Biochemical and Biophysical Research Communications 345 (2006) 1446–1453

BBRC
www.elsevier.com/locate/ybbrc

Central role of endogenous Toll-like receptor-2 activation in regulating

inflammation, reactive oxygen species production, and subsequent
neointimal formation after vascular injury
Tetsuro Shishido a,b,*, Naoki Nozaki a, Hiroki Takahashi a, Takanori Arimoto a,
Takeshi Niizeki a, Yo Koyama a, Jun-ichi Abe b, Yasuchika Takeishi a, Isao Kubota a

a
From Department of Cardiology, Pulmonology, and Nephrology, Yamagata University School of Medicine, Yamagata, Japan
b
Center for Cardiovascular Research, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

Received 25 April 2006

Available online 16 May 2006

Abstract

Background: It is now evident that inflammation after vascular injury has significant impact on the restenosis after revascularization
procedures such as angioplasty, stenting, and bypass grafting. However, the mechanisms that regulate inflammation and repair after vas-
cular injury are incompletely understood. Here, we report that vascular injury-mediated cytokine expression, reactive oxygen species
(ROS) production, as well as subsequent neointimal formation requires Toll-like receptor-2 (TLR-2) mediated signaling pathway in vivo.
Methods and results: Vascular injury was induced by cuff-placement around the femoral artery in non-transgenic littermates (NLC)
and TLR-2 knockout (TLR-2KO) mice. After cuff-placement in NLC mice, expression of TLR-2 was significantly increased in both
smooth muscle medial layer and adventitia. Interestingly, we found that inflammatory genes expression such as tumor necrosis fac-
tor-a, interleukin-1b (IL-1b), IL-6, and monocyte chemoattractant protein-1 were markedly decreased in TLR-2KO mice compared with
NLC mice. In addition, ROS production after vascular injury was attenuated in TLR-2KO mice compared with NLC mice. Since we
observed the significant role of endogenous TLR-2 activation in regulating inflammatory responses and ROS production after vascular
injury, we determined whether inhibition of endogenous TLR-2 activation can inhibit neointimal proliferation after vascular injury.
Neointimal hyperplasia was markedly suppressed in TLR-2KO mice compared with WT mice at both 2 and 4 weeks after vascular injury.
Conclusions: These findings suggested that endogenous TLR-2 activation might play a central role in the regulation of vascular
inflammation as well as subsequent neointimal formation in injured vessels.
 2006 Elsevier Inc. All rights reserved.

Keywords: Immune system; Inflammation; ROS; Toll-like receptor; Vascular injury

There is increasing evidence that inflammation of blood
vessel plays a critical role during the initiation and mainte-
nance of vascular diseases such as atherosclerosis forma-
tion and restenosis after stent implantation and
angioplasty. A number of inflammatory factors were sug-
gested to play a critical role in the regulation of the process
of injury and repair after vascular injury, but the exact role
and function of each inflammatory molecules including
*
Corresponding author. Fax: +1 585 273 3156.
E-mail address: Tetsuro_Shishido@URMC.Rochester.edu (T. Shishi-
do).
TNF-a, IL-1, IL-6, and MCP-1 remains unclear. Especial-
ly, the ‘‘complexity’’ of inflammatory process in cardiovas-
cular disease received attention after the data that
randomized and double-blind clinical study of infliximab
and etanercept, which are antagonists of TNF-a, worsened
the heart failure dose-dependently, even though many ani-
mal studies have shown the benefits in heart. While, the
benefits of antagonizing TNF-a on neointimal formation
after vascular injury [1] or vascular smooth muscle prolifer-
ation in vein graft culture have also been reported [2]. In
contrast, another report showed that TNF-a increased
inflammatory responses, but had no effect on neointimal

