Endothelial microparticles are increased in congenital heart diseases and contribute to endothelial dysfunction
Lin et al. J Transl Med
Endothelial microparticles are increased in congenital heart diseases and contribute to endothelial dysfunction
Ze‑Bang Lin 0 1
Hong‑Bo Ci 0 1
Yan Li 0 1
Tian‑Pu Cheng 0 1
Dong‑Hong Liu 1
Yan‑Sheng Wang 1
Jun Xu 1
HaoX‑iang Yuan 0 1
Hua‑Ming Li 0 1
Jing Chen 1
Li Zhou 0 1
Zhi‑Ping Wang 0 1
Xi Zhang 0 1
ZhiJ‑un Ou 1
Jing‑Song Ou 0 1
0 Division of Cardiac Surgery, The First Affiliated Hospital of Sun Yat‐ sen University , 58 Zhong Shan Er Road, Guangzhou 510080 , People's Republic of China
1 supervision of the research for this article
Background: We previously demonstrated that endothelial microparticles (EMPs) are increased in mitral valve diseases and impair valvular endothelial cell function. Perioperative systemic inflammation is an important risk factor and complication of cardiac surgery. In this study, we investigate whether EMPs increase in congenital heart diseases to promote inflammation and endothelial dysfunction. Methods: The level of plasma EMPs in 20 patients with atrial septal defect (ASD), 23 patients with ventricular septal defect (VSD), and 30 healthy subjects were analyzed by flow cytometry. EMPs generated from human umbilical vascular endothelial cells (HUVECs) were injected into C57BL6 mice, or cultured with HUVECs without or with siRNAs targeting P38 MAPK. The expression and/or phosphorylation of endothelial nitric oxide synthase (eNOS), P38 MAPK, and caveolin‑ 1 in mouse heart and/or in cultured HUVECs were determined. We evaluated generation of nitric oxide (NO) in mouse hearts, and levels of tumor necrosis factor‑ α (TNF‑ α) and interleukin‑ 6 (IL‑ 6) in cultured HUVECs and in mice. Results: EMPs were significantly elevated in patients with ASD and VSD, especially in those with pulmonary hypertension when compared with controls. EMPs increased caveolin‑ 1 expression and P38 MAPK phosphorylation and decreased eNOS phosphorylation and NO production in mouse hearts. EMPs stimulated P38 MAPK expression, TNF‑ α and IL‑ 6 production, which were all inhibited by siRNAs targeting P38 MAPK in cultured HUVECs. Conclusions: EMPs were increased in adult patients with congenital heart diseases and may contribute to increased inflammation leading to endothelial dysfunction via P38 MAPK‑ dependent pathways. This novel data provides a potential therapeutic target to address important complications of surgery of congenial heart disease.
Endothelial microparticles; Congenital heart disease; Inflammation; Endothelial nitric oxide synthase; P38 MAPK pathway
Recently, we demonstrated that circulating microparticles
in patients with valvular heart disease impaired
endothelium dependent vasodilation by uncoupling and inhibiting
endothelial nitric oxide synthase (eNOS) .
Microparticles are generated from a variety of sources
including endothelial cells, platelets, T cells, etc. We previously
showed that microparticles generated from endothelial
cells induce acute lung injury, inhibit angiogenesis, and
impair vasodilation [2, 3]. We also demonstrated that the
number of plasma endothelial microparticles (EMPs) is
significantly higher in patients with mitral valve diseases
leading to impairment of human mitral valve endothelial
cell function . In proteomics studies, we found that
EMPs contain hundreds of proteins, some of which may
impair vasodilation, induce lung injury and/or activate
inflammation [5, 6]. Thus, EMPs are considered both a
marker and a mediator of endothelial injury. Previously
Amabile reported that EMP levels are correlated with
pulmonary arterial pressure in adults with primary and
secondary pulmonary arterial hypertension . However,
Samadja showed no significant difference in EMPs levels
between irreversible and reversible pulmonary arterial
hypertension in children with congenital heart disease .
