Materials & Methods
Study Population. Ten patients (6 men and 4 women) with ACS (comprising unstable
angina pectoris (UAP), ST-segment elevation myocardial infarction (STEMI) and
non-STEMI (NSTEMI)) 1 who had a percutaneous coronary intervention (PCI), but
no evidence of previous myocardial infarction, were recruited. All patients were
admitted to the coronary care unit (CCU) of The First Affiliated Hospital Xi'an
Jiaotong University. All patients experienced ongoing pain despite pharmacotherapy,
including aspirin, intravenous heparin and nitroglycerin, and orally administered
prophylactic anti-anginal agents as indicated in Table 1. In view of these symptoms,
all patients underwent coronary angiography (CAG), and the degree of coronary
artery stenosis was recorded for subsequent correlation with the MP levels. All
patients with disorders, such as severe trauma, multiple sclerosis, hypertension, lupus
anticoagulant, infectious diseases, renal failure, and the acute phase of rheumatic
diseases, were excluded given that these conditions are independent causes of
elevated MPs. Ten age- and sex-matched healthy subjects were enrolled as a control
group. This study was approved by The First Affiliated Hospital Xi'an Jiaotong
University Ethics Review Board. Informed consent was obtained from all patients and
controls.
Blood sampling and MPs concentration. Fasting blood samples were obtained on
the day of hospitalization before any medications were administered. Blood samples
were centrifuged (11,000 g, 2 minutes, 4ºC) to obtain platelet-poor plasma. Then,
MPs were obtained by centrifugation at 13,000 g for 45 minutes (4ºC) 2. After
aspirating the supernatant carefully, the precipitated MPs were resuspended in 100 µl
of RPMI1640. The final MP concentration was determined by a bicinchoninic acid
protein assay (Merck). The MPs were stored at -80°C until further analysis. Our
studies included observations on vasodilation, Western blotting, nitric oxide (NO) and
superoxide (O2˙−) production that collectively required significant numbers of MPs.
Given the limited availability of blood from ACS patients, we pooled MPs from
several patients to create a batch of ‘mixed MPs’ to complete our experimental
protocols. To reduce inter-batch differences, each mixture included samples from
patients with STEMI, NSTEMI, and both high and low degrees of stenosis. Final
microparticles concentrations we used corresponded to their circulating plasma2, as
show in Fig 1A. We suppose 3mg/ml as physiological concentrations(Fig 1A: control
group:2.7±0.4 mg/ml), and 5mg/ml as pathologic concentrations(Fig 1A: ACS day 0:
4.52±0.7 mg/ml; ACS day 2: 4.47±1.1 mg/ml). For the lacking of enough MPs, we
only test the vasodilatation with 5mg/ml concentration.
Flow cytometry analysis. Blood samples were centrifuged (11,000 g, 2 minutes,
4°C), and 50 µl of the supernatant platelet-poor plasma was incubated with both 5 µl
of anti-CD31-PE and 5 µl of anti-CD41-FITC or CD14-FITC (their corresponding
isotypes were used as controls, Beckman Coulter) at room temperature for 30 min3.
Before the samples were analyzed, 50 µl of flow count calibrator beads (Beckman
Coulter) was added into the antibody-labeled tubes. Histograms were gated at <1 µm,
the estimated size of an EMP. CD31(+)/CD41(-) microparticles were identified as
endothelial-derived MPs (EMP), whereas CD31(+)/CD41(+) microparticles were
identified as platelet-derived MPs (PMP).
Vasodilatation detection. All animal experiments were approved by The First
Affiliated Hospital Xi'an Jiaotong University Animal Ethics Committee. The rats
were purchased from the Animal Experimental Center of Xi’an Jiaotong University.
