Content uploaded by Yuji Nadatani
Author content
All content in this area was uploaded by Yuji Nadatani on Apr 02, 2015
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
High-Mobility Group Box 1 Inhibits Gastric Ulcer Healing
through Toll-Like Receptor 4 and Receptor for Advanced
Glycation End Products
Yuji Nadatani1, Toshio Watanabe1*, Tetsuya Tanigawa1, Fumikazu Ohkawa2, Shogo Takeda1, Akira
Higashimori1, Mitsue Sogawa1, Hirokazu Yamagami1, Masatsugu Shiba1, Kenji Watanabe1, Kazunari
Tominaga1, Yasuhiro Fujiwara1, Koji Takeuchi2, Tetsuo Arakawa1
1 Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan, 2 Division of Pathological Sciences, Department of
Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Kyoto, Japan
Abstract
High-mobility group box 1 (HMGB1) was initially discovered as a nuclear protein that interacts with DNA as a
chromatin-associated non-histone protein to stabilize nucleosomes and to regulate the transcription of many genes in
the nucleus. Once leaked or actively secreted into the extracellular environment, HMGB1 activates inflammatory
pathways by stimulating multiple receptors, including Toll-like receptor (TLR) 2, TLR4, and receptor for advanced
glycation end products (RAGE), leading to tissue injury. Although HMGB1’s ability to induce inflammation has been
well documented, no studies have examined the role of HMGB1 in wound healing in the gastrointestinal field. The
aim of this study was to evaluate the role of HMGB1 and its receptors in the healing of gastric ulcers. We also
investigated which receptor among TLR2, TLR4, or RAGE mediates HMGB1’s effects on ulcer healing. Gastric ulcers
were induced by serosal application of acetic acid in mice, and gastric tissues were processed for further evaluation.
The induction of ulcer increased the immunohistochemical staining of cytoplasmic HMGB1 and elevated serum
HMGB1 levels. Ulcer size, myeloperoxidase (MPO) activity, and the expression of tumor necrosis factor α (TNFα)
mRNA peaked on day 4. Intraperitoneal administration of HMGB1 delayed ulcer healing and elevated MPO activity
and TNFα expression. In contrast, administration of anti-HMGB1 antibody promoted ulcer healing and reduced MPO
activity and TNFα expression. TLR4 and RAGE deficiency enhanced ulcer healing and reduced the level of TNFα,
whereas ulcer healing in TLR2 knockout (KO) mice was similar to that in wild-type mice. In TLR4 KO and RAGE KO
mice, exogenous HMGB1 did not affect ulcer healing and TNFα expression. Thus, we showed that HMGB1 is a
complicating factor in the gastric ulcer healing process, which acts through TLR4 and RAGE to induce excessive
inflammatory responses.
Citation: Nadatani Y, Watanabe T, Tanigawa T, Ohkawa F, Takeda S, et al. (2013) High-Mobility Group Box 1 Inhibits Gastric Ulcer Healing through Toll-
Like Receptor 4 and Receptor for Advanced Glycation End Products. PLoS ONE 8(11): e80130. doi:10.1371/journal.pone.0080130
Editor: Mathias Chamaillard, INSERM, France
Received May 29, 2013; Accepted September 30, 2013; Published November 11, 2013
Copyright: © 2013 Nadatani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: Kenji Watanabe - Grant/Research Support from the Mitsubishi Tanabe Pharma Corporation, Abbott Japan Co., LTD.; Mitsubishi
Tanabe Pharma Corporation; Speaking and Teaching: Abbott Japan Co.,LTD. Yasuhiro Fujiwara - Speaking and Teaching: Eisai Co. Ltd Tetsuo Arakawa -
Advisory Committees or Review Panels: Eisai Co. Ltd, Otsuka Pharmaceutical Co. Ltd. The other authors have declared that no competing interests exist.
This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: watanabet@med.osaka-cu.ac.jp
Introduction
High-mobility group box protein 1 (HMGB1), a member of the
high-mobility group protein superfamily, is a nuclear protein [1].
HMGB1 interacts with DNA as a chromatin-associated
nonhistone protein to stabilize nucleosomes and to regulate the
transcription of many genes in the nucleus [2]. When leaked
from a cell during necrotic cell death [3] or actively secreted
into the extracellular environment by monocytes and
macrophages [3,4], HMGB1 acts as an alarmin with potent
proinflammatory properties [5].
The best studied HMGB1 receptors are Toll-like receptor
(TLR) 2 [6,7], TLR 4 [6-9], and receptor for advanced glycation
end products (RAGE) [6,8]. TLR2 and TLR4 are members of
the TLR family, and they play a crucial role in innate immune
responses to pathogen-associated molecular patterns and
damage-associated molecular pattern molecules [10]. TLR2
primarily recognizes components of the gram-positive bacterial
cell wall, and TLR4 primarily recognizes lipopolysaccharide,
which is the major cell wall component of gram-negative
bacteria. Triggering TLR2 and TLR4 signaling pathways leads
to the activation of nuclear factor κB (NF-κB), through the
PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80130
accessory protein MyD88, and the subsequent regulation of
immune and inflammatory genes, including inflammatory
cytokines such as tumor necrosis factor α (TNFα), with the
activation of mitogen-activated protein kinases [11-13].
Receptor for advanced glycation end products (RAGE) is a
multi-ligand receptor that belongs to the immunoglobulin
superfamily [14]. Other known RAGE ligands include amyloid
[15] and S100 [16]. Multiple experiments have suggested that
the ligand-RAGE interaction also activates NF-κB and mitogen-
activated protein kinases [17-20].
Many pathological conditions are related to the
proinflammatory properties of HMGB1. Previous reports
demonstrated that HMGB1 plays a critical role in endotoxemia
[21], acute pancreatitis [22], acute respiratory distress
syndrome [23], some autoimmune diseases [24], cerebral
ischemia injury [25], and ischemia-reperfusion (I-R) injuries of
the liver [26], heart [27], and kidney [28]. With regard to the
gastrointestinal tract, HMGB1 is a complicating factor in
experimental colitis [29,30], and non-steroidal anti-inflammatory
drug induced small intestinal injury [31].
At present, the role of HMGB1 in wound healing is unclear,
although its ability to induce inflammation has been well
documented, as described above. In the gastrointestinal field,
no study has examined the role of HMGB1 in wound healing.
The aim of this study was to investigate the role of HMGB1 in
gastric ulcer healing. We investigated the role of HMGB1 in the
healing process by using an established experimental chronic
gastric ulcer model created in rodent by topical application of
acetic acid from the gastric serosal side. The model closely
mimics human peptic gastric ulcer in histology and morphology
[32]. We also investigated whether HMGB1 affects ulcer
healing through TLR2, TLR4, or RAGE.
