Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells
Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells
Ge Wang 0 2 5 6
Kan Liu 1 5 6
Yue Li 3 5 6
Wei Yi 0 5 6
Yang Yang 0 5 6
Dajun Zhao 0 5 6
Chongxi Fan 4 5 6
Honggang Yang 2 5 6
Ting Geng 2 5 6
Jianzhou Xing 2 5 6
Yu Zhang 0 5 6
Songtao Tan 2 5 6
Dinghua Yi * 0 5 6
0 Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University , 127 Changle West Road, Xi9an 710032, China,
1 School of Basic Medical Sciences, The Fourth Military Medical University , 169 Changle West Road, Xi9an 710032, China,
2 Department of Cardiovascular Surgery, Guangdong Provincial Corps Hospital of Chinese People's Armed Police Forces, Guangzhou Medical University , 268 Yanling Road, Guangzhou 510507, China,
3 Department of Air Logistics, The 463rd Hospital of PLA , 46 Xiaoheyan Road, Shenyang 110042, China,
4 Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University , 1 Xinsi Road, Xi9an 710038 , China
5 Funding: This study was supported by grants from the 12th National Five Years Supporting Project of China (2011BAI11B20), the National Natural Science Foundation of China (81100137 and 81000938). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
6 Editor: Niels Olsen Saraiva Camara, Universidade de Sao Paulo , Brazil
Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-a (TNF-a)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-a treatment not only significantly increased the adhesion of
monocytes to HUVECs and inflammatory cytokine (sICAM1, sE-selectin, MCP-1
and IL-8) production in cell culture supernatants but it also increased ICAM and
MMP9 protein expression in HUVECs. TNF-a also effectively increased the
ERSrelated molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK). EP
treatment effectively reversed the effects of the TNF-a-induced adhesion of
monocytes on HUVECs, inflammatory cytokines and ERS-related molecules.
Furthermore, thapsigargin (THA, an ERS inducer) attenuated the protective effects
of EP against TNF-a-induced inflammatory injury and ERS. The PERK siRNA
treatment not only inhibited ERS-related molecules but also mimicked the
protective effects of EP to decrease TNF-a-induced inflammatory injury. In
summary, we have demonstrated for the first time that EP can effectively reduce
vascular endothelial inflammation and that this effect at least in part depends on the
attenuation of ERS.
The incidence of cardiovascular diseases, such as atherosclerosis (AS), is
increasing globally and has become a costly public health issue . The
endothelium plays a critical role in the regulation of vascular function and in the
development of AS . Increasing evidence suggests that AS is the result of a
prolonged and excessive inflammatory process occurring in the vascular wall,
often beginning with inflammatory changes to the endothelium and characterized
by the expression of adhesion molecules [3, 4]. Multiple cytokines and signaling
pathways have been implicated in inflammation-induced vascular endothelial cell
(EC) injury . However, the underlying pathophysiological mechanisms of EC
inflammatory injury have not been fully elucidated, and more effective treatment
methods and drugs to treat EC inflammatory injury need to be explored.
Pyruvate, which is the anionic form of a simple alpha-keto acid, plays a key role
in intermediary metabolism as a product of glycolysis and as the starting substrate
for the tricarboxylic acid (TCA) cycle . Pyruvate is also an important
endogenous scavenger of reactive oxygen species (ROS) and an anti-inflammatory
agent . However, its poor stability in solution may limit its use as a therapeutic
agent . Ethyl pyruvate (EP), which is a stable and lipophilic derivative of
pyruvate, not only overcomes the disadvantages of pyruvate but also possesses
many important pharmacological effects . EP can effectively increase the
survival rate and/or improve organ dysfunction in animal models of critical
diseases, such as severe sepsis, hemorrhagic shock, acute pancreatitis, and acute
respiratory distress syndrome, and of intestinal injuries in ischemic models .
Notably, EP has been demonstrated to be a potent anti-inflammatory agent in a
variety of in vivo and in vitro model systems . However, the protective effects
and the mechanisms underlying the action of EP against EC inflammatory injury
are not fully understood.
