
If this “vicious cycle” leads to
the reduction of AGER1 levels, the native intracellular
anti-oxidants may also be suppressed. This possibility is
supported by the observed loss in intracellular anti-oxi-
dant GSH potential in wild-type mice, which is associated
with lower levels of AGER1 and increased levels of AGEs
and OS.
Further support for such an effect is that MG,
an active oxidant, was present in most cells of the intima
in wild-type mice, whereas AGER1 was detectable in a
much smaller fraction. This contrasts with the uniform
co-localization of AGER1 and MG in most vascular cells of
AGER1-tg mice. These data suggest that if AGER1 levels
are maintained, despite increased stress, they can effec-
tively enhance AGE removal, even during excessive influx
from exogenous sources. Thus, the maintenance or en-
hancement of AGER1 expression in vivo may help preserve
normal cellular redox status, during periods of systemic
oxidant overload.
The effect of high-level AGER1 expression on vascular
injury responses was examined in a well-known model of
wire-induced acute femoral arterial injury in fat-fed mice.
This model represents a complex pathological process,
involving endothelial cell denudation, vascular smooth mus-
cle cell migration and proliferation, infiltration by inflamma-
tory cells, oxidant overload, and procoagulant respons-
We found that AGER1 was highly expressed in the
cytoplasm of cells in all layers of the injured femoral artery of
AGER1-tg mice. In contrast, AGER1 staining was markedly
attenuated or not detectable in 30 to 40% of cells contained
in the massively thickened intima in wild-type mice. Al-
though the injured arteries in AGER1-tg displayed only a
minimal intimal response, the intima in wild-type mice was
thickened, multilayered, and contained
cells surrounded by a dense layer of connective tissue. Of
note, in the intima of wild-type mice AGEs were mostly
intracellular relative to those AGEs detected in the connec-
tive tissue, suggesting that an increased number of cells in
this region were actively involved in extracellular matrix
turnover. Intimal lesions in AGER1-tg mice, while also
present, were small and few in number. These were largely
composed of connective tissue, which was largely CML-
positive, suggesting that the repaired connective tissue
slowly accumulated stable AGEs. AGEs have been impli-
cated in the inflammatory response, increased proliferation
of vascular smooth muscle cells, and senescence of endo-
thelial cells.
The current study, showing that cells
in the hyperplastic neointima of wild-type controls contained
-SMA staining, is consistent with the suggestion
that these were AGE-activated smooth muscle cells derived
from the media.
The media in AGER1-tg of injured femoral arteries mice
was generally of normal width, and although multifocal
areas resembled healed scars, cells with abundant
AGER1 positive staining material suggested that the vas-
cular injury in the AGER1-tg mice resolved by fibrosis,
rather than by increased smooth muscle cell proliferation
and/or migration. This contrasted to the thinned media in
Figure 6. AGER1 expression and the location and amount of AGEs in
AGER1-tg and wild-type (WT) mice. Analysis of femoral artery sections by
immunohistochemistry after a high-fat diet (8 weeks) and wire-induced
injury (4 weeks) in AGER1-tg mice (n 14) and wild-type mice (n 13).
Representative sections of injured vessels from wild-type (A, C, and E) and
AGER1-tg mice (B, D, and F), stained with anti-AGER1 (A and B), anti-MG (C
and D), and anti-CML (E and F) antibodies, as described. Arrows indicate
cellular localization of AGER1 (A and B), the thinned media, compared with
thickened and cellular intima in wild-type mice (C) and to the normal intima
of AGER1-tg mice (D); and an area of fibrosis (F). A: Many cells in the
hyperplastic intima do not contain AGER1 (arrow). B: The media contains
scattered areas of fibrosis (arrow). C: The media (arrows) underlying the
hyperplastic intima is thinned; intracellular MG staining is present in all cell
layers. D: Cells in the fibrotic areas of the media are smaller, but contain MG
intracellularly. E: CML is present in the cytoplasm of cells of all three layers,
and in the focally dense extracellular matrix of the media (arrow). F: CML is
present in the extracellular matrix of the occasional plaques (arrow) and in
the fibrotic areas of the media (double arrows).