Materials and Methods
Animals
TLR2- and TLR4-knockout (KO) mice, which were originally
generated by Dr. S. Akira (Osaka University, Osaka, Japan)
and backcrossed 8 times onto a C57BL/6 background, were
obtained from Oriental Bioservice, Inc. (Kyoto, Japan). RAGE-
KO mice, which had been backcrossed onto a C57BL/6
background, were originally generated by and a gift from Dr. Y.
Yamamoto (Kanazawa Medical University, Kanazawa, Japan).
Wild-type C57BL/6 mice were purchased from Charles River
Japan, Inc. (Atsugi, Japan) as the control strain for TLR2 KO,
TLR4 KO, and RAGE KO mice. Specific pathogen-free 12-
week-old male animals were used. All animals were housed in
polycarbonate cages with paper chip bedding. The cages were
located in an air-conditioned biohazard room with a 12-h light-
dark cycle. All experimental procedures were approved by the
Animal Care Committee of the Osaka City University Graduate
School of Medicine (Permit Number: 11006). All surgeries were
performed under isoflurane, and all efforts were made to
minimize suffering.
Experimental Induction of Ulcer
Gastric ulcer was induced by a method described in detail
elsewhere [33], with minor modifications. Briefly, under ether
anesthesia, the abdomens of the animals were incised and the
stomach was exposed. A polypropylene tube (4 mm in
diameter) was placed against the serosal side of the stomach.
An 80-μL aliquot of 60% acetic acid was added to the tube,
which was kept in contact with the serosal surface for 30 s.
After immediate removal of acetic acid from the tube by
aspiration, the stomach was returned to its original position,
and the abdomen was closed. Previous reports demonstrated
that the size of the gastric ulcer reached a maximum on day 3
or 4 after ulcer induction, and thereafter, it gradually decreased
[32,34]. The healing phase of the experimental gastric ulcer
starts on day 4 after ulcer induction.
Experimental Groups
To investigate the effect of exogenous HMGB1, mice
received intraperitoneal injections of human recombinant
HMGB1 (rHMGB1; 100–1000 μg/kg; Sigma-Aldrich Co., St.
Louis, MO) or vehicle (phosphate-buffered saline) twice daily,
beginning at 4 days after ulcer induction (from day 4 to day 9).
Next, the effect of immunoneutralization of HMGB1 on
gastric ulcer healing was assessed. Mice were intraperitoneally
administered neutralizing chicken anti-HMGB1 polyclonal
antibody (5 mg/kg; Shino-Test Corporation, Tokyo, Japan) or
normal chicken IgY (5 mg/kg; Sigma-Aldrich Co.) beginning at
4 days after ulcer induction (from day 4 to day 9). Moreover, to
confirm the effect of release of an inhibitor of HMGB1, ethyl
pyruvate or vehicle were injected twice daily, beginning on day
4 after ulcer induction.
Furthermore, to determine the receptor responsible for
HMGB1-related gastric ulcer healing, gastric ulcers were
induced in TLR2 KO, TLR4 KO, and RAGE KO mice with or
without intraperitoneal injection of 1000 μg/kg of rHMGB1 twice
daily beginning on day 4 after ulcer induction.
The stomach was removed and the ulcer size was measured
on day 6 or 9 after ulcer induction. Ulcer size was expressed as
an ulcer index, the product of the maximum length and
minimum length (i.e. maximum length was multiplied by
minimum length). Studies were carried out using 4–8 samples.
The samples of gastric tissue were processed for further
evaluation.
mRNA Expression of Inflammatory Mediators in Gastric
Tissue Determined by Real-time Quantitative Reverse
Transcription-Polymerase Chain Reaction (RT-PCR)
Real-time quantitative RT-PCR was performed as previously
described [35]. In brief, total RNA was isolated from intestinal
tissue by using an ISOGEN kit (Nippon Gene Co., Ltd., Tokyo,
Japan) according to the manufacturer’s protocol.
Complementary DNA was acquired using a High Capacity
RNA-to-cDNA Kit (Life Technologies Corporation, Carlsbad,
CA) according to the manufacturer’s protocol. Real-time
quantitative RT-PCR analyses were performed using an
Applied Biosystems 7500 Fast Real-Time PCR system and
software (Life Technologies Corporation). The reaction mixture
was prepared according to the manufacturer’s protocol by
using the TaqMan Fast Universal PCR master mixture (Life
Technologies Corporation). Thermal cycling conditions were as
follows: 45 cycles of amplification at 95°C for 15 s and 60°C for
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 2 November 2013 | Volume 8 | Issue 11 | e80130
1 min. Total RNA was subjected to real-time quantitative RT-
PCR for the measurement of target genes using TaqMan
glyceraldehyde-3-phosphate dehydrogenase control reagents
(Life Technologies Corporation), which were used as an
internal standard. The expression of mRNA encoding HMGB1,
TLR4, RAGE, vascular endothelial growth factor (VEGF),
interleukin-1-beta (IL-1β), and TNFα in ulcerated and normal
gastric tissues was quantified using real time RT-PCR and
standardized to glyceraldehyde-3-phosphate dehydrogenase
mRNA levels. The expression of each mRNA is indicated as a
ratio, relative to the mean value in normal gastric tissue. The
primers and probes used for RT-PCR are shown in Table 1.
Immunohistochemical and Immunofluorescence
Staining
Tissue samples were fixed with 0.1 M phosphate buffer (pH
7.4) containing 4% paraformaldehyde. Samples were
embedded in paraffin, and serial 5-μm-thick sections were
mounted on silanized slides (Dako, Tokyo, Japan). The
specimens were immersed in a solution of 3% H2O2 in absolute
methanol for 5 min in order to inhibit endogenous peroxidase
activity and then incubated in 5% skim milk for 10 min.
Hematoxylin and eosin staining was performed for the
morphological observations. A rabbit monoclonal anti-HMGB1
antibody (diluted 1:250, Abcam, Cambridge, MA) was applied
as the primary antibody and incubated overnight at 4°C with
the specimens. A Secondary antibody (Histofine Simple Stain
MAX Peroxidase kit; Nichirei Biosciences Inc., Tokyo, Japan)
was incubated with the specimens for 1 h according to the
manufacturer’s instructions. Immunoreactivity was visualized
by treating the sections with Histofine Simple Stain and
diaminobenzidine solution (Nichirei Biosciences Inc.). The
specimens were then counterstained with hematoxylin. Next,
TLR2, TLR4 and RAGE expression was determined by an
immunofluorescence method. The primary antibodies used in
immunofluorescence staining included a mouse monoclonal
antibody against TLR2 (diluted 1:200; Abcam), a mouse
monoclonal antibody against TLR4 (diluted 1:200; Abcam), and
a rat monoclonal antibody against RAGE (diluted 1:250;
Abcam). Tissue samples, which were prepared as described
above, were incubated overnight at 4°C with the primary
antibodies and then reacted with the corresponding fluorescent
dye-conjugated secondary antibodies (Abcam) for 2 h.
Samples were examined with a confocal microscope equipped
with argon and argon-krypton laser sources.