Thus, whether EMPs are consistently elevated in all
congenital heart diseases remains unclear.
Perioperative systemic inflammation is a major risk
factor and complication of cardiac surgery. Cardiac surgery
with cardiopulmonary bypass (CPB) results in systemic
inflammatory responses that contribute to instability
of circulation post-operatively [9, 10]. Preventza et al.
reported that CPB time is related to mortality after
surgery . Although we previously demonstrated that
EMPs contain proteins that can induce inflammation, the
impact and the mechanisms of action of EMPs to
promote an inflammatory response are not fully understood.
In this study, we investigated, for the first time, whether
EMPs increased in two common congenital heart
diseases, atrial septal defect (ASD) and ventricular septal
defect (VSD), and the impact and the mechanisms of
action of EMPs to promote inflammation and subsequent
20 patients diagnosed with ASD, 23 patients with VSD,
and 30 age-matched healthy volunteer control subjects
were recruited at The First Affiliated Hospital of Sun
Yat-sen University. Patients with diseases which may
increase EMPs level, including coronary heart disease,
hypertension, infectious disease, severe trauma, antibiotic
therapy, lupus anticoagulant, multiple sclerosis, renal
failure, rheumatic diseases in acute stage, and valvular heart
disease were explicitly excluded. Healthy volunteers below
18 years old and those who abused alcohol and/or heavy
smokers were excluded. This study was approved by the
Ethics Committee of The First Affiliated Hospital, Sun
Yat-sen University. Informed consent was obtained from
all subjects enrolled in this study. Clinical characteristics,
doppler echocardiographic metrics (the definition and
classification of pulmonary hypertension are referenced
as in the 2014 Nice Pulmonary Hypertension
Classification System) and operation data were collected (Table 1).
Blood sampling and flow cytometry
All patients preoperatively fasted overnight as did healthy
volunteers. Blood samples were drawn and centrifuged to
obtain platelet-poor plasma (PPP) . 50 μL of PPP was
incubated with 4 μl of anti-CD31-PE and 4 μL of
antiCD42b-FITC antibodies at room temperature for 20 min
with gentle orbital shaking in the dark . The samples
incubated with corresponding isotype control (all from
Beckman Coulter, France) were used as controls. After
labeling, samples were analyzed via MoFlo XDP
(Beckman coulter) by an independent examiner who was
blinded to study arm and intentions. Before analysis,
50 μL flow count calibrator beads (Beckman Coulter)
with known concentration provided by the manufacturer
were added into the antibody-labeled tubes. After
excluding non-specific fluorescence, those positively labeled by
anti-CD31-PE and negatively labeled by
anti-CD42bFITC and <1 μm in size were considered to be EMPs .
Generation of EMPs
EMPs were generated ex vivo by incubating human
umbilical vein endothelial cells (HUVECs) with
plasminogen activated inhibitor-1 (PAI-1) as previously
described [2–6]. Briefly, passage 4 HUVECs were grown
to confluence in T75 flasks coated with 1% gelatin in
endothelial cell growth medium-2 (Clonetics) containing
20% fetal bovine serum. Cultured cells were maintained
at 37 °C in 5% humidified CO2. After serum starvation,
cells were stimulated with 10 ng/mL human PAI-1. Three
hours later, the EMPs-rich supernatant was collected
and centrifuged (300×g, 10 min) to remove cell debris.