The endoplasmic reticulum (ER) is an organelle involved in protein folding and
modification, and it acts as a major intracellular calcium store . ER stress
(ERS) is caused by disturbances in the structure and function of the ER and can
result from hypoxia, nutrient deprivation, Ca2+ imbalances or perturbations in
protein glycosylation, leading to the accumulation of unfolded proteins in the ER
and the activation of the unfolded protein response (UPR) pathway [12, 13]. The
UPR pathway is triggered by three sensors, including activating transcription
factor 6 (ATF6), activating transcription factor 4 (ATF4), PKR-like ER kinase
(PERK) and inositol-requiring enzyme 1 (IRE1) . Under normal
conditions, these sensors remainin an inactive state, in which they are bound to
the chaperone glucose-regulated protein 78 (GRP78). ER stress causes misfolded
and unfolded proteins to bind to GRP78, releasing it from the UPR sensors and
triggering the UPR by inducing the transcription of genes encoding relevant
proteins. This UPR activation thereby reduces global protein synthesis and
stimulates ER-associated protein degradation. These activities serve to restore
normal ER function, or, when normal ER function cannot be restored, trigger
apoptosis [16, 17]. Signaling through the PERK, IRE1, ATF4 and ATF6 pathways
can trigger pro-apoptotic signals via the activation of downstream molecules, such
as the C/EBP homologous protein (CHOP), the a-subunit of eukaryotic
translational initiation factor 2 (eIF2a) and members of the apoptotic family [18
20]. Caspase12 is considered to be critical in ERS-induced apoptosis, and it is
activated during the ER stress response. Under significantly elevated ERS, the UPR
is unable to restore normal cellular function, and signaling switches from
prosurvival to pro-apoptotic, in which pro-caspase12 is released, and the apoptotic
response is initiated. The released pro-caspase12 is subsequently cleaved to its
active caspase12 form, which has been proposed to be a key mediator in the
initiation of ERS-induced apoptosis . It is worth noting that the inflammatory
response can induce ERS, and the inhibition of ERS can effectively attenuate EC
inflammatory injury . Remarkably, the pharmacological actions of other
pyruvate derivatives are closely related to ERS .
Most importantly, we assessed the anti-inflammatory effects of EP in human
umbilical vein endothelial cells (HUVECs) and explored the role of ERS in this
EP, thapsigargin (THA, an ERS inducer), tumor necrosis factor-a (TNF-a),
dimethyl sulfoxide (DMSO) and the antibody against TNF-a were purchased
from the Sigma-Aldrich Company (St. Louis, MO, USA). The Cell Counting Kit-8
(CCK8) was purchased from Dojindo (Kumamoto, Japan). PERK siRNA and the
antibodies against GRP78, intercellular cell adhesion molecule 1 (ICAM-1),
matrix metalloproteinase 9 (MMP9) and caspase12 were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Human soluble adhesion molecule
ICAM-1 (sICAM-1), soluble E-selectin (sE-selectin), interleukin (IL-8) and
monocyte chemoattractant protein-1 (MCP-1) ELISA kits were obtained from
R&D Systems (Minneapolis, MN, USA). The antibodies against
phosphorylatedPERK (p-PERK), PERK, ATF4 and b-actin were obtained from Cell Signaling
Technology (Beverly, MA, USA). The rabbit anti-goat, goat anti-rabbit and goat
anti-mouse secondary antibodies were purchased from the Zhongshan Company
Cell culture and treatments
HUVECs (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 medium
(HyClone, UT, USA) supplemented with 10% fetal calf serum, 2 mM
Lglutamine, 100 U/ml penicillin, and 100 g/ml streptomycin at 37C in 5% CO2
and 95% air. The EP solution was prepared in DMSO and diluted with culture
medium immediately prior to the experiment. DMSO (0.01%) was used as the
control group. As described in previous studies , TNF-a was used to mimic
inflammatory-induced EC injury. The cells were first treated with TNF-a (1, 5 or
10 ng/mL) for 6 h to explore the effects of TNF-a on cell viability, inflammatory
cytokines and ERS-related molecules. Further, the cells were treated with EP (1, 5
or 10 mM) for 2 h in the absence or presence of THA (1 mM) and PERK siRNA
(pretreated for 24 h). The cells were then treated with TNF-a (10 ng/mL; the
concentration was chosen based on preliminary experiments). After the
treatments were performed, the cells were harvested for further analysis.
Analysis of cell viability
CCK-8 was used to measure cell viability. Cells were cultured in a 96-well plate
and exposed to various treatments, according to the manufacturers protocols.
The control group was treated with 0.1% DMSO. Then, 10 ml of CCK-8 was
added to each well, and the plate was incubated at 37C for 2 h. Optical density
(OD) values were assessed at 450 nm using a microplate reader (SpectraMax 190,
Molecular Device, USA), and cell viability was expressed in terms of the OD value.
