Atherosclerosis 220 (2012) 581–586
Contents lists available at SciVerse ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Pentraxin 3: A novel and independent prognostic marker in ischemic stroke Wi-Sun Ryu a,b , Chi Kyung Kim a,b , Beom Joon Kim a,b , Chulho Kim a,b,c , Seung-Hoon Lee a,b,∗,1 , Byung-Woo Yoon a,b,∗∗,1 a b c
Department of Neurology, Seoul National University Hospital, Seoul, Republic of Korea Clinical Research Center for Stroke, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea Department of Neurology, Hallym University Sacred Heart Hospital, Chuncheon, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 May 2011 Received in revised form 13 November 2011 Accepted 22 November 2011 Available online 1 December 2011 Keywords: Pentraxin 3 Ischemic stroke Mortality
a b s t r a c t Objective: Pentraxin 3 (PTX3) is one of the pattern-recognition receptors related to the initial step of the immune response with C-reactive protein, but the physiologic and pathologic functions are not fully understood. The purpose of the current study was to determine the impact of PTX3 levels on mortality after ischemic stroke. Methods: We consecutively enrolled 376 patients who had ischemic stroke between September 2004 and September 2006. The patients were divided into tertiles according to PTX3 levels. Cox regression analysis was used to determine hazard ratios (HRs) and 95% confidence intervals (CIs) of the PTX3 tertiles for allcause mortality with adjustment for traditional risk factors and laboratory variables, including C-reactive protein. Results: During the follow-up, 19.4% of the patients were deceased. The median PTX3 levels were higher in the deceased patients (18.0 vs. 6.4 ng/mL, p < 0.001). Based on Cox regression analysis, compared with the first tertile of PTX3, the adjusted HRs of the second and third tertiles for all-cause mortality were 1.24 (95% CI, 0.52–2.98) and 2.64 (95% CI, 1.19–5.85), respectively. When the log-transformed levels of PTX3 were incorporated as continuous variables, higher levels of PTX3 were also associated with an increased mortality (increase per log unit; HR, 1.60; 95% CI, 1.19–2.16). Conclusions: We showed that higher levels of PTX3 are independently associated with increased mortality after ischemic stroke. Our results suggest that PTX3 may be used as a new powerful prognostic biomarker in patients with ischemic stroke. © 2011 Elsevier Ireland Ltd. All rights reserved.
Given the increasing incidence and high cost of stroke, an early risk assessment with an estimate of the severity of disease and prognosis is essential for optimized care and allocation of limited health resources [1]. In this context, a number of blood markers have been investigated as candidates for outcome predictors of stroke [2–4]. Among these markers, C-reactive protein (CRP) has been reported to be an independent prognostic factor in patients with acute ischemic stroke [2,5,6]. The classical short pentraxin, CRP, is a member of the pentraxin superfamily. Pentraxin 3 (PTX3), which is one of the long pentraxins, conserves the C-terminal pentraxin domain with the
∗ Corresponding author at: Department of Neurology, Seoul National University Hospital, 28 Yongon-dong, Jongno-gu, Seoul 110-744, Republic of Korea. Tel.: +82 2 2072 1014; fax: +82 2 3672 7553. ∗∗ Corresponding author at: Department of Neurology, Seoul National University Hospital, 28 Yongon-dong, Jongno-gu, Seoul 110-744, Republic of Korea. Tel.: +82 2 2072 2875; fax: +82 2 3672 7553. E-mail addresses:
[email protected] (W.-S. Ryu),
[email protected] (S.-H. Lee),
[email protected] (B.-W. Yoon). 1 These authors contributed equally. 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.11.036
classical short pentraxins, but differs for the presence of an unrelated long N-terminal domain. A variety of cell types can produce PTX3 upon exposure to primary inflammatory signals, such as interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), oxidized lowdensity lipoprotein, and microbial moieties [7,8]. Because these cells include macrophages, endothelial cells, and vascular smooth muscle cells, PTX3 release appears to be a specific response to vascular damage, indicating that PTX3 may provide more explicit information on development and progression of atherosclerosis than non-specific markers, such as CRP [9]. Several recent studies have suggested that PTX3 might be associated with increased mortality in patients with vascular diseases [10–12]. In patients with myocardial infarctions, higher PTX3 levels (>10.73 ng/mL) are associated with increased 3-month mortality [10]. In addition, in patients with chronic kidney disease, higher PTX3 levels are associated with increased all-cause mortality [13]. However, reports on the relationship between PTX3 and ischemic stroke have been sparse. In this context, we determined whether or not the PTX3 level is associated with poor outcome after ischemic stroke in the current study, especially in terms of mortality.