The supernatant was removed after ultracentrifugation
(105×g, 60 min) and EMPs were resuspended in
phosphate-buffered saline (PBS) at room temperature for
Table 1 Clinical characteristic and Doppler echocardiographic variables
Atrial septal defect group
Ventricular septal defect group
Values are expressed as mean ± standard deviation
PH pulmonary hypertension, NYHA New York Heart Association, CPB cardiopulmonary bypass, LAD left atrial diameter, LVEDD left ventricular end-diastolic diameter,
LVESD left ventricular end-systolic diameter, RAD right atrial diameter, RVD right ventricle diameter, LVEF left ventricular ejection fraction
* P < 0.05 compared with control group
# P < 0.05 compared with atrial septal defect group
SiRNA targeting P38 MAPK
To confirm the relationship between EMPs and the P38
MAPK pathway, we used targeted siRNAs to reduce
expression of P38 MAPK in HUVECs. HUVECs were
cultured in endothelial cell medium (ScienCell)
supplemented with 5% Fetal bovine serum (FBS), 1% growth
factors, and 1% penicillin/streptomycin. Cells were serum
starved in 0.5% FBS over night before experiments.
Targeted SiRNAs and scrambled siRNAs (negative
control) (Dharmacon, MA) were transfected into cells with
siPORT™ NeoFX™ Transfection Agent (Invitrogen, USA)
and Opti-MEM I (Gibco). InitialsiRNA concentrations
used were 70 nM. At 12 h after transfection, medium
was exchanged with endothelial cell medium (Sciencell,
Carlsbad, CA) consisting of 10% Fetal bovine serum and
10 ng/mL epidermal growth factor to remove siRNA. A
separate control group containing only with siPORT™
NeoFX™ Transfection Agent and Opti-MEM I was also
ELISA for cell cultures
After siRNA transfection, HUVECs were cultured for an
additional 48–72 h until the monolayer of cells reached
90–100% confluence. The cultured cells were then serum
starved overnight and stimulated with EMPs (2 × 105/
mL) for 6 h. After centrifuging for 20 min at 12,000×g,
the supernatants were collected and tumor necrosis
factor-α (TNF-α) and interleukin (IL)-6 levels were
evaluated using ELISA kits (ebioscience) as recommended in
the manufacturer’s protocol.
All animal experiments were approved by the Animal
Ethics Commission of the First Affiliated Hospital of
Sun Yat-sen University. The investigation conformed
to the provisions of the Declaration of Helsinki in 1995
(as revised in Edinburgh 2000). Eight-week-old female
C57BL6 mice were obtained from the animal center
of Sun Yat-sen University, north campus. 1 × 105 or
5 × 105/mL EMPs were injected into the mice via tail
vein. Those injected with an equal volume of PBS were
used as controls. 6 h after injection, mice were fully
anesthetized with sodium pentobarbital (50 mg/kg) and
blood samples were drawn for measurement of
proinflammatory factors. In addition, the heart was isolated
and frozen in liquid nitrogen for further immunoblotting
analysis and immunohistochemical staining as previously
described [3, 12, 13].
ELISA for plasma
The blood samples drawn from mice were centrifuged
and plasma was isolated. TNF-α and IL-6 concentrations
were determined using ELISA kits (ebioscience).
To investigate the effects of EMPs on relevant proteins
in the mouse heart, eNOS expression and
phosphorylation at Ser1177, P38 MAPK expression and
phosphorylation, and expression of caveolin-1 were assessed by
immunoblotting with specific antibodies as previously
To investigate the effects of EMPs and siRNAs on
cultured cells, HUVECs were evaluated without and with
transfection of siRNAs targeting P38 MAPK (and
scrambled control siRNAs) as described above. HUVECs were
stimulated with EMPs (2 × 105/mL) for 1 h. Then cellular
proteins were harvested, total and phosphorylated P38
MAPK, and GAPDH protein expression were determined
by immunoblotting as previously described [4, 14].
Antibodies for detection of phosphorylation of eNOS
at Ser1177, P38 MAPK, phosphorylation of P38 MAPK,
and caveolin-1 were purchased from Cell Signaling
Technology (Danvers, MA). Anti-eNOS was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).
AntiGAPDH was obtained from Proteintech Group (Chicago,
IL). Proteins were separated by SDS-PAGE and then
visualized using luminol reagent (Santa Cruz Biotechnology)
according to standard methods.