Measurement of Serum HMGB1 Levels
Blood (1000 μL) samples were obtained in serum separator
tubes by cardiac puncture. After centrifugation at 3,000 rpm for
10 min, the serum was collected and stored at −80°C. Serum
levels of HMGB1 were measured using an HMGB1 sandwich
ELISA kit (Shino-Test Corporation) according to the
manufacturer’s protocol.
Measurement of Myeloperoxidase (MPO) Activity
Methods used to measure MPO activity are described in
detail elsewhere [36]. In brief, the specimens were
homogenized in 50 mM potassium phosphate buffer (pH 6.0)
containing 0.5% hexadecyltrimethylammonium bromide (Sigma
Chemical Co.). Suspensions were centrifuged, and MPO
activity in the resulting supernatant was assayed with a
spectrophotometer. One unit of MPO activity was defined as
the amount of enzyme that degraded 1 μmol peroxide/min at
25°C. The results are expressed as units per gram of gastric
tissue.
Table 1. Primers and Probes.
Gene Primer and Probe
TNF-α Primer
(forward) 5’-TCATGCACCACCATCAAGGA-3’
Primer
(reverse) 5’-GAGGCAACCTGACCACTCTCC-3’
Probe 5’-FAM-AATGGGCTTTCCGAATTCACTGGAGC-
TAMRA-3’
IL-1β Primer
(forward) 5’-ACAGGCTCCGAGATGAACAAC-3’
Primer
(reverse) 5’-CCATTGAGGTGGAGAGCTTTC-3’
Probe 5’-FAM-GAAAAAGCCTCGTGCTGTCGGACCCATAT-
TAMRA-3’
RAGE Primer
(forward) 5’-CCACTGGATAAAGGATGGTGCA-3’
Primer
(reverse) 5’-CAGCTATAGGTGCCCTCATCCTC-3’
Probe 5’-FAM-AGCCCTGTGCTGCTCCTCCCTGAG-
TAMRA-3’
TLR2 Primer
(forward) 5’-CTCTGGAGCATCCGAATTGC-3’
Primer
(reverse) 5’-GCTGAAGAGGACTGTTATGGC-3’
Probe 5’-CCTCAGACAAAGCGTCAAATCTCAGAGGA-
TAMRA-3’
TLR4 Primer
(forward) 5’-GGCTGGATTTATCCAGGTGTGA-3’
Primer
(reverse) 5’-CTGTCAGTATCAAGTTTGAGAGGTG-3’
Probe 5’-AGCCATGCCATGCCTTGTCTTCAATTGT-
TAMRA-3’
HMGB1 Primer
(forward) 5’-CAGCCATTGCAGTACATTGAGC-3’
Primer
(reverse) 5’-TCTCCTTTGCCCATGTTTAGTTG-3’
Probe 5’-GACAGAGTCGCCCAGTGCCCGTCC-TAMRA-3’
VEGF Primer
(forward) 5’-TCCGCAGACGTGTAAATGTTC-3’
Primer
(reverse) 5’-TTAACTCAAGCTGCCTCGCCT-3’
Probe 5’-FAM-TGCAAAAACACACAGACTCGCGTTGC-
TAMRA-3’
doi: 10.1371/journal.pone.0080130.t001
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 3 November 2013 | Volume 8 | Issue 11 | e80130
Statistical Analysis
Values are expressed as the mean ± standard error of the
mean (SEM). One-way analysis of variance (ANOVA) was
used to test the significance of the differences between
treatment group means, and the results were analyzed with
Fisher’s protected least-significant-difference test. P-values
less than 0.05 were considered statistically significant.
Results
Time Course of Gastric Ulcer Healing
To evaluate the healing process of gastric ulcers,
experimental gastric ulcer was induced by topical application of
acetic acid from the gastric serosal side. The ulcers were
evaluated microscopically (Figure 1A, 1B) and macroscopically.
The size of the ulcers reached a maximum on day 4 and
decreased over time thereafter (Figure 1C). MPO activity
(Figure 1D) and the expression of TNFα (Figure 1E) and IL-1β
(Figure 1E) mRNA in the ulcerated gastric tissue also peaked
on day 4, and their levels were higher in ulcerated tissue than
in normal gastric tissue throughout the examination period.
Ulceration also elevated the expression of VEGF mRNA
(Figure 1F).
HMGB1 Expression following Ulceration
We next evaluated whether HMGB1 was involved in gastric
ulcer healing. HMGB1 mRNA levels in gastric tissue (Figure
2A) and serum levels of HMGB1 (Figure 2B) reached their
maximum at day 4. HMGB1 mRNA levels were almost constant
during the examination period, whereas the serum levels of
HMGB1 dropped to normal levels 6 days after the induction of
ulcer. Immunohistochemically, ulceration induced prominent
cytoplasmic staining of HMGB1 in epithelial cells, especially in
injured areas (Figure 2C, 2D). In contrast, in intact gastric
mucosa, HMGB1 localization was limited to inside the nuclei of
epithelial cells (Figure 2E).
Effects of Exogenous HMGB1, HMGB1
Immunoneutralization, and Inhibition of HMGB1
Release on Gastric Ulcer Healing
mRNA expression and MPO activity were evaluated on day
6, and the ulcer index was evaluated on day 9, according to the
time course study. Mice were administered rHMGB1 or anti-
HMGB1 antibody intraperitoneally following the induction of
ulcer. Administration of rHMGB1 at a dose of either 100 μg/kg
or 1000 μg/kg significantly suppressed gastric ulcer healing
(Figure 3A), which was associated with increased MPO activity
(Figure 3B) and TNFα mRNA expression in ulcerated tissues
(Figure 3C). In contrast, HMGB1 neutralizing antibodies
promoted ulcer healing (Figure 4A) with reduced MPO activity
(Figure 4B) and TNFα mRNA expression (Figure 4C). The
complicating effect of exogenous rHMGB1 on gastric ulcer
healing was canceled by coadministration of anti-HMGB1
antibody (Figure 4F). Expression of VEGF mRNA was not
affected by treatment with either rHMGB1 (Figure 3D, 3E) or
anti-HMGB1 antibody (Figure 4D, 4E). In normal gastric tissue,
administration of rHMGB1 or anti-HMGB1 also did not have
any effects on cytokine expression or MPO activity (data not
shown). Furthermore, the administration of ethyl pyruvate, an
inhibitor of HMGB1 release, markedly promoted ulcer healing,
compared with vehicle treatment. Healing was accompanied by
the suppression of TNFα mRNA expression (data not shown).
Role of TLR2, TLR4, and RAGE in Gastric Ulcer Healing
To investigate whether TLR2, TLR4, and RAGE contribute to
HMGB1-mediated gastric ulcer healing, experimental gastric
ulcers were induced in TLR2 KO, TLR4 KO, RAGE KO, and
control wild-type mice. TLR4 and RAGE deficiency promoted
gastric ulcer formation and prevented the increase in TNFα
mRNA expression after ulceration, whereas TLR2 deficiency
affected neither the ulcer index nor the expression of TNFα
mRNA (Figure 5A, 5B). Administration of exogenous HMGB1
affected neither the ulcer index nor the expression of TNFα
mRNA in either TLR4 KO or RAGE KO mice (Figure 5C–5F).
