Journal of the Neurological Sciences 340 (2014) 75–79

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Plasma S100A12 is associated with functional outcome after ischemic stroke: Research for Biomarkers in Ischemic Stroke Yoshinobu Wakisaka a,⁎,1, Tetsuro Ago a,1, Masahiro Kamouchi b, Jyunya Kuroda a, Ryu Matsuo a, Jun Hata a,c, Seiji Gotoh a,c, Tetsu Isomura d,2, Hideto Awano d,3, Kazuo Suzuki d,3, Kenji Fukuda a,e, Yasushi Okada f, Yutaka Kiyohara c, Hiroaki Ooboshi g, Takanari Kitazono a, on behalf of the REBIOS Investigators a

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Department of Health Care Administration and Management, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan c Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan d Molecuence Corporation, Yokohama, Japan e Department of Cerebrovascular Disease, Institute of Neuroscience, St Mary's Hospital, Kurume, Japan f Department of Cerebrovascular Medicine and Neurology, Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan g Department of Internal Medicine, Fukuoka Dental College Medical and Dental Hospital, Japan b

a r t i c l e

i n f o

Article history: Received 10 October 2013 Received in revised form 23 February 2014 Accepted 25 February 2014 Available online 4 March 2014 Keywords: Ischemic stroke Functional outcome Inflammation Biomarker S100A12

a b s t r a c t Background: Ischemic stroke is accompanied by an inflammatory response, which exacerbates brain injury and deteriorates functional outcome. S100A12 is expressed abundantly in granulocytes, and has been implicated to play an important role on inflammatory reactions in various disease states. We aimed to determine the association between plasma S100A12 levels and a functional outcome in patients with acute ischemic stroke. Methods: We prospectively included 171 patients with acute ischemic stroke within 24 h after onset in this study. Plasma samples were collected for the measurement of S100A12 levels. Poor functional outcome was defined as a modified Rankin Scale of 2–6 at day 90 after stroke onset. Results: Of 171 patients, 74 (43.3%) had a poor functional outcome at day 90 after stroke onset. Plasma S100A12 levels on admission were significantly higher in patients with a poor functional outcome (2.1 [1.2–5.1] ng/mL, median [interquartile]) than in those with a favorable outcome (1.1 [0.5–2.0] ng/mL; p b 0.001). Multivariate analysis showed that the highest quartile of plasma S100A12 levels on admission showed a significantly higher risk for a poor functional outcome (odds ratio, 4.01; 95% confidence interval, 1.09–16.10; p = 0.03) than the lowest quartile. Conclusions: High plasma S100A12 levels on admission are associated with a poor functional outcome in patients with acute ischemic stroke. © 2014 Elsevier B.V. All rights reserved.

1. Introduction There is increasing evidence that acute ischemic stroke is accompanied by an inflammatory response in the brain and in the periphery, which exacerbates brain injury and suppresses functional recovery after ischemic stroke [1–3]. Abbreviations: NIHSS, National Institute of Health Stroke Scale; RAGE, receptor for advanced glycation end products; EN-RAGE, extracellular newly identified receptor for advanced glycation end products (RAGE)-binding protein; REBIOS, Research for Biomarkers in Ischemic Stroke; FSR, Fukuoka Stroke Registry; mRS, modified Rankin Scale; OR, odds ratio; CI, confidence interval. ⁎ Corresponding author at: Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: +81 92 642 5256; fax: +81 92 642 5271. E-mail address: [email protected] (Y. Wakisaka). 1 These authors contributed equally to this work. 2 Current affiliation: The KAITEKI Institute, Inc., Tokyo, Japan. 3 Current affiliation: Mitsubishi Tanabe Pharma Corporation, Osaka, Japan.

http://dx.doi.org/10.1016/j.jns.2014.02.031 0022-510X/© 2014 Elsevier B.V. All rights reserved.