To investigate the effects of EMPs on relevant
protein expression in mouse hearts, isolated mouse hearts
treated without or with EMPs were embedded with
paraffin, sectioned and subjected to immunostaining.
The expression of caveolin-1, eNOS, and P38 MAPK
in mouse hearts was detected by
immunohistochemical staining using standard protocols. Polycolonal rabbit
IgG anti-Caveolin-1 antibody (1:100, Abcam), anti-eNOS
antibody (1:100, Abcam) and anti-p38 MAPK antibody
(1:100, Abcam) were used as primary antibodies.
Measurement of nitric oxide (NO) generation
1 × 105 or 5 × 105/mL EMPs or equal volumes of PBS
were injected into the mouse tail vein. 6 h later, hearts
were isolated and placed in 1:9 (wt/vol) cold
homogenization buffer. The hearts were cut into small pieces
with an iris scissors and homogenized five times on ice
(10 s with 30 s intervals between homogenizations). The
homogenates were then centrifuged for 8 min (2000 rpm,
4 °C) and the supernatant was isolated. NO
concentration was determined by measuring total nitrate plus
nitrite (NO3− + NO2−) using an NO detection kit
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China)
according to the manufacturer’s instructions. Briefly,
nitrate was enzymatically converted into nitrite by nitrate
reductase, and nitrite was quantified with Griess
reagent at an absorbance of 550 nm, as previously described
. The range of the detection of nitrate plus nitrite is
0–600 μmol/L according to the manufacturer. In
addition, the total protein concentration of the supernatant
was determined by bicinchoninic acid protein assay
(Merck, Whitehouse Station, NJ).
Statistical analyses were performed using Prism 5
software. For comparison of healthy subjects, ASD patients,
and VSD patients, or the impact of different
concentrations of EMPs on protein expression in hearts and
cultured cells, one-way ANOVA and Newman-Keuls
comparisons were used. Chi-square test was used to
compare proportions between different groups. For
comparison between EMPs and controls, a t test was used.
P < 0.05 was considered to indicated statistical
significance. Data were presented as mean ± SDEM.
Demographic and clinical parameters
Demographic and clinical features are shown in Table 1.
Age and gender were similar among the three groups of
subjects. 15 patients with ASD and 6 patients with VSD
were suffering from pulmonary hypertension.
According to New York heart association classification, of the 20
patients with ASD 16 patients were class II and 4 patients
were class III. Of 23 patients with VSD, 15 patients were
class II and 8 patients were class III, respectively. The
duration of CPB (66 ± 28 vs. 75 ± 23 min) and the time
of aortic cross clamping (33 ± 24 vs. 39 ± 20 min) were
similar among ASD group and VSD group during the
Transthoracic echocardiography differences found
among the three subject groups are shown in Table 1. The
control group had no evidence of morphologic heart
disease. When compared with the control group, left atrial
diameter (LAD), right atrial diameter (RAD) and right
ventricular diameter (RVD) were significantly enlarged
in ASD patients; left ventricular end-diastolic
diameter (LVEDD), and left ventricular end-systolic diameter
(LVESD) were significantly smaller in the ASD patients
group. LAD and LVEDD were significantly enlarged in
the VSD patients group. When compared with the ASD
group, LVEDD and LVESD were significantly enlarged
in the VSD group; RVD was significantly smaller in
VSD group. There were no significant differences in left
ventricular ejection fraction among the three groups of
Levels of plasma EMPs
When compared with the control group, plasma EMPs
levels were significantly elevated in the ASD group and
VSD group (Fig. 1A). The higher levels of plasma EMPs
in ASD and VSD patients were positively correlated with
pulmonary hypertension (Fig. 1B).