Analysis of monocyte adhesion
Monocyte adhesion to ECs was determined using U937 cells (ATCC, Manassas,
VA, USA) as previously described by our group . In brief, HUVECs were
grown to confluence in 96-well plates. After treatment, the HUVECs were gently
washed with serum-free media, and calcein-AM-labeled U937 cells (56104/ml
DMEM medium containing 1% FBS) were then added. After incubation for 1 h,
the HUVEC monolayer was gently washed with phosphate-buffered saline (PBS)
to remove unbound monocytes. The fluorescence was measured to determine the
levels of bound monocytes using a microplate reader (SpectraMax 190, Molecular
Device, USA) at excitation and emission wavelengths of 496 and 520 nm,
After treatment, the cell culture supernatants were collected, and the production
of sICAM1, sE-selectin, MCP-1 and IL-8 by the HUVECs was measured using
special ELISA kits.
Small interfering RNA transfection
For siRNA transfections, HUVECs were plated onto 6-well, 24-well or 96-well
plates and allowed to grow to subconfluency. The cells were transiently transfected
with the negative control or PERK siRNA at 100 pM for 24 h using the
Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) in
OPTIMEM media (Gibco, Carlsbad, CA, USA). The cells were subsequently prepared
for use in further experiments.
After treatment, the HUVECs were lysed in sample buffer (150 mM Tris pH 6.8,
8 M urea, 50 mM DTT, 2% sodium dodecyl sulfate, 15% sucrose, 2 mM EDTA,
0.01% bromophenol blue, 1% protease and phosphatase inhibitor cocktails),
sonicated, boiled, run through an 812% Bis/Tris gel using 56 MES buffer
(Invitrogen) and transferred to an Immobilon NC membrane (Millipore, Billerica,
MA, USA). The membranes were blocked with 5% nonfat milk in TBST (150 mM
NaCl, 50 mM Tris pH 7.5, 0.1% Tween-20) and then probed with antibodies
against GRP78, ICAM1, caspase12 and MMP9 (1:500) and against p-PERK,
TNFa, ATF4 and b-actin (1:1000) overnight at 4C. The membranes were then placed
in blocking buffer, washed with TBST, probed with secondary antibodies (1:5000)
in blocking buffer at room temperature for 90 min and washed. Fluorescence was
detected using a Bio-Rad imaging system (Bio-Rad, USA), and the signals were
quantified using the Image Lab Software (Bio-Rad, USA).
All of the values are presented as the mean standard deviation (SD). Group
comparisons were performed using ANOVA (SPSS 13.0). All of the groups were
analyzed simultaneously using the LSD t-test. A difference of P,0.05 was
considered to be statistically significant. All of the experiments were repeated three
HUVECs were first treated with TNF-a (1, 5 or 10 ng/mL) for 6 h to explore the
effects of TNF-a on cell viability, inflammatory cytokines and ERS-related
molecules. As shown in Fig. 1A, TNF-a treatment had no significant effects on
cell viability (vs. the control group, P.0.05). However, TNF-a increased sICAM-1
production in the cell culture supernatants to 6.140.41, 11.930.85 and
15.011.26 pg/well, for each respective trial (vs. the control group, P,0.01,
Fig. 1B). TNF-a also up-regulated ICAM-1 and MMP9 protein expression in the
HUVECs (vs. the control group, P,0.01, Fig. 1B) in a dose-dependent manner,
and the Western blot results are shown in Fig. 1C (vs. the control group, P,0.01).