S100 proteins are a family of low-molecular-weight proteins that have Ca2+-binding activity and mediate various intracellular signaling pathways [4–6]. A notable feature of S100 proteins is that they can also function extracellularly when secreted [4–6]. Among them, S100A12, formerly known as extracellular newly identified receptor for advanced glycation end products (RAGE)-binding protein (EN-RAGE), is expressed specifically and abundantly in granulocytes [4–6]. When secreted extracellularly, S100A12 can bind to RAGE expressed in endothelial cells, macrophages, monocytes, microglia, and lymphocytes, triggering the inflammatory responses through secretion of pro-inflammatory cytokines [5–7]. Plasma S100A12 is increased in inflammatory disorders and could be a marker of inflammation [6]. Besides inflammatory disorders, plasma S100A12 levels are increased in patients with cardiovascular diseases, such as coronary artery disease and carotid atherosclerosis, and have been suggested to be associated with the pathogenesis and progression of these diseases [8,9].

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2. Methods

measured by multiplexed immunoassays (HumanMAP® v 1.6, RulesBased Medicine, Inc., Austin, TX) [10]. The lower limit of detection of plasma S100A12 was 0.22 ng/mL, and values below the detection limit were replaced by a value at half of the detection limit, as was reported previously [15]. The plasma S100A12 levels on admission were divided into quartile (Q): Q1, 0.11–0.75 ng/mL; Q2, 0.76– 1.59 ng/mL; Q3, 1.60–3.49 ng/mL; and Q4, 3.50–46.6 ng/mL. Hypertension, diabetes mellitus, and dyslipidemia were defined, and atrial fibrillation was diagnosed as previously reported [12,13]. Blood pressure and body mass index were measured on admission. Thrombolytic therapy was defined as intravenous or intra-arterial administration of thrombolytic agents in the acute phase of stroke [13].

2.1. Study subjects

2.4. Evaluation of neurological severity and functional outcome

We performed the Research for Biomarkers in Ischemic Stroke (REBIOS) [12], in which patients with acute ischemic stroke were enrolled from the Fukuoka Stroke Registry (FSR), a multicenter observational study for acute stroke in Japan [13]. On admission, the objectives, study design, risks, and benefits were explained in detail to each patient or surrogate family members, and written informed consent was obtained. Patients who consented to the study were prospectively enrolled and followed up to 3 months after the onset. Inclusion criteria were as follows: 1) ischemic stroke hospitalized within 24 h after onset, 2) patient or surrogate (in case of consciousness disturbance) consents to the study, and 3) aged between 40 and 85 years. Patients with the following complications were excluded from the study: 1) signs of infection, 2) malignancy, 3) anemia, 4) chronic inflammatory diseases, and 5) impairment of daily living before onset. From November 2007 to April 2010, we recruited 171 ischemic stroke patients who were hospitalized at Kyushu University Hospital, National Hospital Organization Kyushu Medical Center, or St. Mary's Hospital. Age- and sex-matched controls (n = 171) without a history of cardiovascular diseases were enrolled as healthy subjects from the participants in the Hisayama Study, Japan [14]. The study protocol and all subsequent amendments were approved by the leading ethic committee of the Kyushu University Hospital (Kyushu University Institutional Review Board for Clinical Research, reference number #20-30, date of approval 9/26/2008) and the local ethics committees of the participating centers, National Hospital Organization Kyushu Medical Center (Kyushu Medical Center Institutional Review Board for Clinical Research, reference number #07-38, date of approval 8/26/2007) and St Mary's Hospital (St Mary's Hospital Institutional Review Board for Clinical Research, date of approval 3/11/2008). The study is performed in accordance with the Declaration of Helsinki and its subsequent amendments, as well as the guidelines of Good Clinical Practice.

The National Institute of Health Stroke Scale (NIHSS) was used to evaluate neurological severity at each time of follow-up. The functional outcome at day 90 was defined as favorable (modified Rankin Scale [mRS] 0–1) or poor (mRS 2–6) [13].

Recently, a gene expression profiling study demonstrated that expression of S100A12 is increased in peripheral leukocyte in patients with acute ischemic stroke [10]. It was also reported that protein expression of S100A12 is induced in granulocytes immediately after hypoxic insults [11]. No studies, however, have examined the significance of S100A12 on the prognosis of acute ischemic stroke. Based on the proinflammatory properties of S100A12, we hypothesized that S100A12 relates to a functional outcome after ischemic stroke. In this study, we measured plasma S100A12 levels and investigated their clinical significance on a functional outcome after ischemic stroke.

