Original Research Cardiology 2014;128:259–265 DOI: 10.1159/000358508
Received: September 9, 2013 Accepted after revision: January 9, 2014 Published online: May 16, 2014
Drug-Eluting Stent, but Not Bare Metal Stent, Accentuates the Systematic Inflammatory Response in Patients Junhui Liu Xiao-Zhen Zhuo Weimin Liu Zhaofei Wan Xinhong Wang Weiping Zhang Shanshan Gao Zuyi Yuan Yue Wu Department of Cardiology, First Affiliated Hospital of Xi’an Jiaotong University Health Science Center, Xi’an, PR China
Abstract Objective: The systematic pro-inflammatory responses after percutaneous coronary intervention with drug-eluting stents (DES) remain poorly defined. Therefore, we compared the systematic pro-inflammatory state of circulating mononuclear cells (MNCs) between DES and bare metal stent (BMS) implantation. Methods: Patients with indications for treatment with stents were randomized in a 1: 1 ratio to placement of DES or BMS. The primary endpoint was a change of pro-inflammatory state at 12 weeks postprocedure. Results: Thirty-six consecutive patients received DES or BMS. At 12 weeks after stent implantation, the lipid profile and high-sensitivity C-reactive protein (hs-CRP) improved significantly in both groups. The mRNA levels and plasma concentrations of interleukin-6, tumor necrosis factor-α and matrix metalloproteinase-9 were significantly elevated in the DES group, which was not observed in the BMS group. An increase in NF-κB binding activity and a decrease in PPAR-γ expression in MNCs were observed in the DES group, along with increases in IκB phosphorylation and p50 expression. However, similar changes were not observed in the BMS group. Conclusions: Systematic inflam-
© 2014 S. Karger AG, Basel 0008–6312/14/1283–0259$39.50/0 E-Mail [email protected] www.karger.com/crd
matory responses were accentuated after the patients were treated percutaneously with DES, despite their improved lipid profile and hs-CRP. These data may provide fundamental information for optimizing therapeutic strategy in the era of DES. © 2014 S. Karger AG, Basel
Introduction
Polymer-based sirolimus and paclitaxel drug-eluting stents (DES) have been demonstrated to successfully reduce the rate of target vessel restenosis [1], which is the major complication of bare metal stent (BMS). Although percutaneous coronary intervention (PCI) with DES has been widely used to treat patients with symptomatic coronary artery disease (CAD) all over the world, recent studies have raised concerns about its long-term clinical safety and efficacy, which remains a challenge for interventional cardiologists, including delayed arterial healing response, in-stent restenosis, late stent thrombosis and the local inflammatory reactions [2–4]. Moreover, the systematic pro-inflammatory response after PCI with DES is still poorly defined.
J.L. and X.-Z.Z. contributed equally to this work.
Zuyi Yuan or Yue Wu Department of Cardiovascular Medicine First Affiliated Hospital of Medical School, Xi’an Jiaotong University 277 West Yanta Road, Xi’an, Shaanxi 710061 (PR China) E-Mail zuyiyuan @ mail.xjtu.edu.cn or imyuewu @ gmail.com
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Key Words Patients · Drug-eluting stent · Pro-inflammation · Mononuclear cells
Circulating mononuclear cells (MNCs) represent a detective model for metabolic diseases. It is widely used for evaluating the systematic pro-inflammatory status in patients [5, 6]. Systematic pro-inflammatory status is defined as elevated plasma concentration of pro-inflammatory factors and their gene expressions in MNCs [5–7]. Moreover, the NF-κB binding activity in the nuclei, expressions of subunits p65 and p50, and the decreased expression of IκB in MNCs are also important characteristics of chronic metabolic pro-inflammation such as diabetes, obesity and atherosclerosis [6–8]. Although it has been reported that the levels of some plasma inflammatory markers and factors are increased for several days after BMS implantation [9, 10], it is still unclear whether percutaneous treatment with DES induces systematic inflammatory responses. In a previous study, we demonstrated that the plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) in CAD patients unexpectedly increased to 141 ± 12, 112 ± 6 and 124 ± 11% of the baseline 3 months after DES implantation [8]. However, these data have only been compared with those of patients with coronary angiography only, and could not be used to determine whether DES or stent induces systematic inflammatory responses. Therefore, in this study, we directly compared the inflammatory responses in MNCs between DES and BMS.
Study Endpoints and Follow-Up The primary endpoint of the study was to compare the change in plasma IL-6 levels between the DES and the BMS groups for 12 weeks of follow-up. Patients were followed by telephone or hospital visits at 4 weeks after PCI. Formal follow-up was scheduled at 12 weeks after PCI. Venous blood samples were drawn from each patient after overnight fasting at the 12-week follow-up. Plasma Concentrations of Pro-Inflammatory Mediators Concentrations of plasma IL-6, TNF-α, IL-4 and MMP-9 were measured with ELISA kits according to the manufacturers’ instructions (ExCell). High-sensitivity C-reactive protein (hs-CRP) assays were performed by our hospital’s clinical laboratory. Statistical Analysis Statistical analysis was performed with SPSS for Windows (version 13.0). Summary values were expressed as mean ± SE and skewed data were reported as median with interquartile range. Analyses of changes from baseline were performed using a paired t test. The Holm-Sidak two-way repeated-measures ANOVA (TWRMANOVA) method was used for all multiple comparisons between the DES and the BMS groups. Correlation analysis was performed using Spearman’s rank order correlation among parameters and the pro-inflammatory markers. Statistical significance was assumed at the 5% α-error level (p < 0.05). The sample size of the study was determined based on an estimation of the primary endpoint of pro-inflammatory state from our previous data [8]. We assumed that IL-6 would increase to 141 ± 12% in the DES group and would not change in the BMS group. Based on a two-sided test for differences in independent binomial proportions with a level of 0.05, 36 patients (18 patients for each group) would have to undergo randomization for the study to have 80% power to detect a difference in the pro-inflammatory state between the two groups.
