Prognostic value and the changes of plasma levels of secretory type II phospholipase A2 in patients with coronary artery disease undergoing percutaneous coronary intervention

Ping-Yen Liua,b, Yi-Heng Lia, Wei-Chuan Tsaia, Ting-Hsing Chaoa, Liang-Miin Tsaia, Hua-Lin Wuc and Jyh-Hong Chena,*

a Division of Cardiology, Department of Internal Medicine, College of Medicine, National Cheng-Kung University Medical Center, Tainan, Taiwan
b Institute of Clinical Medicine, College of Medicine, National Cheng-Kung University Medical Center, Tainan, Taiwan
c Department of Biochemistry, College of Medicine, National Cheng-Kung University Medical Center, Tainan, Taiwan

* Correspondence to: Jyh-Hong Chen, MD, PhD, FACC, Division of Cardiology, Department of Internal Medicine, National Cheng-Kung University Medical Center, 138 Sheng-Li Road, Tainan 704, Taiwan. Tel: +886-6-235-3535 ext. 2389; fax: +886-6-275-3834
E-mail address: jyhhong{at}mail.ncku.edu.tw

Received 22 May 2003; revised 4 July 2003; accepted 17 July 2003

Abstract

Aim To evaluate the serial changes of plasma secretory type II phospholipase A2(sPLA2), C-reactive protein (CRP) and cardiac injury markers in coronary artery disease (CAD) patients undergoing percutaneous coronary intervention (PCI) and their prognostic impacts.

Methods and results Plasma levels of sPLA2, CRP, creatine kinase (CK), CK-MB and troponin-T were measured in 247 consecutive CAD patients receiving PCI procedure and 100 control subjects without CAD. In CAD group, serial blood samples were taken before coronary angiography, after coronary angiography, immediately after PCI, 24-h and 48-h after PCI. The sPLA2and CRP levels did not change after coronary angiography. The level of sPLA2significantly increased immediately after PCI. Creatine kinase and cardiac injury markers did not rise immediately after PCI, but elevated significantly at 24h after intervention. After a 2-year follow up, increased sPLA2(>450ng/dl) after PCI, smoking and diabetes mellitus were the independent risk factors for subsequent coronary events (odds ratios 2.1, 2.3 and 3.1, respectively) in patients with CAD.

Conclusion The present study showed that PCI might cause immediate elevation of circulating levels of sPLA2following the mechanical disruption of coronary plaque, and the elevated level of sPLA2had significant prognostic impact.

Key Words: Acute coronary syndrome • Inflammation • Percutaneous coronaryIntervention • Prognosis

1. Introduction

The discovery of inflammatory cells in the caps of atherosclerotic plaques led to postulation that inflammation plays a key role in the cascade of events leading to plaque rupture.1Recent studies have demonstrated that plasma inflammatory markers, including C-reactiveprotein (CRP), are elevated in patients with acute coronary syndrome and coronary artery disease (CAD).2–5A2phospholipases are a family of enzymes that can hydrolyze phospholipids at the sn-2 position to generate lysophospholipids, fatty acids, and precursors of various pro-inflammatory lipid mediators including leukotrienes, eicosanoids, prostaglandins, and platelet-activating factors.6–8These enzymes, found in the media of normal and diseased arteries, might be involved in modifying low-density lipoprotein (LDL).9,10Secretory nonpancreatic type II phospholipase A2(sPLA2) significantly contribute to the pathogenesis of various inflammatory diseases.7,8The plasma levels of sPLA2were also higher in patients with CAD, and play a role as a prognostic indicator in these patients.11–13

Plasma inflammatory markers, such as CRP, were not only elevated after percutaneous coronary intervention (PCI) but also were predictors of long-term prognosis after procedure.5sPLA2was found to be highly expressed in human atherosclerotic arterial walls,14–16where the inflammatory process is known to have a pathogenic role.1,17The lipid products generated through sPLA2and their related products, including modified LDLs, participate in the development of atherosclerosis and play a significant role in the pathogenesis of CAD.1,12,17,18Recently, sPLA2was found to cooperate with tumour necrosis factor (TNF)-{alpha} receptor superfamily signalling, including CD40/CD40 ligand pair and Fas ligand (CD95), for the inflammatory response and the development in atherogenesis.19,20However, there had been no clinical study to investigate the changes of plasma level of sPLA2after PCI and the association of plasma sPLA2with coronary events after PCI. Thus, this study was designed to evaluate the changes of plasma levels of sPLA2, CRP and troponin-T after coronary intervention and to examine whether the sPLA2levels after interventions had prognostic value for the subsequent events.