Administration of exogenous HMGB1, however, delayed ulcer
healing in wild-type mice and reduced TNFα mRNA expression
(Figure 3).
Expression of TLR2, TLR4 and RAGE during Gastric
Ulcer Healing
We next investigated mRNA expression and
immunoreactivity of TLR2, TLR4 and RAGE in wild-type mice.
A significant up-regulation of TLR2 mRNA was observed
following the induction of an ulcer (Figure 6A). TLR2
immunoreactivity was observed mainly in inflammatory cells
(Figure 6B). The expression of TLR4 mRNA was not affected
by the induction of ulcer (Figure 6C). TLR4 immunoreactivity
was observed in the apical part of the epithelial lining at the
ulcer edge and in some inflammatory cells in the ulcer bed
(Figure 6D).
A significant up-regulation of RAGE mRNA was observed on
day 9 after the induction of ulcer (Figure 6E). RAGE
immunoreactivity was observed mainly in inflammatory cells at
the edge of the ulcer beds and in the vascular endothelial cell
membrane (Figure 6F).
Staining of ulcerated tissue from TLR4 KO or RAGE KO
mice with an anti-TLR4 antibody or anti-RAGE antibody,
respectively, revealed no positive signals, confirming the
specificity of these antibodies (data not shown).
Discussion
In this study, we demonstrated that exogenous HMGB1
delays gastric ulcer healing, while inducing TNFα expression
and MPO activity. Conversely, immunoneutralization of
HMGB1 or inhibiting the release of HMGB1 promotes ulcer
healing while reducing TNFα expression and MPO activity.
Additionally, TLR4 and RAGE deficiency promotes ulcer
healing, and exogenous HMGB1 fails to delay ulcer healing in
TLR4 KO and RAGE KO mice. These results suggest that
HMGB1 is a complicating factor for gastric ulcer healing that
acts through TLR4- and RAGE-dependent pathways. To our
knowledge, this is the first report to clarify the role of HMGB1 in
wound healing within the gastrointestinal tract.
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 4 November 2013 | Volume 8 | Issue 11 | e80130
Figure 1. Ulcer index and cytokine expression following ulceration. A, B: Hematoxylin-eosin staining of gastric ulcer (day 6).
The arrow indicates the ulcer site. B: Higher magnification of Figure 1A. C: Time course of the ulcer index following ulceration. The
size of the macroscopically visible ulcer was expressed as an ulcer index, the product of maximum length and minimum length. D:
Myeloperoxidase (MPO) activity of gastric tissue. One unit of MPO activity was defined as the amount of enzyme that degrades 1
μmol peroxide/min at 25°C. The results are expressed as units per gram of gastric tissue. E–H: The mRNA expression of tumor
necrosis factor α (TNFα) (E), interleukin-1β (IL1-β) (F), and vascular endothelial growth factor (VEGF) (G) were determined by
quantitative reverse transcription-polymerase chain reaction (RT-PCR). mRNA levels are expressed as ratios, relative to the mean
value for normal gastric tissue. Each column represents the mean ± standard error of the mean ± SEM. N = 6–9. **P < 0.01, *P <
0.05 vs. untreated controls. ++P < 0.01, +P < 0.05 vs. day 4 group.
doi: 10.1371/journal.pone.0080130.g001
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 5 November 2013 | Volume 8 | Issue 11 | e80130
Figure 2. High mobility group box 1 (HMGB1) expression following ulceration. Changes in HMGB1 mRNA expression (A)
and HMGB1 serum levels (B) during gastric ulcer healing. The levels of HMGB1 mRNA were determined by quantitative RT-PCR,
and HMGB1 serum levels were determined by ELISA. HMGB1 mRNA levels are expressed as ratios, relative to the mean value of
the gastric tissue in the control group (0-h group). Each column represents the mean ± SEM. N = 3–7. **P < 0.01, *P < 0.05 vs.
control group. C, D: Immunohistochemistry of HMGB1 in ulcerated gastric tissue (day 6). HMGB1 localized to the cytoplasm in
injured epithelial cells as well as to the nuclei of epithelial cells and interstitial cells. D: Higher magnification of Figure 2C. E:
Immunohistochemistry of HMGB1 in untreated gastric tissue. HMGB1 localization was limited to the inside of nuclei of epithelial
cells in intact gastric mucosa.
doi: 10.1371/journal.pone.0080130.g002
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 6 November 2013 | Volume 8 | Issue 11 | e80130
A few studies addressed the role of HMGB1 in wound
healing in organs other than the gastrointestinal tract, but the
results of these studies are controversial [37-40]. Consistent
with our results, some studies demonstrated that HMGB1 has
an inhibitory effect on wound healing [37,38]. Zhang et al.
demonstrated that HMGB1 impairs incision wound healing by
reducing reparative collagen deposition via RAGE [37]. Goova
et al. demonstrated that blocking RAGE ligands such as AGE
and HMGB1 by using a soluble form of receptor for AGE
(sRAGE), accelerates ulcer healing and suppresses the levels
of inflammatory cytokines [38]. Previous studies demonstrated
that excessive inflammation impaired wound healing. For
example, our previous study demonstrated that TNFα over-
expression and excessive neutrophil infiltration are
complicating factors in the formation and healing of gastric
ulcer [41]. Furthermore, in another model of tissue repair,
Goren et al. demonstrated that excessive neutrophil and
macrophage infiltration with TNFα over-expression inhibits the
healing of mouse skin injuries [42]. Thus, it is possible that
HMGB1 delays gastric ulcer healing through TNFα-triggered
inflammatory responses.
In contrast, some in vitro studies suggested that HMGB1 and
its receptors are essential for wound healing [39,40]. Staraino
et al. demonstrated that HMGB1 accelerates the wound
healing process and regeneration by enhancing the migration
of skin fibroblasts and keratinocytes [39]. HMGB1 also
promotes the wound healing of 3T3 fibroblasts by inducing cell
proliferation and migration through the activation of the RAGE/
extracellular signal-regulated kinase pathway [40]. Because
these in vitro studies were performed in the absence of
inflammatory cells, the differences in experimental methods
may result in inconsistent conclusions on the role of HMGB1 in
wound healing.
