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Introduction
Inflammation plays a pathogenic role in restenosis after percutaneous transluminal coronary
angioplasty[1]. Recently, increasing evidence have shown that the
CD40-CD40 ligand (CD40L) interaction plays a crucial role in the
pathogenesis of atherosclerosis and coronary artery
disease[2]. Furthermore, we found that patients with acute coronary
syndromes (ACS) had higher serum concentrations of soluble
CD40L than healthy volunteers or those with stable angina,
and an obvious correlation was also observed between
soluble CD40L (sCD40L) concentration and complex
coronary stenosis[3,4]. Recently, the increasing CD40-CD40L
system has been observed in patients with hypercholesterolemia,
and the circulating sCD40L has strong independent
prognostic value among ACS[5,6]. However, the role of the
CD40-CD40L system in the pathophysiology of restenosis is still
unclear.
Until now, there has been little information on whether
the CD40-CD40L system may influence inflammatory
reaction after vessel injury, which is ultimately responsible for
luminal renarrowing after percutaneous coronary
intervention (PCI) in humans. We hypothesized that the CD40-CD40L
interaction might also contribute to the development of
restenosis after PCI. Therefore, the present study was
designed to investigate the possible role of the CD40-CD40L
system in restenosis after PCI and its correlation with the
C-reactive protein (CRP), as well as lumen loss in PCI patients.
Materials and methods
Reagents The mouse anti-human CD40, mouse
anti-human CD40L, and CD61 fluorescein isothiocyanate
(FITC)-conjugated antibodies were bought from PharMingen (San
Diego, CA, USA). sCD40L, an ELISA kit was purchased
from Bender Medsystems (PharMingen).
Patients and controls One hundred and twenty patients
with stable angina or ACS were enrolled in the trial.
Coronary angiography showed that all the patients had
substantial coronary artery disease with a diameter stenosis of at
least 70% at a culprit lesion that was suitable for angioplasty.
Drug-eluting stents were implanted in the patients with
diabetes or in the patients with the £3.0 mm target vessel
diameter or the >20 mm lesion length. For the comparison, 20 sex- and age-balanced donors served as a control group.
Patients with infection, tumor, liver or kidney diseases were
excluded. Informed written consent was obtained from each
patient and the study was approved by the Ethical
Committee on Human Research. We also excluded patients who
were treated with anti-inflammatory drugs prior to
enroll-ment, for example, statins, clopidogrel, and angiotensin II
type 1 receptor blockers (Table 1).
Angioplasty procedure and follow-up evaluation
PCI and quantitative coronary angiography were performed
according to standard techniques as described
previously[7]. Patients were followed for 6 months for restenosis control.
Restenosis was defined as a loss of 50% or more of the initial
gain during the follow-up period by angiography. The
continuous luminal loss was defined according to the following
equation: late loss=[(post-intervention minimal lumen
diameter-follow-up minimal lumen diameter)/vessel size]×100%.
The vessel size is the value of the reference diameter
function at the minimal position of the obstruction.
Blood sampling protocol A rich-plasma platelet was
prepared as previously described[8]. Briefly, peripheral venous
blood was drawn into tubes containing sodium citrate.
Citrated blood samples were centrifuged at
200×g for 10 min at room temperature to obtain platelet-rich plasma.
Non-citrated blood was immersed in melting ice and allowed to
clot for 1 h before centrifugation
(1500×g for 10 min at 4 oC).
The supernatant was stored at -80 oC until analysis. Samples
were thawed only once.
Detection of CD40 and CD40L on platelets by flow
cytometry Platelet immunostaining was performed as
previously described[9]. Fixed blood was diluted at 1:100 with
PBS and incubated with the first antibody (30 min at 4
oC) diluted with PBS. Then the platelets were incubated with
PE-conjugated secondary antibody (30 min at 4
oC) and analyzed using CELLQUEST software
(BD Bioscience, Bredford, MA, USA). For each treatment, the mean fluorescence
intensity (MFI) value for the control stained population was
subtracted from the MFI value of the positive-stained sample.
Platelets were identified by their positivity for GP IIIa
antigen (CD61) and their characteristic light scatter. The platelet
population evaluated was ³98% positive for CD61.
Enzyme immunoassays Serum and plasma samples were
frozen and thawed only once. Specific immunoassays for
sCD40L (sCD40L detection limit, 95 pg/mL; Bender
Med-systems, PharMingen) and high-sensitive CRP on an
IMMULITE automated analyzer (Diagnostic Product Corporation, Los Angeles, CA, USA) were performed in
triplicate as previously described[7]. At our laboratory, the
intra-assay and interassay coefficients of variation were
less than 5%.
Statistical analysis Statistical evaluations were
performed with Graphpad software (Prism 3.0, La Jolla, CA, USA)
and SAS 8.0 software (SAS Institute Inc. Cary, NC, USA).
Data were expressed as mean±SD and statistically compared
by repeated measures. Correlation was evaluated through a
regressive analysis. The Pearson two-way test and the
Spearman two-way test were used to assess the relationship
between 2 quantitative variables with normal or abnormal
distribution. All P values of less than 0.05 were considered
statistically significant.
Results
Clinical follow-up results No patients developed
myocardial infarction or sudden death during the 6 month follow-up period. Restenosis occurred in 29 of the 120 PCI
patients (24.2%). The incidence of restenosis was 17.7%
and 31.0% in patients who received drug eluting stents or
bare mental stents, respectively.