2. Methods

2.1. Study patients
This study enrolled 292 consecutive patients who had angiographic documentation of CAD and were indicated to undergo revascularization by PCI. They were further divided into two groups according to if they received the procedure of angioplasty alone or angioplasty combined with stent implantation. We excluded 45 patients because they met one or more of the following exclusion criteria: acute myocardial infarction, major surgery, trauma, documented malignancy, hypoxia, rheumatoid arthritis, osteoarthritis, renal insufficiency, and active infection within the previous 4 weeks before catheterization study.

2.2. Control patients
The control group enrolled 100 consecutive patients who underwent diagnostic coronary angiography for atypical chest pain during the same study period as the CAD patients. They had angiographically normal coronary arteries (<10% stenosis), normal left ventriculography and no clinical evidence of coronary artery spasm or syndrome X. None of these patients had any of the same exclusion criteria as described previously for patients with CAD. All study patients gave informed consent before the study. This study was in agreement with the guidelines approved by the ethics committee at our institution.

2.3. Blood sampling
In the CAD group, blood samples were obtained from peripheral veins before and after diagnostic coronary angiography. Additionally, blood samples were also taken immediately after PCI, 24h and 48h after PCI. In the control group, the blood samples were taken before, immediately after, 24h and 48h after diagnostic coronary angiography. The blood samples were drawn into 5-ml EDTA glass tubes. The plasma was then separated by centrifugation at 3000rpm at 4°C for 15min and stored at –70°C until it was assayed for plasma sPLA2, CRP and cardiac injury markers.

2.4. Biochemical measurements
Levels of plasma sPLA2were measured by enzyme immunosorbent assay (EIA) using a monoclonal antibody developed against membrane-associated PLA2, which was purified from human spleen and was identical with type II PLA2 purified from rheumatoid arthritic synovial fluid (sPLA2 EIA Kit, Cayman Chemical Company, Ann Arbor, USA). This monoclonal antibody had no detectable cross-reactivity with human pancreatic PLA2 (type IB). The EIA gave a linear response in a range from 156 to 10 000ng/dl of sPLA2. The inter-assay and intra-assay coefficients of variation were <10%. The plasma level of the immunoreactive sPLA2 had a significant correlation with the calcium-dependent PLA2activity in the citrated plasma, a result that was compatible with those in previous reports.12Serum levels of CRP were measured using an N Latex CRP immuno-detection kit (Dade Behring, Tokyo, Japan).21

2.4. PCI in CAD group
Coronary angioplasty was performed using the standard Judkins technique and a movable guide wire system through the femoral or radial artery. All patients received pre-medication with 10 000U of intravenous heparin before introduction of the guide wire. The dilatation procedure was performed with multiple balloon inflation using steerable non-perfusion balloon dilation catheters (USCI, Inc. USA) ranging in diameter from 2.0 to 3.5mm when inflated. Optimal balloon size was chosen on the basis of estimated reference diameter of normal segments adjacent to the lesion. The duration of the balloon inflation was maintained for 60 to 90s at a pressure ranging from 6 to 14atm. A nonionic iodinated contrast agent was used during the procedure in all patients. Bolus heparin intravenously was administered to keep activated clotting time around 250 to 350ms during the procedure. The interventional cardiologist decided independently the need for stenting after angioplasty. Patients receiving angioplasty were subdivided into angioplasty only group and angioplasty with stent group for further analysis.

2.5. Coronary angiography in control group
Diagnostic coronary angiography was performed using the standard Judkins technique through the femoral or radial artery. No heparin was used during the whole procedure of diagnostic angiography.

