Cyanate is a novel inducer of endothelial icam-1 expression.

Abstract

Recent work has shown that humans are significantly exposed to isocyanic acid/cyanate, which is generated when coal, biomass, or tobacco is burned. In vivo, cyanate is formed by the phagocyte protein myeloperoxidase and by breakdown of urea. Carbamylation of proteins through cyanate has been demonstrated to predict cardiovascular risk and is thought to promote vascular dysfunction; however, the underlying mechanisms remain unclear.
Here, we show that cyanate induces intercellular cell adhesion molecule-1 (ICAM-1) expression with subsequently enhanced neutrophil adhesion in human coronary artery endothelial cells. Cyanate triggers ICAM-1 expression through a mechanism depending on activation of the mitogen-activated protein kinase p38 and nuclear factor-kappaB. Endothelial ICAM-1 expression was not induced when low-molecular-weight substances were removed from cell culture medium, thus ruling out a role of carbamylated (lipo)proteins in ICAM-1 induction. In mice, oral administration of cyanate induced marked endothelial ICAM-1 expression in the aorta. Moreover, in patients with end-stage renal disease, the extent of plasma protein carbamylation (a marker for cyanate exposure) significantly correlated with plasma levels of soluble ICAM-1.
Here, we demonstrate for the first time that cyanate, rather than carbamylated lipoproteins, induces vascular ICAM-1 expression in vivo.
Collectively, our data raise the possibility that cyanate amplifies vascular inflammation, linking inflammation, smoking, and uremia.

Full text

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ORIGINAL RESEARCH COMMUNICATION
Cyanate Is a Novel Inducer
of Endothelial ICAM-1 Expression
Dalia El-Gamal,1Michael Holzer,1Martin Gauster,2Rudolf Schicho,1Veronika Binder,1
Viktoria Konya,1Christian Wadsack,3Rufina Schuligoi,1Akos Heinemann,1and Gunther Marsche1
Abstract
Aim: Recent work has shown that humans are significantly exposed to isocyanic acid/cyanate, which is gen-
erated when coal, biomass, or tobacco is burned. In vivo, cyanate is formed by the phagocyte protein myelo-
peroxidase and by breakdown of urea. Carbamylation of proteins through cyanate has been demonstrated to
predict cardiovascular risk and is thought to promote vascular dysfunction; however, the underlying mecha-
nisms remain unclear. Results: Here, we show that cyanate induces intercellular cell adhesion molecule-1
(ICAM-1) expression with subsequently enhanced neutrophil adhesion in human coronary artery endothelial
cells. Cyanate triggers ICAM-1 expression through a mechanism depending on activation of the mitogen-
activated protein kinase p38 and nuclear factor-kappaB. Endothelial ICAM-1 expression was not induced when
low-molecular-weight substances were removed from cell culture medium, thus ruling out a role of carbamy-
lated (lipo)proteins in ICAM-1 induction. In mice, oral administration of cyanate induced marked endothelial
ICAM-1 expression in the aorta. Moreover, in patients with end-stage renal disease, the extent of plasma protein
carbamylation (a marker for cyanate exposure) significantly correlated with plasma levels of soluble ICAM-1.
Innovation: Here, we demonstrate for the first time that cyanate, rather than carbamylated lipoproteins, induces
vascular ICAM-1 expression in vivo. Conclusion: Collectively, our data raise the possibility that cyanate amplifies
vascular inflammation, linking inflammation, smoking, and uremia. Antioxid. Redox Signal. 16, 129–137.
Introduction
P
(41). Cyanate irreversibly transforms lysine to e-amino-
carbamyllysine, also known as homocitrulline (HCit) (36).
This pathway is of particular relevance, as clinical studies have
shown that carbamylated proteins are independent risk factors
for development of coronary artery disease and stroke (41). A
potential role for protein carbamylation in human disease has
beeninvestigatedmainlyinthecontextofrenaldisease.Cyanate
is formed in vivo by breakdown of urea, and about 0.8% of urea
decomposes to cyanate (11). Since urea levels increase up to
110mM in patients with chronic renal failure, cyanate concen-
trations of about 1mM may be formed (5, 6). In patients who
undergodialysis,cardiovasculardiseaseistheprincipalcauseof
morbidity, and cardiac mortality of patients aged 45 years or
younger is more than 100-fold increased when compared with
the general population (8, 22, 37).