Fig. 1 EMPs increase in patients with atrial septal defect and ventricular septal defect. A Compared with control group, EMPs were increased in
patients with ASD and VSD. B EMPs were increased in patients with ASD and VSD associated with pulmonary hypertension (PH) than those without
PH. *P < 0.05 compared with control group. #P < 0.05 compared with ASD and VSD patients
Effects of EMPs on expression of P38 MAPK, Caveolin‑1
and eNOS in mouse hearts and in HUVECs
In mouse hearts, both immunohistochemical staining
and immunoblotting showed that EMPs significantly
increased expression of caveolin-1 (Figs. 2A, B; 3A).
EMPs dramatically decreased eNOS phosphorylation at
the S1177 site without altering total eNOS expression
(Figs. 2C, D; 3B).
Both immunohistochemical staining and
immunoblotting showed that EMPs dramatically increased P38
MAPK phosphorylation (Fig. 4). EMPs also stimulated
increased expression of P38 MAPK in HUVECs. This was
substantially inhibited by siRNAs targeting P38 MAPK
(Fig. 5A, B).
Effects of EMPs on TNF‑α and IL‑6 concentrations
in cultured HUVECs
EMPs significantly stimulated an increase in TNF-α
concentration in cultured HUVECs. In EMPs stimulated
groups, TNF-α concentration in the group transfected
with siRNA targeting P38 MAPK was significantly lower
than in cells transfected with the scrambled siRNA
control. No differences were observed between control siRNA
and cells not treated with siRNA (Fig. 5C). A similar
pattern was observed with concentrations of IL-6 (Fig 5D).
Effects of EMPs on TNF‑α and IL‑6 concentrations in mouse
As shown in Fig. 6A and B, TNF-α and IL-6
concentrations in the plasma of mice were significantly increased
after intravenous injection with EMPs 5 × 105/mL for 6 h
(when compared with controls).
Effects of EMPs on NO generation in mouse hearts
As shown in Fig. 6C, NO concentration in the mouse
hearts treated with EMPs was significantly decreased
when compared with controls. There was a
dose-dependent effect of EMPs to stimulate NO production in mouse
hearts (Fig. 6C).
We and others have previously demonstrated that EMPs
levels are elevated in patients with valvular heart
diseases and this contributes to impairing mitral valve
endothelial cell function . We further showed that
circulating microparticles generated from patients with
valvular heart disease and cardiac surgery can contribute
to endothelial dysfunction . In the present study, we
are first to show that EMPs levels are also increased in
adult patients with ASD and VSD resulting in increased
inflammation and endothelial dysfunction. This indicates
that EMPs are not only increased in acquired heart
diseases, but also elevated in simple acyanotic congenital
heart diseases where it may play a pathophysiological
The mechanisms underlying increased numbers
of plasma EMPs in ASD and VSD are unclear.
Previous studies demonstrated that EMPs can be released
from endothelial cell activation and/or apoptosis .
The atrium and ventricle sizes were increased in ASD
and VSD patients. These changes and additional septal
defects lead to hemodynamics change and shear stress
that may potentially contribute to EMP formation [15,
16]. Shear stress is a major determinant of
endothelial damage which may induce activation of endothelial
Fig. 2 Effects of EMPs on Caveolin‑1 and eNOS in the mouse hearts. A–D Immunohistochemical staining showed that EMPs can increase the
expression of caveolin‑1 and decrease the expression of phosphorylation of eNOS in the mouse hearts
cells and lead to EMPs release . It is also possible
that abnormal hemodynamic forces including abnormal
blood flow through cardiac defects and increased blood
flow in cardiopulmonary circulation may stimulate EMPs
Pulmonary hypertension is one major complications of
congenital heart diseases [17, 18]. In the present study,
we found that levels of EMPs were higher in patients
with ASD and VSD accompanied by pulmonary
hypertension than in those without pulmonary hypertension.