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.05.056
 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453
formation after vascular injury [3]. These data support the
idea that TNF-a may not be the ‘‘solo’’ factor to regulate
inflammatory responses in the vessels and suggest the par-
ticipation of other critical factor to regulate this vascular
injury process.
During the search of critical and responsible inflamma-
tory response and signaling in vascular injury, we became
interested in Toll-like receptors. Toll-like receptors (TLRs)
have IL-1 receptor-like intracellular signaling pathways
and are widely accepted as a key receptor for immune
response. Activation of TLR pathway leads to nuclear
localization of NF-jB/Rel-type transcription factor and
gene expressions of pro-inflammatory cytokine [4]. Until
now thirteen receptors have been identified with different
or overlapping ligands. TLR-1 and 2 heterodimers can rec-
ognize bacterial lipoproteins, TLR-4 recognizes LPS from
Gram-negative bacteria, and TLR-9 recognizes bacterial
DNA (unmethylated CG motifs). In addition, other studies
have demonstrated that TLR-mediated pathways are also
activated by endogenous ligands, such as heat-shock pro-
tein and oxidative stress [5,6]. Interestingly, recent studies
showed that TLR-2 and 4 signal in response to the extracel-
lular matrix component hyaluronan, which fragments
when lungs are injured [7]. TLR-2 and 4 drive inflamma-
tion in response to the fragment form of hyaluronan. How-
ever, although TLR-2 and 4 double knock out mice
markedly inhibited inflammatory response in bleomycin-
mediated lung injury, lung injury of interstitial thickness
in lung tissue and protein accumulation in bronchoalveolar
lavage fluid in double knockout mice were significantly
increased. These data suggest that inflammation is not a
necessary ‘‘bad’’ process, and also possesses a ‘‘good’’ side.
For example, inflammation counteracts infection and has a
role to repair tissue injury. Therefore, the balance between
injury and repair during inflammation may decide the
‘‘fate’’ of the process of the disease [8].
Previously, Schoneveld et al. have reported that ‘‘exog-
enous’’ ligand stimulation of TLR-2 induced neointimal
formation after vascular injury and atherosclerotic forma-
tion, and these effects were reduced in TLR-2 knockout
mice [9]. Of note, the role of endogenous TLR-2 activation
without the treatment of exogenous TLR-2 ligand in the
process of neointimal formation after vascular injury
remains largely unknown. Since TLR-2 and 4 double
knockout mice enhanced lung injury, it is possible that
the endogenous TLR-2 activation may have a protective
effect and inhibit neointimal formation after vascular
injury.
To determine the role of endogenous TLR-2 activa-
tion after vascular injury, in this study we have created
vascular injury by cuff-placement onto the femoral artery
in non-transgenic littermate control (NLC) and TLR-2
knockout mice. First we found that TLR-2 expression
was significantly increased after vascular injury. Next,
we demonstrated that TLR-2 deficiency resulted in
suppressed production of pro-inflammatory cytokines
including TNF-a, IL-1b, IL-6, and MCP-1, as well as
1447
reactive oxygen species (ROS) in injured vessels. Finally,
although we did not treat the vessels with TLR-2 ligand
in our injury model, we observed marked decrease of
neointimal formation in TLR-2 knockout mice compared
with NLC, which is opposite to the lung injury model as
previously described [7]. To our knowledge, this is the
first report to show the critical role of ‘‘endogenous’’
TLR-2 activation in injured vessel to regulate cytokine
production, ROS production as well as subsequent neo-
intimal formation. Based on our current data, TLR-2
could regulate variety of cytokines production including
TNF-a, IL-1b, IL-6, and MCP-1, and also ROS produc-
tion after vascular injury. These data suggest the central
role of endogenous TLR-2 activation after vascular inju-
ry, and inhibition of TLR-2 may provide a novel and
stronger therapeutic approach to inhibit vascular injury
than antagonizing TNF-a.
Materials and methods
Animal treatment. The TLR-2 knockout (KO) mice were kindly sup-
plied from Drs. Shizuo Akira and Osamu Takeuchi (Osaka University,
Osaka, Japan) [10]. Male TLR-2 KO mice and normal wild-type (WT)
mice with same C57BL/6 background were used. Mice were housed in a
facility with a 12/12 light and dark cycle, and were given free access to
water and standard rodent chow. The room was kept specific pathogen-
free. The animals were handled according to the animal welfare regula-
tions of Yamagata University, and the study protocol was approved by
the Animal Subjects Committee of Yamagata University. The investiga-
tion conforms to the Guide for the Care and Use of Laboratory Animals
published by the US National Institute of Health. The mice were anes-
thetized with ketamine (70 mg/kg) and xylazine (7 mg/kg) by intraperi-
toneal injection. The femoral artery of the mouse was isolated from the
surrounding tissues, and a polyethylene cuff tube (2 mm long PE-90; inner
diameter, 0.86 mm; outer diameter, 1.27 mm; Becton–Dickinson, USA)
was loosely placed around the artery and closed with suture as described
previously [11].
Physiological analysis. Body weight, blood pressure, and heart rate
were measured before and 4 weeks after cuff-placement. Blood pressure
and heart rate were recorded by photoelectric oscillometric method (UR-
5000; Ueda Electric Works, Tokyo, Japan) in an awake state [12].
Extraction of total RNAs and reverse transcriptase-polymerase chain
reaction (RT-PCR). RNA extraction and RT-PCR study were performed
as described previously [13,14]. PCR primers for TLR-2 were 5 0 -GTC
TCT GCG ACC TAG AAG TGG A-3 0 (forward) and 5 0 -CGG AGG
GAA TAG AGG TGA AAG A-3 0 (reverse) (GenBank Accession No.
AF185284), primers for TNF-a were 5 0 -CAG CCT CTT CTC ATT CCT
GCT TGT G-3 0 (forward) and 5 0 -CTG GAA GAC TCC TCC CAG GTA
TAT-3 0 (reverse) (GenBank Accession No. X02611), primers for IL-1b
were 5 0 -ATG GCA ACT GTT CCT GAA-3 0 (forward) and 5 0 -GTA CAA
AGC TCA TGG AGA-3 0 (reverse) (GenBank Accession No. BC011437),
primers for IL-6 were 5 0 -TTC CCT ACT TCA CAA GTC-3 0 (forward)
and 5 0 -GGT TTG CCG AGT AGA TCT-3 0 (reverse) (GenBank Acces-
sion No. X54542), primers for MCP-1 were 5 0 - ACT GAA GCC AGC
TCT CTC TTC CTC-3 0 (forward) and 5 0 -TTC CTT CTT GGG GTC
AGC ACA GAC-3 0 (reverse) (GenBank Accession No. J04467) and
primers for GAPDH were 5 0 -ACT CCA CTC ACG GCA AAT TCA
ACG G-3 0 (forward) and 5 0 -AGG GGC GGA GAT GAT GAC CC-3 0
(reverse) (GenBank Accession No. M32599). The PCR products were
fractionated on a 1–2% agarose gel and visualized by ethidium bromide
staining. The intensities of the bands were normalized for GAPDH and
were expressed as fold increase over WT mice.
Immunohistochemistry. The frozen sections (4 lm thickness) were
immunohistochemically stained by the streptavidin–biotin–peroxidase
1448 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453

