Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells
Chao et al. Journal of Biomedical Science
Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells
Hung-Hsing Chao 0 4 6
Po-Yuan Chen 0 3
Wen-Rui Hao 1
Wei-Ping Chiang 1
Tzu-Hurng Cheng 0 8 9
Shih-Hurng Loh 8
Yuk-Man Leung 7
Ju-Chi Liu 1 2
Jin-Jer Chen 5 10
Li-Chin Sung 1 2
0 Equal contributors
1 Division of Cardiology, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University , No. 291, Zhongzheng RdZhonghe District, New Taipei City 23561 , Taiwan
2 Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University , Taipei 11031 , Taiwan
3 Department of Biological Science and Technology, College of Biopharmaceutical and Food Sciences, China Medical University , Taichung 40402 , Taiwan
4 Department of Surgery, School of Medicine, Taipei Medical University , Taipei 11031 , Taiwan
5 Institute of Biomedical Sciences , Academia Sinica, Taipei 115 , Taiwan
6 Division of Cardiovascular Surgery, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital , Taipei 111 , Taiwan
7 Department of Physiology, School of Medicine, China Medical University , Taichung 40402 , Taiwan
8 Department of Pharmacology & Graduate Institute of Pharmacology, National Defense Medical Center , Taipei 114 , Taiwan
9 Department of Biochemistry, School of Medicine, China Medical University , Taichung 40402 , Taiwan
10 Graduate Institute of Clinical Medicine, College of Medicine, China Medical University , Taichung 40402 , Taiwan
Background: This study investigated whether lipopolysaccharide (LPS) increase protease-activated receptor-2 (PAR-2) expression and enhance the association between PAR-2 expression and chemokine production in human vascular endothelial cells (ECs). Methods: The morphology of ECs was observed through microphotography in cultured human umbilical vein ECs (EA. hy926 cells) treated with various LPS concentrations (0, 0.25, 0.5, 1, and 2 μg/mL) for 24 h, and cell viability was assessed using the MTT assay. Intracellular calcium imaging was performed to assess agonist (trypsin)-induced PAR-2 activity. Western blotting was used to explore the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule monocyte chemoattractant protein-1 (MCP-1) in ECs. Results: Trypsin stimulation increased intracellular calcium release in ECs. The calcium influx was augmented in cells pretreated with a high LPS concentration (1 μg/mL). After 24 h treatment of LPS, no changes in ECs viability or morphology were observed. Western blotting revealed that LPS increased PAR-2 expression and enhanced trypsin-induced extracellular signal-regulated kinase (ERK)/p38 phosphorylation and MCP-1 secretion. However, pretreatment with selective ERK (PD98059) , p38 mitogen-activated protein kinase (MAPK) (SB203580) inhibitors, and the selective PAR-2 antagonist (FSLLRY-NH2) blocked the effects of LPS-activated PAR-2 on MCP-1 secretion. Conclusions: Our findings provide the first evidence that the bacterial endotoxin LPS potentiates calcium mobilization and ERK/p38 MAPK pathway activation and leads to the secretion of the pro-inflammatory chemokine MCP-1 by inducing PAR-2 expression and its associated activity in vascular ECs. Therefore, PAR-2 exerts vascular inflammatory effects and plays an important role in bacterial infection-induced pathological responses.
