Diabetes Care
1
Ratio of Serum Levels of AGEs to
Soluble Form of RAGE Is A
Predictor of Endothelial Function
DOI: 10.2337/dc14-1435
Masato Kajikawa,1 Ayumu Nakashima,2
Noritaka Fujimura,3 Tatsuya Maruhashi,1
Yumiko Iwamoto,1 Akimichi Iwamoto,1
Takeshi Matsumoto,1 Nozomu Oda,1
Takayuki Hidaka,1 Yasuki Kihara,1
Kazuaki Chayama,4 Chikara Goto,5
Yoshiki Aibara,6 Kensuke Noma,6
Masayoshi Takeuchi,7 Takanori Matsui,8
Sho-ichi Yamagishi,8 and
Yukihito Higashi2,6
OBJECTIVE
Advanced glycation end products (AGEs) and their specific receptor, the receptor
for AGEs (RAGE), play an important role in atherosclerosis. Recently, a soluble
form of RAGE (sRAGE) has been identified in human serum. However, the role of
sRAGE in cardiovascular disease is still controversial. There is no information on
the association between simultaneous measurements of AGEs and sRAGE and
vascular function. In this study, we evaluated the associations between serum
levels of AGEs and sRAGE, ratio of AGEs to sRAGE, and vascular function.
RESEARCH DESIGN AND METHODS
We measured serum levels of AGEs and sRAGE and assessed vascular function by
measurement of flow-mediated vasodilation (FMD) and nitroglycerine-induced
vasodilation in 110 subjects who underwent health examinations. Multivariate
regression analyses were performed to identify factors associated with vascular
function.
RESULTS
CONCLUSIONS
These findings suggest that sRAGE may have a counterregulatory mechanism that
is activated to counteract the vasotoxic effect of the AGE–RAGE axis. The ratio of
AGEs to sRAGE may be a new chemical biomarker of endothelial function.
Nonenzymatic modification of proteins by reducing sugars has been reported to play a
crucial role in the pathogenesis of various types of diseases (1,2). It is well established,
in vivo, that early glycation products react repeatedly with the amino groups of proteins and form irreversible cross-link moieties termed advanced glycation end products
(AGEs) (3). There is growing evidence that interaction of AGEs and their specific receptor, the receptor for AGEs (RAGE), stimulates oxidative stress generation and plays
an important role in cardiovascular disease and diabetes complications (4–6).
Recently, a soluble form of RAGE (sRAGE) has been identified in human serum (7,8).
It has been reported that administration of a recombinant sRAGE inhibits the development and progression of atherosclerosis in animal models (8,9). These beneficial
Corresponding author: Yukihito Higashi, yhigashi@
hiroshima-u.ac.jp.
Received 10 June 2014 and accepted 23 September 2014.
This article contains Supplementary Data online
at http://care.diabetesjournals.org/lookup/
suppl/doi:10.2337/dc14-1435/-/DC1.
© 2014 by the American Diabetes Association.
Readers may use this article as long as the work
is properly cited, the use is educational and not
for profit, and the work is not altered.
Diabetes Care Publish Ahead of Print, published online October 21, 2014
CARDIOVASCULAR AND METABOLIC RISK
Univariate regression analysis revealed that FMD correlated with age, BMI, systolic
blood pressure, diastolic blood pressure, heart rate, triglycerides, HDL cholesterol,
glucose, smoking pack-years, nitroglycerine-induced vasodilation, serum levels of
AGEs and sRAGE, and ratio of AGEs to sRAGE. Multivariate analysis revealed that
the ratio of AGEs to sRAGE remained an independent predictor of FMD, while serum
level of AGEs alone or sRAGE alone was not associated with FMD.