method as described previously [11,15]. Briefly, endogenous peroxidase used cuff-placement as a vascular injury model, because
and the non-specific binding of the antibody were blocked with 0.3% the neointimal formation induced by this model is
hydrogen peroxide in methanol and 2% goat serum in PBS, respectively.
The antibody to TLR-2 or non-specific IgG (Santa Cruze Biotechnology,
very reliable and reproducible, especially in mice. Again,
Inc., Santa Cruz, CA, USA) in 1% BSA in PBS was applied to the sections we did not treat the vessels with lipopolysaccharide
and incubated for 16–24 h at 4 C. After washing, biotinylated secondary (TLR-4 ligand) or synthetic TLR-2 ligand, Pam3Cys-
antibody and then streptavidin conjugate were applied to each tissue SK4, which is different from the previous reports [9].
sections. Color development was performed with 3,3 0 -diaminobenzidine, Although we did not add TLR-2 ligand, expression of
and slides were counterstained with hematoxylin [15].
Measurement of superoxide generation by DHE staining. The oxi-
TLR-2 mRNA was significantly increased after one week
dative fluorescent dye dihydroethidium (DHE; Sigma–Aldrich, St. of cuff-mediated vascular injury, and maintained its
Louis, MO, USA) was used to measure in situ production of super- increase until four weeks after injury as shown in Fig. 1.
oxide. Fresh unfixed frozen 30 mm thick sections obtained from the To investigate the localization of TLR-2 expression after
cuffed femoral artery of mice were placed on a glass slide. DHE cuff-mediated vascular injury, we performed immunohisto-
(20 mmol/L) was applied topically to each tissue section, and the sec-
tions were then incubated at 37 C for 30 min in a humidified chamber
chemical staining in vessels after 1 week of vascular injury
protected from light. In the presence of O2  , DHE is converted to the with anti-TLR-2 polyclonal antibody and compared the
fluorescent molecule ethidium bromide, which binds to DNA in the TLR-2 expression with non-injured vessels as shown in
nucleus and fluoresces red. Slides were briefly washed in PBS and Fig. 2. In non-injured vessel we could not detect TLR-2
coverslips were placed over the sections. For quantification of vascular expression in both smooth muscle medial layer and
ethidium fluorescence, fluorescence (intensity · area) was measured only
on the vascular area including endothelial, intimal, and medial
adventitial cells. In contrast, although we did not observe
area [16]. significant neointimal formation after 1 week of injury
Tissue preparation and histology. At 2 and 4 weeks after cuff-placement,
the mice were killed by an overdose of the anesthesia, and thoracic aortas
were perfused with phosphate buffered saline (PBS) and subsequently with A Cuff +
4% formaldehyde. Paraffin sections of the femoral artery were stained with
either elastica-Gorldner or hematoxylin and eosin. With the use of com- Cuff - 1 week 4 weeks
puterized morphometric analysis, the intimal cross-sectional area between
the lumen and the internal elastic lamina and the medial area between the
internal and external elastic lamina were measured as previously reported
TLR-2
[11,17] using Scion image for windowsPlus software (Image, Scion Cor-
poration, MD, USA).
Statistics analysis. All values are expressed as means ± SEM. To
compare histological data and gene expression of TLR-2, one-way GAPDH
ANOVA followed by post hoc procedure was performed. The other data
were compared with Student’s t-test. Statistical significance was accepted
at a value of P < 0.05.
B 3 *
*
Results
TLR-2 expressions
(fold increase)