After decapitation, the thoracic aortas were dissected and placed in ice-cold Krebs
solution with the following composition (in mmol/L, Sigma- Aldrich, St. Louis, MO):
NaCl 119.0, NaHCO3 25.0, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, and
MgSO4 1.2 (37°C, pH 7.4). After removing tissue surrounding the aortas carefully,
four or five 2 to 3 mm-wide aortic rings were obtained from each rat and connected to
an isometric force transducer (EMKA Technologies, Paris, France) as previously
described4. The aortic rings, with a pre-tension of 1 g, were suspended in organ
chambers filled with Krebs solution and aerated continuously with 95% O2 and 5%
CO2. After 60 min of equilibration, rings were exposed to 60 mmol/l KCl at least
thrice to assess their stability, and only preparations with a maximum tension value
that remained unchanged during these trials were used for the experiments. The rings
were subsequently treated with MPs from controls at 3 mg/ml or ACS patients at 5
mg/ml for 30 min before pre-constriction with 10−6 mol/l phenylephrine (PE,
Sigma-Aldrich, St. Louis, MO). Endothelium-dependent relaxation to acetylcholine
(Ach: 10−8–10−4 mol/l; Sigma-Aldrich, St. Louis, MO) was recorded with or without
pre-incubation with 1 mmol/l NG-nitro- L-arginine methyl ester (L-NAME; an
inhibitor of eNOS, Sigma-Aldrich, St. Louis, MO) for 30 min. The vascular relaxation
curves were produced by Graph-Pad Prism, version 5 (Graph-Pad Software, San
Diego, CA, USA).
Measurement of superoxide production. Lucigenin-enhanced chemiluminescence
was measured as previously described5. Briefly, the aorta was cut into rings and
incubated in Krebs solution for 30 min for equilibration. Then, L-NAME (1 mmol/l)
was added to certain samples for an additional 30 min. After 30 min of pre-incubation,
vessels were treated with or without MPs (3 mg/ml) from controls or patients with
ACS. After incubation with the MPs, the rings were placed into 96-well plates with
Krebs containing 5 μmol/l lucigenin (Sigma- Aldrich, St. Louis, MO). The rings were
allowed to adapt in the dark for 10 min at 37°C before O2˙− production was measured
using a SpectraMax M5/M5e multi-detection reader (Molecular Devices). After
measurement, the aortic samples were dried and weighed, and O2˙− generation was
reported as relative light units (RLU)/second/mg protein.
Measurement of nitric oxide (NO) generation. Briefly, aortas were cut into rings
and equilibrated for 30 min in Krebs solution in 12-well plates. The tissues were
maintained in this solution for an additional 30 min with or without L-NAME (1
mmol/l) and then treated with or without MPs (3 mg/ml) from controls or ACS
patients. After incubation with the MPs, the supernatant was assessed for NO
generation using a specific detection kit (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China). The aortic samples were dried and weighed, and NO generation was
reported as nmol/mg protein3.
Western blot analysis. Whole rat thoracic aortas were treated with or without MPs
(3 mg/ml) from controls or ACS patients for 1 h and then washed thrice with Hanks’
balanced salt solution (HBSS). The aortic proteins were then harvested, and
immunoblots were performed as previously described3. Antibodies to Akt (Cell
Signaling Technology, Beverly, MA, USA), phosphorylated Akt (Cell Signaling
Technology, Beverly, MA, USA ), endothelial nitric oxide synthase (eNOS; Santa
Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated at Ser1177 eNOS (Cell
Signaling Technology, Beverly, MA, USA ), and β-actin (Cell Signaling Technology,
Beverly, MA, USA ) were used for Western blot analyses.
Immunoprecipitation. Aortas were treated with or without MPs (3 mg/ml) from
controls or ACS patients for 1 h and then washed three times with HBSS before the
aortic proteins were harvested. The aortic protein lysates were incubated for 24 h with
an anti-eNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) to
immunoprecipitate eNOS. The eNOS immunocomplex was mixed with Laemmle
buffer, heated (95°C, 5 min), mixed, and stored on ice until fractionation. The
separated proteins were immunoblotted for eNOS and heat shock protein 90 (Hsp90,
Santa Cruz Biotechnology, Santa Cruz, CA, USA) as previously described3.