2.6. Follow-up study
The CAD patients were regularly followed up in our cardiology ward or clinics for a maximum of 24 months or until occurrence of one of the following coronary events: repeat angioplasty or bypass surgery due to recurrent angina pectoris, nonfatal myocardial infarction, or cardiac death. Recurrent angina pectoris was defined as recurrent chest pain with ischemic electrocardiographic changes lasting >10min despite full medications. Diagnosis of myocardial infarction was based on chest pain, appearance of a new Q wave on the electrocardiogram, and an increase in CK-MB isoenzyme to twice the upper limit of normal. Cause of death was determined from hospital records.

2.7. Statistical analysis
Values were expressed as mean±SD if they were normally distributed or median with range if not normal distribution. For normal distribution variables, the frequencies between CAD patients and the controls were compared by chi-squared analysis. One-way analysis of variance followed by Fisher's protected least-significant difference test was used for comparison of mean values of continuous variables (expressed as mean±SD) among groups. For nonparametric analysis, we used Mann–Whitney U-test to evaluate the difference of levels between CAD and control groups, and between subgroups (angioplasty only or with stent). Receiver operator characteristic (ROC) analysis was performed to determine the best cutoff values to detect subsequent cardiac endpoints after PCI, defined as reported above. Friedman one-way analysis of variance (Friedman ANOVA) was used to assess differences in measured parameters before and at various time intervals after PCI. Because the levels of sPLA2were not normally distributed, we used Spearman's rank correlation test to access the relation between the levels of sPLA2and CRP.

The Kaplan–Meier method (log-rank test) was applied in survival analysis according to the levels of sPLA2. The predictive value for coronary events during the follow-up period was assessed by Cox's proportional hazard analysis with the following factors as categorical variates: baseline sPLA2levels (>290ng/dl), post-PCI sPLA2levels (>450ng/dl), high CRP level (>0.5mg/dl), multiple (>double-vessel CAD) coronary arteries with stenosis, previous myocardial infarction, lower left ventricular ejection fraction on baseline left ventriculography or echocardiography (<50%), age (≥70 years), sex (male), smoking history (define as smoking ≥10cigarettes/day for ≥10 years), hypertension (>140/90mmHg or requiring antihypertensive medication), diabetes mellitus, hypercholesterolaemia (>200mg/dl or the use of lipid-lowing medications), high low-density lipoprotein (LDL) cholesterol (>130mg/dl), low high-density lipoprotein (HDL) cholesterol (<40mg/dl), ST-segment depression and/or T wave inversion electrocardiogram at baseline or during PCI.

The multivariate Cox analysis using forward stepwise method included only the covariates that predicted coronary events and their p values were less than 0.2 in the forward stepwise selection univariate analysis. Statistical significance was defined as P<0.05. Analyses were assessed using SPSS 8.0 and Excel 2000 for Microsoft Word.

3. Results

Two hundred and forty-seven patients were included in the study group with CAD (single-vessel disease, 120 patients; double-vessel disease, 98 patients; triple-vessel disease, 74 patients). We also enrolled 100 patients with normal coronary arteries as the control group. All patients’ characteristics are listed in Table 1. The prevalence of diabetes mellitus, hypertension and the levels of cholesterol were higher in CAD group. The baseline CRP and sPLA2levels were all higher in the CAD group. We subdivided the patients with CAD who underwent PCI into angioplasty only (n=137) and angioplasty with stent implantation (n=110) respectively. The procedural and angiographic characteristics of the study group of patients with PCI are listed in Table 2. Most of our patients were one-vessel or two-vessel coronaryartery disease. The number of diseased CAD and procedure time were similar in both groups. The final minimal lumen diameter and lumen were larger in angioplasty with stent group than the angioplasty only group (P<0.05). The target intervention lesion, the balloon size, maximal pressure and total inflation time of balloon were all similar in both groups. Only dissection events happened more in the angioplasty alone group (11.6% vs 4.5%, P=0.01)


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Table 1 Baseline characteristics of control and study patientsa

 

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Table 2 Angiographic characteristics and procedure related data in patients with PCIa

 
3.1. PLA2as a coronary risk factor
Median sPLA2in CAD group before PCI were higher than in the control group (245, range: 220 to 368ng/dl vs 180, range: 130 to 242ng/dl, P<0.0001). The baseline levels of sPLA2had a significant and positive correlation with baseline CRP levels (Fig. 1).