In addition, TNF-a increased the expression of the ERS-related protein GRP78, as
well as ATF4, cleaved-caspase12 and p-PERK (vs. the control group, P,0.01,
Fig. 1C). The effects of TNF-a on these proteins were the most obvious at 10 ng/
mL, which led to the increased expression of ICAM1, MMP9, GRP78, ATF4,
cleaved-caspase12 and p-PERK to 484.0925.80%, 314.1520.73%,
290.4216.46%, 436.4826.93%, 519.0431.78% and 406.5124.18%,
respectively. These results suggest that ERS may be involved in EC inflammatory
injury. TNF-a at a concentration of 10 ng/mL was selected for further
Effects of EP on TNF-a-induced cell viability, monocyte adhesion
and inflammatory cytokines
HUVECs were treated with EP (1, 5 or 10 mM) for 2 h and then subjected to
TNF-a(10 ng/mL) treatment. As shown in Fig. 2A, there were no significant
differences in cell viability among the groups (P.0.05). Exposure of the HUVECs
to TNF-a significantly increased their binding to the U937 monocytes to
874.0251.69% (vs. the control group, P,0.01, Fig. 2B). Pretreatment with EP
significantly inhibited the TNF-a-induced binding of the U937 monocytes to the
HUVECs to 672.1742.65%, 560.0834.01% and 348.1023.84% for each trial,
respectively (vs. the TNF-a group, P,0.01). In addition, exposure of the HUVECs
to TNF-a significantly increased the production of sICAM1, IL-8, MCP-1 and
sEselectin to 14.591.30 pg/well, 9.170.75 ng/well, 14.711.21 ng/well and
1.7150.128 ng/well in the cell culture supernatants (vs. the control group,
P,0.01, Figs. 2C2F), respectively. The EP treatment reversed the
TNF-ainduced sICAM1, IL-8, MCP-1 and sE-selectin production (vs. the TNF-a group,
P,0.01), and the effects were the most obvious at 10 mM EP, which led to
decreased sICAM1, IL-8, MCP-1 and sE-selectin production to 6.950.57 pg/
well, 4.150.41 ng/well, 6.940.65 ng/well and 0.8100.064 ng/well. TNF-a
(10 ng/mL) significantly up-regulated the expression of the ICAM-1 and MMP9
proteins to 496.1532.87% and 320.4719.95% in the HUVECs (vs. the control
group, P,0.01, Fig. 3). EP treatment reversed the effects of TNF-a, and these
effects were the most obvious at10 mM EP, which decreased the ICAM1 and
MMP9 expression to 150.238.18% and 132.098.02% (vs. the TNF-a group,
As shown in Fig. 3, TNF-a (10 ng/mL) significantly up-regulated the expression
of the GRP78, ATF4, cleaved-caspase12 and p-PERK proteins to 291.1617.31%,
397.1522.74%, 503.7733.60% and 414.0623.10% in HUVECs (vs. the
control group, P,0.01). EP treatment reversed the effects of TNF-a, decreasing
the GRP78, cleaved-caspase12, ATF4 and p-PERK expression to 96.445.50%,
52.944.26%, 67.444.31% and 128.606.88% at 10 mM EP (vs. the TNF-a
Effects of EP and THA co-treatment on TNF-a-induced cell viability
and inflammatory cytokines
To explore the role of ERS in the anti-inflammatory effects of EP, HUVECs were
treated with EP (10 mM) for 2 h in the absence or presence of THA (1 mM, based
on preliminary experiments) and then subjected to TNF-a (10 ng/mL) treatment.
EP treatment significantly inhibited the TNF-a-induced binding of U937
monocytes to the HUVECs to 39.814.05% and decreased the production of
sICAM1 and MCP-1 in the cell culture supernatants to 6.800.57 pg/well and
7.030.60 ng/well (vs. the TNF-a group, P,0.01, Figs. 4A4C). However, THA
and EP co-treatment increased the binding of U937 monocytes to HUVECs to
60.554.90% and reversed the down-regulation of sICAM1 and MCP-1
production to 12.590.94 pg/well and 10.340.75 ng/well (vs. the EP+TNF-a
group, P,0.01). In addition, EP treatment also decreased ICAM-1, MMP9 and
TNF-a protein expression to 31.093.80%, 25.143.30% and 45.614.50% in
the HUVECs (vs. the TNF-a group, P,0.01, Fig. 5), while THA and EP
cotreatment reversed the down-regulation of the expression of the ICAM-1, MMP9
and TNF-a proteins to 67.104.65%, 80.954.71% and 86.295.02% in the
HUVECs (vs. the EP+TNF-a group, P,0.01). Compared with the TNF-a group,
THA+TNF-a treatment not only significantly increased the binding of U937
monocytes to HUVECs and increased sICAM1 and MCP-1 production in the cell
culture supernatants (P,0.01, Figs. 4A4C) but also increased the expression of
the ICAM-1, MMP9 and TNF-a proteins in the HUVECs (P,0.01, Fig. 5).
Effects of EP and THA co-treatment on the ERS-related molecules
As shown in Fig. 5, EP treatment significantly decreased the expression of the
GRP78, ATF4, cleaved-caspase12 and p-PERK proteins to 34.634.02%,
11.902.68%, 15.693.05% and 23.293.47% in the HUVECs (vs. the TNF-a
group, P,0.01), while THA and EP co-treatment reversed the down-regulation of
GRP78, ATF4, cleaved-caspase12, and p-PERK protein expression to
78.074.35%, 32.293.75%, 24.803.48% and 138.147.02% (vs. the
EP+TNF-a group, P,0.01). Compared with the TNF-a group, THA+TNF-a
treatment significantly increased GRP78, ATF4, cleaved-caspase12 and p-PERK
protein expression to 168.428.90%, 167.149.01%, 269.6615.89% and
205.6011.87% in the HUVECs (vs. the TNF-a group, P,0.01).