Lipopolysaccharides; Protease-activated receptor-2; Endothelial cells; Monocyte chemoattractant protein-1; Mitogen-activated protein kinases
The important role of bacterial endotoxins in the
pathophysiology of sepsis was recognized in the 1960s and
]. Lipopolysaccharides (LPS), also called
endotoxins, are expressed by most gram-negative bacteria
and play an important role in the function and structural
integrity of the outer lipid membrane [
]. LPSs are a
family of large molecules containing three structural
elements: a core oligosaccharide, an O-antigen, and a
lipid A component [
]. High LPS levels have certain
toxic effects on cells, whereas low LPS levels promote
cell proliferation. Epidemiological studies have indicated
that LPS constitute a risk factor for diseases such as
atherosclerosis and diabetes [
The endothelium also plays a major role in the
pathogenesis of sepsis. Endothelial cells (ECs) line the inner wall
of blood vessels, lying at the interface between circulating
blood and the surrounding tissue [
]. During infection,
LPS bind to the surface of ECs, resulting in the activation
of endothelial signaling pathways and the release of
inflammatory mediators [
3, 5, 6
]. These mediators induce
the production of reactive oxygen species, secretion of
chemokines and adhesion molecules, reduction of
antiinflammatory mediators, and transmigration of leukocytes
5, 7, 8
]. The infection-induced inflammatory reaction is
further mediated by complex interactions between
circulating leukocytes and the vascular endothelium [
3, 7, 9, 10
The adherence of monocytes to the activated endothelium
and their subsequent proliferation are critical for
atherosclerotic plaque formation [
produced by ECs are vital for promoting the movement of
circulating monocytes to atherosclerotic vessels and the
infection site [
9, 14, 16
]. Monocyte chemoattractant
protein-1 (MCP-1), a potent chemoattractant for
monocytes, is closely involved in atherosclerosis development [
11, 12, 17, 18
]. Studies have observed elevated plasma
MCP-1 levels in patients with coronary artery disease, with
the highest levels being observed in those with acute
coronary syndrome and diabetes [
17, 19, 20
LPSinduced MCP-1 secretion from the vascular endothelium
has been reported to recruit circulating monocytes, the
underlying mechanism remains largely unexplained [
Protease-activated receptor-2 (PAR-2) is a member of
the G protein-coupled receptor family with seven
transmembrane-spanning domains, and it is mainly
activated by trypsin [
]. PAR-2 is a key mediator of
innate immunity and inflammatory response propagation
. Endothelial PAR-2 is mainly activated by the locally
released trypsin that accompanies tissue injury or
inflammation. PAR-2 is widely expressed in nearly all cell
types in the vascular wall (ECs, myocytes, and
]. Several studies have revealed that PAR-2
is involved in inflammation and endotoxin shock [
]. The expression of PAR-2 was increased 5- to
10fold in ECs after LPS exposure in vitro, thus suggesting
the possible involvement of PAR-2 in endotoxemia .
Immunohistochemical studies have demonstrated
preferential and localized increases in the expression of PAR-2
in the aorta and jugular vein, and these increases were
associated with endotoxin shock [
enhanced PAR-2 expression has been observed in human
coronary atherosclerotic lesions, suggesting that PAR-2
regulates signaling during vascular injures [
Increasing bodies of evidence from cellular and animal
studies reveal that PAR-2 activation is associated with
increased MCP-1 secretion [
]. However, the
relationship between the PAR-2 signaling pathway and
LPSactivated MCP-1 secretion remains unclear [
The present study investigated whether LPS activates
PAR-2 expression and consequently enhances
trypsininduced PAR-2 signaling and subsequent MCP-1
secretion in human vascular ECs.
Unless stated otherwise, trypsin, purified LPS (obtained
through phenol extraction) from Escherichia coli (serotype
O26:B6), salts, buffers, and all other chemicals of reagent
grade were purchased from Sigma-Aldrich (St. Louis, MO,
USA). The specific PAR-1 agonist (TRAP6), PAR-2
agonist (AC 55541), PAR-4 agonist (AY-NH2) and the selective
PAR-2 antagonist (FSLLRY-NH2) were purchased from
Tocris Bioscience (Bristol, UK). Antibody-directed
phosphorylated ERK was purchased from Novus (St. Charles,
MO, USA), and anti-ERK was purchased from BD
(Franklin Lakes, NJ, USA). Antiphosphorylated p38, anti-p38,
and anti-c-JUN N-terminal kinase (JNK) were purchased
from Calbiochem (San Diego, CA, USA). Anti-MCP-1
was purchased from Sigma-Aldrich. Monoclonal
antiphosphorylated JNK, anti-PAR-2 (Additional file 1: Figure S1),
and anti-β-actin antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA).