2.2. Diagnosis of ischemic stroke and subtype Ischemic stroke was defined and classified into four subtypes (i.e., atherothrombotic infarction, cardioembolic infarction, lacunar infarction, and unclassified infarction), as previously reported [12,13]. The diagnosis of ischemic stroke was confirmed by brain imaging, including computed tomography and magnetic resonance imaging, in all patients. Non-cardioembolic infarction was defined either as lacunar, atherothrombotic, or unclassified infarction. 2.3. Clinical assessment Peripheral blood samples were collected from patients at five time points (days 0 [within 24 h], 3, 7, 14, and 90) after the onset. Blood samples were mixed in an EDTA-containing tube, and were centrifuged immediately at 1400 ×g for 10 min at 4 °C to separate plasma from blood cells. Plasma samples were frozen at −80 °C within 10 min, and were stocked for about 2 weeks until analysis. Plasma S100A12 values were

2.5. Statistical analyses Statistical analyses were performed using JMP software ver.10 (SAS Institute, Cary, NC). The plasma S100A12 levels between patients and controls were compared by the Mann–Whitney U test. Baseline characteristics of patients according to plasma S100A12 levels on admission and clinical outcomes were compared by the χ2 test or a logistic analysis for categorical variables, and by the unpaired Student's t-test, the Mann–Whitney U test, the Tukey–Kramer test, the Steel–Dwass test, or analysis of variance for continuous or scoring variables, as appropriate. Multivariate analysis for the association between plasma S100A12 levels and NIHSS, and between plasma S100A12 levels and the frequency of thrombolytic therapy was estimated by multiple logistic regression analysis and logistic regression analysis, respectively. Age- and sex-adjusted or multivariate-adjusted odds ratios (ORs) and their 95% confidence intervals (CIs) for a poor functional outcome were estimated by logistic regression analysis. Probability values b0.05 were considered statistically significant. 3. Results Clinical characteristics of 171 patients with acute ischemic stroke are summarized in Table 1. Plasma S100A12 levels on admission in patients with ischemic stroke were significantly higher (1.6 [0.8–3.5] ng/mL, median [interquartile range]) than those in age- and sex-matched healthy controls (0.9 [0.4–1.6] ng/mL, p b 0.001). We stratified the patients into quartile groups (Q1 to Q4) according to plasma S100A12 levels on admission (Table 1). The frequency of males was significantly decreased, and the frequencies of hypertension and leukocyte count were significantly increased with a higher plasma S100A12 status. Although NIHSS tended to increase with a higher plasma S100A12 status, this trend disappeared after adjustment for possible confounding factors including age, sex, stroke subtype, systolic blood pressure, hypertension, dyslipidemia, diabetes mellitus, atrial fibrillation, body mass index, leukocyte count, casual blood glucose, C-reactive protein, fibrinogen, prior anticoagulant therapy, and prior antiplatelet therapy (p for trend = 0.32). Similarly, multivariate analysis showed that there was no association between plasma S100A12 levels on admission and frequency of thrombolytic therapy after adjustment for possible confounding factors, such as age, sex, baseline NIHSS, stroke subtype, systolic blood pressure, hypertension, dyslipidemia, diabetes mellitus, atrial fibrillation, body mass index, and casual blood glucose (p for trend = 0.14). Age, stroke subtype, and frequency of edaravone therapy (a free radical scavenger approved by the Japanese health authorities as a neuroprotective agent for the treatment of acute ischemic stroke) [16] were not different among the groups (Table 1).

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Table 1 Baseline characteristics of patients.