Endothelial cell activation and apoptosis is observed in
patients with pulmonary hypertension [19, 20] leading
to increases in pulmonary blood flow that exert
abnormal shear stress and result in endothelial dysfunction in
lamb models . These abnormalities may cause more
EMPs release in ASD and VSD patients with pulmonary
hypertension than in isolated ASD and VSD patients
without pulmonary hypertension. Samadja reported no
significant differences in EMPs level between irreversible
and reversible pulmonary arterial hypertension in
children with congenital heart diseases. Thus, it is possible
that EMPs levels increase when pulmonary
hypertension occurs but does not further increase upon
progression to irreversible pulmonary arterial hypertension. This
may be due to the fact that endothelial cells are already
activated to release EMPs at a maximal rate in reversible
pulmonary hypertension and further increases to
generate more EMPs do not occur as disease progresses to
the irreversible state. Taken together, this data suggests
that increased level of EMPs may be a predictor for
pulmonary hypertension in congenital heart disease .
Indeed, EMPs are already considered as marker of many
other diseases. Our findings extend these previous results
to suggest that EMPs may also be a biomarker of
pulmonary hypertension in adult congenital heart diseases and
congenital heart diseases per se.
We and others have been demonstrated that EMPs may
play an important role in exacerbating endothelial
dysfunction [2–4, 7]. It is well known that eNOS-mediated
NO production plays a critical role in the regulation of
endothelial function [23, 24]. To investigate the
mechanisms by which EMPs impair endothelial function, we
performed some basic studies in mice and endothelial
cells. We found that EMPs significantly inhibited eNOS
phosphorylation and NO production in mouse hearts
in a dose-dependent manner. Caveolin-1 binds to eNOS
to keep eNOS in an inactive state . We found that
caveolin-1 expression in mouse hearts was increased by
Fig. 3 Effects of EMPs on Caveolin‑1 and eNOS in the mouse hearts. A, B Immunoblotting showed that EMPs were significantly increased expres‑
sion of caveolin‑1 and decreased eNOS phosphorylation at S1177 site with a dose ‑ dependent effect without altering the eNOS expression in the
mouse hearts. (*P < 0.05 compared with control group. #P < 0.05 compared with group of EMPs 105/mL, n = 8)
EMPs in a dose-dependent manner. This suggests that
EMPs may inhibit eNOS activity to block NO generation
in mice hearts through an increase in caveolin-1
expression. As reduction of NO bioactivity is a hallmark of
endothelial dysfunction, our findings indicate that EMPs
may inhibit eNOS activity to reduce NO to contribute
to endothelial dysfunction in hearts. It should be noted
that iNOS is another major source of NO. EMPs may
also inhibit iNOS to produce NO. However, we found
that EMPs didn’t inhibit iNOS expression in the healthy
and hypercholesterolemic mice hearts in both the
current study and previous study (data not shown) . This
suggests that iNOS is probably not a major contributor to
NO reduction observed in our studies. We recently
demonstrated that circulating microparticles from valvular
heart diseases and cardiac surgery can inhibit NO
generation to impair endothelium dependent vasodilation .
As EMPs are part of circulating microparticles, our
findings that plasma EMPs level increased and EMPs inhibit
NO generation in the hearts suggest that EMPs may
affect hemodynamic stability in patients with ASD and
VSD post cardiac surgery. In addition, as EMPs inhibit
NO production, this may also exacerbate the
development of pulmonary hypertension leading to a further
increase in EMPs.
Perioperative systemic inflammation is one of the
major risk factors and complications in cardiac surgery.
P38 MAPK signaling mediates myocardial inflammatory
responses and endothelial dysfunction that can
contribute to myocardial damage following acute injury [25,
26]. P38 MAPK signaling pathway also plays a vital role
in the formation and maturation process of EMPs .