Importantly, isocyanic acid was recently identified as a
component of smoke from coal, biomass, or tobacco, thus
ost-translationalcarbamylationofproteinsthrough
cyanate is thought to promote vascular dysfunction
causing protein carbamylation at physiologically significant
levels (31). Moreover, it was recently observed that cyanate is
a major product of the phagocyte protein myeloperoxidase
(MPO) (3, 41). In human atherosclerotic lesions, MPO selec-
tively carbamylates high-density lipoprotein (HDL), thus
rendering HDL dysfunctional (15). Of particular interest,
MPO released by degranulation of activated neutrophils av-
idly associates with endothelial cells and accumulates in the
subendothelial matrix of vascular tissues (4). Thus, it can be
assumed that vascular endothelial cells might be exposed to
high local concentrations of cyanate.
One key event in the development of atherosclerosis is the
adhesion of leukocytes to the vascular endothelium. In large
part, these processes are mediated by a diverse group of cel-
lular adhesion molecules such as intercellular cell adhesion
molecule-1 (ICAM-1), vascular cell adhesion molecule-1
(VCAM-1), and E-selectin, which are expressed on the surface
ofactivatedvascularendothelialcells(9,23).Recentdatafrom
patients with renal failure strongly suggest that high serum
levels of adhesion molecules may predict future cardiovas-
cular events (29, 38, 39). In the current study, we demonstrate
Institutes of1Experimental and Clinical Pharmacology and2Cell Biology, Histology, and Embryology, Medical University of Graz, Graz,
Austria.
3Department of Obstetrics and Gynecology, Medical University of Graz, Graz, Austria.
ANTIOXIDANTS & REDOX SIGNALING
Volume 16, Number 2, 2012
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2011.4090
129

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that cyanate induces endothelial ICAM-1 expression in vivo
and in vitro. We observed that the carbamyllysine content of
plasma, a marker for cyanate formation, significantly corre-
lates with soluble ICAM-1 (sICAM-1) levels in patients with
end-stage renal disease. Since ICAM-1 is known for its im-
portance in mediating cell-cell interactions and facilitating
leukocyte endothelial transmigration, our results suggest that
cyanate promotes vascular inflammation.
Results
Cyanate induces ICAM-1 expression
in human coronary artery endothelial cells
Vascular endothelial cells might be locally exposed to high
cyanate concentrations. Treatment of human coronary artery
endothelial cells (HCAEC) for 48h with increasing concentra-
tionsofcyanateinducedaconcentration-dependentexpression
of ICAM-1, whereas VCAM-1 (Fig. 1A) and E-selectin expres-
sion (Fig. 1C) was unaltered. Cyanate-induced ICAM-1 ex-
pression was time dependent and substantially increased from
24to48h(Fig.1B).Cyanatetreatmenthadnoeffectonviability
of endothelial cells (Fig. 1D).
Cyanate-induced (lipo)protein carbamylation
does not mediate endothelial ICAM-1 expression
Our experiments on HCAEC were performed in the pres-
ence of serum; therefore, cyanate-driven carbamylation of
(lipo)proteins may have contributed to ICAM-1 expression.
To test whether carbamylated (lipo)proteins are involved in
ICAM-1 expression in our experiments, serum-containing cell
culture medium was incubated with cyanate (1mM) for 48h
in the absence of cells to induce protein carbamylation (pre-
conditioned medium). To remove cyanate, an aliquot of the
preconditioned medium was dialyzed against fresh cell cul-
ture medium. Cells were then treated with preconditioned
medium or dialyzed preconditioned medium for 48h. In
contrast to the cyanate-containing preconditioned medium,
dialyzed medium failed to induce ICAM-1 expression
(Fig. 2A). When cyanate was removed by a different method
(gel filtration on Sephadex PD-10 columns), then similar re-
sults were observed (Supplementary Fig. S1; Supplementary
Data are available online at www.liebertonline.com/ars).
Previous investigations have shown that carbamylated
low-density lipoprotein (LDL) induces adhesion molecule
expression in endothelial cells (2). In a further set of experi-
ments,LDLwasexposedtocyanate(1,2,or10mM)for48hto
FIG. 1.
cule expression in endothelial cells. (A) Human coronary
artery endothelial cells (HCAEC) were treated for 48h with
increasing concentrations of sodium cyanate (0.5 up to 2mM)
added to cell culture medium. Subsequently, expression of
cell adhesion molecules was assessed by flow cytometry. (B)
HCAEC were treated with 1mM sodium cyanate for 24h
or 48h, and intercellular cell adhesion molecule-1 (ICAM-1)
and vascular cell adhesion molecule-1 (VCAM-1) expression
was determined. (C) HCAEC were treated with sodium cy-
anate (1mM) for the indicated time points, and E-selectin
expression was determined. (D) HCAEC were treated with
increasing concentrations of sodium cyanate for 48h, and
cell viability was assessed by performing an MTT (3-(4, 5-
methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) re-
duction assay. All experiments were performed in duplicate.