1730 Torreggiani et al
AJP October 2009, Vol. 175, No. 4
Page 9
wild-type mice, which contained many hypocellular, fi-
brotic areas that contained CML-positive connective tis-
sue. The significant inflammatory infiltrate present in the
media and adventitia of wild-type controls was not seen
in AGER1-tg mice, consistent with the anti-inflammatory
properties previously attributed to AGER1.
Although the present findings are the first in vivo evi-
dence of the ability of AGER1 to control AGE levels and
AGE-induced cellular responses, the beneficial effects of
efficient AGE removal are well established. For instance,
studies in ApoE-deficient mice crossed with mice trans-
genic for soluble AGE-binding molecules thought to aid
in their clearance, such as lysozyme,
or mice treated
with soluble RAGE,
have linked low AGEs to attenu
ated responses to injury and to exogenous oxidant
burden, despite hyperlipidemia. Furthermore, preven-
tion of the age-dependent loss in AGER1 expression
and efficacy, by restriction of dietary AGEs, or calories,
preserved anti-oxidant defenses, delayed cardiovas-
cular and renal changes, and prolonged lifespan in
aging mice.
Thus, both the absolute AGER1 levels
and its responsiveness to increased levels of AGEs
may be important.
A link between high AGER1 levels and improved
glucose metabolism, previously found in aging mice
kept for life on reduced AGE intake,
was con
firmed in the present study. We found that AGER1-tg
mice had increased resistance to the metabolic effects
of the high-fat diet, whereas the majority of wild-type
mice became glucose-intolerant and/or stably hyper-
glycemic. The changes seen in wild-type mice, but not in
AGER1-tg mice were related to the higher oxidant and/or
AGE burden found in wild-type mice compared with
AGER1-tg mice. These involved impaired peripheral glu-
cose uptake and utilization in wild-type mice, because
neither plasma insulin nor lipid levels differed significantly
from those in AGER1-tg mice. Two recent articles sug-
gest that high levels of AGEs may have direct effects on
glucose metabolism, and could explain the higher glu-
cose levels and the impaired responses to glucose and
insulin we found in wild-type mice after the high-fat diet.
One study showed that the injection of AGEs reduced
insulin secretion in mice, presumably by inhibiting ATP
production via inhibition of cytochrome c oxidase, which
resulted in an impairment of glucose-stimulated insulin se-
cretion through inducible nitric oxide synthase-dependent
nitric oxide production.
A second study showed that
increased levels of AGEs contribute to peripheral insulin
resistance in mice by impairing insulin action via the
formation of a multimolecular complex that includes insu-
lin receptor substrate 1, RAGE, and SRC in striated mus-
cle cells.
In both of these studies, high levels of AGEs
were shown to result in hyperglycemia. Given that pro-
oxidants such as AGEs are toxic to pancreatic islets
and that anti-AGE agents and diets protect against islet
-cell injury and dysfunction,
it is possible that
AGER1 overexpression could protect against
-cell injury
as a matter of course. Further studies are required to
elucidate the mechanisms involved in these effects. How-
ever, in an elegant study on bone marrow-derived vas-
cular wall progenitors in a mouse model of diabetes
and obesity, both insulin resistance and vascular func-
tion were restored toward normal by either decreasing
OS with a selenorganic antioxidant or the introduction
of normal progenitors.
In conclusion, the current study confirms in vitro data
and correlations in animal and clinical studies, indicat-
ing that AGER1 is an important inhibitor of the systemic
toxicity of AGEs and OS. While focusing on vascular
injury, this study provides the first direct evidence of a
beneficial relationship between AGER1 expression,
systemic anti-AGE actions, and vascular responses to
injury. The mechanisms by which these occur in vivo
remain to be established, but may converge on the
timely neutralization of pro-inflammatory effects of
AGEs and ROS.