Methods
Patients Patients were eligible and consecutively enrolled from our hospital if they had a de novo lesion with diameter stenosis of more than 50% in a native coronary artery with a reference diameter of 2.5–4.0 mm. Patients were randomized in a 1:1 ratio to two groups: the Everolimus DES (Abbott Vascular, Santa Clara, Calif., USA) group and the BMS (Bx-Velocity; Cordis Corp., Bridgewater, N.J., USA) group. The exclusion criteria included clinical evidence of acute inflammation, a previous stent implant, and tumor and connective tissue diseases. Online supplementary table 1 summarizes all the demographic data of the subjects. Fasting blood samples were collected after hospitalization. All patients were given the optimal standard treatment from the beginning of hospitalization, including aspirin, clopidogrel, β-receptor-blockers, angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers and statins. This study complied with the Declaration of Helsinki, and the research protocol was approved by the Ethics Committee of Xi’an Jiaotong University.
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Cardiology 2014;128:259–265 DOI: 10.1159/000358508
Results
Clinical Data and Results A total of 36 patients were consecutively enrolled and randomized to the DES (n = 18) and BMS (n = 18) groups. Online supplementary table 1 summarizes the demographic data of all subjects. The baseline clinical and angiographic characteristics were similar between the two groups (online suppl. table 1). All 36 patients fulfilled the 12 weeks of follow-up. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol and hs-CRP were improved significantly after standard medication in both the DES and the BMS groups, with no differences between the two groups observed (table 1). The body mass index (BMI), waist to hip ratio, blood pressure, fasting glucose, N-terminal pro-brain natriuretic peptide and ejection fraction showed no significant differences compared with the baseline in both groups (table 1). Liu/Zhuo/Liu/Wan/Wang/Zhang/Gao/ Yuan/Wu
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A detailed description of the methods and tables is provided in the online supplementary file (for all online suppl. material, see www.karger.com/doi/10.1159/000358508).
Table 1. Subject characteristics at baseline and after 12 weeks of intervention
Marker
BMI, kg/m2 Waist to hip ratio, % Systolic BP, mm Hg Diastolic BP, mm Hg Fasting glucose, mmol/l Total cholesterol, mmol/l Triglycerides, mmol/l HDL cholesterol, mmol/l LDL cholesterol, mmol/l hsCRP, mg/l NT-pro BNP, pg/ml EF, %
DES group (n = 18)
BMS group (n = 18)
week 0
week 12
week 0
week 12
24.3±4.1 88.3±5.9 131±21 79±11 5.76±0.71 3.74±0.62 1.69±1.01 0.89±0.18 2.34±0.76 3.96 (1.09, 12.9) 210 (89, 991) 59.0±10.3
24.2±3.5 88.7±6.6 126±17 77±11 5.45±0.62 3.24±0.91* 1.19±0.51* 1.15±0.49* 1.74±0.61** 2.78 (0.77, 8.71)** 189 (112, 756) 58.6±10.1
24.5±3.9 89.5±5.7 135±17 80±10 5.82±0.59 3.69±0.52 1.70±1.06 0.86±0.19 2.21±0.53 4.11 (1.06, 11.8) 192 (103, 897) 57.9±8.8
24.3±3.8 89.2±6.7 126±16 76±9 5.49±0.57 3.22±0.81* 1.25±0.41* 1.15±0.41* 1.69±0.48** 2.56 (0.59, 5.39)** 187 (108, 801) 58.7±9.6
Values are mean ± SD or median (interquartile range). * p < 0.05; ** p < 0.01 compared with baseline. BP = Blood pressure; NT-pro BNP = N-terminal pro-brain natriuretic peptide; EF = ejection fraction.
DES Increased the mRNA Levels of Inflammatory Factors in MNCs To investigate whether the changes in plasma pro-inflammatory factors were due to changes in mRNA expression in MNCs, we performed quantitative real-time PCR and used the comparative threshold cycle method to calculate the relative gene expressions. In the DES-implanted group, the mRNA levels of IL-6, TNF-α and MMP-9 increased by 2.04 ± 0.43, 1.15 ± 0.29 and 0.54 ± 0.21 units compared with the baseline (p = 0.0002, 0.001 and 0.020, respectively; fig. 1b), whereas in the BMS group, the mRNA levels of these factors did not change significantly (p = 0.309, 0.773 and 0.049, respectively). DES Accentuates Proinflammation
Compared with the BMS group, the mRNA levels of these factors were significantly elevated after the percutaneous treatment with DES (TWRMANOVA, p = 0.0001, 0.022 and 0.004, respectively; fig. 1b), which was consistent with their plasma protein levels. We also observed IL-4 expression at both protein and mRNA levels, but there were no differences in the baseline and follow-up data between the DES and BMS groups (fig. 1). NF-κB Binding Activity Was Increased after DES Implantation To investigate whether pro-inflammatory factors were activated by the transcription factor NF-κB, we measured the nuclear NF-κB DNA binding activity in MNCs (fig. 2a). The NF-κB activity significantly increased to 158 ± 27% of the baseline after DES implantation (p = 0.016; fig. 2a). Compared with the unchanged BMS group (102 ± 6.5%, p = 0.779; fig. 2a), the inter-group change was also statistically significant (TWRMANOVA, p = 0.017), suggesting that the systematic inflammatory responses were induced by the activated NF-κB after DES implantation. PPAR-γ Binding Activity Was Decreased after DES Implantation Next, we measured the PPAR-γ binding activity in MNCs. PPAR-γ is an anti-inflammatory transcriptional factor [11]. As shown in figure 2a, we found that PPAR-γ activity was significantly decreased in the DES group (p = 0.007; fig. 2a) and unchanged in the BMS group Cardiology 2014;128:259–265 DOI: 10.1159/000358508
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DES Accentuated the Levels of Systematic Inflammatory Mediators Twelve weeks after percutaneous treatment with DES, the plasma concentrations of IL-6, TNF-α and MMP-9 increased to 153 ± 21, 136 ± 15 and 108 ± 3.5% of the baseline, respectively (p = 0.012, 0.009 and 0.021, respectively; fig. 1a), whereas in the BMS group, the plasma concentrations of these factors changed to 105 ± 10, 103 ± 8 and 87 ± 4.4% of the baseline, respectively (p = 0.491, 0.575 and 0.010, respectively; fig. 1a). Compared with the control group, plasma IL-6, TNF-α and MMP-9 concentrations were significantly elevated in the DES group (TWRMANOVA, p = 0.005, 0.023 and 0.001, respectively; fig. 1a), suggesting that the pro-inflammatory response was enhanced after DES implantation.