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Fig. 1 Correlation between the baseline levels of sPLA2and the CRP levels. sPLA2, secretory non-pancreatic type II phospholipase A2; CRP, C-reactive protein.

 
In multiple logistic regression analysis with forward stepwise selection, the baseline higher levels of sPLA2(>290ng/dl), smoking, diabetes mellitus and hypertension were the variables that differed significantly and independently between the control group and the CAD group, as shown in Table 3.


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Table 3 Multiple logistic regression analysis: final significant variables differences between patients with CAD and control subjects

 
3.2. sPLA2changes before and after PCI
The sequential changes of levels of sPLA2, CRP, CK, CK-MB and troponin-T in control and CAD group are shown in Table 4. Before diagnostic angiography, the levels of sPLA2in patients with CAD were higher than in the control subjects without CAD. In both control group and CAD group, the sPLA2levels did not change after the procedures of diagnostic angiography. The level of sPLA2significantly increased immediately after coronary intervention. However, the levels of CRP, CK, MB-CK and troponin-T did not rise immediately after PCI but elevated significantly later at 24h after intervention. In control group, there were no significant changes of the levels of sPLA2, CRP and cardiac injury markers at 24h and 48h after the diagnostic angiography.


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Table 4 The serial changes of levels of biochemical markers in patients receiving coronary angiography or PCI

 
To compare the different effects of intervention methods, we further divided the CAD patients into two groups based on whether they received angioplasty only or both angioplasty and stent implantation procedure. However, there were no significant differences in the elevation of these biochemical markers in each subgroups with angioplasty only or both angioplasty and stent implantation (Table 5).


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Table 5 The serial changes of levels of biochemical markers in CAD patients: compared in angioplasty only and both angioplasty and stent implantation subgroups

 
3.3. Levels of sPLA2after PCI as a predictor of future coronary events
All of the patients received the standard medical therapy, including calcium channel blockers (75% of patients), ß-blockers (47%), nitrates (76%), angiotensin-converting enzyme inhibitors (55%), aspirin and/or ticlopidine (94%), and lipid-lowering agents (32%), during the clinical follow-up. Only 11 patients after PCI were lost to follow-up. The remaining 236 patients after PCI were followed for a mean duration of 19.3 months (range 1 to 24 months).

The best cutoff values of baseline sPLA2level, sPLA2level after PCI and CRP level for the prediction of subsequent adverse coronary events were 290ng/dl, 450ng/dl and 7.5mg/dl respectively by the ROC analysis. The patients with higher levels of sPLA2after PCI (>450ng/dl, n=146) had 67 coronary events (15 repeated angioplasty, 10 bypass surgery, 21 myocardial infarction, seven coronary death, 14 unstable angina) during the follow-up period, whereas the patients with lower levels (≤449ng/dl, n=90) had only 20 events (eight angioplasty, four bypass surgery, three coronary death, five unstable angina) (45.9% vs 22.2%, P<0.001). The prevalence rates of hypertension, diabetes mellitus and chronic smoking behaviour were all significantly higher among the patients with sPLA2levels >450ng/dl after PCI (hypertension: 48% vs 36%, P=0.01; diabetes mellitus: 38% vs 28%, P< 0.01 and smoking: 60% vs 46%, P<0.01, respectively). There was no significant difference in the rates of each of the medications used between the patients with and without coronary events during the follow-up period. The percentage of stent implantation after angioplasty among patients with higher sPLA2levels after PCI was similar to those with lower sPLA2after PCI (83 vs 80%, P=0.45). However, the coronary events in the angioplasty only group were higher than the angioplasty with stent implantation group during this 2-year follow-up (44.8% vs 30.7%, P=0.02). With this cutoff value, the sensitivity was 45.9%, specificity was 77.8%. Using the incidence (37.8%) of subsequent cardiac events observed in our cohort, the positive and negative predictive values were 77.0% and 53.0%, respectively.