1
Department of Cardiovascular Medicine, Graduate School of Biomedical and Health Sciences,
Hiroshima University, Hiroshima, Japan
2
Division of Regeneration and Medicine, Medical
Center for Translational and Clinical Research,
Hiroshima University Hospital, Hiroshima, Japan
3
Department of Cardiology, Chugoku Rosai Hospital, Hiroshima, Japan
4
Department of Gastroenterology and Metabolism, Institute of Biomedical and Health Sciences,
Graduate School of Biomedical and Health Sciences, Hiroshima University Hiroshima, Japan
5
Hiroshima International University, Hiroshima,
Japan
6
Department of Cardiovascular Regeneration and
Medicine, Research Institute for Radiation Biology
and Medicine, Hiroshima University, Hiroshima,
Japan
7
Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University,
Ishikawa, Japan
8
Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume
University School of Medicine, Kurume, Japan
2
Ratio of AGEs to sRAGE and Endothelial Function
effects of exogenous administration of
sRAGE on the vasculature are assumed
to be due to capture of circulating AGEs
by acting as a decoy receptor.
Flow-mediated vasodilation (FMD) as
an index of endothelium-dependent
vasodilation and nitroglycerine-induced
vasodilation as an index of endotheliumindependent vasodilation in the brachial
artery using high-resolution ultrasound to
provide useful information on vascular
function (10–16). Endothelial dysfunction
occurs in the early stage of atherosclerosis, leading to the maintenance and progression of atherosclerosis, resulting in
cardiovascular complications (17). Evaluation of FMD is noninvasive and reflects
nitric oxide production. Furthermore,
previous studies have shown that endothelial function measured by FMD can
serve as an independent predictor of cardiovascular events (18–21).
Recent studies have shown that serum levels of sRAGE are decreased in
cardiovascular diseases, while other
studies have shown that serum level of
sRAGE reflects tissue RAGE expression
and predicts future cardiovascular
events (22–24). The reason for the controversial results remains unclear. Simultaneous measurements of serum
levels of AGEs and sRAGE would enable
more specific conclusions concerning
the role of the AGE–RAGE axis in cardiovascular disease to be drawn. Ratio of
AGEs to sRAGE may be a more useful
biomarker reflecting the AGE–RAGE
axis (25). In this study, we examined
the association between the ratio of serum level of AGEs to sRAGE and vascular
function.
RESEARCH DESIGN AND METHODS
Diabetes Care
calcium, and free thyroxine concentrations, and 24-h urinary excretion of 17hydroxycorticosteroids, 17-ketogenic
steroids, and vanillymandelic acid. Diabetes mellitus was defined according to
the American Diabetes Association (26).
Dyslipidemia was defined according to
the third report of the National Cholesterol Education Program (27). The ethical
committees in our institutions approved
the study protocol. Written informed
consent for participation in the study
was obtained from all subjects.
Study Protocol
We measured serum levels of AGEs and
sRAGE and assessed vascular function by
measurements of FMD and nitroglycerineinduced vasodilation in all subjects.
Subjects fasted the previous night for
at least 12 h. Subjects were instructed
to abstain from smoking at least 12 h
prior to the measurements. The study
began at 8:30 A.M. The subjects were
kept in the supine position in a quiet,
dark, air-conditioned room (constant
temperature of 22–258C) throughout
the study. A 23-gauge polyethylene
catheter was inserted into the left
deep antecubital vein to obtain blood
samples. Thirty minutes after maintaining the supine position, FMD and
nitroglycerine-induced vasodilation
were measured. The observers were
blind to the form of examination. Serum
levels of glyceraldehyde-derived AGEs
were measured with an ELISA as described previously (28). Serum sRAGE
levels were determined with a commercially available ELISA kit (R&D Systems,
Minneapolis, MN).