0w
12w DES
5
0w
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100
0
0w 12w BMS
0w
12w DES
p = 0.022 15
**
10 5 0
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Plasma IL-4 (pg/ml)
5
p = 0.0001
***
Plasma MMP-9 (ng/ml)
10
0
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10
0
15
*
* 200
*
*
6 4 2 0w
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p = 0.384
4
8
0
2
0
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p = 0.004 10
4
3 2 1 0
0w
12w DES
0w 12w BMS
Fig. 1. Changes in pro-inflammatory mediators after standard drug medication for 12 weeks in the DES (n = 18) and BMS (n = 18) groups. a Plasma concentrations of IL-6, TNF-α, MMP-9 and IL-4 at baseline (0w) and 12 weeks (12w) were presented as raw
data. b The mRNA expression of peripheral blood MNCs were presented and the comparative threshold cycle methods were used to calculate the relative gene expression (arbitrary units, au). Compared with baseline: * p < 0.05; ** p < 0.01; *** p < 0.001.
(TWRMANOVA, p = 0.001; fig. 2a), suggesting that the impaired PPAR-γ signaling also contributed to DES-related pro-inflammation.
were observed in the DES group, but not in the BMS group (TWRMANOVA, p = 0.036 and 0.011, respectively; fig. 2b). These results suggested that the activated NFκB signal cascade plays an important role in DES-related pro-inflammatory responses. Interestingly, we also found that PPAR-γ were dramatically decreased in the DES group (p = 0.008; fig. 2c) and were unchanged in the BMS group (TWRMANOVA, p = 0.002; fig. 2c), suggesting that the ratio of NF-κB to PPAR-γ level may be the key regulator of DES-related pro-inflammation.
NF-κB and PPAR-γ Signaling Imbalance May Contribute to Systematic Pro-Inflammation We further examined the expressions of the p50 subunit and p65 subunit of NF-κB in MNCs. The protein level of p65 remained unchanged compared with the baselines of the two groups (fig. 2b). The p50 protein increased significantly in the DES group to 140 ± 17% of the baseline (p = 0.042), but it decreased to 63 ± 14% (p = 0.033; fig. 2b) of the baseline in the BMS group (TWRMANOVA, p < 0.001; fig. 2b). These data indicated that standard drug therapy restrained the systematic inflammation by the NF-κB subunit p50 pathway in the BMS group, but not in the DES group. Next, we examined the IκB-α and its phosphorylation in MNCs. As shown in figure 2b the decreased IκB-α (52 ± 21% of baseline, p = 0.022; fig. 2b) and increased phosphorylated IκB-α (203 ± 49% of baseline, p = 0.015) 262
Cardiology 2014;128:259–265 DOI: 10.1159/000358508
Improved Metabolic Parameters Failed to Correlate with the Changes in Pro-Inflammatory Factors After analyzing the correlation among the changes of metabolic parameters and inflammatory factors such as total cholesterol, triglycerides, HDL and LDL, we found that the changes to metabolic parameters significantly correlated with one another (table 2). There were also strong correlations among the changes in IL-6, TNF-α, MMP-9 and NF-κB activities (table 2), which showed sigLiu/Zhuo/Liu/Wan/Wang/Zhang/Gao/ Yuan/Wu
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IL-6 mRNA (au)
15
12w DES
**
p = 0.569
IL-4 mRNA (au)
a
0w
20
MMP-9 mRNA (au)
10
0
b
Plasma 71)į (pg/ml)
20
TNF-į mRNA (au)
Plasma IL-6 (pg/ml)
*
p = 0.001
p = 0.023
p = 0.005
30
p = 0.016
p = 0.001
a
PPAR-Dž activity (score)
NF-NJB activity (score)
200 150 100 50 0
0w
12w
0w
DES BMS
200 150 100
*
50 0
12w
0w
DES Patient Timing
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2 0w
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BMS
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2 12w
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p50
p65 GAPDH
Protein level (au)
INJB-DŽ
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p65 p50 INJB-DŽ
*
0w
12w
###
*
100 50 0
b
0w
12w
p-INJB-į INJB-į DŽ-Actin
Fig. 2. Imbalanced NF-κB and PPAR-γ sig-
250
c
200
p-INJB-į INJB-į PPAR-Dž
*
150
#
100
*
50 0
0w
#
##
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Cardiology 2014;128:259–265 DOI: 10.1159/000358508
0w
12w
263
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DES Accentuates Proinflammation
PPAR-Dž
Protein level (au)
naling of MNCs in patients after coronary DES implantation. a The NF-κB and PPAR-γ binding activity from nuclear extracts at baseline (0w) and 12 weeks (12w) is shown. b Western blot analysis for cytoplasmic NF-κB (p65 and p50), phosphorIκB-α, IκB-α, IκB-β and PPAR-γ in MNCs, and densitometry are shown as arbitrary units (au). c Densitometry of EMSA and mRNA expressions of p50, IκB-α and PPAR-γ as arbitrary units (au). n = 18 for each group. Compared with baseline: * p < 0.05; ** p < 0.01. Compared between the DES and the BMS group: # p < 0.05; ## p < 0.01; ### p < 0.001.