Statistical analysis. Data are presented as the means±SD, and analyzed using
GraphPad Prism, version 5.0 (GraphPad Software, San Diego, CA, USA). Normally
distributed continuous variables were compared using an independent-samples t-test.
Non-normally distributed continuous variables were evaluated with a Mann-Whitney
U-test. An independent-samples t-test was used for two-group comparisons, and a
one-way analysis of variance was used for multigroup comparison. Dichotomous data
are presented as absolute numbers. Correlations between selected variables were
estimated by Spearman’s rank correlation coefficient. Differences were considered
significant when P < 0.05.
Results
Plasma MPs. Plasma MPs concentrations were elevated in patients with ACS (day
0: 4.52±0.7; day 2: 4.47±1.1, n = 10) compared with the control group (2.7±0.4, n
= 10; Fig. 1A). Much more EMP (43.5%±1.5%, Fig. 1B,C) were detected in day2
MPs compared with day0 MPs (PMP, 32.3%±2.3%, Fig. 1B,C). EMP in day0
contained 38.5%±1.5%, and PMP occupied 35.3%±2.3%.
Effects of MPs on endothelium-dependent vasodilatation. Endothelium-dependent
relaxations in response to Ach were impaired after incubation with MPs (3mg/ml)
from both day0 and day2 ACS patients, but there were no significant difference
between day0 and day2 (Fig. 2A). And this effect was enhanced at the concentration
of 5mg/ml, and there weren’t statistical difference between day0 and day2 either (Fig.
2B).
Effects of MPs on NO and O2˙− generation. NO and O2˙− generation were
measured to detect whether MPs from patients with ACS of day0 and day2 have
difference on increasing oxidative stress. There were no statistical difference between
day0 and day2 on decreasing NO (Fig. 3A) or production of O2˙− (Fig. 3B).
Effects of MPs on the Akt/eNOS pathway.
Western blotting was used to
investigate the different effects on phosphorylation of Akt and eNOS between day0
and day2 MPs from ACS patients. We found that MPs from day2 ACS patients can
reduce eNOS phosphorylation at the Ser1177 site (Fig. 4A) and Akt phosphorylation
(Fig. 4B) much more, but the difference weren’t significant compared with day0 MPs.
Effects of MPs on eNOS and Hsp90 association. The immunoprecipitation
experiment revealed that both day0 and day2 MPs would decrease the association of
eNOS and Hsp90. And there were no statistical difference between day0 and day2
on the effects of eNOS associated with Hsp90 (Fig. 5).
*
*
4
2
AC
S(
da
y
0)
AC
S(
co
nt
da
y
ro
2)
0
l
Concentration of MPs (mg/ml)
6
B
50
40
#
*
30
20
10
rs
ot
he
PM
EM
P
0
P
Mean percentage of MPs(%)
A
C
Fig. 1. Origins of day2 MPs from ACS patients. Wen-Qi Han
a: MPs were increased in patients with ACS (day 0: 4.52±0.7; day 2: 4.47±1.1, n = 10) compared
with control group(2.7±0.4, n=10). Data are means ± SD, * vs. control, P<0.05.
b/c: Origins of MPs from day 2 ACS patients. Endothelial-derived MPs (EMP, CD31(+)/CD41(-), blue
area, b) occupied the most (43.5%±1.5%,c); then platelet-derived MPs (PMP, CD31(+)/ CD41(+),
green area, b; 32.3%±2.3%, c ); MPs that we didn’t test their origins held 24.1%±2.3% (other, c ). *
vs. EMP and PMP, # vs. EMP P<0.05, n=10.
control
ACS day0 (3mg/ml)
ACS day2 (3mg/ml)
80
100
*
60
40
control
ACS day0 (5mg/ml)
ACS day2 (5mg/ml)
80
Relaxation (%)
Relaxation (%)
100
*
60
40
20
20
0
0
8
7
6
5
Acetylcholine (-log M)
A
4
8
7
6
5
4
Acetylcholine (-log M)
B
Fig. 2. Effects of MPs from day0 and day2 ACS patients on endothelium-dependent dilatation.