Subjects
Measurement of FMD and
Nitroglycerine-Induced Vasodilation
A total of 110 subjects were enrolled
from people who underwent healthscreening examinations at Hiroshima
University Hospital. Hypertension was
defined as systolic blood pressure of
.140 mmHg or diastolic blood pressure
of .90 mmHg, in a sitting position, on at
least three different occasions. Patients
with secondary forms of hypertension
were excluded in all patients with hypertension on the basis of complete history,
physical examination, radiological and
ultrasound examinations, urinalysis,
plasma renin activity, plasma aldosterone and norepinephrine concentrations, serum creatinine, potassium,
Vascular response to reactive hyperemia
in the brachial artery was used for assessment of endothelium-dependent FMD. A
high-resolution linear artery transducer
was coupled to computer-assisted analysis software (UNEXEF18G; UNEX Co, Nagoya, Japan) that used an automated
edge detection system for measurement
of brachial artery diameter (29). A blood
pressure cuff was placed around the forearm. The brachial artery was scanned longitudinally 5–10 cm above the elbow.
When the clearest B-mode image of the
anterior and posterior intimal interfaces
between the lumen and vessel wall was
obtained, the transducer was held at the
same point throughout the scan by a special probe holder (UNEX Co) to ensure
consistency of the image. Depth and
gain setting were set to optimize the images of the arterial lumen wall interface.
When the tracking gate was placed on the
intima, the artery diameter was automatically tracked, and the waveform of diameter changes over the cardiac cycle was
displayed in real time using the FMD
mode of the tracking system. This allowed
the ultrasound images to be optimized at
the start of the scan and the transducer
position to be adjusted immediately for
optimal tracking performance throughout the scan. Pulsed Doppler flow was
assessed at baseline and during peak hyperemic flow, which was confirmed to
occur within 15 s after cuff deflation.
Blood flow velocity was calculated from
the color Doppler data and displayed as a
waveform in real time. The baseline longitudinal image of the artery was acquired for 30 s, and then the blood
pressure cuff was inflated to 50 mmHg
above systolic pressure for 5 min. The
longitudinal image of the artery was recorded continuously until 5 min after
cuff deflation. Pulsed Doppler velocity
signals were obtained for 20 s at baseline
and for 10 s immediately after cuff deflation. Changes in brachial artery diameter were immediately expressed as
percentage change relative to the vessel
diameter before cuff inflation. FMD was
automatically calculated as the percentage change in peak vessel diameter from
the baseline value. Percentage of FMD
([peak diameter 2 baseline diameter]/
baseline diameter) was used for analysis.
Blood flow volume was calculated by
multiplying the Doppler flow velocity
(corrected for the angle) by heart rate
and vessel cross-sectional area (2r2).
Reactive hyperemia was calculated as
the maximum percentage increase in
flow after cuff deflation compared with
baseline flow.
The response to nitroglycerine was
used for assessment of endotheliumindependent vasodilation. Nitroglycerineinduced vasodilation was measured as
described previously (29). Briefly, after
acquiring baseline rest images for 30 s,
a sublingual tablet (75 mg nitroglycerine)
was given, and images of the artery
were recorded continuously until the dilation reached a plateau after administration of nitroglycerine. Subjects who had
received nitrate treatment and subjects
care.diabetesjournals.org
in whom the sublingually administered
nitroglycerine tablet was not dissolved
during the measurement were excluded
from this study. Nitroglycerine-induced
vasodilation was automatically calculated as a percent change in peak vessel
diameter from the baseline value. Percentage of nitroglycerine-induced vasodilation ([peak diameter 2 baseline
diameter]/baseline diameter) was used
for analysis.
Statistical Analysis
We calculated the sample size based on
previous data for the ratio of AGEs to
sRAGE (25). For the current study, we
estimated that 86 subjects were needed
with an a = 0.05 and a power of 0.9.
Finally, we enrolled 110 subjects with
consideration for 20% dropouts. Results
are presented as means 6 SD for continuous variables and as percentages for
categorical variables. Statistical significance was set at a level of P , 0.05.
Spearman rank correlation analysis was
used to determine associations between FMD and selected parameters.
Multivariate linear regression analyses
Kajikawa and Associates
were performed to determine relationships between vascular function and serum levels of AGEs, sRAGE, and ratio of
AGEs to sRAGE before (model 1) and
after adjustment for age and sex (model
2), further adjusted for BMI, presence of
hypertension, dyslipidemia, diabetes,
and current smoking (model 3), and further adjusted for BMI, presence of hypertension, dyslipidemia, glucose levels,
and current smoking (model 4). The data
were processed using the software
package Stata version 9 (Stata Co., College Station, TX).