Table 2. Associations between changes in metabolic and inflammatory parameters (n = 18)
ΔTC ΔTG ΔLDL ΔHDL ΔCRP ΔGlu ΔIL-6 ΔTNF-α ΔMMP9 ΔNF-κB ΔPPAR-γ
ΔTC
ΔTG
ΔLDL
ΔHDL
ΔCRP
ΔGlu
ΔIL-6
ΔTNF-α
ΔMMP9
ΔNF-κB
– 0.509b 0.795d –0.401 0.586c 0.185 –0.204 –0.109 –0.094 –0.113 0.259
– 0.516b –0.178 0.480b 0.056 –0.112 –0.126 –0.107 –0.079 0.126
– –0.458a 0.512b 0.186 –0.125 –0.189 –0.136 –0.152 0.271
– –0.459a –0.115 0.102 0.077 0.151 0.077 –0.191
– –0.101 –0.086 –0.161 –0.121 –0.203 0.379
– 0.062 0.079 –0.132 –0.092 0.122
– 0.626c 0.526b 0.701d –0.523b
– 0.591c 0.604c –0.611c
– 0.491b –0.531b
– –0.688c
The change from baseline after 12 weeks is designated by ‘Δ’. IL-6, TNF-α and MMP-9 are presented as plasma levels; NF-κB and PPAR-γ are presented as DNA binding activity. Correlation analysis was performed using Spearman’s rank order correlation. a 0.05 < p < 0.10; b p < 0.05; c p < 0.01; d p < 0.001. TC = Total cholesterol; TG = triglycerides; Glu = fasting glucose.
Discussion
In this study, we provided evidence to support the hypothesis that circulating inflammatory responses were unimproved or partly accentuated after DES implantation, but not BMS implantation, because of the increase in NF-κB activity via the p50 induction and IκB-α phosphorylation pathway, suppressed PPAR-γ expression and elevated plasma inflammatory factors. According to the guidelines [12], these ACS patients should also be treated with optimal drug combinations, including aspirin, clopidogrel, β-receptor-blockers, statins and angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers, which may exert many beneficial effects such as antiplatelet effect, metabolic improvement, endothelium protection and the prevention of vascular remodeling [12, 13]. However, it remains poorly understood whether the combination of these drugs inhibit systematic inflammation in patients after DES implantation. Our results also suggest that the standard medications can partly treat pro-inflammation after BMS implantation, which is evidenced by the decreased MMP-9 level and inhibited p50 expression, but cannot reverse the systematic inflamma264
Cardiology 2014;128:259–265 DOI: 10.1159/000358508
tion after DES implantation. These findings indicate that further optimizations of the present drug therapy are required in the era of DES. CRP, which is widely accepted as an important inflammatory marker in CAD [12], is dramatically improved but uncorrelated with other inflammatory factors and NF-κB. The important role of statins in regulating hepatic lipid and CRP secretion has been reported previously, which explains the parallel improvement of lipid profile and CRP [12, 14]. However, whether the level of IL-6 and other inflammatory factors can be used to predict the clinical cardiovascular events of these patients should be further explored because the CRP level cannot represent pro-inflammatory states in DES-implanted patients. NF-κB composed of p65/p50 dimmers is inactive in the cytoplasm when in association with IkB proteins [15]. Inducing stimuli or inflammation status triggers the phosphorylation, ubiquitination and degradation of IκB proteins. Then the released NF-κB dimers translocate to the nucleus, bind specific DNA sequences and promote the transcription of target genes such as TNF-α, IL-6 and MMP-9 [15–17]. Our data also suggest that the phosphorylation of IκB-α facilitates its degradation and, thus, NF-κB cannot be restricted to the cytoplasm and shuttle into the nucleus to induce the transcription of pro-inflammatory genes. Moreover, our results demonstrate that the increased level of p50 further activates the NF-κB pathway in the MNCs. It has been demonstrated that the nuclear receptor PPAR-γ can inhibit the function of NF-κB, and the imbalLiu/Zhuo/Liu/Wan/Wang/Zhang/Gao/ Yuan/Wu
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nificant inverse correlations with the change in PPAR-γ activity. However, there was no significant correlation between pro-inflammatory factors and metabolic parameters. Moreover, the change of hs-CRP significantly correlated with metabolic parameters, but not pro-inflammatory factors (table 2).
ance between PPAR-γ and NF-κB disturbs the homeostasis and may trigger inflammatory diseases, including vascular inflammation [18, 19]. In the present study, we also found a dramatic PPAR-γ loss in the MNCs in parallel with the increasing levels of inflammatory factors and the enhanced NF-κB activity. These indicated that the effective strategy for increasing PPAR-γ could be further explored to restrain NF-κB-related pro-inflammatory responses in these patients. Several previous studies have suggested that DES may trigger local inflammatory responses. Histopathological studies have also revealed that the polymer induced inflammatory cell accumulation, such as T lymphocytes and macrophages around the DES strut 9 months after implantation [3, 4], which is longer than the observation time in the present study (3 months). It is still unknown whether systematic pro-inflammation contributes to the local inflammatory responses. Thus, long-time and multitime-point observations for systematic pro-inflammation warrant further investigation.
We conclude that the systematic inflammatory responses are accentuated for at least 3 months after percutaneous treatment with DES, as evidenced by the enhanced NF-κB activity, suppressed PPAR-γ level, and the elevated plasma level of inflammatory factors. These data can provide fundamental information for optimizing our therapeutic strategy in the era of DES.
Acknowledgements This study was supported in part by the National Natural Science Foundation of China (81100209 to Y.W. and 30871043 to Z.Y.); the National Science Fund for Distinguished Young Scholars (81025002 to Z.Y.), and the National Basic Research Program of China (‘973 Project’ No. 2012CB517804 to Z.Y.).
Conflict of Interest All authors have no disclosures.