Control was set at 100%, and values are expressed as % of
control. Results shown are mean–SEM (n=3–5). *p<0.05
versus control.
Flow-cytometric quantification of adhesion mole-
Innovation
Humansareexposed tosignificantamounts ofisocyanic
acid/cyanate formed by pyrolysis/combustion of coal,
biomass, or tobacco. Important endogenous sources of
cyanate include the breakdown of urea and myeloperox-
idase (MPO)-catalyzed oxidation of thiocyanate. Carba-
mylationof proteins through
demonstrated topredictcardiovascularriskand isthought
to promote vascular dysfunction; however, the underlying
mechanisms remain unclear.
Here,weshowfor thefirsttimethatcyanate,rather than
carbamylated (lipo)proteins, triggers vascular intercellular
cell adhesion molecule-1 (ICAM-1) expression. Cyanate
induces ICAM-1 expression through a mechanism de-
pending on activation of p38 mitogen-activated protein
kinase (MAPK) and nuclear factor-kappaB. In mice, oral
administration of cyanate induces marked endothelial
ICAM-1 expression in the aortic arch. Most importantly, in
patients with end-stage renal disease, the extent of plasma
protein carbamylation (a marker for cyanate exposure)
significantly correlates with plasma levels of soluble
ICAM-1. Since ICAM-1 is known for its importance in
mediating cell–cell interactions and facilitating leukocyte-
endothelial transmigration, our results provide further
insights to explain a part of the underlying mechanisms
that contribute to the enhanced cardiovascular risk asso-
ciated with smoking and chronic renal failure.
cyanate hasbeen
130EL-GAMAL ET AL.

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induce protein carbamylation. The carbamyllysine content of
LDL was assessed by mass spectrometry (Supplementary
Fig. S2). However, when HCAEC were subsequently exposed
to carbamylated LDL, then no change in adhesion molecule
expression was observed. This clearly indicates that carba-
mylated LDL does not contribute to ICAM-1 expression un-
der our experimental conditions (Fig. 2B). Notably, we cannot
exclude that more extensively carbamylated LDL, as used in
a previous study (2), may trigger adhesion molecule
expression.
Cyanate-induced ICAM-1 expression
is mediated by the p38 MAPK - NF-kB signaling
To elucidate the molecular mechanisms involved in endo-
thelial activation, cyanate-induced ICAM-1 expression was
examined in the presence of inhibitors of the mitogen-
activated protein kinase (MAPK) family members extracel-
lular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and
c-Jun N-terminal kinase (JNK). As shown in Figure 3A, two
different, specific p38 MAPK inhibitors (SB203580 or
SB202190) significantly suppressed cyanate-induced ICAM-1
expression in HCAEC, whereas inhibitors of the JNK or
ERK1/2 signaling pathways had no effect. Nuclear factor-jB
(NF-jB) is a well-characterized transcription factor that is
pivotal in ICAM-1 expression. To examine the contribution of
NF-jB, experiments were performed in the presence of a
specific NF-jB inhibitor (BAY-11 7082). On inhibition of NF-
jB, cyanate-induced ICAM-1 expression was completely
blocked,thus demonstrating adirectinvolvement ofNF-jBin
cyanate-induced ICAM-1 expression (Fig. 3B).
Next, we assessed whether cyanate-stimulated ICAM-1
expression induces leukocyte adhesion, a key event in the
development of atherosclerosis. For that purpose, human
polymorphonuclear leukocytes were isolated and allowed to
adhere to cyanate-treated endothelial cells. A significant in-
crease of neutrophil adhesion to cyanate-stimulated HCAEC
as compared with control HCAEC was observed (Fig. 3C).
Importantly, the increase in neutrophil adhesion was com-
pletely reversed in the presence of the NF-jB inhibitor.
Cyanate induces endothelial ICAM-1
expression in vivo
To test the physiologic relevance of our in vitro observa-
tions, we examined whether oral administration of cyanate
increases ICAM-1 expression in mice. Male C57BL/6 mice
were assigned to three groups receiving normal drinking
water (control), drinking water containing 0.2mg/ml sodium
cyanate(low-cyanate),anddrinkingwatercontaining1mg/ml
sodium cyanate (high-cyanate), respectively, for a period of 9
weeks. General characteristics of mice are shown in Table 1.