In the present study, our data showed that
phosphorylation of p38 MAPK in mouse hearts was increased by
EMPs in a dose-dependent manner. This suggests that
EMPs can activate the P38 MAPK pathway. We further
found that EMPs also increased P38 MAPK expression
and stimulated increases in TNF-α and IL-6 levels both
in cultured endothelial cells and mice. The increased
P38 MAPK expression as well as downstream TNF-α
and IL-6 production was inhibited by disrupting the p38
MAPK pathway with targeted specific siRNAs. This
suggests that EMPs stimulate TNF-α and IL-6 production
using a P38 MAPK-dependent pathway consistent with
Fig. 4 EMPs increased P38 phosphorylation in the mouse hearts. A, B Immunohistochemical staining showed that EMPs can increase the phospho‑
rylation of P38 in the mouse hearts. C, D Immunoblotting showed that EMPs were dramatically increased P38 phosphorylation with a dose‑
dependent effect. (*P < 0.05 compared with control group, #P < 0.05 compared with the group of EMPs 105/mL, n = 8)
other studies of pro-inflammatory signaling related to
TNF-α and IL-6 production [26, 28]. In some studies,
EMPs level is correlated with serum IL-6 level, in both
healthy subjects and in patients with coronary heart
disease [29, 30]. In vitro studies show that
inflammatory factors induce endothelial cell apoptosis leading to
production of EMPs. Our study showed that EMPs can
induce endothelial cells to release pro-inflammatory
factors in a P38 MAPK-dependent fashion. Thus, EMPs may
be both the cause and consequence of pro-inflammatory
responses in a feed-forward mechanism that amplifies
inflammation and its pathophysiological consequences.
This raises the possibility that blocking EMPs’ function
or reducing EMPs’ level may be a potential therapeutic
strategy for controlling inflammation in congenital heart
disease. Since TNF-α and IL-6 are secretory mediators
and proinflammatory cytokines that increase endothelial
cell permeability and promote endothelial cell activation,
dysfunction, and apoptosis [25, 26], our data suggests
that EMPs may induce inflammation to impair
endothelial function and damage vascular endothelial cells by
activating the P38 MAPK pathway to promote TNF-α
and IL-6 production. Thus, future studies will be
investigated whether EMPs upregulate some gene expressions
to activate P38 MAPK pathway in endothelial cells.
Study Limitations: Due to the limitations in current
technology, we are unable to extract EMPs from patients’
plasma directly to perform animal or cell studies. Instead,
in the present study, we have relied on a published
strategy to obtain EMPs from cultured endothelial cells
Fig. 5 EMPs can stimulate TNF‑α and IL ‑6 release in HUVECs, which can be down‑regulated by P38‑siRNAs. A, B Immunoblotting showed EMPs can
increase P38 expression, which can be reduced by siRNAs targeting P38 MAPK (Si‑P38). C, D TNF‑α and IL ‑6 concentration were relative low in con‑
trol (C) and negative (Neg) group. After 6 h stimulated with EMPs, TNF‑α and IL ‑6 level increased in each group. TNF‑α and IL ‑6 level in siRNAs P38
MAPK group was significantly lower than that in non‑P38 siRNAs interfering groups. (*P < 0.05 compared with control group, **P < 0.05 compared
with negative group, ***P < 0.05 compared with the group of EMPs 105/mL, n = 8)
stimulated with PAI-1 [2, 4, 6] that may cause
xenotransplant-type reactions. In addition, HUVECs used in the
present study may not be representative of all human
endothelial cell types and components of EMPs from
different endothelial cell sources may have different
functional abilities with diverse biological functions [5, 6].
Thus, EMPs used in the current study may have some
differences from EMPs in congenital heart disease patients.
Furthermore, the mice used in the current study were
healthy mice and its physiology may not represent human
congenital heart disease (patho) physiology. However,
the primary goal of the present study was to determine
effects of EMPs on mouse heart, independent of
pathology. If EMPs can adversely affect healthy hearts, it seems
plausible that effects of EMPs in congenital heart disease
may be even more severe. Our data clearly demonstrated
EMPs impaired heart function. We only tested effects of
EMPs on endothelial cells because we wanted to focus
on endothelial function, a major line of investigation in
our lab. This represents an additional study limitation.