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DES Accentuates Proinflammation
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Received: September 9, 2013 Accepted after revision: January 9, 2014 Published online: May 16, 2014
Drug-Eluting Stent, but Not Bare Metal Stent, Accentuates the Systematic Inflammatory Response in Patients Junhui Liu Xiao-Zhen Zhuo Weimin Liu Zhaofei Wan Xinhong Wang Weiping Zhang Shanshan Gao Zuyi Yuan Yue Wu Department of Cardiology, First Affiliated Hospital of Xi’an Jiaotong University Health Science Center, Xi’an, PR China
Abstract Objective: The systematic pro-inflammatory responses after percutaneous coronary intervention with drug-eluting stents (DES) remain poorly defined. Therefore, we compared the systematic pro-inflammatory state of circulating mononuclear cells (MNCs) between DES and bare metal stent (BMS) implantation. Methods: Patients with indications for treatment with stents were randomized in a 1: 1 ratio to placement of DES or BMS. The primary endpoint was a change of pro-inflammatory state at 12 weeks postprocedure. Results: Thirty-six consecutive patients received DES or BMS. At 12 weeks after stent implantation, the lipid profile and high-sensitivity C-reactive protein (hs-CRP) improved significantly in both groups. The mRNA levels and plasma concentrations of interleukin-6, tumor necrosis factor-α and matrix metalloproteinase-9 were significantly elevated in the DES group, which was not observed in the BMS group. An increase in NF-κB binding activity and a decrease in PPAR-γ expression in MNCs were observed in the DES group, along with increases in IκB phosphorylation and p50 expression. However, similar changes were not observed in the BMS group. Conclusions: Systematic inflam-
© 2014 S. Karger AG, Basel 0008–6312/14/1283–0259$39.50/0 E-Mail [email protected] www.karger.com/crd
matory responses were accentuated after the patients were treated percutaneously with DES, despite their improved lipid profile and hs-CRP. These data may provide fundamental information for optimizing therapeutic strategy in the era of DES. © 2014 S. Karger AG, Basel
Introduction
Polymer-based sirolimus and paclitaxel drug-eluting stents (DES) have been demonstrated to successfully reduce the rate of target vessel restenosis [1], which is the major complication of bare metal stent (BMS). Although percutaneous coronary intervention (PCI) with DES has been widely used to treat patients with symptomatic coronary artery disease (CAD) all over the world, recent studies have raised concerns about its long-term clinical safety and efficacy, which remains a challenge for interventional cardiologists, including delayed arterial healing response, in-stent restenosis, late stent thrombosis and the local inflammatory reactions [2–4]. Moreover, the systematic pro-inflammatory response after PCI with DES is still poorly defined.
J.L. and X.-Z.Z. contributed equally to this work.
Zuyi Yuan or Yue Wu Department of Cardiovascular Medicine First Affiliated Hospital of Medical School, Xi’an Jiaotong University 277 West Yanta Road, Xi’an, Shaanxi 710061 (PR China) E-Mail zuyiyuan @ mail.xjtu.edu.cn or imyuewu @ gmail.com
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Key Words Patients · Drug-eluting stent · Pro-inflammation · Mononuclear cells
Circulating mononuclear cells (MNCs) represent a detective model for metabolic diseases. It is widely used for evaluating the systematic pro-inflammatory status in patients [5, 6]. Systematic pro-inflammatory status is defined as elevated plasma concentration of pro-inflammatory factors and their gene expressions in MNCs [5–7]. Moreover, the NF-κB binding activity in the nuclei, expressions of subunits p65 and p50, and the decreased expression of IκB in MNCs are also important characteristics of chronic metabolic pro-inflammation such as diabetes, obesity and atherosclerosis [6–8]. Although it has been reported that the levels of some plasma inflammatory markers and factors are increased for several days after BMS implantation [9, 10], it is still unclear whether percutaneous treatment with DES induces systematic inflammatory responses. In a previous study, we demonstrated that the plasma concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) in CAD patients unexpectedly increased to 141 ± 12, 112 ± 6 and 124 ± 11% of the baseline 3 months after DES implantation [8]. However, these data have only been compared with those of patients with coronary angiography only, and could not be used to determine whether DES or stent induces systematic inflammatory responses. Therefore, in this study, we directly compared the inflammatory responses in MNCs between DES and BMS.
Study Endpoints and Follow-Up The primary endpoint of the study was to compare the change in plasma IL-6 levels between the DES and the BMS groups for 12 weeks of follow-up. Patients were followed by telephone or hospital visits at 4 weeks after PCI. Formal follow-up was scheduled at 12 weeks after PCI. Venous blood samples were drawn from each patient after overnight fasting at the 12-week follow-up. Plasma Concentrations of Pro-Inflammatory Mediators Concentrations of plasma IL-6, TNF-α, IL-4 and MMP-9 were measured with ELISA kits according to the manufacturers’ instructions (ExCell). High-sensitivity C-reactive protein (hs-CRP) assays were performed by our hospital’s clinical laboratory. Statistical Analysis Statistical analysis was performed with SPSS for Windows (version 13.0). Summary values were expressed as mean ± SE and skewed data were reported as median with interquartile range. Analyses of changes from baseline were performed using a paired t test. The Holm-Sidak two-way repeated-measures ANOVA (TWRMANOVA) method was used for all multiple comparisons between the DES and the BMS groups. Correlation analysis was performed using Spearman’s rank order correlation among parameters and the pro-inflammatory markers. Statistical significance was assumed at the 5% α-error level (p < 0.05). The sample size of the study was determined based on an estimation of the primary endpoint of pro-inflammatory state from our previous data [8]. We assumed that IL-6 would increase to 141 ± 12% in the DES group and would not change in the BMS group. Based on a two-sided test for differences in independent binomial proportions with a level of 0.05, 36 patients (18 patients for each group) would have to undergo randomization for the study to have 80% power to detect a difference in the pro-inflammatory state between the two groups.