RESULTS
Clinical Characteristics
The baseline characteristics of the 110
subjects are summarized in Table 1. Of
the 110 subjects, 80 (72.7%) were men
and 30 (27.3%) were women. Fortyeight (43.6%) had hypertension, 37
(33.6%) had dyslipidemia, 13 (11.8%)
had diabetes mellitus, and 49 (44.5%)
were current smokers. The mean serum levels of AGEs and sRAGE were
9.5 6 3.5 U/mL and 823 6 388 pg/mL,
respectively.
Table 1—Clinical characteristics of the subjects
Variables
n = 110
Age, years
46 6 19
Sex, men/women
BMI, kg/m2
80/30
23.0 6 3.7
Systolic blood pressure, mmHg
127 6 19
Diastolic blood pressure, mmHg
74 6 14
Heart rate, bpm
70 6 13
Total cholesterol, mmol/L
4.89 6 0.91
Triglycerides, mmol/L
1.49 6 1.15
HDL cholesterol, mmol/L
1.50 6 0.44
LDL cholesterol, mmol/L
2.79 6 0.80
Glucose, mmol/L
HbA1c, %
5.88 6 1.33
5.6 6 0.7
HbA1c, mmol/mol
37.8 6 7.3
Hypertension, n (%)
48 (43.6)
Dyslipidemia, n (%)
37 (33.6)
Diabetes mellitus, n (%)
13 (11.8)
Previous coronary heart disease, n (%)
6 (5.5)
Previous stroke, n (%)
3 (2.7)
Smoker, n (%)
Smoking, pack-years
49 (44.5)
15.1 6 20.5
FMD, %
5.4 6 2.9
Nitroglycerine-induced vasodilation, %
14.1 6 6.2
AGEs, U/mL
9.5 6 3.5
sRAGE, pg/mL
823 6 388
Ratio of AGEs to sRAGE (31022), U/pg
1.43 6 0.90
Results are presented as means 6 SD for continuous variables and percentages for categorical
variables.
Relationships of Vascular Function
With Cardiovascular Risk Factors,
Serum Levels of AGEs and sRAGE,
and Ratio of AGEs to sRAGE
FMD was negatively correlated with age,
BMI, systolic blood pressure, diastolic
blood pressure, heart rate, triglycerides,
glucose, HbA1c, and smoking pack-years
and was positively correlated with HDL
cholesterol and nitroglycerine-induced
vasodilation (Table 2).
There were significant relationships
between FMD and serum level of AGEs
(r = 20.30; P = 0.001; Fig. 1A), serum level
of sRAGE (r = 0.22; P = 0.02; Fig. 1B), and
ratio of serum levels of AGEs to sRAGE (r =
20.33; P , 0.001; Fig. 1C). After adjustment for confounders, neither AGEs nor
sRAGE correlated with FMD (Table 3).
Multiple logistic regression analysis revealed that age and ratio of serum levels
of AGEs to sRAGE remained independently associated with FMD (Table 3).
Nitroglycerine-induced vasodilation
was negatively correlated with age,
BMI, systolic blood pressure, diastolic
blood pressure, heart rate, glucose,
HbA1c, and smoking pack-years and positively correlated with HDL cholesterol
and FMD (Table 2).
There was a significant relationship between nitroglycerine-induced vasodilation and serum level of AGEs (r = 20.20;
P = 0.04; Fig. 2A). Nitroglycerine-induced
vasodilation was not correlated with serum level of sRAGE (r = 0.13; P = 0.20; Fig.
2B) or ratio of serum levels of AGEs to
sRAGE (r = 20.17; P = 0.08; Fig. 2C). After
adjustment for confounders, AGEs did
not correlate with nitroglycerine-induced
vasodilation (Table 3).