VEGF, a potent angiogenic growth factor, plays an important
role in gastric ulcer healing [43,44]. Previous reports
demonstrated that HMGB1 could induce the expression of
Figure 3. Effect of exogenous HMGB1 on the healing of gastric ulcers. Mice received intraperitoneal injections of human
rHMGB1 (100–1000 μg/kg) or vehicle after ulceration. Ulcer index, mRNA expression, and myeloperoxidase (MPO) activity were
measured in gastric tissues. Effects of exogenous HMGB1 on the ulcer index (A), MPO activity (B), and cytokine expression (C, D)
following ulceration. Ulcer size was expressed as an ulcer index, the product of maximum length and minimum length. MPO
activities (B) were measured according to Bradley’s methods, and the expression of TNFα (C) and VEGF (D) was determined by
quantitative RT-PCR. Each column represents the mean ± SEM. N = 5–8. **P < 0.01, *P < 0.05 vs. vehicle-treated control group.
doi: 10.1371/journal.pone.0080130.g003
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 7 November 2013 | Volume 8 | Issue 11 | e80130
VEGF in several tissues and animal models [45-47]. However,
in this study, neither rHMGB1 nor anti-HMGB1 antibodies
affected VEGF expression in ulcerous tissue. The reason for
the disparity between our findings and those of earlier
investigators is not clear, but it may be associated with the use
of different organs and experimental models.
There are 2 possible sources of extracellular HMGB1:
passive release from necrotic cells [48] and active secretion
from inflammatory cells [5]. In this study, HMGB1
immunoreactivity was observed in the cytoplasm as well as the
nucleus, suggesting that necrotic cells are a source of HMGB1.
Gastric HMGB1 mRNA levels increased during ulcer healing,
suggesting that inflammatory cells produce and release
HMGB1. Collectively, the elevation of serum HMGB1 resulted
was due to passive release from injured cells at the gastric
ulcer and active secretion from inflammatory cells.
Serum HMGB1 levels increased following the induction of
gastric ulcer but declined at the late phase of healing, although
the expression of HMGB1 and TNFα remained high. These
data raise the possibility of a systemic HMGB1 trapping
system. To this end, the existence of an HMGB1 binding
protein has been reported. Thrombomodulin, a cell-surface
glycoprotein, is one example of an HMGB1 binding protein.
Recombinant human soluble thrombomodulin inhibited the
increase in plasma HMGB1 induced by lipopolysaccharide in a
rat model [49] and bound to HMGB1 through its lectin domain
[50] to prevent HMGB1 from interacting with other receptors
[51]. In clinical applications, thrombomodulin is also useful in
HMGB1-related diseases and conditions such as sepsis
because of its anti-HMGB1 properties [52]. sRAGE, found in
the circulation, is another example of an HMGB1 binding
protein. sRAGE is the soluble form of RAGE; it acts as a decoy
to prevent interaction between cell surface RAGE and its
Figure 4. Effect of HMGB1 immunoneutralization on the healing of gastric ulcers. Mice received intraperitoneal injections of
neutralizing chicken anti-HMGB1 polyclonal antibody (50 mg/kg) or vehicle after ulceration. Ulcer index, mRNA expression, and
myeloperoxidase (MPO) activity were measured in gastric tissues. Effects of the anti-HMGB1 antibody on the ulcer index (A), MPO
activity (B), and cytokine expression (C, D) following ulceration. Ulcer size was expressed as an ulcer index, the product of
maximum length and minimum length. MPO activities (B) were measured according to Bradley’s methods, and the expression of
TNFα (C) and VEGF (D) mRNA was determined by quantitative RT-PCR. Each column represents the mean ± SEM. N = 5–8. **P <
0.01, *P < 0.05 vs. vehicle-treated control group.
doi: 10.1371/journal.pone.0080130.g004
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 8 November 2013 | Volume 8 | Issue 11 | e80130
ligands, such HMGB1 [53]. In the present study, such trapping
systems might also play a protective role in preventing the
spread of inflammation, thereby promoting ulcer healing.
It is known that many peptic ulcer patients are infected with
Helicobacter pylori. Although in the present study we did not
investigate the role of HMGB1 in gastric ulcer healing in mice
infected with H. pylori, clinical and experimental studies
suggest that the deleterious effect of HMGB1 on gastric ulcer
healing would be more pronounced in patients with an H. pylori
infection than in those without it. We previously showed that H.
pylori infection increases neutrophil infiltration into ulcerated
tissues in Mongolian gerbils [54]. Shimizu et al. also
demonstrated that neutrophils and macrophages infiltrate ulcer
margins to a higher degree in patients with H. pylori infection
than in those without the infection [55]. A large amount of
HMGB1 is, therefore, likely present in the ulcerated tissue
infected with H. pylori, since it would be secreted by those
inflammatory cells. Furthermore, Radin et al. reported that
VacA, a major virulence factor of this organism, causes
programmed necrosis of gastric epithelial cells and subsequent
release of HMGB1 [56]. Thus, we expect that the deleterious of
HMGB1 on ulcer healing would be more prominent in H. pylori-
infected patients.
TLR2, TLR4, and RAGE, which mediate proinflammatory
responses, are commonly known HMGB1 receptors. Our
results clearly showed that TLR4 and RAGE play crucial roles
in gastric ulcer healing. This result is consistent with previous
findings on the inflammatory responses induced by HMGB1.
One of the most established models involving the interaction
between HMGB1 and these receptors is I-R injury. In hepatic I-
R injury, TLR4-deficient mice exhibited less liver I-R damage;
the damage in TLR4-deficient mice was not affected by
rHMGB1 or anti-HMGB1 antibody [26]. Moreover, blocking
RAGE protected against hepatocellular death and necrosis in
the hepatic I-R injury model [57]. In a model of cardiac I-R
injury, Andrassy et al. showed that RAGE-deficient mice
Figure 5. Role of TLR2, TLR4, and RAGE in gastric ulcer healing. Gastric ulcers were induced in TLR2 KO, TLR4 KO, and
RAGE KO mice. The ulcer index (A) and TNFα mRNA levels (B) were measured in gastric tissue. C–F: Effect of recombinant
HMGB1 on gastric ulcer healing in TLR4 KO and RAGE KO mice. Gastric ulcers were induced in TLR4 KO (C, D) and RAGE KO
(E, F) mice with or without intraperitoneal injections of 1000 μg/kg rHMGB1. The ulcer index (A, C, E) and TNFα mRNA levels (B, D,
F) were measured in gastric tissues. The mRNA levels assessed by RT-PCR are expressed as ratios, relative to the mean value for
normal gastric tissue. Each column represents the mean ± SEM. N = 4–6. **P < 0.01, *P < 0.05 vs. wild-type mice.
doi: 10.1371/journal.pone.0080130.g005
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 9 November 2013 | Volume 8 | Issue 11 | e80130
Figure 6. Expression of TLR2, TLR4 and RAGE during gastric ulcer healing. A, C, E: The expression of TLR2 (A), TLR4 (C)
and RAGE (E) mRNA was determined by quantitative RT-PCR. mRNA levels are expressed as ratios, relative to the mean value for
normal small gastric tissue. Each column represents the mean ± SEM. N = 6. **P < 0.01, *P < 0.05 vs. untreated control group. +P <
0.05 vs. day 4 group. B, D, F: Immunohistochemical staining of TLR2 (B), TLR4 (D) and RAGE (F) in gastric ulcer tissue on day 6.