EA. hy926 cells
The human EC line, EA. hy926, was originally derived
from a human umbilical vein obtained from the
American Type Culture Collection (Manassas, VA, USA). The
cells were grown in Dulbecco’s Modified Eagle’s
Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12;
1:1, Life Technologies, Grand Island, NY, USA)
supplemented with 10% fetal bovine serum (FBS), 1%
Lglutamine, and 1% penicillin–streptomycin in a
humidified atmosphere of 5% CO2 at 37 °C. During cell culture,
the medium was changed every 3 days until the cells
reached 90% confluence. To prevent FBS-induced
trypsin inactivation, all cells were incubated in a FBS-free
DMEM with 1% penicillin–streptomycin solution during
Intracellular calcium release measurement
Intracellular calcium release in ECs was assessed
through microfluorimetric measurements of the
cytosolic Ca2+ concentration by using fura-2 as described
]. In brief, ECs were incubated with
5 μM fura-2 AM (Invitrogen, Carlsbad, CA, USA) for
1 h at 37 °C and subsequently washed and bathed in
DMEM supplemented with 10% FBS and penicillin–
streptomycin solution (100 units/mL, 100 μg/mL;
Invitrogen) under 5% CO2. The cells were alternately
excited at 340 and 380 nm using an optical filter
changer (Lambda 10-2, Sutter Instruments, Novato,
CA, USA). Emission was measured at 500 nm, and
images were captured using a charge-coupled device
camera (CoolSnap HQ2, Photometrics) attached to an
inverted Nikon TE 2000-U microscope. The captured
images were analyzed using MAG Biosystems
Software. All experiments were performed at room
temperature (approximately 25 °C).
Reverse transcription polymerase chain reaction
Total RNA was isolated using RNAzol solution
(Biogenesis, Poole, Dorset, UK), according to the manufacturer’s
instructions. RNA purity was estimated though optical
density measurements at 260/280 nm. The derived total
RNA (5 μg) was subjected to first-strand cDNA
synthesis in a 10-μL reaction volume containing 250 mM
TrisHCl (pH 8.3 at 20 °C), 375 mM KCl, 15 mM MgCl2,
1 mM 1,4-dithiothreitol (DTT), 1 mM of each dNTP,
and 20 U of an RNase inhibitor in the presence of 1.5 μg
of an oligo dT primer and 200 U of Superscriptase (all
chemicals were obtained from Life Technologies). After
the completion of the first-strand cDNA synthesis
process, the reaction was terminated by heat inactivation
(5 min, 95 °C) and the derived total RNA was diluted
with water to obtain 50 ng/μL of RNA equivalent. cDNA
equivalent to 100 ng of the total RNA was subjected to
polymerase chain reaction (PCR) in a 50-μL reaction
volume, containing 10 mM Tris-HCl (pH 9 at 25 °C),
50 M KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 0.1% (v/
v) Triton X-100, 2 mM DTT, 200 μM of each dNTP,
1 μM of each primer, and 0.2 U of TaqDNA polymerase
(AB Biotechnology) under the following conditions:
denaturation, 30 s at 94 °C; primer annealing, 1 min at
58 °C; and primer extension, 1 min at 72 °C. The PCR
products (10 μL) were electrophoresed in 1% agarose
gels and visualized through ultraviolet illumination. The
PAR-2 forward and reverse primers were 5′-TGGC
ACCATCCAAGGAAC-3′ and 5′-GTCAGCCAAGGC
CAGATT-3′, respectively. The glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) forward and reverse primers were
5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCAC
Cells were lysed in a buffer containing 50 mM Tris
(pH 8.0), 5 mM ethylenediaminetetraacetic acid,
5 mM ethylene glycol tetraacetic acid, 0.2% sodium
dodecyl sulfate (SDS), 0.5% Nonidet P-40, 1 mM sodium
orthovanadate, 20 mM sodium pyrophosphate, and Roche
complete protease inhibitor mixture (Roche, Mannheim,
Germany). The protein in the medium was precipitated
with 10% trichloroacetic acid and 0.1% sodium
deoxycholate. The precipitates were redissolved in the SDS sample
buffer. The extracted protein was separated through
SDSpolyacrylamide gel electrophoresis and transferred onto a
polyvinylidene fluoride membrane (Bio-Rad Laboratories,
Hercules, CA, USA). The membrane was blocked with
2%–3% skim milk in Tris-buffered saline (TBS) containing
0.05% Tween20; subsequently, the membrane was probed
with the anti-PAR-2, anti-phospho-ERK (p-ERK),
antitotal ERK or anti-phospho-p38 MAPK (p-p38),
antitotal p38 MAPK, anti-phospho-JNK (p-JNK),
antitotal JNK, anti-MCP-1, and anti-β-actin antibodies.
After overnight incubation with different antibodies at
4 °C, the membrane was washed three times with TBS and
incubated with horseradish peroxidase-conjugated
secondary antibodies for 1 h at room temperature. Finally,
the immunoblots were quantified using Image J
densitometry analysis software (National Institutes of Health,
Bethesda, MD, USA).