Effects of PERK siRNA on TNF-a-induced cell viability and
To further explore the role of ERS in the anti-inflammatory effects, HUVECs were
treated with PERK siRNA for 24 h and then subjected to TNF-a (10 ng/mL)
treatment. PERK siRNA treatment significantly inhibited the TNF-a-induced
binding of U937 monocytes to HUVECs to 619.4736.92% and decreased
sICAM1 and MCP-1 production in the cell culture supernatants to 8.410.66 pg/
well and 8.520.71 ng/well (vs. the control siRNA+TNF-a group, P,0.01,
Figs. 6A and 6B). In addition, PERK siRNA treatment also decreased ICAM-1 and
MMP9 protein expression to 214.0112.70% and 220.9011.84% in the
HUVECs (vs. the control siRNA+TNF-a group, P,0.01, Fig. 7). Compared with
the control siRNA group, PERK siRNA treatment alone had no effect on the
binding of U937 monocytes to HUVECs and had no effect on sICAM1 and
MCP1 production in the cell culture supernatants (P.0.05, Figs. 6A6B). It also did
not affect ICAM-1 orMMP9 protein expression in the HUVECs (P.0.05, Fig. 7).
Effects of PERK siRNA on the TNF-a-induced ERS-related
molecules in HUVECs
As shown in Fig. 7, control siRNA+TNF-a treatment significantly increased
GRP78, ATF4, cleaved-caspase12 and p-PERK protein expression to
303.9017.59%, 405.4321.38%, 494.6226.05% and 387.6221.26% in the
HUVECs (vs. the control siRNA group, P,0.01), while PERK siRNA+TNF-a
treatment significantly decreased GRP78, ATF4, cleaved-caspase12 and p-PERK
protein expression to 191.2810.37%, 235.7114.02%, 293.2016.07% and
125.097.80% (vs. the control siRNA+TNF-a group, P,0.01). Compared with
the control siRNA group, PERK siRNA treatment alone significantly decreased
GRP78 and p-PERK protein expression to 41.524.70%, 82.955.27%,
70.284.81% and 22.133.46% (vs. the control siRNA group, P,0.01).
Although the pathogenesis of atherosclerotic vascular disease involves
multifactorial processes, accumulating evidence demonstrates that inflammation and
subsequent endothelial dysfunction play fundamental roles in the initiation and
progression of atherosclerosis . Apoptotic endothelial cells contribute to
endothelial dysfunction, the destabilization of atherosclerotic plaques, and
thrombosis during the initiation and progression of atherosclerosis . Among
several proapoptotic factors present in atherosclerotic plaques, oxidized
lowdensity lipoproteins (ox-LDLs) participate in the formation and progression of
lesions by triggering lipid storage, local inflammation, TNF-a production, ERS
activation, and toxic events, which can cause vascular wall injury and death;
plaque transition from stable to vulnerable; erosion and rupture; and subsequent
athero-thrombosis [30, 31].
Accumulating evidence suggests that the inflammatory cytokine TNF-a, which
is a pleiotropic proinflammatory cytokine, plays an important role in the
disruption of vascular function and the subsequent development of vascular
disease . Epidemiological studies have demonstrated that TNF-a is
remarkably elevated in the plasma and arteries of humans with vascular
complications . In addition, TNF-a has been evaluated as an injury-related
factor in many studies involving inflammation [4, 27, 28, 32]. Therefore, TNF-a
was selected to mimic EC inflammatory injury in this study. Furthermore, TNF-a
has been shown to induce the expression and release of a series of adhesion
molecules and chemokines involved in the inflammatory response in ECs [34, 35].
Chemokines such as MCP-1 and IL-8 are key mediators in the regulation of
enhanced EC-monocyte interactions and subsequent monocyte recruitment to
vascular tissues . Cytokines such as TNF-a induce the expression of adhesion
molecules on the surface of ECs, resulting in the adhesion and migration of
monocytes to the subendothelial space . Adhesion molecules such as ICAM1,
MMP9 and E-selectin have been suggested to be atherosclerotic inflammatory
EP has been demonstrated to protect against the inflammatory response in
some cardiovascular diseases. For example, it has the ability to inhibit neutrophil
activation, inflammatory cytokine release, and nuclear factor kB translocation. EP
has been associated with a delayed myocardial protective effect after regional
ischemia and reperfusion (IR) injury in an in vivo rat heart model . Liu and
colleagues found that EP ameliorates monocrotaline-induced pulmonary arterial
hypertension and reverses pulmonary vascular remolding in rats by inhibiting the
release of TNF-a and IL-6 and by reducing the expression of endothelin-1 .