In the current study, 14 of the 110 subjects were healthy subjects. Serum levels
of AGEs and ratio of AGEs to sRAGE were
significantly higher in diabetic patients
than in healthy subjects (6.8 6 1.7 vs.
12.5 6 3.5 U/mL and 0.83 6 0.40 vs.
1.78 6 0.81 31022 U/pg; P , 0.001
and P = 0.001, respectively; Supplementary Fig. 1A and C). There was no significant difference in serum level of sRAGE
between the two groups (920 6 247 vs.
767 6 242 pg/mL; P = 0.11; Supplementary Fig. 1B). FMD and nitroglycerininduced vasodilation were significantly
lower in diabetic patients than in healthy
subjects (3.6 6 2.8 vs. 8.7 6 2.6% and
8.4 6 3.4 vs. 15.6 6 4.0%; P , 0.001
and P , 0.001, respectively; Supplementary Fig. 1D and E).
3
4
Ratio of AGEs to sRAGE and Endothelial Function
Diabetes Care
Table 2—Univariate analysis of relationships among FMD, nitroglycerine-induced
vasodilation, and variables (Spearman rank correlation analysis)
Nitroglycerine-induced
Variables
FMD
vasodilation
Age, years
20.381**
20.454**
0.017
20.038
BMI, kg/m2
20.281**
20.207*
Systolic blood pressure, mmHg
Diastolic blood pressure, mmHg
20.443**
20.460**
20.418**
20.352**
Heart rate, bpm
20.285**
20.288**
20.165
20.098
20.371**
20.191
Sex, men/women
Total cholesterol, mmol/L
Triglycerides, mmol/L
HDL cholesterol, mmol/L
0.193*
0.206*
LDL cholesterol, mmol/L
20.102
20.076
Glucose, mmol/L
20.204*
20.290**
HbA1c, %
Smoking, pack-years
20.330**
20.318**
20.298*
20.293**
FMD, %
Nitroglycerine-induced vasodilation, %
AGEs, U/mL
sRAGE, pg/mL
Ratio of AGEs to sRAGE (31022), U/pg
0.400**
0.400**
20.340**
20.260**
0.200*
0.119
20.384**
20.227*
*P , 0.05, **P , 0.01.
CONCLUSIONS
In the current study, we demonstrated
that: 1) the ratio of serum levels of
AGEs to sRAGE was an independent
predictor of endothelial dysfunction
evaluated by FMD; and 2) while serum
levels of AGEs and sRAGE were positively and negatively associated with
endothelial dysfunction, respectively,
the significance was lost after adjusting
for various confounders.
Hypertension, dyslipidemia, diabetes,
aging, smoking, and obesity are commonly known cardiovascular risk factors
(10–12). These cardiovascular risk factors
are independent contributing factors of
endothelial dysfunction (10–12). Some investigators have shown the relationships
among serum levels of carboxymethyllysine (CML), glucose-derived AGEs and
sRAGE, and endothelial function (30–
32). Furthermore, single oral AGEs challenge acutely impaired endothelial function evaluated by FMD in both diabetic
and nondiabetic subjects, which was associated with increase in serum CML levels (30). These observations suggest that
both AGEs and sRAGE levels are biomarkers for endothelial dysfunction.
However, the potential problem with
these studies is that they did not simultaneously measure AGEs and sRAGE levels.
In this study, multivariate regression analysis revealed that the ratio of AGEs to
sRAGE remained an independent predictor of endothelial dysfunction, although
each value was not correlated with endothelial function. The present findings
have extended our previous observation
showing that the ratio of serum levels of
AGEs to sRAGE was inversely associated
with adiponectin, an anti-inflammatory
and vascular protective adipocytokine
with insulin-sensitizing properties (25).
The ratio of AGEs to sRAGE rather than
each parameter alone might be a useful
biomarker of endothelial dysfunction in
humans.