Methods
Patients Patients were eligible and consecutively enrolled from our hospital if they had a de novo lesion with diameter stenosis of more than 50% in a native coronary artery with a reference diameter of 2.5–4.0 mm. Patients were randomized in a 1:1 ratio to two groups: the Everolimus DES (Abbott Vascular, Santa Clara, Calif., USA) group and the BMS (Bx-Velocity; Cordis Corp., Bridgewater, N.J., USA) group. The exclusion criteria included clinical evidence of acute inflammation, a previous stent implant, and tumor and connective tissue diseases. Online supplementary table 1 summarizes all the demographic data of the subjects. Fasting blood samples were collected after hospitalization. All patients were given the optimal standard treatment from the beginning of hospitalization, including aspirin, clopidogrel, β-receptor-blockers, angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers and statins. This study complied with the Declaration of Helsinki, and the research protocol was approved by the Ethics Committee of Xi’an Jiaotong University.
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Results
Clinical Data and Results A total of 36 patients were consecutively enrolled and randomized to the DES (n = 18) and BMS (n = 18) groups. Online supplementary table 1 summarizes the demographic data of all subjects. The baseline clinical and angiographic characteristics were similar between the two groups (online suppl. table 1). All 36 patients fulfilled the 12 weeks of follow-up. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol and hs-CRP were improved significantly after standard medication in both the DES and the BMS groups, with no differences between the two groups observed (table 1). The body mass index (BMI), waist to hip ratio, blood pressure, fasting glucose, N-terminal pro-brain natriuretic peptide and ejection fraction showed no significant differences compared with the baseline in both groups (table 1). Liu/Zhuo/Liu/Wan/Wang/Zhang/Gao/ Yuan/Wu
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A detailed description of the methods and tables is provided in the online supplementary file (for all online suppl. material, see www.karger.com/doi/10.1159/000358508).
Table 1. Subject characteristics at baseline and after 12 weeks of intervention
Marker
BMI, kg/m2 Waist to hip ratio, % Systolic BP, mm Hg Diastolic BP, mm Hg Fasting glucose, mmol/l Total cholesterol, mmol/l Triglycerides, mmol/l HDL cholesterol, mmol/l LDL cholesterol, mmol/l hsCRP, mg/l NT-pro BNP, pg/ml EF, %
DES group (n = 18)
BMS group (n = 18)
week 0
week 12
week 0
week 12
24.3±4.1 88.3±5.9 131±21 79±11 5.76±0.71 3.74±0.62 1.69±1.01 0.89±0.18 2.34±0.76 3.96 (1.09, 12.9) 210 (89, 991) 59.0±10.3
24.2±3.5 88.7±6.6 126±17 77±11 5.45±0.62 3.24±0.91* 1.19±0.51* 1.15±0.49* 1.74±0.61** 2.78 (0.77, 8.71)** 189 (112, 756) 58.6±10.1
24.5±3.9 89.5±5.7 135±17 80±10 5.82±0.59 3.69±0.52 1.70±1.06 0.86±0.19 2.21±0.53 4.11 (1.06, 11.8) 192 (103, 897) 57.9±8.8
24.3±3.8 89.2±6.7 126±16 76±9 5.49±0.57 3.22±0.81* 1.25±0.41* 1.15±0.41* 1.69±0.48** 2.56 (0.59, 5.39)** 187 (108, 801) 58.7±9.6
Values are mean ± SD or median (interquartile range). * p < 0.05; ** p < 0.01 compared with baseline. BP = Blood pressure; NT-pro BNP = N-terminal pro-brain natriuretic peptide; EF = ejection fraction.
DES Increased the mRNA Levels of Inflammatory Factors in MNCs To investigate whether the changes in plasma pro-inflammatory factors were due to changes in mRNA expression in MNCs, we performed quantitative real-time PCR and used the comparative threshold cycle method to calculate the relative gene expressions. In the DES-implanted group, the mRNA levels of IL-6, TNF-α and MMP-9 increased by 2.04 ± 0.43, 1.15 ± 0.29 and 0.54 ± 0.21 units compared with the baseline (p = 0.0002, 0.001 and 0.020, respectively; fig. 1b), whereas in the BMS group, the mRNA levels of these factors did not change significantly (p = 0.309, 0.773 and 0.049, respectively). DES Accentuates Proinflammation
Compared with the BMS group, the mRNA levels of these factors were significantly elevated after the percutaneous treatment with DES (TWRMANOVA, p = 0.0001, 0.022 and 0.004, respectively; fig. 1b), which was consistent with their plasma protein levels. We also observed IL-4 expression at both protein and mRNA levels, but there were no differences in the baseline and follow-up data between the DES and BMS groups (fig. 1). NF-κB Binding Activity Was Increased after DES Implantation To investigate whether pro-inflammatory factors were activated by the transcription factor NF-κB, we measured the nuclear NF-κB DNA binding activity in MNCs (fig. 2a). The NF-κB activity significantly increased to 158 ± 27% of the baseline after DES implantation (p = 0.016; fig. 2a). Compared with the unchanged BMS group (102 ± 6.5%, p = 0.779; fig. 2a), the inter-group change was also statistically significant (TWRMANOVA, p = 0.017), suggesting that the systematic inflammatory responses were induced by the activated NF-κB after DES implantation. PPAR-γ Binding Activity Was Decreased after DES Implantation Next, we measured the PPAR-γ binding activity in MNCs. PPAR-γ is an anti-inflammatory transcriptional factor [11]. As shown in figure 2a, we found that PPAR-γ activity was significantly decreased in the DES group (p = 0.007; fig. 2a) and unchanged in the BMS group Cardiology 2014;128:259–265 DOI: 10.1159/000358508
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DES Accentuated the Levels of Systematic Inflammatory Mediators Twelve weeks after percutaneous treatment with DES, the plasma concentrations of IL-6, TNF-α and MMP-9 increased to 153 ± 21, 136 ± 15 and 108 ± 3.5% of the baseline, respectively (p = 0.012, 0.009 and 0.021, respectively; fig. 1a), whereas in the BMS group, the plasma concentrations of these factors changed to 105 ± 10, 103 ± 8 and 87 ± 4.4% of the baseline, respectively (p = 0.491, 0.575 and 0.010, respectively; fig. 1a). Compared with the control group, plasma IL-6, TNF-α and MMP-9 concentrations were significantly elevated in the DES group (TWRMANOVA, p = 0.005, 0.023 and 0.001, respectively; fig. 1a), suggesting that the pro-inflammatory response was enhanced after DES implantation.