Mass spectrometry analysis of plasma proteins was per-
formed to assess plasma protein carbamylation as a marker
for cyanateexposure. Cyanate-treated mice showedincreased
carbamyllysine levels compared with controls, whereas
plasma total cholesterol and urea values were not altered
(Table 1). To investigate the involvement of lipid peroxida-
tion, plasma malondialdehyde levels were measured, but no
significant difference was observed between treatment
groups (Table 1).
Consistent with our in vitro findings, cyanate treatment
significantly increased the expression of ICAM-1 in vascular
endothelial cells of the aortic arch in mice (Fig. 4A, 4B).
Increased sICAM-1 in patients with renal failure
Significantly elevated MPO-activity and high urea con-
centrations lead to increased cyanate formation in patients
FIG. 2.
expression in HCAEC. (A) Growth medium was incubated
with sodium cyanate (1mM) for 48h (preconditioned me-
dium), and low-molecular-weight substances were subse-
quently removed by dialysis. HCAEC were then treated for
48h with preconditioned medium or dialyzed preconditioned
medium as described in the Materials and Methods section. (B)
Low-density lipoprotein (LDL) was exposed to cyanate (1, 2, or
10mM)for48htoinduceproteincarbamylationfollowedbygel
filtration to remove residual cyanate. HCAEC were then treated
with control LDL, carbamylated LDL (400lg/ml), or cyanate
(1–2mM) for 48h. Adhesion molecule expression was deter-
mined by flow cytometry. All experiments were performed in
duplicate. Control was set at 100%, and values are expressed as
% of control. Results shown are mean–SEM (n=3). *p<0.05
versus control.
Carbamylated lipoproteins do not induce ICAM-1
CYANATE INDUCES VASCULAR ICAM-1 EXPRESSION 131

Page 4

with chronic renal failure. Therefore, we next assessed whe-
ther plasma carbamyllysine levels in patients with end-stage
renal disease correlate with plasma sICAM-1 concentrations,
a proteolytic cleavage product of vascular ICAM-1 (21, 42).
We measured sICAM-1 concentrations in plasma from pa-
tients with end-stage renal disease on maintenance hemodi-
alysis (n=23) and age-matched healthy control subjects
(n=19). The general characteristics of both groups are shown
in Table 2. In line with others (29, 38, 39), we observed that
sICAM-1 concentrations were significantly higher in patients
who have undergone hemodialysis compared with control
subjects (Fig.5A).Patientswhohaveundergonehemodialysis
showed elevated levels of both HCit and carboxymethyl-
lysine (CML) (Fig. 5B). Importantly, sICAM-1 levels signifi-
cantly correlated with plasma protein carbamylation (HCit
content), whereas no correlation was found with plasma
levels of the major advanced glycation end product CML,
the formation of which is closely linked to increased local
oxidative stress (40). In addition, sICAM-1 correlated with C-
reactive protein in patients who have undergone hemodial-
ysis, but not with creatinine or uric acid (Table 3).
Discussion
In the current study, we demonstrate that cyanate exacer-
bates vascular endothelial inflammation. Cyanate induced a
time- and concentration-dependent upregulation of endo-
thelial ICAM-1 expression and subsequently increased neu-
trophil adhesion. Our results indicate that cyanate-induced
ICAM-1expressioninHCAECismediatedthroughactivation
of the stress-activated p38 MAPK and NF-jB signaling
pathways. In line with our in vitro results, oral administration
of cyanate dose dependently increased ICAM-1 expression in
vascular endothelial cells in the aortic arch of mice. Im-
portantly, plasma levels of carbamyllysine in mice of the low-
cyanate group reached levels that we observed in patients
who have undergone hemodialysis (276–31 vs. 290–30lmol
HCit/mol lysine, respectively), thus indicating that cyanate
concentrations used for mouse experiments are biologically
relevant. In agreement with our observation, previous studies
have shown that intraperitoneal injection of cyanate into rats
induces infiltration of mononuclear cells (1, 27).