Additionally, EP has been shown to reduce the systemic inflammatory response
and lung injury resulting from shock and IR in an experimental model of
ruptured abdominal aortic aneurysm . However, the protective effects and
mechanisms underlying the action of EP against EC inflammatory injury have not
been fully elucidated. In our study, exposure of HUVECs to TNF-a significantly
increased their adhesion to U937 monocytes, while pretreatment with EP
significantly inhibited the TNF-a-induced adhesion of U937 monocytes to
HUVECs. In addition, exposure of HUVECs to TNF-a significantly increased
sICAM1, IL-8, MCP-1 and sE-selectin production in the cell culture supernatants.
TNF-a also significantly up-regulated ICAM-1 and MMP9 protein expression in
HUVECs. In addition, EP treatment reversed the effects of TNF-a, and the effects
were the most obvious at 10 mM EP.
Previous studies have confirmed that ERS plays an important role in regulating
the pathological process of EC inflammation. Shinozaki and colleagues have
confirmed that a deficiency in Herp (an ERS-related protein) suppresses
atherosclerosis in apolipoprotein E knockout mice by attenuating the
inflammatory response . Remarkably, Zeng and colleagues have found that
4phenylbutyric acid (an ERS inhibitor) suppresses inflammation by regulating the
ERS in endothelial cells stimulated by uremic serum . Furthermore, the
pharmacological actions of other pyruvate derivatives are closely related to ERS.
For example, 3-bromopyruvate induces ERS, overcomes autophagy and causes
apoptosis in human hepatocellular carcinoma cell lines [24, 25]. Considering the
above, we speculated that ERS may be involved in the anti-inflammatory effects of
EP in ECs. In this study, TNF-a treatment significantly increased the ERS-related
molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK). EP treatment
effectively reversed the TNF-a-induced up-regulation of the ERS-related
molecules and inflammatory injury. Furthermore, THA (an ERS inducer) not
only up-regulated the ERS-related molecules (GRP78, ATF4, caspase12 and
pPERK) but also attenuated the protective effects of EP against TNF-a-induced
inflammatory injury. PERK siRNA treatment not only inhibited the ERS-related
molecules (GRP78, ATF4, caspase12 and p-PERK) but also mimicked the
protective effects of EP in the attenuation of TNF-a-induced inflammatory injury.
In summary, we have demonstrated for the first time that EP can effectively
reduce vascular endothelial inflammation and that this effect at least in part
depends on the attenuation of ERS. Furthermore, the inhibition of the ERS
pathway confers a protective effect against endothelial inflammatory injury,
indicating that ERS is a crucial mediator of endothelial inflammation.
Conceived and designed the experiments: DHY STT. Performed the experiments:
GW KL YL. Analyzed the data: WY YY DJZ. Contributed reagents/materials/
analysis tools: CXF HGY TG JZX YZ. Wrote the paper: GW KL YL.
9. Jang M, Lee MJ, Cho IH (2014) Ethyl pyruvate ameliorates 3-nitropropionic acid-induced striatal toxicity
through anti-neuronal cell death and anti-inflammatory mechanisms. Brain Behav Immun 38: 151165.
1. Li X , Zhu M , Penfold ME , Koenen RR , Thiemann A , et al. ( 2014 ) Activation of CXCR7 limits atherosclerosis and improves hyperlipidemia by increasing cholesterol uptake in adipose tissue . Circulation 129 ( 11 ): 1244 - 1253 .
2. Duan W , Yang Y , Yi W , Yan J , Liang Z , et al. ( 2013 ) New role of JAK2/STAT3 signaling in endothelial cell oxidative stress injury and protective effect of melatonin . PLoS One 8 ( 3 ): e57941 .
3. Kassi E , Adamopoulos C , Basdra EK , Papavassiliou AG ( 2013 ) Role of vitamin D in atherosclerosis . Circulation 128 ( 23 ): 2517 - 2531 .
4. Chen ML , Yi L , Jin X , Liang XY , Zhou Y , et al. ( 2013 ) Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway . Autophagy 9 ( 12 ): 2033 - 2045 .