1. Ross R: Atherosclerosis: an inflammatory disease. N Engl J Med
1999, 340:115–126
2. Madamanchi NR, Runge MS: Mitochondrial dysfunction in atheroscle-
rosis. Circ Res 2007, 100:460 473
3. Liu SX, Hou FF, Guo ZJ, Nagai R, Zhang WR, Liu ZQ, Zhou ZM, Zhou
M, Xie D, Wang GB, Zhang X: Advanced oxidation protein products
accelerate atherosclerosis through promoting oxidative stress and
inflammation. Arterioscler Thromb Vasc Biol 2006, 26:1156 –1162
4. Chen J, Li H, Addabbo F, Zhang F, Pelger E, Patschan D, Park HC,
Kuo MC, Ni J, Gobe G, Chander PN, Nasjletti A, Goligorsky MS:
Adoptive transfer of syngeneic bone marrow-derived cells in mice
with obesity-induced diabetes: selenoorganic antioxidant ebselen
restores stem cell competence. Am J Pathol 2009, 174:701–711
5. Chen J, Park HC, Addabbo F, Ni J, Pelger E, Li H, Plotkin M, Goligorsky
MS: Kidney-derived mesenchymal stem cells contribute to vasculogen-
esis, angiogenesis and endothelial repair. Kidney Int 2008, 74:879 889
6. Brownlee M: Biochemistry and molecular cell biology of diabetic
complications. Nature 2001, 414:813– 820
7. Stadtman ER: Protein oxidation and aging. Science 1992, 257:
1220 –1224
8. Vlassara H, Cai W, Crandall J, Goldberg T, Oberstein R, Dardaine V,
Peppa M, Rayfield EJ: Inflammatory mediators are induced by dietary
glycotoxins, a major risk factor for diabetic angiopathy. Proc Natl
Acad Sci USA 2002, 99:15596 –15601
9. Huebschmann AG, Regensteiner JG, Vlassara H, Reusch JE: Diabe-
tes and advanced glycoxidation end products. Diabetes Care 2006,
29:1420 –1432
10. Schleicher E, Friess U: Oxidative stress AGE, and atherosclerosis.
Kidney Int Suppl 2007, 106:S17–S26
11. Uribarri J, Cai W, Peppa M, Goodman S, Ferrucci L, Striker G,
Vlassara H: Circulating glycotoxins and dietary advanced glycation
endproducts: two links to inflammatory response, oxidative stress,
and aging. J Gerontol A Biol Sci Med Sci 2007, 62:427– 433
12. Uribarri J, Stirban A, Sander D, Cai W, Negrean M, Buenting CE,
Koschinsky T, Vlassara H: Single oral challenge by advanced glyca-
tion end products acutely impairs endothelial function in diabetic and
nondiabetic subjects. Diabetes Care 2007, 30:2579 –2582
13. Vlassara H, Cai W, Goodman S, Pyzik R, Yong A, Chen X, Zhu L,
Neade T, Beeri M, Silverman JM, Ferrucci L, Tansman L, Striker GE,
Uribarri J: Protection against loss of innate defenses in adulthood by
low AGE intake; role of an anti-inflammatory Age-receptor-1. J Clin
Endo Met 2009 (in press)
14. Uribarri J, Peppa M, Cai W, Goldberg T, Lu M, He C, Vlassara H:
Restriction of dietary glycotoxins reduces excessive advanced glycation
end products in renal failure patients. J Am Soc Nephrol 2003, 14:728–731
15. Linden E, Cai W, He JC, Xue C, Li Z, Winston J, Vlassara H, Uribarri
J: Endothelial dysfunction in patients with chronic kidney disease
results from advanced glycation end products (AGE)-mediated inhi-
bition of endothelial nitric oxide synthase through RAGE activation.