Several studies have implicated a role of p38 MAPK and
NF-jB in the regulation of ICAM-1 expression by various
inducers, such as lipopolysaccharide, bile acids, or sphingo-
sine 1-phosphate (30). Recently, it was shown that ketoalde-
hyde-modified phosphatidylethanolamine
MAPK and adhesion molecule expression in endothelial cells
(13). Of particular interest, we could demonstrate in a previ-
ous study that phosphatidylethanolamine is targeted by cy-
anate (15). Thus, it is tempting to speculate whether
phosphatidylethanolamine modified by cyanate could acti-
vate endothelial cells.
inducesp38
Table 1. Biochemical Characteristics of Mice
Receiving Cyanate in Drinking Water for 9 Weeks
CharacteristicControl Low cyanateHigh cyanate
Total cholesterol
(mg/dl)
Urea (mg/dl)
Malondialdehyde
(nmol/ml)
HCit (lmol/mol Lys)
108–4 123–10 118–14
74–5
28.5–5.5
78–4
31.0–3.7
88–7
32.5–4.8
2.9–0.8 276.8–30.8a1311.0–128.2a
Plasma levels of total cholesterol, urea, and malondialdehyde were
measured by using commercial kits; carbamyllysine quantification
was performed by liquid chromatography tandem mass spectrom-
etry and expressed as HCit lmol/mol Lys. Results are given as
mean–SEM (n=5 mice per group).
ap<0.05 versus control.
HCit, homocitrulline; Lys, lysine.
FIG. 3.
by p38 mitogen-activated protein kinase (MAPK) and nu-
clearfactor-kappaB(NF-jB)
HCAEC were treated for 48h with sodium cyanate (1mM) in
the presence of the following MAPK family member inhibi-
tors: p38 MAPK inhibitors (SB203580; 5lM or SB202190;
2lM), c-Jun N-terminal kinase (JNK) inhibitors (JNK-2 in-
hibitor; 5lM or JNK-8 inhibitor; 5lM), and extracellular
signal-regulated kinase 1/2 inhibitors (PD98059; 10lM or
U0126; 5lM). (B) The NF-jB inhibitor, BAY-11 7082 (BAY,
5lM). Subsequently, ICAM-1 expression was determined by
flow cytometry. (C) HCAEC monolayers were grown on 96-
well plates and treated for 48h with 1mM sodium cyanate in
the presence or absence of BAY-11 7082 (5lM). Subse-
quently, fluorochrome-labeled neutrophils were added to the
confluent monolayers, and percentage of adhesion was de-
termined after a 30min incubation period at 37?C. All ex-
periments were performed in duplicate. Control was set at
100%, and values are expressed as % of control. Results
shown are mean–SEM (n=3–5).*p<0.05 versus control;
#p<0.05 versus cyanate-treated cells.
Cyanate-induced ICAM-1 expression is mediated
signaling pathway.(A)
132 EL-GAMAL ET AL.

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Many studies have evaluated the impact of protein carba-
mylation on structure and/or function of proteins, enzymes,
or hormones (16, 20,26). It waslong thought that formation of
cyanate occurs to a significant extent only during renal dys-
function. Recent studies unambiguously have demonstrated
that humans are exposed to significant amounts of isocyanic
acid and cyanate formed by pyrolysis/combustion of coal,
biomass, or tobacco (31) and that cyanate formation is also
catalyzed by the leukocyte heme peroxidase MPO (3, 41).
Importantly, in patients who have undergone hemodialysis
with high-grade persistent inflammation, significantly ele-
vated MPO activity was recently demonstrated, thus
Table 2. Clinical Chemistry of Control Subjects
and Patients Who Have Undergone Hemodialysis
Control Hemodialysis
n
Age (year)
Male/female
Plasma parameter
Cholesterol (mg/dl)
Triglycerides (mg/dl)
Urea (mg/dl)
CRP (mg/l)
Fibrinogen (mg/dl)
Creatinine (mg/dl)
Uric acid (mg/dl)
1923
53 (45–68)
9/10
68 (48–74)
13/10
188 (176–195)
122 (83–169)
28 (25–31)
1 (0–3)
272 (211–403)
0.92 (0.84–1.14)
4.8 (4.4–5.4)
161 (124–196)a
147 (90–200)
118 (98–143)b
9 (3–17)b
490 (411–619)b
6.91 (6.28–9.81)b
5.8 (5.1–7.1)b
Results are given as median with the interquartile range.
Significance was accepted atap<0.05;bp<0.01 (Mann–Whitney test).
CRP, C-reactive protein.
FIG. 5.
CAM-1), carbamyllysine (HCit), and carboxymethyl-lysine
(CML) in patients who have undergone hemodialysis. (A)
Plasma sICAM-1 concentrations of patients who have un-
dergone hemodialysis (n=23) and controls (n=19) were
quantified by ELISA. (B) Plasma levels of HCit and CML of
patients who have undergone hemodialysis (n=23) and
controls (n=19) were quantified by liquid chromatography
tandem mass spectrometry. *p<0.001.