0w
12w DES
5
0w
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100
0
0w 12w BMS
0w
12w DES
p = 0.022 15
**
10 5 0
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Plasma IL-4 (pg/ml)
5
p = 0.0001
***
Plasma MMP-9 (ng/ml)
10
0
0w 12w BMS
10
0
15
*
* 200
*
*
6 4 2 0w
12w DES
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p = 0.384
4
8
0
2
0
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p = 0.004 10
4
3 2 1 0
0w
12w DES
0w 12w BMS
Fig. 1. Changes in pro-inflammatory mediators after standard drug medication for 12 weeks in the DES (n = 18) and BMS (n = 18) groups. a Plasma concentrations of IL-6, TNF-α, MMP-9 and IL-4 at baseline (0w) and 12 weeks (12w) were presented as raw
data. b The mRNA expression of peripheral blood MNCs were presented and the comparative threshold cycle methods were used to calculate the relative gene expression (arbitrary units, au). Compared with baseline: * p < 0.05; ** p < 0.01; *** p < 0.001.
(TWRMANOVA, p = 0.001; fig. 2a), suggesting that the impaired PPAR-γ signaling also contributed to DES-related pro-inflammation.
were observed in the DES group, but not in the BMS group (TWRMANOVA, p = 0.036 and 0.011, respectively; fig. 2b). These results suggested that the activated NFκB signal cascade plays an important role in DES-related pro-inflammatory responses. Interestingly, we also found that PPAR-γ were dramatically decreased in the DES group (p = 0.008; fig. 2c) and were unchanged in the BMS group (TWRMANOVA, p = 0.002; fig. 2c), suggesting that the ratio of NF-κB to PPAR-γ level may be the key regulator of DES-related pro-inflammation.
NF-κB and PPAR-γ Signaling Imbalance May Contribute to Systematic Pro-Inflammation We further examined the expressions of the p50 subunit and p65 subunit of NF-κB in MNCs. The protein level of p65 remained unchanged compared with the baselines of the two groups (fig. 2b). The p50 protein increased significantly in the DES group to 140 ± 17% of the baseline (p = 0.042), but it decreased to 63 ± 14% (p = 0.033; fig. 2b) of the baseline in the BMS group (TWRMANOVA, p < 0.001; fig. 2b). These data indicated that standard drug therapy restrained the systematic inflammation by the NF-κB subunit p50 pathway in the BMS group, but not in the DES group. Next, we examined the IκB-α and its phosphorylation in MNCs. As shown in figure 2b the decreased IκB-α (52 ± 21% of baseline, p = 0.022; fig. 2b) and increased phosphorylated IκB-α (203 ± 49% of baseline, p = 0.015) 262
Cardiology 2014;128:259–265 DOI: 10.1159/000358508
Improved Metabolic Parameters Failed to Correlate with the Changes in Pro-Inflammatory Factors After analyzing the correlation among the changes of metabolic parameters and inflammatory factors such as total cholesterol, triglycerides, HDL and LDL, we found that the changes to metabolic parameters significantly correlated with one another (table 2). There were also strong correlations among the changes in IL-6, TNF-α, MMP-9 and NF-κB activities (table 2), which showed sigLiu/Zhuo/Liu/Wan/Wang/Zhang/Gao/ Yuan/Wu
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IL-6 mRNA (au)
15
12w DES
**
p = 0.569
IL-4 mRNA (au)
a
0w
20
MMP-9 mRNA (au)
10
0
b
Plasma 71)į (pg/ml)
20
TNF-į mRNA (au)
Plasma IL-6 (pg/ml)
*
p = 0.001
p = 0.023
p = 0.005
30
p = 0.016
p = 0.001
a
PPAR-Dž activity (score)
NF-NJB activity (score)
200 150 100 50 0
0w
12w
0w
DES BMS
200 150 100
*
50 0
12w
0w
DES Patient Timing
1 0w
2 0w
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BMS
1 12w
2 12w
3 0w
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p50
p65 GAPDH
Protein level (au)
INJB-DŽ
150
p65 p50 INJB-DŽ
*
0w
12w
###
*
100 50 0
b
0w
12w
p-INJB-į INJB-į DŽ-Actin
Fig. 2. Imbalanced NF-κB and PPAR-γ sig-
250
c
200
p-INJB-į INJB-į PPAR-Dž
*
150
#
100
*
50 0
0w
#
##
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Cardiology 2014;128:259–265 DOI: 10.1159/000358508
0w
12w
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DES Accentuates Proinflammation
PPAR-Dž
Protein level (au)
naling of MNCs in patients after coronary DES implantation. a The NF-κB and PPAR-γ binding activity from nuclear extracts at baseline (0w) and 12 weeks (12w) is shown. b Western blot analysis for cytoplasmic NF-κB (p65 and p50), phosphorIκB-α, IκB-α, IκB-β and PPAR-γ in MNCs, and densitometry are shown as arbitrary units (au). c Densitometry of EMSA and mRNA expressions of p50, IκB-α and PPAR-γ as arbitrary units (au). n = 18 for each group. Compared with baseline: * p < 0.05; ** p < 0.01. Compared between the DES and the BMS group: # p < 0.05; ## p < 0.01; ### p < 0.001.