There is a growing body of evidence
that the AGE–RAGE axis plays an important role in the development and progression of atherosclerosis (4–6). Engagement
of RAGE with AGEs elicits oxidative stress
generation, which could reduce NO production and/or bioavailability, thereby
evoking inflammatory and thrombogenic
reactions in a variety of cells (33). These
observations support the active participation of the AGE–RAGE axis in endothelial
dysfunction. In univariate analysis in the
current study, besides an increased AGE
level, a low sRAGE value was associated
with endothelial dysfunction. However, it
is unlikely that endogenous sRAGE could
work as a decoy receptor for AGEs and
play a protective role against atherosclerosis in humans because sRAGE level is
1,000 times lower than that needed for
Figure 1—Scatter plots show the relationships between FMD and AGEs (A), sRAGE
(B), and ratio of AGEs to sRAGE (C).
efficiently binding to and capturing serum
AGEs (7). sRAGE is mainly generated from
the cleavage of cell-surface full-length
RAGE, the process of which is promoted
by the engagement of RAGE with ligands
such as AGEs and high-mobility group
protein box-1 (7). Therefore, a low sRAGE
level might be a consequence of impaired
shedding of RAGE by ligands. In these circumstances, the AGE–RAGE axis could be
further augmented. This is one possible
reason why the ratio of AGEs to sRAGE is a
more sensitive marker of endothelial dysfunction than each parameter alone.
Several prospective studies have
shown that elevated serum level of
sRAGE is associated with incidence of cardiovascular disease or all-cause mortality
in diabetic subjects (7,22,23). However,
there is some controversy about the
role of sRAGE in cardiovascular disease
in humans. Indeed, Falcone et al. (24) reported that a decreased sRAGE level was
correlated with a higher prevalence of
cardiovascular risk factors and coronary
artery disease in nondiabetic subjects.
Furthermore, a low plasma level of sRAGE
at baseline was independently associated
care.diabetesjournals.org
Kajikawa and Associates
Table 3—Multivariate analysis of relationships between vascular function and
AGEs, sRAGE, and ratio of AGEs to sRAGE
NitroglycerineFMD
induced vasodilation
Models used
Model 1
AGEs, U/mL
sRAGE, pg/mL
Ratio of AGEs to sRAGE (31022), U/pg
Model 2
AGEs, U/mL
sRAGE, pg/mL
Ratio of AGEs to sRAGE (31022), U/pg
Model 3
AGEs, U/mL
sRAGE, pg/mL
Ratio of AGEs to sRAGE (31022), U/pg
Model 4
AGEs, U/mL
sRAGE, pg/mL
Ratio of AGEs to sRAGE (31022), U/pg
b
b
P value
20.30 0.001
0.22
0.02
20.33 ,0.001
20.20
0.13
20.17
0.04
0.20
0.08
20.18
0.15
20.21
0.06
0.09
0.02
20.08
0.06
20.04
0.41
0.53
0.67
20.14
0.13
20.22
0.13
0.15
0.03
0.003
0.02
0.02
0.97
0.84
0.84
20.15
0.14
20.23
0.11
0.13
0.02
0.009
0.03
0.04
0.92
0.78
0.70
P value
Model 1: unadjusted model; model 2: adjusted for age and sex; model 3: adjusted for age, sex,
BMI, presence of hypertension, dyslipidemia, diabetes, and current smoking; and model 4:
adjusted for age, sex, BMI, presence of hypertension, dyslipidemia, glucose levels, and current
smoking.
with the risk of coronary heart disease and
all-cause mortality during a median followup period of 18 years in a communitybased population (34). At present, the
exact reason for the discrepant results
about the relationship between sRAGE
and cardiovascular disease is unclear.
Clinical significance of sRAGE as a biomarker might differ considerably depending on the patients’ background.
The characterization of AGEs measured is important to interpret the present data and to make a detailed
comparison with previous reports.