Increased plasma levels of soluble ICAM-1 (sI-
FIG. 4.
expression in mice. (A) Cyanate induces ICAM-1 expression
in aortas of mice. Sections of paraffin-embedded aortic ar-
ches stained with polyclonal anti-CD54 (anti-ICAM-1) or
rabbit control IgG using immunohistochemistry. In contrast
to control mice, mice receiving low cyanate (0.2mg/ml) and
mice receiving high cyanate (1mg/ml) in drinking water ex-
pressed detectable levels of ICAM-1 in vascular endothelial
cells (black arrows). Control IgG showed no staining. Positive
immunohistochemical staining is indicated by a red im-
munoreaction product. Images are representatives of the
treatment groups (five mice/group). Scale bar indicates 50lm.
(B) Quantification of endothelial ICAM-1 immunostaining in
each group expressed as mean–SD. *p<0.05, **p<0.001 versus
control.
Oral administration of cyanate induces ICAM-1
CYANATE INDUCES VASCULAR ICAM-1 EXPRESSION133

Page 6

supporting the association between inflammation and cu-
mulative oxidative stress (32). The high carbamyllysine con-
tent of HDL in human atherosclerotic lesions correlated with
the MPO-specific oxidation product 3-chlorotyrosine, which
strongly supports the notion that macrophage-associated
MPO generates significant amounts of cyanate (15). Hence,
inflammation-driven formation of cyanate is a quantitatively
important mechanism for cyanate formation and protein
carbamylation.
A most important finding of the current study is that sI-
CAM-1 plasma levels in patients who have undergone he-
modialysis significantly correlate with plasma concentrations
ofcarbamyllysine.Notably, thetime-average concentration of
urea in plasma of patients with renal failure, which correlates
with plasma levels of protein-bound carbamyllysine, is asso-
ciated with an increased odds ratio for death (28). We made
the interesting observation that plasma concentrations of the
advancedglycationendproductCML,theformationofwhich
is closely linked to oxidative stress in patients who have un-
dergone hemodialysis, showed no correlation with sICAM
levels(40).Inthisregard,itneedstobenotedthatthepresence
of high serum levels of advanced glycation end products, as
measured by CML, may not be linked to increased mortality
in patients who have undergone hemodialysis (35).
Circulatingadhesionmoleculesarefoundtobeincreasedin
a variety of inflammatory disorders (12), indicating endo-
thelial activation and enhanced endothelial-leukocyte inter-
action. Recent data from patients with renal failure strongly
suggest that high serum levels of soluble adhesion molecules
may predict future cardiovascular events (29, 38, 39). Serum
concentrations of adhesion molecules may also increase with
the progression of renal dysfunction, thus suggesting that
inadequate clearance contributes to elevated serum levels of
adhesion molecules in chronic renal failure (7). However, in
the current study, no significant relationship was observed
between residual renal function and plasma levels of sICAM-
1 (Table 3), which is in agreement with a previous study (38).
The localization of phagocytes in the immediate vicinity of
endothelial cells at sites of inflammation may contribute to
cyanate-induced endothelial activation, as it was previously
shown that MPO-containing neutrophils are markedly
enriched with carbamylated proteins (19). Therefore, cyanate-
induced expression of endothelial ICAM-1 and the subse-
quently enhanced endothelial-neutrophil interaction might
form a vicious circle inducing endothelial dysfunction. In this
regard, it was shown that serum MPO levels correlate with
levels of inflammatory markers and mortality in patients who
have undergone hemodialysis (17).
The present findings provide further insight into the un-
derlying mechanisms that contribute to the enhanced car-
diovascular risk associated with smoking and chronic renal
failure. Monitoring of plasma carbamyllysine levels may offer
a basis for identification of humans at increased risk of car-
diovascular disease. Anti-ICAM-1 antibodies and/or inter-
ventions aimed at reducing levels of cyanate are potential
promising approaches in reducing vascular disease in pa-
tients with renal failure.
Materials and Methods
Reagents used arelisted in the SupplementaryMaterials and
Methods (available online at www.liebertonline.com/ars).
Blood collection
Blood was taken from patients who have undergone he-
modialysis before the dialysis session and from age-matched
control subjects at the time of routine laboratory investiga-
tions in agreement with the Institutional Review Board of the
Medical University of Graz as described (24).