Wen-Qi Han
MPs from day0 and day2 ACS patients both at 3mg/ml (a) and 5mg/ml (b) could significantly impair
endothelium-dependent dilatation stimulated by acetylcholine (Ach).But there were no significantly
difference between day0 and day2 at the same concentration of MPs stimulation. Data are means ± SD,
* vs. control p<0.05, n=5.
4
#
3
2
1
2
*
A
o
c
n
la
b
y2
C
S
da
y0
A
C
S
da
ro
A
co
nt
an
bl
l
k
+
0
L
b
la
0
n
-N k
n
tr co AM
o nt
A
l+ r E
C
o
l
S A Ld CS NA
a
M
y
0 da E
A
+
C
y
S A L- 0
d CS NA
a
M
y
2 da
E
+
y
L
-N 2
A
M
E
*
RL U/s e c o n d /mg
*
4
k
nitrite+nitrate(mol/g protein)
6
B
Fig. 3. Effects of MPs on nitric oxide (NO) and superoxide anion (O2 ˙-) generation. Wen-Qi Han
a: a significant decreasing in NO production was evident with MPs from day0 ACS patient in cultured
aorta. The future descending of NO in day2 wasn’t significant compared with day0. *P <0.05 vs. blank
and control.
b: O2˙- generation was increased in both day0 and day2 ACS groups. Although O2˙- production was
higher in day2 group, there was no statistical difference compared with day0. The increased O2˙generation in both groups can be inhibited by NG-nitro- L-arginine methyl ester (L-NAME). * vs. ACS
day0 and ACS day2 ; # vs. ACS day0+ L-NAME and ACS day2+ L-NAME, P < 0.05, n = 5.
B
C
D0
IB eNOS
IB AKT
IB S1177
IB P-AKT
IB β-actin
IB β-actin
D2
B
C
D0
p-AKT/AKT
p-eNOS/eNOS
0.25
0.6
0.4
* *
0.2
Relative Density
0.0
0.20
0.15
0.10
*
0.05
*
eNOS/-actin
y2
AC
S
da
AC
S
da
y0
l
ro
co
nt
bl
an
k
da
y2
AC
S
S
da
y0
l
ro
AC
co
nt
bl
an
k
0.00
AKT/-actin
0.8
Relative Density
1.0
0.8
0.6
0.4
0.2
0.4
0.2
y2
AC
S
da
S
AC
da
y0
l
ro
k
an
bl
y2
AC
S
da
y0
S
da
ro
AC
co
nt
an
bl
l
0.0
k
0.0
0.6
co
nt
Relative Density
0.8
Relative Density
D2
Fig. 4. MPs of day0 and day2 from patients with ACS had no significant difference on decreasing
endothelial nitric oxide synthase (eNOS) and phosphorylation of Akt (p-Akt) in rat thoracic aorta.
Wen-Qi Han
Representative immunoblot (IB) showing that MPs of day0 and day2 from patients with ACS
decreased eNOS phosphorylation at the Ser1177 site (p-eNOS) and p-AKT with the same degree in rat
thoracic aorta. * vs. blank and control P <0.05, n= 5.
IP eNOS
IB eNOS
IB Hsp90
B
C
D0
D2
Hsp90/eNOS
Relative Density
0.8
0.6
0.4
*
*
0.2
y2
AC
S
da
S
AC
da
y0
l
ro
co
nt
bl
an
k
0.0
Fig. 5. Effects of MPs from day0 and day2 ACS patients on the association of eNOS with heat
shock protein 90 (Hsp90) in rat thoracic aortas. Wen-Qi Han
MPs of day0 and day2 from patients with ACS significantly decrease the association between eNOS
and Hsp90, but there were no statistical difference between them. * vs. blank and control P < 0.05, n
=5.
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