Cell adhesion assay
EA. hy926 monolayers, grown as described previously, were
established in culture dishes and subsequently treated with
LPS (0, 0.25, 0.5, 1, and 2 μg/mL). After a 24 h of
incubation, the EA. hy926 cells in each well were treated with
trypsin (5 μg/mL) and cultured for 12 h, followed by
incubation with 2 × 105 peripheral blood mononuclear cells for
30 min in a humidified atmosphere of 5% CO2 at 37 °C
]. After incubation, non-adherent cells were removed by
washing two times with PBS. Six random high-power
microscopic fields (100×) were photographed, and the
number of adhered cells was directly calculated.
All experiments were performed at least in triplicate. Data
are presented as mean ± standard error of the mean
(SEM). Statistical analysis was performed using the
Student t test or analysis of variance, followed by the Dunnett
multiple comparison test by using Prism software (version
3.00 for Windows GraphPad, San Diego, CA, USA). A P
value of <0.05 was considered statistically significant.
Analysis of PAR-2 expression in LPS-treated ECs
PAR-2 is highly expressed in ECs and plays an important
role in inflammation [
]. In this study, PAR-2 expression
after LPS treatment was examined in vitro. Reverse
transcription (RT)-PCR and Western blotting revealed
increased PAR-2 mRNA and protein expression levels in
EA. hy926 cells (Fig. 1). Notably, LPS increased PAR-2
expression in EA. hy926 cells. The PAR-2 mRNA levels in
EA. hy926 cells were significantly elevated after LPS
treatment (1 μg/mL, 5 min; P < 0.01 compared with the control
group; Fig. 1a and b). However, the stimulating effects of
LPS pretreatment on PAR-2 protein levels were not
apparent after short-term LPS treatment (<12 h). By contrast,
1 μg/mL of LPS resulted in increased PAR-2 protein
levels, with a peak at 20 h of treatment (Fig. 1c and d).
LPS pretreatment enhances trypsin-induced intracellular calcium release in ECs
Trypsin is an endogenous PAR-2 activator [
examined the effects of LPS pretreatment on
trypsininduced intracellular calcium release in ECs. The relative
changes in intracellular calcium release were determined
using the fura-2 F340/F380 nm ratio. Basal intracellular
calcium release (prior to trypsin exposure) did not differ
between the control and LPS-pretreatment groups. In
addition, treatment with LPS alone (1 μg/mL, 24 h) did
not alter EC viability (data not shown). In the absence of
LPS pretreatment, trypsin (2 μg/mL) induced a rapid
and transient increase in intracellular Ca2+ release in
ECs (Fig. 2a). However, after LPS pretreatment (1 μg/
mL, 24 h), the trypsin-induced intracellular Ca2+ release
increased to 40% (Fig. 2c). Figure 2b presents a typical
example of the averaged maximal intracellular Ca2+
levels induced by trypsin in the absence or presence of
LPS pretreatment. The maximal increase in the
trypsininduced intracellular Ca2+ release was higher in the
LPSpretreatment group than in the control group (Fig. 2d).
The concentration–response curve for the
trypsininduced Ca2+ release in the absence or presence of LPS
pretreatment (1 μg/mL, 24 h) is shown in Fig. 2e. In the
concentration-response curve for the trypsin-induced
Ca2+ release after LPS pretreatment, a left shift with
changing maximum response suggests the stimulating
effects of LPS pretreatment on PAR-2.
LPS pretreatment enhances trypsin-induced ERK/p38 phosphorylation in ECs
A study demonstrated that PAR-2 activation can
influence cellular functions through several signal
transduction pathways [
]. We thus investigated the
trypsin-induced phosphorylation of ERK and observed
that p-ERK levels were significantly higher in EA.
hy926 cells stimulated with trypsin (5 μg/mL, 10 min)
than in untreated cells (Fig. 3). Subsequently, the
observed ERK phosphorylation subsided gradually.