There are multiple types of immunologically distinct and structurally identified
AGEs such as glyoxal and methylglyoxal
hydroimidazolones, pyrraline, and pentosidine in humans (35,36). However,
they constitute a small percentage of
circulating AGEs in vivo, and their biological relevance in endothelial dysfunction has remained unclear (35,36). In this
study, we measured glyceraldehydederived AGEs to examine the role of
AGEs and sRAGE in endothelial dysfunction because we previously found that:
1) serum levels of glyceraldehyde-derived
AGEs are elevated under inflammatory
and/or diabetic conditions and correlated
with vascular inflammation evaluated by
18F-fluorodeoxyglucose–positron emission tomography in humans; 2) this type
of AGE mimics the biological effects of
AGE-rich serum purified from diabetic patients on hemodialysis; and 3) deleterious
and cytopathic effects of the AGE-rich serum were neutralized by addition of an
antiglyceraldehyde-derived AGE-specific
antibody but not by other types of antiAGE antibodies (35–39). Therefore, although glyceraldehyde, which could be
derived from glucose metabolism, is
not a major sugar in vivo, and its incubation with proteins will generate a
large number of structurally unidentified AGEs, and we cannot identify the
structure of AGEs measured in this
study, our study suggests the clinical
utility of measurement of the ratio of
serum levels of glyceraldehyde-derived
AGEs to sRAGE for detecting endothelial
dysfunction in humans. In regard to the
characterization of AGEs measured, we
previously reported that antibodies
raised against glyceraldehyde-derived
AGEs used for the ELISA did not crossreact with early glycation products
(HbA1c and/or fructoselysine) or structurally identified AGEs such as glyoxal
and methylglyoxal hydroimidazolones,
CML, carboxyethyllysine, or pentosidine
(35,36). So, these structurally identified AGEs were not evaluated in our
ELISA system, although methylglyoxal
hydroimidazolone could be formed
through both glyceraldehyde-related
and methylglyoxal-related pathways.
Figure 2—Scatter plots show the relationships between nitroglycerine-induced vasodilation and AGEs (A), sRAGE (B), and ratio of
AGEs to sRAGE (C).
Thus, besides glyceraldehyde-derived
AGEs, it would be interesting to further
examine whether the ratios of serum levels of these structurally identified AGEs to
sRAGE are also associated with endothelial dysfunction.
Study Limitations
First, circulating sRAGE could be generated from cleavage of cell-surface RAGE
or novel splice variants of RAGE (esRAGE)
(7). In this study, we measured total levels
of circulating sRAGE by ELISA. Therefore,
although esRAGE levels are approximately three- to fivefold lower than levels
of sRAGE (7), we did not know whether
the ratio of AGEs to esRAGE was also associated with endothelial dysfunction.
Second, to evaluate what percent of serum sRAGE was bound form with AGEs,
we performed additional experiments
using a Dynabeads Protein A Immunoprecipitation kit (Life Technologies, Carlsbad,
CA). In brief, polyclonal antibody raised
against AGEs used for ELISA or nonimmune rabbit IgG was first bound to Dynabeads Protein A, and then beads and
5
6
Ratio of AGEs to sRAGE and Endothelial Function
serum were mixed and incubated at 48C
with tilting and rotation. After 1 h, the
captured immune complexes were removed from the supernatant by magnetic
separation according to the supplier’s
recommendation. Immunoprecipitation
of AGEs from serum did not affect sRAGE
level; sRAGE values in AGE-cleared and
noncleared serum were 100.0 6 22.4
and 101.8 6 21.7%, respectively (n = 5;
P = 0.79). So, almost all of the sRAGE
would be an unbound form with AGEs.