Clin J Am Soc Nephrol 2008, 3:691– 698
16. Yang Z, Makita Z, Horii Y, Brunelle S, Cerami A, Sehajpal P, Suthanthiran
M, Vlassara H: Two novel rat liver membrane proteins that bind ad-
AGER1 Reduces OS and Vascular Inflammation 1731
AJP October 2009, Vol. 175, No. 4
Page 10
vanced glycosylation endproducts: relationship to macrophage recep-
tor for glucose-modified proteins. J Exp Med 1991, 174:515–524
17. Lu C, He JC, Cai W, Liu H, Zhu L, Vlassara H: Advanced glycation
endproduct (AGE) receptor 1 is a negative regulator of the inflamma-
tory response to AGE in mesangial cells. Proc Natl Acad Sci USA
2004, 101:11767–11772
18. Cai W, He JC, Zhu L, Lu C, Vlassara H: Advanced glycation end
product (AGE) receptor 1 suppresses cell oxidant stress and activa-
tion signaling via EGF receptor. Proc Natl Acad Sci USA 2006,
19. Sakaguchi T, Yan SF, Yan SD, Belov D, Rong LL, Sousa M, Andrassy
M, Marso SP, Duda S, Arnold B, Liliensiek B, Nawroth PP, Stern DM,
Schmidt AM, Naka Y: Central role of RAGE-dependent neointimal
expansion in arterial restenosis. J Clin Invest 2003, 111:959 –972
20. Harja E, Bu DX, Hudson BI, Chang JS, Shen X, Hallam K, Kalea AZ,
Lu Y, Rosario RH, Oruganti S, Nikolla Z, Belov D, Lalla E, Ramasamy
R, Yan SF, Schmidt AM: Vascular and inflammatory stresses mediate
atherosclerosis via RAGE and its ligands in apoE
mice. J Clin
Invest 2008, 118:183–194
21. Kelleher DJ, Kreibich G, Gilmore R: Oligosaccharyltransferase activ-
ity is associated with a protein complex composed of ribophorins I
and II and a 48 kd protein. Cell 1992, 69:55– 65
22. Cai W, He JC, Zhu L, Chen X, Striker GE, Vlassara H: AGE-receptor-1
counteracts cellular oxidant stress induced by AGEs via negative
regulation of p66shc-dependent FKHRL1 phosphorylation. Am J
Physiol Cell Physiol 2008, 294:C145–C152
23. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP,
Lanfrancone L, Pelicci PG: The p66shc adaptor protein controls
oxidative stress response and life span in mammals. Nature 1999,
402:309 –313
24. Napoli C, Martin-Padura I, de Nigris F, Giorgio M, Mansueto G,
Somma P, Condorelli M, Sica G, De Rosa G, Pelicci P: Deletion of the
p66Shc longevity gene reduces systemic and tissue oxidative stress,
vascular cell apoptosis, and early atherogenesis in mice fed a high-
fat diet. Proc Natl Acad Sci USA 2003, 100:2112–2116
25. Camici GG, Schiavoni M, Francia P, Bachschmid M, Martin-Padura I,
Hersberger M, Tanner FC, Pelicci P, Volpe M, Anversa P, Luscher TF,
Cosentino F: Genetic deletion of p66(Shc) adaptor protein prevents
hyperglycemia-induced endothelial dysfunction and oxidative stress.
Proc Natl Acad Sci USA 2007, 104:5217–5222
26. He C, Zheng F, Sabol J, Stitt A, Striker L, Hattori M, Vlassara H:
Differential expression of renal AGE-receptor genes in NOD mouse
kidneys: possible role in non-obese diabetic renal disease. Kidney Int
2000, 58:1931–1940
27. He CJ, Koschinsky T, Buenting C, Vlassara H: Presence of diabetic
complications in type 1 diabetic patients correlates with low expres-
sion of mononuclear cell AGE-receptor-1 and elevated serum AGE.
Mol Med 2001, 7:159 –168
28. Cai W, He JC, Zhu L, Chen X, Wallenstein S, Striker GE, Vlassara H:
Reduced oxidant stress and extended lifespan in mice exposed to a
low glycotoxin diet: association with increased AGER1 expression.