5. Abe J , Berk BC ( 2013 ) Atheroprone flow activation of the sterol regulatory element binding protein 2 and nod-like receptor protein 3 inflammasome mediates focal atherosclerosis . Circulation 128 ( 6 ): 579 - 582 .
6. Kaplon J , Zheng L , Meissl K , Chaneton B , Selivanov VA , et al. ( 2013 ) A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence . Nature 498 ( 7452 ): 109 - 112 .
7. Das UN ( 2006 ) Pyruvate is an endogenous anti-inflammatory and anti-oxidant molecule . Med Sci Monit 12 ( 5 ): RA79 - 84 .
8. Sims CA , Wattanasirichaigoon S , Menconi MJ , Ajami AM , Fink MP ( 2001 ) Ringer's ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats . Crit Care Med 29 ( 8 ): 1513 - 1518 .
10. Jang HJ , Kim YM , Tsoyi K , Park EJ , Lee YS , et al. ( 2012 ) Ethyl pyruvate induces heme oxygenase-1 through p38 mitogen-activated protein kinase activation by depletion of glutathione in RAW 264.7 cells and improves survival in septic animals . Antioxid Redox Signal 17 ( 6 ): 878 - 889 .
11. Meli G , Lecci A , Manca A , Krako N , Albertini V , et al. ( 2014 ) Conformational targeting of intracellular Ab oligomers demonstrates their pathological oligomerization inside the endoplasmic reticulum . Nat Commun 5 : 3867 .
12. Fu S , Yang L , Li P , Hofmann O , Dicker L , et al. ( 2011 ) Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity . Nature 473 ( 7348 ): 528 - 531 .
13. Korennykh AV , Egea PF , Korostelev AA , Finer-Moore J , Zhang C , et al. ( 2009 ) The unfolded protein response signals through high-order assembly of Ire1 . Nature 457 ( 7230 ): 687 - 693 .
14. Chen Y , Wang JJ , Li J , Hosoya KI , Ratan R , et al. ( 2012 ) Activating transcription factor 4 mediates hyperglycaemia-induced endothelial inflammation and retinal vascular leakage through activation of STAT3 in a mouse model of type 1 diabetes . Diabetologia 55 ( 9 ): 2533 - 2545 .
15. Arago n T , van Anken E , Pincus D , Serafimova IM , Korennykh AV , et al. ( 2009 ) Messenger RNA targeting to endoplasmic reticulum stress signalling sites . Nature 457 ( 7230 ): 736 - 740 .
16. Beck D , Niessner H , Smalley KS , Flaherty K , Paraiso KH , et al. ( 2013 ) Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells . Sci Signal 6 ( 260 ): ra7 .
17. Lee JH , Kwon EJ , Kim do H ( 2013 ) Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes . Biochem Biophys Res Commun 439 ( 3 ): 327 - 332 .
18. Pan MY , Shen YC , Lu CH , Yang SY , Ho TF , et al. ( 2012 ) Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines . Toxicol Appl Pharmacol 265 ( 3 ): 325 - 334 .
19. John L , Thomas S , Herchenr oder O , P utzer BM, Schaefer S ( 2011 ) Hepatitis E virus ORF2 protein activates the pro-apoptotic gene CHOP and anti-apoptotic heat shock proteins . PLoS One 6 ( 9 ): e25378 .
20. Chen X , Fu XS , Li CP , Zhao HX ( 2014 ) ER stress and ER stress-induced apoptosis are activated in gastric SMCs in diabetic rats . World J Gastroenterol 20 ( 25 ): 8260 - 8267 .
21. Shinozaki S , Chiba T , Kokame K , Miyata T , Kaneko E , et al. ( 2013 ) A deficiency of Herp, an endoplasmic reticulum stress protein, suppresses atherosclerosis in ApoE knockout mice by attenuating inflammatory responses . PLoS One 8 ( 10 ): e75249 .
22. Kujiraoka T , Satoh Y , Ayaori M , Shiraishi Y , Arai-Nakaya Y , et al. ( 2013 ) Hepatic extracellular signalregulated kinase 2 suppresses endoplasmic reticulum stress and protects from oxidative stress and endothelial dysfunction . J Am Heart Assoc 2 ( 4 ): e000361 .
23. Zeng W , Guo YH , Qi W , Chen JG , Yang LL , et al. ( 2014 ) 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulum stress of endothelial cells stimulated by uremic serum . Life Sci 103 ( 1 ): 15 - 24 .