2
TLR-2 mRNA expression was markedly increased after
cuff-mediated vascular injury
1

There are contradictory data on TLR-2 expression in

atherosclerotic formation [13,14]. In contrast, TLR-ex- 0
pression after vascular injury remains unclear. Therefore,
ks
-

k
ee
ff

ee
Cu

first we investigated the time-dependent TLR-2 mRNA

1w

4w

expression after cuff-mediated vascular injury. At base-

line, body weight, heart rate, and blood pressure were
similar between NLC and TLR-2 KO mice (data not
shown) as previously reported [15,18]. At 4 weeks after
cuff-placement, there were no significant differences in
the body weight, heart rate, or blood pressure between
NLC and TLR-2 KO mice as shown in Table 1. We
Fig. 1. Effect of cuff-placement on expression of Toll-like receptor (TLR)-
2 in femoral artery. (A) RT-PCR band of TLR-2 and GAPDH in wild-
type (WT) mice. (B) Results were quantified by densitometry and
expressed as means ± SEM of six separate experiments. Each sample
was obtained from 4–6 femoral arteries. Gene expressions of TLR-2 were
increased 1 week after cuff-placement and subsequently slightly decreased
in cuff-injured artery in WT mice. *P < 0.05 vs. cuff.

Table 1
Physiological findings at 4 weeks after cuff-placement
Body weight (g) Heart rate (bpm) SBP (mmHg) DBP (mmHg)
WT mice 31.0 ± 0.4 556 ± 25 119 + 2 53 ± 4
TLR-2 KO mice 32.3 ± 0.9 564 ± 21 116 ± 1 52 ± 3
SBP and DBP indicate blood pressure in systolic and diastolic, respectively. All data are expressed as means ± SEM from six experiments.
 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453 1449

TLR-2 Control IgG

Fig. 2. Representative immunohistochemical photomicrographs of cross-sections of femoral arteries stained with antibody to TLR-2 (original
magnification 400·). After cuff-placement, TLR-2 immunoreactivity (brown staining) is markedly increased in cuffed-artery obtained from WT mice (left).
Right figure represents a negative control. Arrow and arrowhead indicate external elastic lamina and endothelium, respectively.

yet, we did observe significant increase of TLR-2 expres- superoxide production was markedly increased in injured
sion in both medial layer of smooth muscle cells and adven- vessels and adventitia (Fig. 4B). However, in TLR-2
titial cells. These data suggest that not only adventitial knockout mice superoxide production after one week of
fibroblasts, but also smooth muscle cells, could express a
functional TLR-2, which is different from TLR-4 expres- A Cuff 1W
sion [9].
TNF-α
Critical role of endogenous TLR-2 activation on
inflammatory genes expression after vascular injury
IL-1α
We examined inflammatory gene expressions of TNF-a,
IL-1b, IL-6, and MCP-1 after vascular injury. We selected
IL-6
one week after vascular injury to investigate these genes
expression, because (1) we and other group found that
growth rate of neointimal formation is maximum at one MCP-1
week after injury [11,19,20] the difference of cell compo-
nents in the injured vessels (smooth muscle vs. adventitial
GAPDH
cells) should not affect the genes expression results because
of limited amount of neointimal formation until one week
O
O
R- T

T
2K
2K
TL W

after vascular injury. After one week of cuff-placement,

R-
TL

TNF-a, IL-1b, IL-6, and MCP-1 mRNA expressions were

significantly increased in NLC mice, although we did not B TNF-α IL-1α IL-6 MCP-1
observe marked neointimal formation at this moment. 1.2
(Fold increase)

Interestingly, we found that all cytokine expressions, which 1.0

* * ** *
we investigated in this study, were significantly diminished 0.8
in TLR-2 knockout mice (Fig. 3). We did not treat any 0.6
0.4
exogenous TLR-2 ligand in the vessels, which is different
0.2
from the previous report [9], suggesting the central role
0
of endogenous TLR-2 activation on regulating cytokine
O
O

O
T

T
T

T
2K

2K

2K

2K
W

W
W

expressions after vascular injury.