Am J Pathol 2007, 170:1893–1902
29. Cai W, He JC, Zhu L, Chen X, Zheng F, Striker GE, Vlassara H: Oral
glycotoxins determine the effects of calorie restriction on oxidant stress,
age-related diseases, and lifespan. Am J Pathol 2008, 173:327–336
30. Sandu O, Song K, Cai W, Zheng F, Uribarri J, Vlassara H: Insulin
resistance and type 2 diabetes in high-fat-fed mice are linked to high
glycotoxin intake. Diabetes 2005, 54:2314 –2319
31. Cai W, Gao QD, Zhu L, Peppa M, He C, Vlassara H: Oxidative
stress-inducing carbonyl compounds from common foods: novel me-
diators of cellular dysfunction. Mol Med 2002, 8:337–346
32. He C, Sabol J, Mitsuhashi T, Vlassara H: Dietary glycotoxins: inhibition of
reactive products by aminoguanidine facilitates renal clearance and
reduces tissue sequestration. Diabetes 1999, 48: 1308 –1315
33. Roque M, Fallon JT, Badimon JJ, Zhang WX, Taubman MB, Reis ED: Mouse
model of femoral artery denudation injury associated with the rapid accu-
mulation of adhesion molecules on the luminal surface and recruit-
ment of neutrophils. Arterioscler Thromb Vasc Biol 2000, 20:335–342
34. Lin RY, Choudhury RP, Cai W, Lu M, Fallon JT, Fisher EA, Vlassara H:
Dietary glycotoxins promote diabetic atherosclerosis in apolipopro-
tein E-deficient mice. Atherosclerosis 2003, 168:213–220
35. Lin RY, Reis ED, Dore AT, Lu M, Ghodsi N, Fallon JT, Fisher EA,
Vlassara H: Lowering of dietary advanced glycation endproducts
(AGE) reduces neointimal formation after arterial injury in genetically
hypercholesterolemic mice. Atherosclerosis 2002, 163:303–311
36. Liu H, Zheng F, Li Z, Uribarri J, Ren B, Hutter R, Tunstead JR,
Badimon J, Striker GE, Vlassara H: Reduced acute vascular injury
and atherosclerosis in hyperlipidemic mice transgenic for lysozyme.
Am J Pathol 2006, 169:303–313
37. Madamanchi NR, Vendrov A, Runge MS: Oxidative stress and vas-
cular disease. Arterioscler Thromb Vasc Biol 2005, 25:29 –38
38. Fox CS, Coady S, Sorlie PD, D’Agostino RB Sr, Pencina MJ, Vasan RS,
Meigs JB, Levy D, Savage PJ: Increasing cardiovascular disease bur-
den due to diabetes mellitus: the Framingham Heart Study. Circulation
2007, 115:1544 –1550
39. Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J, Halsey J,
Qizilbash N, Peto R, Collins R: Blood cholesterol and vascular mor-
tality by age, sex, and blood pressure: a meta-analysis of individual
data from 61 prospective studies with 55,000 vascular deaths. Lancet
2007, 370:1829 –1839
40. Vlassara H: The AG: E-receptor in the pathogenesis of diabetic
complications. Diabetes Metab Res Rev 2001, 17:436 443
41. Zheng F, He C, Cai W, Hattori M, Steffes M, Vlassara H: Prevention of
diabetic nephropathy in mice by a diet low in glycoxidation products.
Diabetes Metab Res Rev 2002, 18:224 –237
42. Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B,
Stern DM, Nawroth PP: Understanding RAGE, the receptor for ad-
vanced glycation end products. J Mol Med 2005, 83:876 886
43. Stitt AW, Burke GA, Chen F, McMullen CB, Vlassara H: Advanced
glycation end-product receptor interactions on microvascular cells oc-
cur within caveolin-rich membrane domains. FASEB J 2000,
14:2390 –2392
44. Vlassara H, Moldawer L, Chan B: Macrophage/monocyte receptor for
nonenzymatically glycosylated protein is upregulated by cachectin/
tumor necrosis factor. J Clin Invest 1989, 84:1813–1820
45. Chen J, Brodsky SV, Goligorsky DM, Hampel DJ, Li H, Gross SS,
Goligorsky MS: Glycated collagen I induces premature senescence-like
phenotypic changes in endothelial cells. Circ Res 2002, 90:1290 –1298
46. Satoh H, Togo M, Hara M, Miyata T, Han K, Maekawa H, Ohno M,
Hashimoto Y, Kurokawa K, Watanabe T: Advanced glycation end-
products stimulate mitogen-activated protein kinase and proliferation
in rabbit vascular smooth muscle cells. Biochem Biophys Res Com-
mun 1997, 239:111–115
47. Sata M, Maejima Y, Adachi F, Fukino K, Saiura A, Sugiura S, Aoyagi
T, Imai Y, Kurihara H, Kimura K, Omata M, Makuuchi M, Hirata Y,
Nagai R: A mouse model of vascular injury that induces rapid onset
of medial cell apoptosis followed by reproducible neointimal hyper-
plasia. J Mol Cell Cardiol 2000, 32:2097–2104