In univariate forward stepwise Cox proportional hazard model analysis, higher levels of sPLA2after PCI (>450ng/dl) (Odds, 2.5, 95% CI, 1.2 to 3.5, P=0.001), higher levels of baseline sPLA2(>290ng/dl) (Odds, 2.0, 95% CI, 1.1 to 3.9, P=0.04), higher levels of CRP after PCI (>7.5mg/dl), severe CAD (more than two-vessel CAD), acute coronary dissection during PCI, smaller size of stent implanted (≤2.5mm), diabetes mellitus, smoking were all significant predictors of clinical coronary events in patients after PCI (Table 6).


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Table 6 Univariate and multiple Cox proportional hazard model analysis for future coronary events after percutaneous coronary interventiona

 
Multivariate forward stepwise Cox proportional hazard analysis showed that higher levels of sPLA2after PCI (>450ng/dl, Odds 2.1, 95% CI 1.4 to 7.0, P=0.025), history of diabetes mellitus (Odds 2.3, 95% CI 1.6–9.5, P=0.001) and smoking (Odds 3.1, 95% CI 1.2–6.2, P<0.001) were independent predictors of subsequent coronary events.

Kaplan–Meier analysis had demonstrated a significantly higher probability of developing the clinical coronary events in the patients with higher levels of sPLA2after PCI (>450ng/dl) than those with the lower levels after PCI (≤449ng/dl), as shown in Fig. 2.



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Fig. 2 Kaplan–Meier survival curve of the proportion of patients remaining free of subsequent coronary events after PCI according to the levels of sPLA2after PCI (cutoff value 450ng/dl). sPLA2, secretory non-pancreatic type II phospholipase A2; PCI, percutaneous coronary intervention.

 
4. Discussion

Data from the present study had demonstrated that serum sPLA2levels were higher among CAD patients than normal subjects. Moreover, the sPLA2levels increased immediately after mechanical disruption of the coronary artery plaque by PCI. Inflammatory markers such as CRP levels had a similar but delayed response to the intervention process. Furthermore, the increased level of sPLA2after PCI was a significant and independent predictor of subsequent coronary events. Those patients with stronger inflammatory response manifested by higher levels of both sPLA2and CRP after PCI were more likely to be associated with subsequent coronary events.

Previous study13had shown that the baseline levels of sPLA2could be a predictor for the future coronary events in patients with CAD without intervention. However, after multivariate Cox hazard analysis, we found that the sPLA2levels after PCI, rather than CRP levels or baseline sPLA2levels, seemed to be a more significant marker of inflammation and coronary events. This might indicate that the elevated levels of sPLA2after PCI would be a more accurate indicator of the artificial plaque rupture and intima injury caused by PCI and thus played as a predictor for further coronary events.

The serial changes of these inflammatory markers, like sPLA2and CRP, and other cardiac enzymes, like CK, CK-MB and troponin-T, are of great clinical significance in the pathogenesis of atherosclerosis and restenosis after PCI. CRP is hepatically derived and is well studied as an acute phase reactant to inflammation in coronary atherosclerosis.7,8,21After PCI, there was a more rapid elevation of sPLA2in response to the vessel plaque injury. CRP and the markers of myocardial injury, including CK, CK-MB and troponin-T, had a more delayed response to the PCI and increased later than the sPLA2.

sPLA2had been shown to be induced by cytokines such as interleukin-1 and tumour necrosis factor-{alpha}.7,8,22,23These factors are found abundantly existing in the atherosclerotic arterial walls and can induce the release of sPLA2from the smooth muscle cells (SMCs), and thus have important roles in the inflammatory and immunological processes in atherosclerotic development.1,24,25PCI can cause the rupture of arterial plaque artificially and thereby release the atherothrombogenic lipid content within the plaque, including plasminogen activators inhibitor-1, adhesion molecules, cytokines, growth factors, and oxygen free radicals into the circulation.26,27SMC proliferation and migration are the most important factors for the pathogenesis of restenosis after PCI. SMC can be induced and then migrate into the subendothelial space by the induction of cytokines, growth factors and several matrix metalloproteinases.5In general, the CRP level is a very useful indicator for the degree of inflammation in human tissue. Our data also showed that the CRP had a positive correlation with the levels of sPLA2. This result indicates that higher levels of sPLA2(>450ng/dl) after PCI can cause a more extensive inflammation process in the arterial wall, and may be related with the subsequent cardiac events.