582
W.-S. Ryu et al. / Atherosclerosis 220 (2012) 581–586
1. Methods 1.1. Study population Between September 2004 and September 2006, there were 849 consecutive patients with acute ischemic strokes whose symptom onset was within 7 days before admission. Of these patients, 401 agreed to provide blood samples. We excluded 15 patients who had unavailable clinical or laboratory data, and further excluded 10 patients who were lost to follow-up. As a result, 376 patients were included in the current study. We ascertained ischemic stroke and determined stroke classification by brain magnetic resonance imaging or computed tomography. The Institutional Review Board in our hospital approved the study protocol. Informed consent was obtained from the patient or the patient’s next of kin according to the clinical status before enrollment. 1.2. Data collection We recorded demographic data (age and sex), traditional risk factors, and basic laboratory data for all subjects. Traditional risk factors included hypertension, diabetes, hypercholesterolemia, heart diseases, and smoking history. The patients were coded as current or non-current smokers. Fasting blood samples were drawn within 24 h of admission, examined for glucose and lipid levels, and also used in a standard battery of biochemical, hematologic tests and PTX3 assay. The time from symptom onset to sampling ranged from 10 to 171 h with median of 49 h. The mortality information was based on computerized searches of death certificate data from the “Statistics Korea” of the Korean National Statistical Office; these data were verifiably linked to the subjects in this study based on a unified 13-digit identification number (the Korean resident registration number) [14,15]. In Korea, professionally trained and certified medical chart recorders summarize, chart, and complete death certificates using information provided by physicians. All death certificates are registered by the Statistics Korea, even when individuals die outside of Korea [16]. Vascular death was defined as death caused by stroke, myocardial infarction, heart failure, pulmonary embolism, cardiac arrhythmia, or other definite vascular causes. Non-vascular death was defined as death brought on by non-vascular causes – accidents, cancer, pulmonary issues (such as pneumonia or chronic obstructive pulmonary disease), and other miscellaneous causes. We censored the mortality data on 31 March 2010. 1.3. PTX3 assay To obtain levels of PTX3, we used an enzyme-linked immunosorbent assay method, as established elsewhere [11,17]. Immediately following venipuncture, the blood samples were centrifuged for 15 min at 2000 × g and 20 ◦ C. Plasma in ethylene diamine tetraacetic acid was frozen and stored at −80 ◦ C until the analyses were performed. PTX3 was measured by a human PTX3 detection set from Alexis Biochemicals (Lausen, Switzerland). In brief, 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with 100 mL of anti-PTX3 monoclonal antibody MNB4 (ascites diluted 1:5000 in coating buffer) and incubated overnight at 4 ◦ C. After incubation, the plates were extensively washed with Dulbecco’s phosphate buffered saline containing 0.05% Tween 20 (washing buffer), and 200 mL of 5% dry milk in washing buffer to block non-specific binding sites. After a 2-h incubation at room temperature, the plates were again washed 3 times with washing buffer. Fifty microliters of purified human recombinant PTX3 standards (100 pg/mL to 40 ng/mL) diluted in RPMI 1640 medium (Seromed, Berlin, Germany) and 2% bovine serum albumin (Sigma Chemicals, St. Louis, MO, USA), or unknown plasma samples were added in
triplicate to each well and incubated for 2 h at 37 ◦ C. The plates were washed 3 times with washing buffer and 100 mL of biotin conjugated anti-PTX3 rabbit serum diluted 1:2000 in washing buffer were added. The plates were incubated for 1 h at 37 ◦ C, then washed 3 times with 200 mL of washing buffer. One hundred microliters of streptavidin-peroxidase conjugated to dextran back-bone (Amdex, Copenhagen, Denmark) diluted 1:4000 was subsequently added to each well and the plates were incubated for 1 h at room temperature. After incubation, the plates were washed 4 times before the addition of 100 mL of the chromogen substrate. Plates were read at 405 nm in an automatic ELISA reader. The analytic coefficient of variation was 10.2% [18].
1.4. Statistical analyses For statistical analyses, the patients were divided into tertiles according to the PTX3 levels. Approximately normally distributed variables were presented as the mean with standard deviation (SD), while variables with skewed distributions were presented as the median with the interquartile range (IQR). The 2 test for association was applied for analyses across the PTX3 tertiles and categorical variables at baseline. One-way analysis of variance (ANOVA) and the Kruskal–Wallis test were used to test for equality of continuous variables as appropriate. When PTX3 levels were incorporated into the statistical model as a continuous variable, log-transformed PTX3 levels were used. The associations between the PTX3 levels and traditional risk factors and laboratory variables were examined by multiple linear regression analysis. The survival rates of patients by PTX3 tertiles were estimated using the Kaplan–Meier product-limit method, and compared using the log-rank test. To examine the effects of PTX3 tertiles on mortality during the follow-up, the Cox proportional hazard regression analysis was used to calculate the crude and adjusted hazard ratios (HRs) with 95% confidence intervals (CIs). The first tertile of PTX3 quartile was used as a reference. We separately carried out the Cox proportional regression tests for PTX3 tertiles and log-transformed PTX3 (per 1 log unit increase). Analyses were adjusted for the following covariates: age (per year), sex, previous stroke, hypertension, diabetes, hypercholesterolemia, smoking, heart disease, National Institute of Health Stroke Scale (NIHSS) scores at the time of admission, log-transformed CRP (per 1 log unit), and serum glucose (per 1 mmol/L). Proportional-hazards assumptions were assessed by visually inspecting log-minus-log plots and plots of Schoenfeld residuals vs. the survival time. No evidence of violations was observed. To examine the prognostic value of PTX3, we constructed a receiver operating curve (ROC) model and calculated the area under the curve (AUC) values. In this analysis, we used the 3-year mortality as a binary outcome variable. Two-tailed p values