48. Liu H, Zheng F, Cao Q, Ren B, Zhu L, Striker G, Vlassara H: Amelio-
ration of oxidant stress by the defensin lysozyme. Am J Physiol
Endocrinol Metab 2006, 290:E824 –E832
49. Hofmann SM, Dong HJ, Li Z, Cai W, Altomonte J, Thung SN, Zeng F,
Fisher EA, Vlassara H: Improved insulin sensitivity is associated with
restricted intake of dietary glycoxidation products in the db/db
mouse. Diabetes 2002, 51:2082–2089
50. Peppa M, He C, Hattori M, McEvoy R, Zheng F, Vlassara H: Fetal or
neonatal low-glycotoxin environment prevents autoimmune diabetes
in NOD mice. Diabetes 2003, 52:1441–1448
51. Zhao Z, Zhao C, Zhang XH, Zheng F, Cai W, Vlassara H, Ma ZA:
Advanced glycation end products inhibit glucose-stimulated insulin
secretion through nitric oxide-dependent inhibition of cytochrome c
oxidase and ATP synthesis. Endocrinology 2009, 150:2569 –2576
52. Cassese A, Esposito I, Fiory F, Barbagallo AP, Paturzo F, Mirra P,
Ulianich L, Giacco F, Iadicicco C, Lombardi A, Oriente F, Van
Obberghen E, Beguinot F, Formisano P, Miele C: In skeletal muscle
advanced glycation end products (AGEs) inhibit insulin action and in-
duce the formation of multimolecular complexes including the receptor
for AGEs. J Biol Chem 2008, 283:36088 –36099
53. Takatori A, Ishii Y, Itagaki S, Kyuwa S, Yoshikawa Y: Amelioration of the
beta-cell dysfunction in diabetic APA hamsters by antioxidants and AGE
inhibitor treatments. Diabetes Metab Res Rev 2004, 20:211–218
54. Sanz J, Fayad ZA: Imaging of atherosclerotic cardiovascular disease.
Nature 2008, 451:953–957
55. Lim M, Park L, Shin G, Hong H, Kang I, Park Y: Induction of apoptosis
of Beta cells of the pancreas by advanced glycation end-products,
important mediators of chronic complications of diabetes mellitus.
Ann NY Acad Sci 2008, 1150:311–315
1732 Torreggiani et al
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    • "One possibility to prevent RAGE-mediated damage, is enabled by the upregulation of advanced glycation end products receptor 1 (AGER1) after AGE exposure. Although AGER1 was first only associated with the AGE turnover, studies observed that its upregulation suppresses RAGE-mediated pathways: AGER1 inhibits the activity of NADPH oxidase and weakens oxidative stress generation as well as ROS-mediated signaling70717273. Moreover, it has been described that AGER1 is linked to sirtuin1 (SIRT1) [50,52], a NAD + -dependent deacetylase. "
    [Show abstract] [Hide abstract] ABSTRACT: Type 2 diabetes mellitus (T2DM) is a very complex and multifactorial metabolic disease characterized by insulin resistance and β cell failure leading to elevated blood glucose levels. Hyperglycemia is suggested to be the main cause of diabetic complications, which not only decrease life quality and expectancy, but are also becoming a problem regarding the financial burden for health care systems. Therefore, and to counteract the continually increasing prevalence of diabetes, understanding the pathogenesis, the main risk factors, and the underlying molecular mechanisms may establish a basis for prevention and therapy. In this regard, research was performed revealing further evidence that oxidative stress has an important role in hyperglycemia-induced tissue injury as well as in early events relevant for the development of T2DM. The formation of advanced glycation end products (AGEs), a group of modified proteins and/or lipids with damaging potential, is one contributing factor. On the one hand it has been reported that AGEs increase reactive oxygen species formation and impair antioxidant systems, on the other hand the formation of some AGEs is induced per se under oxidative conditions. Thus, AGEs contribute at least partly to chronic stress conditions in diabetes. As AGEs are not only formed endogenously, but also derive from exogenous sources, i.e., food, they have been assumed as risk factors for T2DM. However, the role of AGEs in the pathogenesis of T2DM and diabetic complications-if they are causal or simply an effect-is only partly understood. This review will highlight the involvement of AGEs in the development and progression of T2DM and their role in diabetic complications.