Whether EMPs have similar effects on human
cardiomyocytes needs to be evaluated in subsequent studies
beyond the scope of the present study. Based on our
previous findings and other publications [1–6], we speculate
that EMPs may induce inflammation leading to
endothelial dysfunction in patients with congenital heart diseases.
Identifying and evaluating safe P38 MAPK inhibitors to
block P38 MAPK-dependent pathways in vivo as a
potential therapeutic strategy to decrease EMPs are beyond the
scope of the present study but are the subject of future
Fig. 6 EMPs increased TNF‑α and IL ‑6 release in mouse and decreased NO generation in the mouse hearts. A, B The levels of TNF‑α and IL ‑6 in
mouse plasma in EMPs group (5 × 105/mL) were significantly higher than that in control groups. (*P < 0.05 compared with control group, n = 8).
C EMPs significantly decrease NO production in the mouse hearts. The group of EMPs (5 × 105/mL) was released NO less than the group of EMPs
(105/mL). (*P < 0.05 compared with control group, #P < 0.05 compared with group of EMPs (105/mL), n = 12)
studies in our lab. Other future related studies include
evaluating relationships between EMPs level and
cardiopulmonary bypass, deep hypothermic circulatory arrest,
and postoperative complications.
In summary, our study demonstrated that EMPs were
increased in adult patients with congenital heart
diseases. These increased EMPs inhibited eNOS activity
to reduce NO production and activated P38
MAPKdependent signaling pathways to promote production
of TNF-α and IL-6. These mediators of
pro-inflammatory response impaired endothelial function. These
changes may contribute to impairment of myocardial
and vascular function during the perioperative period
to affect hemodynamic stability after cardiac surgery.
In addition, the consequences of increased EMPs may
also promote development of pulmonary
hypertension. We conclude that the P38 MAPK pathway may
play a role in EMPs-induced inflammation and provide
a potential therapeutic target in surgery of congenial
Additional file 1. Online supplemental materials and methods.
EMPs: endothelial microparticles; ASD: atrial septal defect; VSD: ventricular
septal defect; HUVECs: human umbilical vascular endothelial cells; eNOS:
endothelial nitric oxide synthase; TNF‑α: tumor necrosis factor ‑α; IL ‑1: interleu‑
kin‑1; CPB: cardiopulmonary bypass; NO: nitric oxide; NYHA: New York Heart
Association; LAD: left atrial diameter; LVEDD: left ventricular end‑ diastolic
diameter; LVESD: left ventricular end‑systolic diameter; RAD: right atrial diam‑
eter; RVD: right ventricle diameter; LVEF: left ventricular ejection fraction.
ZBL, HBC mainly performed the cell culture and animal experiments. They
also drafted the manuscript. YL, TPC, HML, JC, YSW and HXY performed part
of the cell culture and animal experiments and analyzed part of the data. DHL
performed the Doppler echocardiography study. LZ, ZPW and XZ performed
the human study and analyzed and interpreted this part of data. ZJO, JSO and
JX mainly contributed to the conception and design of the work, interpreted
the data, and revised the manuscript. All authors read and approved the final
Availability of data and materials section
The dataset(s) supporting the conclusions of this article is (are) included
within the article. All clinical data are stored by information system at Division
of Cardiac Surgery, The First Affiliated Hospital of Sun Yat‑sen University.
Ethics approval and consent to participate
The research was permitted by the ethics committee of The First Affiliated
Hospital of Sun Yat‑sen University and written informed consents were
obtained from all patients.
This study was supported by the National Natural Science Foundation of
China (81170271, 81370370, 81500215, 81600382, 81670392 and Dis‑
tinguished Young Scholar 81325001), the Changjiang Scholars Program
from Ministry of Education of China, the Guangdong Pearl River Scholars
Program, 973 project (2009CB522104) and International Cooperation Project
(2015DFA31070) from the Ministry of Science and Technology of China,
Guangdong Natural Science Fund Committee, China (2015A030312009), Sun
Yat‑Sen University Clinical Research 5010 Program; Program of National Key
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