TLR2 immunoreactivity was observed mainly in inflammatory cells (B). TLR4 immunoreactivity (D) was observed in the apical part of
the epithelial lining at the ulcer edge and in some inflammatory cells in the ulcer bed. RAGE immunoreactivity (F) was observed
mainly in inflammatory cells at the edge of ulcer beds and in the vascular endothelial cell membrane (arrow).
doi: 10.1371/journal.pone.0080130.g006
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 10 November 2013 | Volume 8 | Issue 11 | e80130
displayed only slight inflammation resulting from cardiac I-R
injury; the inflammation was not affected by the induction of
rHMGB1 [27]. These findings suggest that the TLR4-HMGB1
and RAGE-HMGB1 interactions play a crucial role in I-R injury,
although it is necessary to consider the differences in each
organ. The other established model is systemic inflammation,
such as sepsis [5,8,58]. In an experimental model of intra-
abdominal sepsis, Susa et al. demonstrated that the HMGB1-
RAGE interaction was closely associated with sepsis-induced
diaphragmatic dysfunction [58]. In an in vivo systemic
inflammation model generated by injection of exogenous
HMGB1, Zoelen et al. demonstrated that HMGB1 induces the
release of cytokines, activation of coagulation, and neutrophil
recruitment through TLR4 and RAGE [8]. Thus, TLR4 and
RAGE play critical roles in pathogenesis mediated by the
HMGB1-associated pathway, including the pathway in our ulcer
healing model.
Our results indicate that TLR2 has no relationship to gastric
ulcer healing: ulcer healing in TLR2 KO mice resembled that in
wild-type mice. Although in vitro studies using macrophage cell
lines indicated that the TLR2-HMGB1 pathway induces
inflammatory responses [6,9], in vivo effects of TLR2 in
HMGB1-mediated pathologies have not been reported. Thus,
based on previous findings [8] and our results, the HMGB1-
TLR2 pathway may play a minor role in the repair and
pathogenesis of tissue injuries and inflammation.
In conclusion, we have shown that HMGB1 is a complicating
factor in the healing process of gastric ulcer as well as in other
pathological conditions. Moreover, we have shown that
HMGB1 inhibits ulcer healing through a mechanism that
involves TLR4, RAGE, and excessive inflammatory responses.
Although proton pomp inhibitors are commonly prescribed for
gastric ulcers, intractable ulcers still pose a clinical problem.
Our present study supports a new concept for the treatment of
intractable gastric ulcers, besides proton pomp inhibitors
therapy.
Acknowledgements
We thank Emi Suzuki-Yoshioka for technical assistance. I
would also like to express my sincere gratitude to the staff
members of the Osaka City University who supported us.
Author Contributions
Conceived and designed the experiments: YN TW K. Takeuchi
TA. Performed the experiments: YN TT FO HY M. Sogawa ST
AH. Analyzed the data: YN TW M. Shiba. Contributed
reagents/materials/analysis tools: KW. Wrote the manuscript:
YN TW K. Tominaga YF.
References
1. Bustin M (1999) Regulation of DNA-dependent activities by the
functional motifs of the high-mobility-group chromosomal proteins. Mol
Cell Biol 19: 5237-5246. PubMed: 10409715.
2. Park JS, Arcaroli J, Yum HK, Yang H, Wang H et al. (2003) Activation
of gene expression in human neutrophils by high mobility group box 1
protein. Am J Physiol Cell Physiol 284: C870-C879. doi:10.1152/ajpcell.
00322.2002. PubMed: 12620891.
3. Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein
HMGB1 by necrotic cells triggers inflammation. Nature 418: 191-195.
doi:10.1038/nature00858. PubMed: 12110890.
4. Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O et al.
(2000) High mobility group 1 protein (HMG-1) stimulates
proinflammatory cytokine synthesis in human monocytes. J Exp Med
192: 565-570. doi:10.1084/jem.192.4.565. PubMed: 10952726.
5. Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M et al.
(1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science
285: 248-251. doi:10.1126/science.285.5425.248. PubMed: 10398600.
6. Yu M, Wang H, Ding A, Golenbock DT, Latz E et al. (2006) HMGB1
signals through toll-like receptor (TLR) 4 and TLR2. Shock 26: 174-179.
doi:10.1097/01.shk.0000225404.51320.82. PubMed: 16878026.
7. Park JS, Gamboni-Robertson F, He Q, Svetkauskaite D, Kim JY et al.
(2006) High mobility group box 1 protein interacts with multiple Toll-like
receptors. Am J Physiol Cell Physiol 290: C917-C924. PubMed:
16267105.
8. van Zoelen MA, Yang H, Florquin S, Meijers JC, Akira S et al. (2009)
Role of toll-like receptors 2 and 4, and the receptor for advanced
glycation end products in high-mobility group box 1-induced
inflammation in vivo. Shock 31: 280-284. doi:10.1097/SHK.
0b013e318186262d. PubMed: 19218854.
9. Dobrovolskaia MA, Medvedev AE, Thomas KE, Cuesta N, Toshchakov
V et al. (2003) Induction of in vitro reprogramming by Toll-like receptor
(TLR)2 and TLR4 agonists in murine macrophages: effects of TLR
"homotolerance" versus "heterotolerance" on NF-kappa B signaling
pathway components. J Immunol 170: 508-519. PubMed: 12496438.
10. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T et al. (1999)
Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are
hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps
gene product. J Immunol 162: 3749-3752. PubMed: 10201887.
11. Qin YH, Dai SM, Tang GS, Zhang J, Ren D et al. (2009) HMGB1
enhances the proinflammatory activity of lipopolysaccharide by
promoting the phosphorylation of MAPK p38 through receptor for
advanced glycation end products. J Immunol 183: 6244-6250. doi:
10.4049/jimmunol.0900390. PubMed: 19890065.
12. Watanabe T, Higuchi K, Kobata A, Nishio H, Tanigawa T et al. (2008)
Non-steroidal anti-inflammatory drug-induced small intestinal damage
is Toll-like receptor 4 dependent. Gut 57: 181-187. doi:10.1136/gut.
2007.125963. PubMed: 17639086.
13. Watanabe T, Nishio H, Tanigawa T, Yamagami H, Okazaki H et al.
(2009) Probiotic Lactobacillus casei strain Shirota prevents
indomethacin-induced small intestinal injury: involvement of lactic acid.
Am J Physiol Gastrointest Liver Physiol 297: G506-G513. doi:10.1152/
ajpgi.90553.2008. PubMed: 19589943.
14. Herold K, Moser B, Chen Y, Zeng S, Yan SF et al. (2007) Receptor for
advanced glycation end products (RAGE) in a dash to the rescue:
inflammatory signals gone awry in the primal response to stress. J
Leukoc Biol 82: 204-212. doi:10.1189/jlb.1206751. PubMed: 17513693.
15. Yan SD, Chen X, Fu J, Chen M, Zhu H et al. (1996) RAGE and
amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 382:
685-691. doi:10.1038/382685a0. PubMed: 8751438.