24. Ganapathy-Kanniappan S , Geschwind JF , Kunjithapatham R , Buijs M , Syed LH , et al. ( 2010 ) 3- Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines . Anticancer Res 30 ( 3 ): 923 - 935 .
25. Yu SJ , Yoon JH , Yang JI , Cho EJ , Kwak MS , et al. ( 2012 ) Enhancement of hexokinase II inhibitorinduced apoptosis in hepatocellular carcinoma cells via augmenting ER stress and anti-angiogenesis by protein disulfide isomerase inhibition . J Bioenerg Biomembr 44 ( 1 ): 101 - 115 .
26. Ganapathy-Kanniappan S , Vali M , Kunjithapatham R , Buijs M , Syed LH , et al. ( 2010 ) 3- bromopyruvate: a new targeted antiglycolytic agent and a promise for cancer therapy . Curr Pharm Biotechnol 11 ( 5 ): 510 - 517 .
27. Baslio J , Hoeth M , Holper-Schichl YM , Resch U , Mayer H , et al. ( 2013 ) TNFa-induced downregulation of Sox18 in endothelial cells is dependent on NF-kB . Biochem Biophys Res Commun 442 ( 3- 4 ): 221 - 226 .
28. Jia Z , Babu PV , Si H , Nallasamy P , Zhu H , et al. ( 2013 ) Genistein inhibits TNF-a-induced endothelial inflammation through the protein kinase pathway A and improves vascular inflammation in C57BL/6 mice . Int J Cardiol 168 ( 3 ): 2637 - 2645 .
29. Hong D , Bai YP , Gao HC , Wang X , Li LF , et al. ( 2014 ) Ox-LDL induces endothelial cell apoptosis via the LOX-1-dependent endoplasmic reticulum stress pathway . Atherosclerosis 235 ( 2 ): 310 - 317 .
30. Ito TK , Yokoyama M , Yoshida Y , Nojima A , Kassai H , et al. ( 2014 ) A Crucial Role for CDC42 in Senescence-Associated Inflammation and Atherosclerosis . PLoS One 9 ( 7 ): e102186 .
31. Aluganti Narasimhulu C , Selvarajan K , Brown M , Parthasarathy S ( 2014 ) Cationic peptides neutralize Ox-LDL, prevent its uptake by macrophages, and attenuate inflammatory response . Atherosclerosis 236 ( 1 ): 133 - 141
32. Nallasamy P , Si H , Babu PV , Pan D , Fu Y , et al. ( 2014 ) Sulforaphane reduces vascular inflammation in mice and prevents TNF-a-induced monocyte adhesion to primary endothelial cells through interfering with the NF-kB pathway . J Nutr Biochem [Epub ahead of print].
33. Ridker PM , Hennekens CH , Buring JE , Rifai N ( 2000 ) C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women . N Engl J Med 342 ( 12 ): 836 - 843 .
34. Liu X , Pan L , Wang X , Gong Q , Zhu YZ ( 2012 ) Leonurine protects against tumor necrosis factor-amediated inflammation in human umbilical vein endothelial cells . Atherosclerosis 222 ( 1 ): 34 - 42 .
35. Roy A , Kolattukudy PE ( 2012 ) Monocyte chemotactic protein-induced protein (MCPIP) promotes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy . Cell Signal 24 ( 11 ): 2123 - 2131 .
36. Desai A , Darland G , Bland JS , Tripp ML , Konda VR ( 2012 ) META060 attenuates TNF-a-activated inflammation, endothelial-monocyte interactions, and matrix metalloproteinase-9 expression, and inhibits NF-kB and AP-1 in THP-1 monocytes . Atherosclerosis 223 ( 1 ): 130 - 136 .
37. Jang IS , Park MY , Shin IW , Sohn JT , Lee HK , et al. ( 2010 ) Ethyl pyruvate has anti-inflammatory and delayed myocardial protective effects after regional ischemia/reperfusion injury . Yonsei Med J 51 ( 6 ): 838 - 844 .
38. Liu C , Fang C , Cao G , Liu K , Wang B , et al. ( 2014 ) Ethyl pyruvate ameliorates monocrotaline-induced pulmonary arterial hypertension in rats . J Cardiovasc Pharmacol [Epub ahead of print].
39. Pulathan Z , Altun G , Hem sinli D , Mente se A, Yulu g E, et al. ( 2014 ) Role of ethyl pyruvate in systemic inflammatory response and lung injury in an experimental model of ruptured abdominal aortic aneurysm . Biomed Res Int 2014 : 857109 .