R-

R-

R-

R-
TL

TL

TL

TL

ROS production after vascular injury was abolished in
TLR-2 knockout mice
Several studies have demonstrated that ROS produc-
tion was increased in cuff-injured artery [21]. In this
study, we used dihydroethidium (DHE), an oxidative
fluorescent dye, to detect ROS production in injured
vessels. The chemiluminescent signal attributable to
Fig. 3. (A) RT-PCR analysis for TNF-a, IL-1b, IL-6, and MCP-1
mRNA. Cuffed-arteries at 1 week after operation and control intact
arteries were harvested and frozen. Each sample was obtained from 4–6
arteries. One representative result of four independent RT-PCR is
shown. (B) Group data for relative band intensity of TNF-a, IL-1b,
IL-6, and MCP-1. Expressions of pro-atherosclerotic genes were
suppressed in TLR-2 KO mice compared with WT mice. All data are
expressed as means ± SEM from six experiments. *P < 0.05 and
**P < 0.01 vs. WT.
1450 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453

A TLR-2 KO-
WT-Sham WT-Cuff Cuff

B TLR-2 KO-
WT-Cuff Cuff

vessel wall

adventitia

C 1.2
(Relative intensity)

1.0
Vascular DHE
fluorescence

0.8
0.6
*
0.4
0.2
0
O
T
W

2K
R-
TL

Fig. 4. In situ detection of superoxide by dihydroethidium fluorescence. (A) Cross-section of non-cuffed femoral artery obtained from control WT mice
and of cuffed-femoral artery in WT and TLR-2 KO mice. (Original magnification 100·). Although fluorescent detection was slight in non-cuffed artery,
dihydroethidium fluorescence was increased after cuff-placement. (B) Boxed region in (A) at vascular and adventitial area. (Original magnification 400·).
Arrow and arrowhead indicate external elastic lamina and endothelium, respectively. (C) Quantification of reactive oxygen species production in vascular
wall. Superoxide production from cuffed-artery was attenuated in TLR-2 KO mice compared with WT mice. Data are expressed as means ± SEM.
*P < 0.05 vs. WT.

injury was markedly suppressed compared with NLC
mice (Fig. 4B and C).
Neointimal formation was markedly suppressed in TLR-2
knock out mice
Since we found that both cytokine expressions and ROS
production were significantly decreased in TLR-2 knockout
mice, we examined whether the deficiency of TLR-2 could
affect the neointimal formation after vascular injury,
although we did not add TLR-2 ligand. There was no obvi-
ous difference in the morphological appearance of the medial
area and adventitial area between NLC and TLR-2 KO mice
in non-injured vessels (data not shown). After vascular
injury, we found significant neointimal formation after
vascular injury in NLC mice (Fig. 5, left). In contrast, the
neointimal formation was markedly suppressed in TLR-2
KO mice compared to NLC mice at 2 weeks (P < 0.05) and
also at 4 weeks (P < 0.01) after vascular injury (Fig. 5, right).
No significant difference in medial area between NLC and
TLR-2 KO mice after vascular injury was observed
(Fig. 6A), and the ratio of the neointimal/medial area was
significantly attenuated at 2 and 4 week after vascular injury
in TLR-2 KO mice as shown in Fig. 6C (P < 0.05 at 2 weeks
and P < 0.01 at 4 weeks after injury). These data suggest that
‘‘endogenous’’ TLR-2 activation is critical for neointimal
formation after vascular injury.
Discussion
In the present study, we described the important role of
endogenous TLR-2 activation on cytokine expression and
ROS production as well as subsequent neointimal forma-
tion after vascular injury. TLR-2 expression was signifi-
cantly increased in both medial layer of smooth muscle
and adventitial cells after vascular injury. Interestingly,
we found that the deficiency of TLR-2 markedly decreased
all cytokine expression, which we investigated, and ROS
production. Furthermore, we found that neointimal forma-
tion was significantly decreased in TLR-2 knockout mice
 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453 1451

WT TLR-2 KO

2 weeks

neointima

WT TLR-2 KO

4 weeks

neointima

Fig. 5. Histology of WT and TLR-2 KO mice. Representative histological photomicrographs of cross-sections of femoral arteries stained with elastica-
Gorldner in WT and TLR-2 KO mice are shown. Although neointimal hyperplasia was occurred in both WT and TLR-2 KO mouse, the neointimal
formation was inhibited in TLR-2 KO mouse compared with WT mouse. Scale bars, 100 lm.