16. Huttunen HJ, Kuja-Panula J, Sorci G, Agneletti AL, Donato R et al.
(2000) Coregulation of neurite outgrowth and cell survival by
amphoterin and S100 proteins through receptor for advanced glycation
end products (RAGE) activation. J Biol Chem 275: 40096-40105. doi:
10.1074/jbc.M006993200. PubMed: 11007787.
17. Lander HM, Tauras JM, Ogiste JS, Hori O, Moss RA et al. (1997)
Activation of the receptor for advanced glycation end products triggers
a p21(ras)-dependent mitogen-activated protein kinase pathway
regulated by oxidant stress. J Biol Chem 272: 17810-17814. doi:
10.1074/jbc.272.28.17810. PubMed: 9211935.
18. Marsche G, Semlitsch M, Hammer A, Frank S, Weigle B et al. (2007)
Hypochlorite-modified albumin colocalizes with RAGE in the artery wall
and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase
pathway. FASEB J 21: 1145-1152. doi:10.1096/fj.06-7439com.
PubMed: 17218539.
19. Zeng S, Dun H, Ippagunta N, Rosario R, Zhang QY et al. (2009)
Receptor for advanced glycation end product (RAGE)-dependent
modulation of early growth response-1 in hepatic ischemia/reperfusion
injury. J Hepatol 50: 929-936. doi:10.1002/hep.23104. PubMed:
19303658.
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 11 November 2013 | Volume 8 | Issue 11 | e80130
20. Taguchi A, Blood DC, del Toro G, Canet A, Lee DC et al. (2000)
Blockade of RAGE-amphoterin signalling suppresses tumour growth
and metastases. Nature 405: 354-360. doi:10.1038/35012626.
PubMed: 10830965.
21. Huang W, Tang Y, Li L (2010) HMGB1, a potent proinflammatory
cytokine in sepsis. Cytokine 51: 119-126. doi:10.1016/j.cyto.
2010.02.021. PubMed: 20347329.
22. Sawa H, Ueda T, Takeyama Y, Yasuda T, Shinzeki M et al. (2006)
Blockade of high mobility group box-1 protein attenuates experimental
severe acute pancreatitis. World J Gastroenterol 12: 7666-7670.
PubMed: 17171797.
23. Kim JY, Park JS, Strassheim D, Douglas I, Diaz del Valle F et al.
(2005) HMGB1 contributes to the development of acute lung injury after
hemorrhage. Am J Physiol Lung Cell Mol Physiol 288: L958-L965. doi:
10.1152/ajplung.00359.2004. PubMed: 15640285.
24. Urbonaviciute V, Voll RE (2011) High-mobility group box 1 represents a
potential marker of disease activity and novel therapeutic target in
systemic lupus erythematosus. J Intern Med 270: 309-318. doi:
10.1111/j.1365-2796.2011.02432.x. PubMed: 21793951.
25. Qiu J, Xu J, Zheng Y, Wei Y, Zhu X et al. (2010) High-mobility group
box 1 promotes metalloproteinase-9 upregulation through Toll-like
receptor 4 after cerebral ischemia. Stroke 41: 2077-2082. doi:10.1161/
STROKEAHA.110.590463. PubMed: 20671243.
26. Tsung A, Sahai R, Tanaka H, Nakao A, Fink MP et al. (2005) The
nuclear factor HMGB1 mediates hepatic injury after murine liver
ischemia-reperfusion. J Exp Med 201: 1135-1143. doi:10.1084/jem.
20042614. PubMed: 15795240.
27. Andrassy M, Volz HC, Igwe JC, Funke B, Eichberger SN et al. (2008)
High-mobility group box-1 in ischemia-reperfusion injury of the heart.
Circulation 117: 3216-3226. doi:10.1161/CIRCULATIONAHA.
108.769331. PubMed: 18574060.
28. Wu H, Chen G, Wyburn KR, Yin J, Bertolino P et al. (2007) TLR4
activation mediates kidney ischemia/reperfusion injury. J Clin Invest
117: 2847-2859. doi:10.1172/JCI31008. PubMed: 17853945.
29. Maeda S, Hikiba Y, Shibata W, Ohmae T, Yanai A et al. (2007)
Essential roles of high-mobility group box 1 in the development of
murine colitis and colitis-associated cancer. Biochem Biophys Res
Commun 360: 394-400. doi:10.1016/j.bbrc.2007.06.065. PubMed:
17599806.
30. Davé SH, Tilstra JS, Matsuoka K, Li F, DeMarco RA et al. (2009) Ethyl
pyruvate decreases HMGB1 release and ameliorates murine colitis. J
Leukoc Biol 86: 633-643. doi:10.1189/jlb.1008662. PubMed: 19454652.
31. Nadatani Y, Watanabe T, Tanigawa T, Machida H, Okazaki H et al.
(2012) High mobility group box 1 promotes small intestinal damage
induced by nonsteroidal anti-inflammatory drugs through Toll-like
receptor 4. Am J Pathol 181: 98-110. doi:10.1016/j.ajpath.2012.03.039.
PubMed: 22634181.
32. Okabe S, Roth JL, Pfeiffer CJ (1971) A method for experimental,
penetrating gastric and duodenal ulcers in rats. Observations on normal
healing. Am J Dig Dis 16: 277-284. doi:10.1007/BF02235252. PubMed:
5554507.
33. Tanigawa T, Watanabe T, Otani K, Nadatani Y, Machida H et al. (2010)
Leptin promotes gastric ulcer healing via upregulation of vascular
endothelial growth factor. Digestion 81: 86-95. doi:10.1159/000243719.
PubMed: 20068308.
34. Watanabe T, Arakawa T, Fukuda T, Higuchi K, Kobayashi K (1995)
Zinc deficiency delays gastric ulcer healing in rats. Dig Dis Sci 40:
1340-1344. doi:10.1007/BF02065548. PubMed: 7781457.
35. Watanabe T, Kobata A, Tanigawa T, Nadatani Y, Yamagami H et al.
(2012) Activation of the MyD88 signaling pathway inhibits ischemia-
reperfusion injury in the small intestine. Am J Physiol Gastrointest Liver
Physiol 303: G324-G334. doi:10.1152/ajpgi.00075.2012. PubMed:
22628037.
36. Otani K, Tanigawa T, Watanabe T, Nadatani Y, Sogawa M et al. (2012)
Toll-like receptor 9 signaling has anti-inflammatory effects on the early
phase of Helicobacter pylori-induced gastritis. Biochem Biophys Res
Commun 426: 342-349. doi:10.1016/j.bbrc.2012.08.080. PubMed:
22940550.
37. Zhang Q, O'Hearn S, Kavalukas SL, Barbul A (2012) Role of high
mobility group box 1 (HMGB1) in wound healing. J Surg Res 176:
343-347. doi:10.1016/j.jss.2011.06.069. PubMed: 21872885.