However, after a 24 h of LPS pretreatment, trypsin enhanced
the phosphorylation of ERK and p38 (Fig. 4a). The
pERK and p-p38 levels were significantly enhanced in
the LPS-pretreatment group supplemented with
trypsin (Fig. 4b and c). However, compared with
trypsin alone, the combined treatment of trypsin and LPS
did not significantly enhance JNK phosphorylation
LPS pretreatment enhances trypsin-induced MCP-1 secretion and cell adhesion in ECs
PAR-2 activation is associated with the adhesion of
leukocytes to the vascular endothelium [
whether LPS pretreatment regulates trypsin-induced
cell adhesion molecule expression and cell adhesion
functions remains unclear. Our results revealed that
compared with trypsin alone, the combined treatment
of LPS (0.25, 0.5, 1, and 2 μg/mL; 24 h) and trypsin
(5 μg/mL) enhanced MCP-1 protein secretion (Fig. 5a).
To assess the effects of LPS pretreatment on
trypsininduced mononuclear cell adhesion, EA. hy926 cells
were treated with different LPS concentrations (0,
0.25, 0.5, 1, and 2 μg/mL) for 24 h, followed by treatment
with trypsin (5 μg/mL) for 12 h. Mononuclear cells
were added to the EC culture to assess cell adhesion
functions. When trypsin stimulation was not applied
very few mononuclear cells adhered to the ECs;
however, trypsin clearly increased mononuclear cell–EC
adhesion. In addition, LPS pretreatment enhanced
trypsin-induced mononuclear cell–EC adhesion in a
concentration-dependent manner (Fig. 5b). Notably,
the combined treatment of LPS and trypsin exerted
more significant regulatory effects on cell adhesion
functions than did trypsin alone. Furthermore, to
investigate the role of trypsin-induced ERK and p38
phosphorylation, LPS-pretreated EA. hy926 cells were
pre-incubated with PD98059 (25 μM), SB203580
(10 μM), or PD98059 (25 μM) and SB203580
(10 μM) for 30 min (Fig. 6a), which significantly
inhibited the enhancement effects of LPS
pretreatment on trypsin-induced MCP-1 secretion. Similarly,
pre-incubation with PD98059 (25 μM) or SB203580
(10 μM) inhibited the promotive effects of LPS
pretreatment on trypsin-induced cell adhesion functions
(Fig. 6b and c). PD98059 and SB203580 also
influenced the regulatory effects of LPS pretreatment on
MCP-1 secretion and cell adhesion functions in
trypsin-treated EA. hy926 cells.
The relationship of PAR-2 activity and the levels of MCP-1 production in response to the combined treatment of trypsin and LPS
There are four known protease-activated receptors (PAR
1-4). PAR-1, PAR-3, and PAR-4 can be activated by
thrombin, and PAR-2 can be mainly activated by trypsin
and numerous studies have demonstrated that PAR-2 is
highly expressed in ECs [
23–25, 27, 39
]. To further rule
out the possibility of the signaling coming from either
PAR-1 or PAR-4 activation in ECs since trypsin can also
activate both of these receptors, selective peptide
agonists were used. PAR-1 agonist (TRAP6, 100 nM) or
PAR-4 agonist (AY-NH2, 50 μM) was used to examine
its effect on ERK/p38 phosphorylation and MCP-1
secretion in ECs (Fig. 7). TRAP6 failed to show increased
ERK/p38 phosphorylation and MCP-1 secretion. However,
the addition of AY-NH2 mildly increased MCP-1
secretion (Fig. 7d). The underlying mechanism of PAR-4
activation in the modulation of MCP-1 secretion is unknown,
which should be validated in future study. In the same
time, we also used a specific PAR-2 agonist (AC 55541,
10 μM) to compare with the effect of LPS plus trypsin on
the stimulation of ERK/p38 phosphorylation and MCP-1
secretion in ECs. The result showed a similar pattern of
stimulatory effect between PAR-2 agonist treated group
and the LPS plus trypsin treated group. In addition, we
also examined the effect of the selective PAR-2 antagonist
(FSLLRY-NH2, 50 μM) on the induction of MCP-1
secretion by LPS plus trypsin treatment. As shown in Fig. 7d,
the application of the PAR-2 antagonist specifically
inhibited the induction of MCP-1 secretion by LPS plus trypsin
treatment. These findings suggest that LPS plus trypsin
treatment regulated the related signaling pathway mainly
through PAR-2 activation.
The present study provides the first evidence that LPS
pretreatment potentiates calcium mobilization and ERK/
p38 MAPK pathway activation and subsequently leads
to MCP-1 secretion by inducing PAR-2 gene expression
in vascular ECs. In addition, pretreatment with selective
inhibitors of ERK (PD98059), p38 (SB203580), or both
suppressed LPS-induced MCP-1 secretion and cell
adhesion functions in ECs (Fig. 8).