Although the levels of sPLA2after PCI were similar in angioplasty-only group and in angioplasty with stent implantation group, the coronary event rates were higher in the patients receiving angioplasty alone than those combined with stent implantation. This could be partially explained by the different pathogeneses of restenosis in these two groups. Proliferation and migration of SMC are responsible for the majority of restenosis in coronary artery after balloon angioplasty. The restenosis after stent implantation in the coronary artery is almost found to be neointima formation at the inner side of the stent. Under the structural protection of a stent, the detrimental effects caused by cytokines, growth factors or sPLA2may probably become insignificant.

The interaction between heparin and the release of phospholipase still remains controversial. We know that heparin can degrade the proteoglycans binding at the surface of endothelial cells and thus release amounts of lipoprotein lipase into the blood. 28Some studies reported that the phospholipase activity increased after large-dose of heparin treatment.29On the contrary, Dua and Cho30found that heparin had specific competitive inhibition to human secretory class II PLA2than other types of PLA2. The different surface cationic characteristics are considered as a key factor. As a result, the real dose-response of heparin and sPLA2is still not clear in humans. In our study, though the patients who had undertaken PCI did receive heparin treatment, the effect of heparin on the levels of sPLA2was not studied. However, the half-life of heparin was short and it is possible that the effect of the heparin might be very minimal during such a long period observation in the present investigation.

Recent studies had shown that lipid-lowering agent, like 3-hydro-3-methylglutaryl-coenzyme A reductase inhibitor or statin, could reduce the incidence of subsequent coronary events either after acute coronary syndrome or after PCI.31,32The vascular protective effects of statin have been well recognized by the effects of anti-inflammation and prevention of smooth muscle proliferation or migration in the vascular wall.33However, in our study cohort, with average value of LDL-C as high as 133±35mg/dl, lower rate with only 32% CAD patients were treated with lipid-lowering agents. It seems that the predictive value of this pro-inflammatory marker sPLA2needs further investigation under more aggressive lipid-lowering treatment. In addition, smoking is an important risk factor in acute coronary event.34It causes advanced pro-coagulation and inflammation status,including higher plasminogen activator-1 levels, lower tissue plasminogen activator levels and even endothelial dysfunction.35,36Nearly 55% of our CAD patients had smoking behaviour. It also needs more cohort studies to evaluate the benefit of quitting smoking among patients at more advanced inflammatory status, especially those with higher baseline CRP or sPLA2levels.

This study was limited by the small sample size. Based on this case-controlled and prospective study, some selection bias would happen because of the cross-sectional nature of our study method. During the PCI procedure, heparin seemed to have partial effect to the release of subsequent sPLA2levels. This effect might be able to make the elevation of sPLA2levels after PCI difficult in interpretation. Additionally, a longer follow-up should be carried on to observe the delayed cardiovascular events after PCI.

5. Conclusion

In conclusion, sPLA2played an important role and had rapid response to the inflammatory activity in atherosclerotic arteries and its plaque rupture. The higher levels of sPLA2after PCI might predict risk of future coronary events.

Acknowledgments

We thank Associate Prof. How-Ran Guo for his suggestions and expert opinions in the statistic analyses. We also thank the staffs in cardiology for their excellent works at our catheterization laboratory in NCKU medical center. This study was supported in part by the MOE Program for Promoting Academic Excellent of Universities under grant number 91-B-FA09-2-4, and by grants NCKUH-90-03 and NCKUH-91-43 from National Cheng Kung University Hospital, Tainan, Taiwan.

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