compared to NLC mice. To our knowledge, these data pro-
vided the first direct evidence of the involvement of endog-
enous TLR-2 activation in inflammation as well as
subsequent neointimal formation after vascular injury.
Based on this work and previous investigator’s data, we
proposed a working model as shown in Fig. 7. Although
the exact mechanism remains unclear, vascular injury
activates endogenous TLR-2 activation. As previously
reported [4,10], TLR-2 activation can significantly increase
NF-jB activation and subsequent cytokine production.
While it is also reported that TLR-2 expression is strongly
regulated by NF-jB activation [22]. Therefore, it is possible
that this positive feedback loop of TLR-2 and NF-jB
worsened the inflammatory responses after vascular injury
(Fig. 7). In addition, we found that ROS production after
vascular injury was also regulated by TLR-2 activation.
ROS also affects vascular inflammation and involves in
cytokine production. Thus, it is also intriguing to hypoth-
esize the existence of direct and/or indirect positive feed-
back loop between TLR-2 activation and inflammatory
responses in injured vessels (Fig. 7). We believe that these
positive feedback loops of TLR-2 signaling pathways have
a central role in regulating inflammatory responses in
injured vessels. Therefore, the specific deletion of TLR-2
1452 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453

A (μm2) vascular injury

18000
+
Medial Area

12000
oxidative stress TLR-2

6000

NF- B activation
+
0
2W

4W
W

+
2

O-

O-
T-

T-
W

2K

2K
W

inflammatory cytokine
B (μm2)
Fig. 7. Schematic representation of the TLR-2 mediated vascular inflam-
8000
mation. After vascular injury, many stimuli including oxidative stress
Neo-intimal Area

induce TLR-2 mediated inflammatory response at the injured artery.

Increase of inflammatory cytokine causes NF-jB activation, oxidative
stress production, and TLR-2 expressions. Up-regulated TLR-2 expres-
4000 sions elicit vascular inflammation prominently.
* **
vascular injury remains unclear. TLR-2 is activated by bac-
0
terial lipoproteins such as components of Gram-positive
2W

4W
W
W

bacteria [23], mycobacteria [24], and Borelia [25]. Recently,

4
2

O-

O-
T-
T-

Jiang et al. have reported that the fragment form of the

W

2K

2K
W

extracellular matrix component of hyaluronan activates

C TLR-2 and TLR-4 in epithelial cells [25]. Hyaluronan is a
60 massive sugar polymer and a key ‘‘shock-absorbing’’ mate-
rial in the extracellular matrix. During inflammation, hyal-
Intima / Media
Volume Ratio

uronidase breaks down hyaluronan, and generates pro-

40 inflammatory fragments of hyaluronan, which activates
NF-jB activation [8], and Jiang et al. reported that hyalu-
20
* ** ronan-mediated NF-jB activation was completely abol-
ished in TLR-4 and TLR-2 double knockout mice.
Therefore, the fragmentation of hyaluronan may involve
0 in endogenous TLR-2 activation after vascular injury. Fur-
ther investigation of the involvement of hyaluronan
2W

4W
2W

4W

fragmentation in vascular injury will be required.