Isolation and carbamylation of LDL
LDL was isolated as described (15) and carbamylated with
potassium cyanate (1, 2, or 10mM) in phosphate buffered sa-
line (pH 7.4) containing 100lM diethylenetriaminepentaacetic
acidfor48hat 37?CfollowedbygelfiltrationonSephadexPD-
10 columns (Amersham Biosciences) to remove residual cya-
nate.Control LDL was incubated under sameconditions inthe
absence of cyanate.
Cell culture
HCAEC were purchased from Lonza (Verviers, Belgium)
and cultured in EGM-2 MV Bullet medium (Lonza) contain-
ing FBS (5%). All experiments were performed without serum
starvation. Endothelial cells were passaged at 80%–90% con-
fluence and were used within 4 passages for experiments.
Removal of cyanate from preconditioned
cell culture medium
Serum containing cell culture medium was incubated with
cyanate (1mM) for 48h to induce protein carbamylation
(preconditioned medium). Subsequently, low-molecular-
weight substances (i.e., cyanate) were removed by either di-
alysis (molecular-weight cut off of 3000 Da) or gel filtration on
Sephadex PD-10 columns.
Table 3. Correlation Matrix of Soluble Intercellular Cell Adhesion Molecule-1, HCit,
Carboxymethyl-Lysine, C-Reactive Protein, Creatinine, and Uric Acid in Patients
Who Have Undergone Hemodialysis
sICAM-1HCitCMLCRP Creatinine
sICAM-1
HCit
CML
CRP
Creatinine
Uric acid
—
0.588a
-0.026
0.609a
-0.304
-0.163
—
—
—
—
—
—
—
—
—
—
—
—
—
—
-0.143
0.360
-0.090
0.004
-0.296
0.421b
0.084
-0.531a
-0.045 0.400b
Spearman rank correlation coefficients are noted atap<0.01;bp<0.05.
HCit, carbamyllysine; CML, carboxymethyl-lysine; sICAM-1, soluble ICAM-1.
134EL-GAMAL ET AL.

Page 7

Flow cytometry
The surfaceexpression ofICAM-1,VCAM-1, andE-selectin
on HCAEC was assessed by flow cytometry as described (14,
18). HCAEC were harvested by using an EDTA buffer
(10mM) and subsequently incubated with anti-CD54 (PE,
1:40), anti-CD106 (FITC, 1:40), or anti-CD62E (PE, 1:40) at 4?C
for 30min. The negative isotype-matched control was FITC
mouse IgG1 or PE mouse IgG1 isotype control. After immu-
nofluorescencestaining,cellswererinsed,fixed,andanalyzed
by flow cytometry.
Cell viability
To assess effects on cell viability, an MTT (3-(4, 5-methyl-
thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) reduction
assay was performed. After treatment, cells were incubated
with fresh medium containing MTT (0.5mg/ml) for 4h at
37?C. Acid-isopropanol (100lL of 0.04 N HCI in isopropanol)
wasthenaddedtodissolvetheformazancrystals.Absorbance
was evaluated at 560nm by using a microplate spectropho-
tometer (xMark?; BioRAD).
Preparation of polymorphonuclear leukocytes
Blood was sampled from healthy volunteers after an in-
formed consent, according to a protocol approved by the In-
stitutional Review Board of the Medical University of Graz.
Polymorphonuclear leukocytes (containing eosinophils and
neutrophils) were prepared as previously described (33, 34).
The resulting purity and viability of neutrophils (i.e., poly-
morphonuclear leukocytes) preparation was typically greater
than 95%.
Leukocyte adhesion assay
HCAEC monolayers grown on 96-well plates were treated
for 48h with cyanate (1mM) in the presence or absence of an
NF-jB inhibitor BAY-11 7082. The fluorescent dye, calcein-
AM, was used to stain freshly isolated neutrophils. Fluor-
ochrome-labeled neutrophils were then added to the conflu-
ent monolayers, and percentage of adhesion was determined
after a 30min incubation period at 37?C (FlexStation? II;
Molecular Devices).