Third, we have previously shown that addition of 10 U/ml glyceraldehyde-derived
AGEs (which is comparable to the serum
concentration in our subjects) to serum
samples does not interfere with the assay
system for sRAGE (37). Likewise, we also
confirmed that addition of 800 pg/mL, a
level comparable to the serum concentration, to assay samples did not affect circulating AGE values; addition of sRAGE
decreased serum AGE levels to 91.3 6
19.8% (n = 5; P = 0.42). Therefore, it is
unlikely that binding of AGEs to sRAGE
in serum could confound the present
findings. Fourth, there are multiple other
classes of RAGE ligands such as S100s or
high-mobility group protein box-1 (7). It
would be interesting to study the relationships among AGEs, these ligands,
and endothelial function. Fifth, although
several cell culture and animal models
have shown that soluble AGEs could impair endothelial function (30–33), crosslinked/less soluble AGEs might be linked
more specifically to changes in endothelial function. However, due to the difficulty in obtaining tissue samples from
all patients, we could not evaluate the
correlation of cross-linked AGEs to endothelial function. Sixth, although there was
no significant difference in FMD between
males and females in a previous study
(29), it has been reported that FMD was
significantly smaller in males than in females (14). In the current study, there
were no significant differences in the serum levels of AGEs (9.6 6 2.9 vs. 9.2 6 4.7
U/mL; P = 0.67), sRAGE (824 6 397 vs.
821 6 369 pg/mL; P = 0.98), ratio of
AGEs to sRAGE (1.44 6 0.88 vs. 1.39 6
0.96; P = 0.77), FMD (5.2 6 2.7 vs. 5.7 6
3.4%; P = 0.55), and nitroglycerineinduced vasodilation (14.5 6 6.4 vs.
13.1 6 5.3%; P = 0.31) between males
and females. In addition, after adjustment
for sex, our findings did not change. However, we cannot deny the possibility that
the results might be confounded with
Diabetes Care
more females in the cohort. Seventh,
measurement of FMD was performed
in a fasted state and with medication, although medication was discontinued for
at least 12 h prior the measurement of
FMD. In the current study, 12 (10.9%) of
the subjects received antihypertensive
drugs, 9 (8.2%) received lipid-lowering
drugs, and 11 (10.0%) received hypoglycemic drugs. Since we enrolled subjects who
underwent health examinations, it would
have been inappropriate to stop medications. Gokce et al. (16) demonstrated that
administration of vasoactive medication
with the exception of nitrates did not
significantly influence the value of FMD
and nitroglycerine-induced vasodilation.
However, we cannot also exclude the
possibility that medication affects FMD
and nitroglycerine-induced vasodilation.
Finally, this was a cross-sectional study
design and did not allow us to establish a
definitive causal relationship among serum levels of AGEs and sRAGE, ratio of
AGEs to sRAGE, and endothelial function.
Previous studies have demonstrated associations between serum levels of AGEs
and sRAGE and endothelial function
(32,33). However, in the current study,
neither the serum level of AGEs nor that
of sRAGE correlated with endothelial
function after adjustment for confounders. Future studies are needed to confirm the relationships among serum
levels of AGEs and sRAGE, ratio of AGEs
to sRAGE, and endothelial function in a
larger population.
In conclusion, sRAGE might have a
counterregulatory mechanism that is
activated to counteract the vasotoxic effect of the AGE–RAGE axis. Our present
study suggests that the ratio of AGEs
to sRAGE may be a new biochemical
marker of endothelial function.
Acknowledgments. The authors thank Miki
Kumiji, Megumi Wakisaka, Ki-ichiro Kawano,
and Satoko Michiyama of Hiroshima University,
Research Institute for Radiation Biology and
Medicine, for excellent secretarial assistance.
Funding. This study was supported in part by a
Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of
Japan (1859081500 and 21590898).
Duality of Interest. No potential conflicts of
interest relevant to this article were reported.
Author Contributors. M.K., N.F., and Y.H.
drafted the article and conceived the study.
A.N., T.Maruhashi, Y.I., A.I., T.Matsumoto, N.O.,
T.H., C.G., Y.A., and K.N. performed the ultrasonography. M.T. and T.Matsui measured the
levels of AGEs and sRAGE. Y.K. and K.C. revised
the article critically for important intellectual
content. S.Y. measured the levels of AGEs and
sRAGE and revised the article critically for
important intellectual content. Y.H. is the guarantor of this work and, as such, had full access to
all the data in the study and takes responsibility
for the integrity of the data and the accuracy of
the data analysis.
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