O-

O-
T-

T-
W

2K

2K
W

Previously, Schoneveld et al. have reported that

Fig. 6. Comparison of the lesion formation after cuff-placement. Cross-
sectional areas of media (A) and intima (B) were evaluated at 2 and 4
weeks after cuff-placement. There was no significant difference between
WT and TLR-2 KO mice in medial area, however, neointimal hyperplasia
was suppressed in TLR-2 KO mice compared with WT mice at 2 and 4
weeks after cuff-injury. Cuff-injury increased the intimal/medial area ratio
(C) in both WT and TLR-2 KO mice, however, TLR-2 KO mice had less
increases in intimal/medial area ratio than WT mice. *P < 0.05 and
**P < 0.01 vs. WT in same time point. Open and black bar represents WT
and TLR-2 KO mice, respectively (n = 9, means ± SEM).
expression can also inhibit other cytokines expression and
show a marked decrease of neointimal formation after
vascular injury. Further investigation is necessary to deter-
mine the role of these positive feedback loops in vascular
injury.
Although this study clearly shows the involvement of
endogenous TLR-2 activation in neointimal formation,
the exact mechanism of endogenous TLR-2 activation after
Pam(3)Cys-SK(4), which is one of the synthetic TLR-2
ligand, directly progresses neointimal formation and ath-
erosclerotic plaque [9]. They also showed that, in vitro
study using cultured fibroblasts, TLR-2 ligand induced
inflammatory cytokines, such as IL-1a, IL1b, IL-6, and
IL-8. In the present study, we showed that productions
of pro-inflammatory cytokines, such as TNF-a, IL-1b,
IL-6, during vascular inflammation were suppressed in
TLR-2 KO mice than in NLC mice. Moreover, ROS pro-
duction in the injured vessels was attenuated in TLR-2
KO mice compared with NLC mice. These findings indicat-
ed that TLR-2 signaling pathway forms a central role in
regulating inflammatory responses as well as subsequent
neointimal formation, and may provide a novel and
stronger therapeutic strategy to inhibit inflammatory
responses and subsequent neointimal formation than other
anti-inflammatory therapies such as dexamethazone or
antagonizing TNF-a.
 T. Shishido et al. / Biochemical and Biophysical Research Communications 345 (2006) 1446–1453
Acknowledgments
We thank Mr. Eiji Tsuchida, Ms. Shuku Takahashi, and
Ms. Sachi Adachi for their excellent technical assistance.
This study was supported in part by Grants-in-Aid for Sci-
entific Research (Nos. 16590657 and 14570635) and for the
21st Century Center of Excellence (COE) program of the
Japan Society for the Promotion of Science (both from
the Ministry of Education, Science, Sports and Culture,
Japan) and grants from the Mochida Memorial Founda-
tion and the Japan Heart Foundation.
References
[1] A.M. Miller, A.R. McPhaden, A. Preston, R.M. Wadsworth, C.L.
Wainwright, TNFalpha increases the inflammatory response to
vascular balloon injury without accelerating neointimal formation,
Atherosclerosis 179 (2005) 51–59.
[2] Q. Javed, N. Swanson, H. Vohra, H. Thurston, A.H. Gershlick,
Tumor necrosis factor-alpha antibody eluting stents reduce vascular
smooth muscle cell proliferation in saphenous vein organ culture,
Exp. Mol. Pathol. 73 (2002) 104–111.
[3] A. Niemann-Jonsson, M.P. Ares, Z.Q. Yan, D.X. Bu, G.N. Fredrik-
son, L. Branen, I. Porn-Ares, A.H. Nilsson, J. Nilsson, Increased rate
of apoptosis in intimal arterial smooth muscle cells through endog-
enous activation of TNF receptors, Arterioscler. Thromb. Vasc. Biol.
21 (2001) 1909–1914.
[4] S. Akira, Toll-like receptor signaling, J. Biol. Chem. 278 (2003)
38105–38108.
[5] R.M. Vabulas, P. Ahmad-Nejad, C. da Costa, T. Miethke, C.J.
Kirschning, H. Hacker, H. Wagner, Endocytosed HSP60s use toll-like
receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1
receptor signaling pathway in innate immune cells, J. Biol. Chem.
276 (2001) 31332–31339.
[6] S. Frantz, R.A. Kelly, T. Bourcier, Role of TLR-2 in the activation of
nuclear factor kappaB by oxidative stress in cardiac myocytes, J. Biol.
Chem. 276 (2001) 5197–5203.
[7] D. Jiang, J. Liang, J. Fan, S. Yu, S. Chen, Y. Luo, G.D. Prestwich,
M.M. Mascarenhas, H.G. Garg, D.A. Quinn, R.J. Homer, D.R.
Goldstein, R. Bucala, P.J. Lee, R. Medzhitov, P.W. Noble, Regula-
tion of lung injury and repair by Toll-like receptors and hyaluronan,