In vivo study
C57BL/6 mice (males, 20–22g, 5 weeks old) were pur-
chased from Charles River (Sulzfeld) and housed in plastic
sawdust floor cages at constant temperature (22?C) and a
12:12-h light–dark cycle with free access to standard labora-
tory chow and tap water. Experimental protocols were ap-
proved by the Animal Care Committee of the Austrian State
Department of Science and Research. Mice (n=15) were
equally assigned to three groups and received normal
drinking water (control group), drinking water containing
0.2mg/ml sodium cyanate (low-cyanate group), or drinking
water containing 1mg/ml sodium cyanate (high-cyanate
group). Treatment continued for a period of 9 weeks, after
which mice were deeply anesthetized with isoflurane, and
0.5ml blood was collected by cardiac puncture using citrate
(3.8%) as an anticoagulant. Plasma was stored at -70?C for
further analysis. After blood collection, mice were killed by
cervical dislocation. The ascending part of the aortic arch,
closer to the lesser curvature, was removed, cleaned of adi-
pose and connective tissue under a dissection microscope,
and immediately fixed in 4% paraformaldehyde.
Plasma parameters
Malondialdehyde, total cholesterol, and urea concentra-
tions were measured by commercial assay kits obtained from
Cayman (Ann Arbor) and BioVision (Mountain View), re-
spectively. For determination of sICAM-1 plasma concentra-
tion, a platinum ELISA kit for human sICAM-1 was used
(eBioscience).
Carbamyllysine and CML quantification
Proteins were hydrolyzed with a fast, low-volume hydro-
lysis method as described (10).
To quantify LDL and plasma protein carbamylation, elec-
trospray ionization liquid chromatography tandem mass
spectrometry was used for HCit and CML quantification as
previously described (15).
Immunohistochemistry
Aortic arches fixed in 4% paraformaldehyde were embed-
ded in paraffin. Serial cross sections (5lm) of the aortic arch,
proximal to the origin of innominate artery, were generated
on a microtome and processed by standard technique for all
mice. Briefly, endogenous peroxidase was blocked with 3%
H2O2, and immunolocalization at the inner curvature of the
cross sections was visualized by using the Ultravision-labeled
polymer-horseradish peroxidase detection system specific for
rabbit antibodies from Lab Vision according to the manufac-
turer’s protocol. Rabbit anti-CD54 to detect ICAM-1 (1:100)
and nonimmune rabbit IgG (isotype control; 1:100) were di-
luted in a protein-protecting diluent buffer (Lab Vision). Be-
tween incubation steps, slides were washed in Tris-buffered
saline containing 0.05% (vol/vol) Tween 20. Slides were
counterstained with Mayer’s hemalaun from Merck.
Cross sections were studied to quantify endothelial ICAM-
1immunostainingattheluminalsurface.Percentagecoverage
with ICAM-1 was measured by using Xcellence imaging
software Version 1.1 from Olympus soft imaging solutions
(Munich).
Statistical analyses
Data are shown as mean–SEM for n observations unless
stated otherwise. Comparison of groups was performed by
using one-way ANOVA with Tukey’s multiple-comparison
post hoc test. Data from hemodialysis and control subjects are
shown as median with the interquartile range, and Mann–
Whitney test was used to test for differences. Correlations
were determined by using Spearman rank correlation. Sig-
nificance was accepted at p<0.05. Statistical analyses were
performed with GraphPad Prism Version 4.03.
Acknowledgments
M.H. was funded by the PhD Program Molecular Medicine
of the Medical University of Graz. This work was supported
by the Austrian Science Fund FWF (Grants P21004-B02, P-
22521-B18, P 22771-B18, and P22976-B18).
CYANATE INDUCES VASCULAR ICAM-1 EXPRESSION 135

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Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Gunther Marsche
Institute for Experimental and Clinical Pharmacology
Medical University of Graz
Universita ¨tsplatz 4
8010 Graz
Austria
E-mail: gunther.marsche@medunigraz.at
Date of first submission to ARS Central, May 27, 2011; date of
final revised submission, July 21, 2011; date of acceptance,
August 12, 2011.
Abbreviations Used
BAY¼BAY-11 7082
CML¼carboxymethyl-lysine
CRP¼C-reactive protein
ERK1/2¼extracellular signal-regulated kinase 1/2
H2O2¼hydrogen peroxide
HCAEC¼human coronary artery endothelial cells
HCit¼homocitrulline, carbamyllysine
HDL¼high-density lipoprotein
ICAM-1¼intercellular cell adhesion molecule-1
JNK¼c-Jun N-terminal kinase
LDL¼low-density lipoprotein
Lys¼lysine
MAPK¼mitogen-activated protein kinase
MPO¼myeloperoxidase
MTT¼3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-
tetrazolium bromide
NF-jB¼nuclear factor-kappaB
sICAM-1¼soluble intercellular cell adhesion molecule-1
VCAM-1¼vascular cell adhesion molecule-1
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