    Full-text · Article · Mar 2015
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    • "Nowadays, accumulating evidence showed that hyperglycemia could mediate the alteration of extra and intracellular metabolism, such as the function of AGEs. The high level of OS associated with cardiovascular disease was linked to prooxidants such as AGEs [27]. AGEs have been regarded as one of the most toxic substances and resulted in OS response in diabetic vascular dysfunction [28–30]. "
    [Show abstract] [Hide abstract] ABSTRACT: Oxidative stress (OS) has been regarded as one of the major pathogeneses of diabetic nephropathy (DN) through damaging kidney which is associated with renal cells dysfunction. The aim of this study was to investigate whether Moutan Cortex (MC) could protect kidney function against oxidative stress in vitro or in vivo. The compounds in MC extract were analyzed by HPLC-ESI-MS. High-glucose-fat diet and STZ (30 mg kg−1) were used to induce DN rats model, while 200 μ g mL−1 AGEs were for HBZY-1 mesangial cell damage. The treatment with MC could significantly increase the activity of SOD, glutathione peroxidase (GSH-PX), and catalase (CAT). However, lipid peroxidation malondialdehyde (MDA) was reduced markedly in vitro or in vivo. Furthermore, MC decreased markedly the levels of blood glucose, serum creatinine, and urine protein in DN rats. Immunohistochemical assay showed that MC downregulated significantly transforming growth factor beta 2 (TGF- β 2) protein expression in renal tissue. Our data provided evidence to support this fact that MC attenuated OS in AGEs-induced mesangial cell dysfunction and also in high-glucose-fat diet and STZ-induced DN rats.
    Full-text · Article · Apr 2014 · Oxidative medicine and cellular longevity
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    • "A potential link tying together high carbohydrate diet and a maladaptive pro-inflammatory state is the increased formation of circulating and tissue-associated AGEs. The presence of increased AGEs is tied to both a pro-oxidative and pro-inflammatory state where oxidative stress mediates the persistent inflammatory response [40,41]. In patients with poorly controlled diabetes and increased glycated hemoglobin (HbA1c), peripheral blood leukocytes express increased innate and type 1 cytokines indicative of a pro-inflammatory phenotype [42,43]. "
    [Show abstract] [Hide abstract] ABSTRACT: Hyperglycemia, the diagnostic feature of diabetes also occurs in non-diabetics associated with chronic inflammation and systemic insulin resistance. Since the increased risk of active TB in diabetics has been linked to the severity and duration of hyperglycemia, we investigated what effect diet-induced hyperglycemia had on the severity of Mycobacterium tuberculosis (Mtb) infection in non-diabetic guinea pigs. Post-prandial hyperglycemia was induced in guinea pigs on normal chow by feeding a 40% sucrose solution daily or water as a carrier control. Sucrose feeding was initiated on the day of aerosol exposure to the H37Rv strain of Mtb and continued for 30 or 60 days of infection. Despite more severe hyperglycemia in sucrose-fed animals on day 30, there was no significant difference in lung bacterial or lesion burden until day 60. However the higher spleen and lymph node bacterial and lesion burden at day 30 indicated earlier and more severe extrapulmonary TB in sucrose-fed animals. In both sucrose- and water-fed animals, serum free fatty acids, important mediators of insulin resistance, were increased by day 30 and remained elevated until day 60 of infection. Hyperglycemia mediated by Mtb infection resulted in accumulation of advanced glycation end products (AGEs) in lung granulomas, which was exacerbated by sucrose feeding. However, tissue and serum AGEs were elevated in both sucrose and water-fed guinea pigs by day 60. These data indicate that Mtb infection alone induces insulin resistance and chronic hyperglycemia, which is exacerbated by sucrose feeding. Moreover, Mtb infection alone resulted in the accumulation tissue and serum AGEs, which are also central to the pathogenesis of diabetes and diabetic complications. The exacerbation of insulin resistance and hyperglycemia by Mtb infection alone may explain why TB is more severe in diabetics with poorly controlled hyperglycemia compared to non-diabetics and patients with properly controlled blood glucose levels.
    Full-text · Article · Oct 2012 · PLoS ONE
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