Table 2. Associations between changes in metabolic and inflammatory parameters (n = 18)
ΔTC ΔTG ΔLDL ΔHDL ΔCRP ΔGlu ΔIL-6 ΔTNF-α ΔMMP9 ΔNF-κB ΔPPAR-γ
ΔTC
ΔTG
ΔLDL
ΔHDL
ΔCRP
ΔGlu
ΔIL-6
ΔTNF-α
ΔMMP9
ΔNF-κB
– 0.509b 0.795d –0.401 0.586c 0.185 –0.204 –0.109 –0.094 –0.113 0.259
– 0.516b –0.178 0.480b 0.056 –0.112 –0.126 –0.107 –0.079 0.126
– –0.458a 0.512b 0.186 –0.125 –0.189 –0.136 –0.152 0.271
– –0.459a –0.115 0.102 0.077 0.151 0.077 –0.191
– –0.101 –0.086 –0.161 –0.121 –0.203 0.379
– 0.062 0.079 –0.132 –0.092 0.122
– 0.626c 0.526b 0.701d –0.523b
– 0.591c 0.604c –0.611c
– 0.491b –0.531b
– –0.688c
The change from baseline after 12 weeks is designated by ‘Δ’. IL-6, TNF-α and MMP-9 are presented as plasma levels; NF-κB and PPAR-γ are presented as DNA binding activity. Correlation analysis was performed using Spearman’s rank order correlation. a 0.05 < p < 0.10; b p < 0.05; c p < 0.01; d p < 0.001. TC = Total cholesterol; TG = triglycerides; Glu = fasting glucose.
Discussion
In this study, we provided evidence to support the hypothesis that circulating inflammatory responses were unimproved or partly accentuated after DES implantation, but not BMS implantation, because of the increase in NF-κB activity via the p50 induction and IκB-α phosphorylation pathway, suppressed PPAR-γ expression and elevated plasma inflammatory factors. According to the guidelines [12], these ACS patients should also be treated with optimal drug combinations, including aspirin, clopidogrel, β-receptor-blockers, statins and angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers, which may exert many beneficial effects such as antiplatelet effect, metabolic improvement, endothelium protection and the prevention of vascular remodeling [12, 13]. However, it remains poorly understood whether the combination of these drugs inhibit systematic inflammation in patients after DES implantation. Our results also suggest that the standard medications can partly treat pro-inflammation after BMS implantation, which is evidenced by the decreased MMP-9 level and inhibited p50 expression, but cannot reverse the systematic inflamma264
Cardiology 2014;128:259–265 DOI: 10.1159/000358508
tion after DES implantation. These findings indicate that further optimizations of the present drug therapy are required in the era of DES. CRP, which is widely accepted as an important inflammatory marker in CAD [12], is dramatically improved but uncorrelated with other inflammatory factors and NF-κB. The important role of statins in regulating hepatic lipid and CRP secretion has been reported previously, which explains the parallel improvement of lipid profile and CRP [12, 14]. However, whether the level of IL-6 and other inflammatory factors can be used to predict the clinical cardiovascular events of these patients should be further explored because the CRP level cannot represent pro-inflammatory states in DES-implanted patients. NF-κB composed of p65/p50 dimmers is inactive in the cytoplasm when in association with IkB proteins [15]. Inducing stimuli or inflammation status triggers the phosphorylation, ubiquitination and degradation of IκB proteins. Then the released NF-κB dimers translocate to the nucleus, bind specific DNA sequences and promote the transcription of target genes such as TNF-α, IL-6 and MMP-9 [15–17]. Our data also suggest that the phosphorylation of IκB-α facilitates its degradation and, thus, NF-κB cannot be restricted to the cytoplasm and shuttle into the nucleus to induce the transcription of pro-inflammatory genes. Moreover, our results demonstrate that the increased level of p50 further activates the NF-κB pathway in the MNCs. It has been demonstrated that the nuclear receptor PPAR-γ can inhibit the function of NF-κB, and the imbalLiu/Zhuo/Liu/Wan/Wang/Zhang/Gao/ Yuan/Wu
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nificant inverse correlations with the change in PPAR-γ activity. However, there was no significant correlation between pro-inflammatory factors and metabolic parameters. Moreover, the change of hs-CRP significantly correlated with metabolic parameters, but not pro-inflammatory factors (table 2).
ance between PPAR-γ and NF-κB disturbs the homeostasis and may trigger inflammatory diseases, including vascular inflammation [18, 19]. In the present study, we also found a dramatic PPAR-γ loss in the MNCs in parallel with the increasing levels of inflammatory factors and the enhanced NF-κB activity. These indicated that the effective strategy for increasing PPAR-γ could be further explored to restrain NF-κB-related pro-inflammatory responses in these patients. Several previous studies have suggested that DES may trigger local inflammatory responses. Histopathological studies have also revealed that the polymer induced inflammatory cell accumulation, such as T lymphocytes and macrophages around the DES strut 9 months after implantation [3, 4], which is longer than the observation time in the present study (3 months). It is still unknown whether systematic pro-inflammation contributes to the local inflammatory responses. Thus, long-time and multitime-point observations for systematic pro-inflammation warrant further investigation.
We conclude that the systematic inflammatory responses are accentuated for at least 3 months after percutaneous treatment with DES, as evidenced by the enhanced NF-κB activity, suppressed PPAR-γ level, and the elevated plasma level of inflammatory factors. These data can provide fundamental information for optimizing our therapeutic strategy in the era of DES.
Acknowledgements This study was supported in part by the National Natural Science Foundation of China (81100209 to Y.W. and 30871043 to Z.Y.); the National Science Fund for Distinguished Young Scholars (81025002 to Z.Y.), and the National Basic Research Program of China (‘973 Project’ No. 2012CB517804 to Z.Y.).
Conflict of Interest All authors have no disclosures.
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DES Accentuates Proinflammation
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