According to the previous studies, LPS has potent
proinflammatory properties, which can activate recognition
receptors on ECs, leading to the release of inflammatory
]. Inflammatory mediators function in
autocrine and paracrine loops to further activate the
monocyte and local endothelium . The combined
effects of LPS and inflammatory mediators on the
endothelium may engender significant pathological changes.
The Bruneck study provided the first epidemiological
evidence that circulating LPS constitute strong risk factor for
carotid atherosclerosis [
]. Moreover, LPS accelerated the
development of atherosclerotic plaques in rabbits on
hypercholesterolemic diets and in mice with
apolipoprotein E-deficient [
]. In healthy humans, an LPS dose of
1 ng/kg is sufficient to induce symptoms including fever
and nausea [
]. In a clinical observational study, the
median endotoxin level in patients with sepsis was 300 pg/
]. In in vitro studies on ECs, the LPS concentration
range used in the basic experiments was 0.1–10 μg/mL [
]. Therefore, the use of different LPS concentrations in
our experiment is reasonable. However, the data presented
in the present in vitro study of an LPS-induced
inflammatory model do not fully represent the in vivo action of LPS;
hence, the results of this study warrant further validation in
additional animal models.
PAR-2 was originally cloned in 1994 and plays major
pathophysiological roles in angiogenesis, tissue regeneration,
and inflammation [
]. Several studies have reported that
PAR-2 exerts extensive effects on inflammatory responses in
vascular tissues, and that LPS exposure results in increased
PAR-2 levels, both in vitro and in vivo, thus suggesting the
possible role of PAR-2 in endotoxemia [
10, 24, 43
demonstrated that LPS increases PAR-2 mRNA and protein
expression in ECs. Moreover, trypsin is a potent PAR-2
activator that cleaves and triggers PAR-2 activation [
Trypsin-induced PAR-2 activation increases intracellular
calcium release through the activation of phospholipase C
isoforms by using several Gq/G11-coupled
receptormodulated intracellular targets [
]. Furthermore, Ca2+
signaling activates tyrosine kinases, which contribute to
mitogen-activated protein kinase (MAPK) activation in ECs
]. Previous studies have demonstrated that PAR-2 can
activate multiple kinase pathways, including the extracellular
signal-regulated kinase (ERK)/p38 MAPK pathway, in a cell
type-specific manner [
]. In the present study, we
analyzed the combined effects of LPS and trypsin on PAR-2
activation in ECs. LPS pretreatment enhances trypsin-induced
intracellular Ca2+ release, ERK/p38 phosphorylation, and
MCP-1 secretion. To elucidate the role of cytosolic calcium
in the signaling pathway, we added BAPTA-AM, an
intracellular calcium chelator, to identify it. The addition of
BAPTA-AM slightly suppressed the LPS-induced ERK/p38
phosphorylation and significantly inhibited MCP-1 synthesis
(Additional file 2: Figure S2), implying that calcium signaling
is involved in the pathway. The inhibition of PAR-2 activity
by a selective PAR-2 peptide antagonist (FSLLRY-NH2) also
blocked the induction of MCP-1 secretion by LPS plus
trypsin treatment, supporting our hypothesis that PAR-2 plays
an important role in the process. Therefore, we concluded
that LPS and trypsin can synergistically stimulate the PAR-2
Previous studies have demonstrated that PAR-2 activation
in vascular ECs significantly increases monocyte
recruitment, possibly through chemokine induction [
The transmigration of monocytes to sub-endothelial
lesions is the initial step of atherosclerotic plaque
12, 31, 47
]. MCP-1, a glycoprotein with an apparent
molecular mass of 14 kDa, is produced by smooth muscle
cells, ECs, and macrophages; MCP-1 is thus a highly
potent chemoattractant for monocytes [
]. MCP-1 is
highly expressed in human atherosclerotic plaques and is
crucial in monocyte recruitment into sub-endothelial
5, 18, 19
]. Some studies have reported that LPS
induces MCP-1 secretion from the vascular endothelium;
however, the underlying mechanism is not yet clearly
]. According to our study results, we
speculate that LPS and trypsin-activated PAR-2 can
induce MCP-1 secretion.