38. Goova MT, Li J, Kislinger T, Qu W, Lu Y et al. (2001) Blockade of
receptor for advanced glycation end-products restores effective wound
healing in diabetic mice. Am J Pathol 159: 513-525. doi:10.1016/
S0002-9440(10)61723-3. PubMed: 11485910.
39. Straino S, Di Carlo A, Mangoni A, De Mori R, Guerra L et al. (2008)
High-mobility group box 1 protein in human and murine skin:
involvement in wound healing. J Invest Dermatol 128: 1545-1553. doi:
10.1038/sj.jid.5701212. PubMed: 18239618.
40. Ranzato E, Patrone M, Pedrazzi M, Burlando B (2010) Hmgb1
promotes wound healing of 3T3 mouse fibroblasts via RAGE-
dependent ERK1/2 activation. Cell Biochem Biophys 57: 9-17. doi:
10.1007/s12013-010-9077-0. PubMed: 20361273.
41. Shimizu N, Watanabe T, Arakawa T, Fujiwara Y, Higuchi K et al. (2000)
Pentoxifylline accelerates gastric ulcer healing in rats: roles of tumor
necrosis factor alpha and neutrophils during the early phase of ulcer
healing. Digestion 61: 157-164. doi:10.1159/000007752. PubMed:
10773720.
42. Goren I, Müller E, Schiefelbein D, Christen U, Pfeilschifter J et al.
(2007) Systemic anti-TNFalpha treatment restores diabetes-impaired
skin repair in ob/ob mice by inactivation of macrophages. J Invest
Dermatol 127: 2259-2267. doi:10.1038/sj.jid.5700842. PubMed:
17460730.
43. Suzuki N, Takahashi S, Okabe S (1998) Relationship between vascular
endothelial growth factor and angiogenesis in spontaneous and
indomethacin-delayed healing of acetic acid-induced gastric ulcers in
rats. J Physiol Pharmacol 49: 515-527. PubMed: 10069693.
44. Takahashi M, Kawabe T, Ogura K, Maeda S, Mikami Y et al. (1997)
Expression of vascular endothelial growth factor at the human gastric
ulcer margin and in cultured gastric fibroblasts: a new angiogenic factor
for gastric ulcer healing. Biochem Biophys Res Commun 234: 493-498.
doi:10.1006/bbrc.1997.5974. PubMed: 9177300.
45. Biscetti F, Straface G, De Cristofaro R, Lancellotti S, Rizzo P et al.
(2010) High-mobility group box-1 protein promotes angiogenesis after
peripheral ischemia in diabetic mice through a VEGF-dependent
mechanism. Diabetes 59: 1496-1505. doi:10.2337/db09-1507.
PubMed: 20200317.
46. Rossini A, Zacheo A, Mocini D, Totta P, Facchiano A et al. (2008)
HMGB1-stimulated human primary cardiac fibroblasts exert a paracrine
action on human and murine cardiac stem cells. J Mol Cell Cardiol 44:
683-693. doi:10.1016/j.yjmcc.2008.01.009. PubMed: 18328501.
47. Lei C, Lin S, Zhang C, Tao W, Dong W et al. (2013) Effects of high-
mobility group box1 on cerebral angiogenesis and neurogenesis after
intracerebral hemorrhage. Neuroscience 229: 12-19. doi:10.1016/
j.neuroscience.2012.10.054. PubMed: 23137544.
48. Degryse B, Bonaldi T, Scaffidi P, Müller S, Resnati M et al. (2001) The
high mobility group (HMG) boxes of the nuclear protein HMG1 induce
chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.
J Cell Biol 152: 1197-1206. doi:10.1083/jcb.152.6.1197. PubMed:
11257120.
49. Nagato M, Okamoto K, Abe Y, Higure A, Yamaguchi K (2009)
Recombinant human soluble thrombomodulin decreases the plasma
high-mobility group box-1 protein levels, whereas improving the acute
liver injury and survival rates in experimental endotoxemia. Crit Care
Med 37: 2181-2186. doi:10.1097/CCM.0b013e3181a55184. PubMed:
19487933.
50. Esmon C (2005) Do-all receptor takes on coagulation, inflammation.
Nat Med 11: 475-477. doi:10.1038/nm0505-475. PubMed: 15875050.
51. Abeyama K, Stern DM, Ito Y, Kawahara K, Yoshimoto Y et al. (2005)
The N-terminal domain of thrombomodulin sequesters high-mobility
group-B1 protein, a novel antiinflammatory mechanism. J Clin Invest
115: 1267-1274. doi:10.1172/JCI200522782. PubMed: 15841214.
52. Inoue Y, Saito T, Ogawa K, Nishio Y, Kosugi S et al. (2013) Role of
serum high mobility group box 1 in hematological malignancies
complicated with systemic inflammatory response syndrome and effect
of recombinant thrombomodulin. Leuk: Lymphoma.
53. Liliensiek B, Weigand MA, Bierhaus A, Nicklas W, Kasper M et al.
(2004) Receptor for advanced glycation end products (RAGE)
regulates sepsis but not the adaptive immune response. J Clin Invest
113: 1641-1650. doi:10.1172/JCI200418704. PubMed: 15173891.
54. Watanabe T, Higuchi K, Hamaguchi M, Tanigawa T, Wada R et al.
(2002) Rebamipide prevents delay of acetic acid-induced gastric ulcer
healing caused by Helicobacter pylori infection in Mongolian gerbils.
Dig Dis Sci 47: 1582-1589. doi:10.1023/A:1015879421739. PubMed:
12141820.
55. Shimizu T, Kusugami K, Ina K, Imada A, Nishio Y et al. (2000)
Helicobacter pylori-associated gastric ulcer exhibits enhanced mucosal
chemokine activity at the ulcer site. Digestion 62: 87-94. doi:
10.1159/000007800. PubMed: 11025355.
56. Radin JN, González-Rivera C, Ivie SE, McClain MS, Cover TL (2011)
Helicobacter pylori VacA induces programmed necrosis in gastric
epithelial cells. Infect Immun 79: 2535-2543. doi:10.1128/IAI.01370-10.
PubMed: 21482684.
57. Zeng S, Feirt N, Goldstein M, Guarrera J, Ippagunta N et al. (2004)
Blockade of receptor for advanced glycation end product (RAGE)
attenuates ischemia and reperfusion injury to the liver in mice.
Hepatology 39: 422-432. doi:10.1002/hep.20045. PubMed: 14767995.
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 12 November 2013 | Volume 8 | Issue 11 | e80130
58. Susa Y, Masuda Y, Imaizumi H, Namiki A (2009) Neutralization of
receptor for advanced glycation end-products and high mobility group
box-1 attenuates septic diaphragm dysfunction in rats with peritonitis.
Crit Care Med 37: 2619-2624. doi:10.1097/CCM.0b013e3181a930f7.
PubMed: 19623040.
HMGB1 Delays Ulcer Healing
PLOS ONE | www.plosone.org 13 November 2013 | Volume 8 | Issue 11 | e80130