CD40-CD40L system expression
Restenotic patients who received either drug eluting stent or bare metal stent
showed higher levels of CD40 (MFI: 60.1±6.0
vs 54.1±6.3) and CD40L (MFI: 11.2±2.8
vs 9.2±1.8, P<0.01) expression on
platelets as well as sCD40L (11.3±2.4 vs
9.4±1.6 ng/mL, P<0.01) compared with preoperative non-restenotic patients (all
P<0.01; Figure 1A, 1B).
Furthermore, the CD40-CD40L system was analyzed in
all patients preoperatively and postoperatively (24 h, 1
week, 2 weeks, 1 month, and 6 months after PCI).
Persistent increases in the CD40 system were observed in restenotic
patients after PCI during the whole 6 month follow-up period
(P<0.01 vs before PCI), whereas they were normalized at 2
weeks after PCI in the non-restenotic patients (Table 2).
Relationship between CRP levels and CD40 system
Patients who developed restenosis showed significantly
higher CRP levels (1.48±0.34 vs 0.92±0.19 ng/mL,
P=0.0001) than non-restenotic patients. Moreover, the CRP kept
considerable levels in the restenotic patients during the whole
follow-up period (Figure 2). The level of CRP was positively
correlated with expression of sCD40L and CD40L in restenotic patients at different time points (Table 3). But it
was not associated with the expression of sCD40L and CD40L
in non-restenotic patients. The level of CRP had no
correlation with CD40 expression for all patients.
Correlation of lumen loss with CD40 system in
reste-notic patients To investigate whether the CD40 system
contributes to luminal restenosis after PCI, we assessed the
association between the expression of the CD40 system and
the degree of luminal renarrowing. The levels of the CD40
system were measured for 6 months after PCI. Lumen loss
showed a significant positive correlation with sCD40L and
CD40L by 6 months (Figure 3). However, there was no
significant correlation of luminal loss with the expression of
CD40 on platelets in the patients (R2=0.02513, P=0.3418,
n=19).
Discussion
Restenosis is a serious complication of
PCI[10]. PCI activates platelets and white cells and triggers an acute
inflammatory response that plays a major role in the pathogenesis
of complications. Increasing evidence support the role of
inflammation in the restenosis after
PCI[11]. Recently, Cipollone et
al[12] reported that the pre-procedural level of
soluble CD40L was associated with late restenosis after
percutanerous transluminal coronary angioplasty
(PTCA). We found that PCI resulted in significant increases in CD40,
CD40L, sCD40L, and CRP in a short time for all patients, and
patients who developed restenosis showed sustained higher
levels of CD40L, sCD40L, and CRP than non-restenotic
patients.
Platelets are major contributors to the elevated level of
circulating sCD40L in patients with acute coronary syndrome
and hypercholesterolemia, and more than 95% of the
circulating sCD40L exists in
platelets[13,14]. We demonstrated that
the increased circulating sCD40L was from activated
platelets in hypertension patients[15]. The disrupted endothelium
by PCI resulted in the exposure of thrombogenic surfaces
that support the adhesion, activation, and aggregation of
platelets. Therefore, the platelet-rich thrombi may be the
major intravascular source of enhanced expression of the
CD40 system both on the platelet surface and in the
circulating environment as they shed sCD40L.
CRP is an acute-phase reactant and a strong marker for
inflammation. Elevated hs-CRP levels have a close
correlation with the occurrence of restenosis or major
cardiovascular events[16,17]. Most studies have focused on the
importance of elevated hs-CRP levels before PCI and immediately
after PCI. Our study showed significantly sustained high
hs-CRP levels in restenotic patients. This result indicates
that patients with long-term enhanced levels of CRP have a
higher incidence of restenosis.
The CD40 system is a predictor of enhanced
inflammatory response. Considerable evidence implicate that the
CD40-CD40L interaction plays a crucial role in multiple stages
of atherosclerosis[18]. Recently, Turker
et al[19] reported that an increased pre-procedural sCD40L level was an
independent predictor of stent restenosis during the 6 month
follow-up period. Therefore, we suspected that the CD40 system
was associated with restenosis after PCI. In this study, we
demonstrated that the CD40L expression was not only
significantly correlated with CRP levels, but also with the
luminal loss in restenotic patients. This result may indicate an
association between CD40L changes and the occurrence of
restenosis. It also suggested that CD40L was a reliable risk
predictor for restenosis and provided a potential preventive
strategy against restenosis, such as using anti-CD40L
antibody to prevent lumen renarrowing after PCI. Further
large-scale cohort studies should be performed to illustrate the
clinical implication of expression of the CD40 and CD40L
system in patients after PTCA.
In this study, it was interesting to note that there was a
correlation between restenosis and CD40L expression rather
than the CD40 levels. This result was similar to our
previously findings in which the increased CD40L levels
indicated a significantly increased risk of major adverse
cardiovascular events[6]. This interesting phenomenon may not
only be due to the cause of activated platelets releasing
circulating sCD40L in these patients, but also
previous in vitro and in vivo studies which demonstrated a crucial role
of CD40L in multiple stages of atherosclerosis and
ACS[20]. Further study remains to be elucidated.
Limitations of our study were the small number of cohort
patients and the limited sample collecting time which may
have affected the results. Therefore, further large-scale
studies and prolonged follow-up should be performed to
illustrate the clinical value of CD40L levels independently or in
combination with other markers for predicting the risk of
restenosis. Simultaneous assessment of the CD40 system
and other factors yields independent and complementary
prognostic information and thus enabled a more powerful
prediction of restenosis.
In conclusion, CD40L expression may provide a useful
marker for risk assessment of restenosis after PCI. However,
the precise role of the systemic inflammation in restenosis
remains to be determined.
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