Previous studies have showed that PAR-2 activates
ERK and p38 MAPK in non-ECs [
25, 29, 33, 46
]. In the
present study, incubation with trypsin or LPS resulted in
significant ERK and p38 MAPK phosphorylation and
activation in EA. hy926 cells. However, pretreatment with
selective ERK and p38 MAPK inhibitors blocked the
promotive effects of trypsin and/or LPS-activated PAR-2
on MCP-1 secretion. The p38 MAPK signaling pathway
plays an important role in mediating pro-inflammatory
responses in ECs [
]. Additionally, previous studies
have demonstrated that the oral administration of a specific
p38 MAPK inhibitor reduces cytokine production, leukocyte
responses, and inflammation in a human endotoxemia
]. Taken together, these data suggest that PAR-2
signaling through the MAPK pathways results in increased
MCP-1 secretion in ECs.
The present in vitro model of LPS-induced MCP-1
secretion through the PAR-2 signaling pathway may not
be directly translatable to clinical investigations of
atherosclerotic cardiovascular disease. Nevertheless, the presented
preliminary results may encourage further research on
identifying the molecular mechanisms underlying
PAR-2mediated MCP-1 secretion and vascular inflammation.
that PAR-2 directly modulates endothelial functions and
EC–monocyte interactions by regulating MCP-1 protein
release. Our findings also provide evidence that the
PAR-2 signaling pathway exerts inflammatory effects on
vascular ECs, leading to the initiation of
infectioninduced pro-atherogenic inflammatory responses. This
information can be used to develop new strategies for
preventing the development of atherosclerotic
Additional file 1: Figure S1. Western blot – Anti-PAR-2 antibody. Lane
A: Control cell lysate at 10 μg. Lane B: PAR-2 agonist (AC 55541, 10
μM)treated cell lysate at 10 μg. Predicted band size: 43~55 kDa. (TIFF 823 kb)
Additional file 2: Figure S2. The effect of calcium chelator (BAPTA-AM)
on the LPS-plus-trypsin-induced ERK/p38 MAPK phosphorylation and
MCP-1 synthesis. EA. hy926 cells (1 × 106/mL) were pretreated with
BAPTA-AM (50 nM) for 30 min, after which they were stimulated with LPS
(2 μg/mL) for 24 h and with trypsin (5 μg/mL) for 10 min. Control cells
were treated with 0.1% DMSO. a Representative data of the p-ERK, ERK,
p38, pp38 and MCP-1 protein levels, and β-actin was used as the loading
control. b Normalization of the p-ERK and total ERK levels. c
Normalization of the p-p38 and total p38 levels. d Normalization of the
MCP-1 and total β-actin levels. Bar graphs represent means ± SEM from
three independent experiments. *p < 0.05 compared with the control
group; #p < 0.05 compared with the LPS-plus-trypsin treatment group.
(TIFF 4147 kb)
DMEM: Dulbecco’s Modified Eagle’s Medium; ECs: Endothelial cells;
ERK: Extracellular signal-regulated kinase; FBS: Fetal bovine serum;
GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; JNK: c-Jun N-terminal
kinase; LPS: Lipopolysaccharides; MAPK: Mitogen-activated protein kinase;
MCP-1: Monocyte chemoattractant protein-1; PAR-2: Protease-activated
receptor-2; PCR: Polymerase chain reaction; SDS: Sodium dodecyl sulfate;
SEMs: Standard errors of the means; TBS: Tris-buffered saline
This study was supported in part by research grants from Shin Kong Wu
Ho-Su Memorial Hospital (SKH-TMU-101-07; SKH-8302-99-DR-15) and
Taipei Medical University (TMU105-AE1-B21), Taipei, Taiwan.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
Conceived and designed the experiments: HHC, PYC, and THC. Analysis and
interpretation of data: JCL, YML, and SHL. Performed the experiments: PYC,
WRH and WPC. Administrative, technical, or material support: PYC, JJC, LCS,
and THC. Wrote the paper: HHC, THC, and LCS. All authors read and
approved the final manuscript.
Ethics approval and consent to participate
In summary, our results reveal that PAR-2 plays an
important role in regulating MCP-1 secretion through the
ERK/p38 MAPK signaling pathway, thus demonstrating
Consent for publication
All the authors have read and approved the paper for publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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