Nat. Med. 11 (2005) 1173–1179.
[8] L.A. O’Neill, TLRs play good cop, bad cop in the lung, Nat. Med. 11
(2005) 1161–1162.
[9] A.H. Schoneveld, M.M. Oude Nijhuis, B. van Middelaar, J.D.
Laman, D.P. de Kleijn, G. Pasterkamp, Toll-like receptor 2 stimu-
lation induces intimal hyperplasia and atherosclerotic lesion devel-
opment, Cardiovasc. Res. 66 (2005) 162–169.
[10] O. Takeuchi, K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa,
K. Takeda, S. Akira, Differential roles of TLR2 and TLR4 in
recognition of Gram-negative and Gram-positive bacterial cell wall
components, Immunity 11 (1999) 443–451.
[11] M. Akishita, M. Horiuchi, H. Yamada, L. Zhang, G. Shirakami, K.
Tamura, Y. Ouchi, V.J. Dzau, Inflammation influences vascular
remodeling through AT2 receptor expression and signaling, Physiol.
Genomics 2 (2000) 13–20.
1453
[12] H. Takahashi, Y. Takeishi, T. Miyamoto, T. Shishido, T. Arimoto, T.
Konta, T. Miyashita, M. Ito, I. Kubota, Protein kinase C and
extracellular signal regulated kinase are involved in cardiac hyper-
trophy of rats with progressive renal injury, Eur. J. Clin. Invest. 34
(2004) 85–93.
[13] K. Edfeldt, J. Swedenborg, G.K. Hansson, Z.Q. Yan, Expression
of toll-like receptors in human atherosclerotic lesions: a possible
pathway for plaque activation, Circulation 105 (2002)
1158–1161.
[14] F.C. Gibson 3rd, C. Hong, H.H. Chou, H. Yumoto, J. Chen, E. Lien,
J. Wong, C.A. Genco, Innate immune recognition of invasive bacteria
accelerates atherosclerosis in apolipoprotein E-deficient mice, Circu-
lation 109 (2004) 2801–2806.
[15] T. Shishido, N. Nozaki, S. Yamaguchi, Y. Shibata, J. Nitobe, T.
Miyamoto, H. Takahashi, T. Arimoto, K. Maeda, M. Yamakawa, O.
Takeuchi, S. Akira, Y. Takeishi, I. Kubota, Toll-like receptor-2
modulates ventricular remodeling after myocardial infarction, Circu-
lation 108 (2003) 2905–2910.
[16] N.J. Alp, M.A. McAteer, J. Khoo, R.P. Choudhury, K.M. Channon,
Increased endothelial tetrahydrobiopterin synthesis by targeted
transgenic GTP-cyclohydrolase I overexpression reduces endothelial
dysfunction and atherosclerosis in ApoE-knockout mice, Arterioscler.
Thromb. Vasc. Biol. 24 (2004) 445–450.
[17] A. Vink, A.H. Schoneveld, J.J. van der Meer, B.J. van
Middelaar, J.P. Sluijter, M.B. Smeets, P.H. Quax, S.K. Lim, C.
Borst, G. Pasterkamp, D.P. de Kleijn, In vivo evidence for a role
of toll-like receptor 4 in the development of intimal lesions,
Circulation 106 (2002) 1985–1990.
[18] N. Nozaki, T. Shishido, Y. Takeishi, I. Kubota, Modulation of
doxorubicin-induced cardiac dysfunction in toll-like receptor-2-
knockout mice, Circulation 110 (2004) 2869–2874.
[19] H.W. Liu, M. Iwai, Y. Takeda-Matsubara, L. Wu, J.M. Li, M.
Okumura, T.X. Cui, M. Horiuchi, Effect of estrogen and AT1
receptor blocker on neointima formation, Hypertension 40 (2002)
451–457, discussion 448–450.
[20] J. Suzuki, M. Iwai, H. Nakagami, L. Wu, R. Chen, T. Sugaya, M.
Hamada, K. Hiwada, M. Horiuchi, Role of angiotensin II-regulated
apoptosis through distinct AT1 and AT2 receptors in neointimal
formation, Circulation 106 (2002) 847–853.
[21] J. Kawai, K. Ando, A. Tojo, T. Shimosawa, K. Takahashi, M.L.
Onozato, M. Yamasaki, T. Ogita, T. Nakaoka, T. Fujita, Endoge-
nous adrenomedullin protects against vascular response to injury in
mice, Circulation 109 (2004) 1147–1153.
[22] S.J. Kammanadiminti, B.J. Mann, L. Dutil, K. Chadee, Regulation
of Toll-like receptor-2 expression by the Gal-lectin of Entamoeba
histolytica, FASEB J. 18 (2004) 155–157.
[23] R. Schwandner, R. Dziarski, H. Wesche, M. Rothe, C.J. Kirsch-
ning, Peptidoglycan- and lipoteichoic acid-induced cell activation is
mediated by toll-like receptor 2, J. Biol. Chem. 274 (1999)
17406–17409.
[24] H.D. Brightbill, D.H. Libraty, S.R. Krutzik, R.B. Yang, J.T. Belisle,
J.R. Bleharski, M. Maitland, M.V. Norgard, S.E. Plevy, S.T. Smale,
P.J. Brennan, B.R. Bloom, P.J. Godowski, R.L. Modlin, Host defense
mechanisms triggered by microbial lipoproteins through toll-like
receptors, Science 285 (1999) 732–736.
[25] M. Hirschfeld, C.J. Kirschning, R. Schwandner, H. Wesche, J.H.
Weis, R.M. Wooten, J.J. Weis, Cutting edge: inflammatory signaling
by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor
2, J. Immunol. 163 (1999) 2382–2386.