Age, y Male Hypertension Dyslipidemia Diabetes mellitus Atrial fibrillation Cardioembolic infarction Body mass index, kg/m2 Systolic blood pressure, mm Hg NIHSS Prior antiplatelet therapy Prior anticoagulant therapy Thrombolytic therapy Edaravone therapy Casual blood glucose, mmol/L Leukocyte, ×109/L C-reactive protein, mg/L Fibrinogen, μg/mL S100A12, ng/mL

Total

Plasma S100A12 levels on admission

(n = 171)

Q1 (n = 42)

Q2 (n = 42)

Q3 (n = 44)

Q4 (n = 43)

p for trend

68.3 ± 10.1 115 (67.3) 132 (77.2) 100 (58.5) 54 (31.6) 59 (34.5) 49 (28.7) 23.6 ± 3.8 161.2 ± 29.1 4 [2–7] 56 (32.7) 18 (10.5) 27 (15.8) 162 (94.7) 7.6 ± 3.3 7.4 ± 2.2 1.0 [0.5–3.0] 3.8 ± 1.2 1.6 [0.8–3.5]

66.4 ± 10.0 32 (76.2) 25 (59.5) 22 (52.4) 8 (19.1) 13 (31.0) 11 (26.2) 23.5 ± 4.1 162.4 ± 34.3 4 [2–6] 13 (31.0) 3 (7.1) 2 (4.8) 39 (92.9) 6.8 ± 1.9 6.7 ± 2.1 0.9 [0.3–2.0] 3.7 ± 1.2 0.3 [0.11–0.50]

68.4 ± 9.7 29 (69.1) 33 (78.6) 24 (57.1) 13 (31.0) 14 (33.3) 13 (31.0) 23.3 ± 3.7 158.2 ± 26.0 4 [2–7] 13 (31.0) 6 (14.3) 7 (16.7) 40 (95.2) 7.5 ± 2.6 7.1 ± 2.1 0.9 [0.4–2.0] 3.8 ± 1.0 1.1 [0.9–1.2]

68.9 ± 10.9 32 (72.7) 37 (84.1)⁎ 26 (59.1) 20 (45.5) 19 (43.2) 10 (22.7) 24.7 ± 3.8 161.5 ± 35.1 4 [2–7] 18 (40.9) 5 (11.4) 9 (20.5)⁎ 41 (93.2) 8.4 ± 4.8 7.3 ± 1.9 1.0 [0.5–3.1] 3.5 ± 1.1 2.0 [1.7–2.2]⁎

70.2 ± 9.6 22 (51.2)⁎ 37 (86.1)⁎ 28 (65.1) 13 (30.2) 13 (30.2) 15 (34.9) 23.1 ± 3.6 162.5 ± 18.6 4 [2–12] 12 (27.9) 4 (9.3) 9 (20.9)⁎ 42 (97.7) 7.9 ± 3.0 8.3 ± 2.4⁎ 2.0 [0.9–5.3]⁎ 4.2 ± 1.4 6.0 [4.5–8.5]⁎

0.10 0.03 0.003 0.23 0.13 0.81 0.57 0.92 0.85 0.09 0.98 0.87 0.03 0.42 0.06 0.001 0.07 0.15 b0.0001

The plasma S100A12 levels on admission were divided into quartile: Q1, 0.11–0.75 ng/mL; Q2, 0.76–1.59 ng/mL; Q3, 1.60–3.49 ng/mL; and Q4, 3.50–46.6 ng/mL. NIHSS: National Institute of Health Stroke Scale. Edaravone: a free radical scavenger approved by the Japanese health authorities as a neuroprotective agent for the treatment of acute ischemic stroke. Baseline data were obtained on admission. Values are expressed as mean ± SD, number of patients (%), or median [interquartile]. ⁎ p b 0.05 vs Q1 group.

Among 171 patients, 74 (43.3%) had a poor functional outcome at day 90 after stroke onset. Plasma S100A12 levels on admission were significantly higher in patients with a poor functional outcome than in those with a favorable outcome (2.1 [1.2–5.1] vs 1.1 [0.5–2.0], p b 0.001) (Table 2). In addition, plasma S100A12 levels in patients with a poor functional outcome were significantly higher than in those with a favorable outcome, at least, until day 14 after stroke onset (Fig. 1). After day 14, plasma S100A12 levels in patients with a poor functional outcome were decreased to levels compatible with those in patients with a favorable outcome (Fig. 1). In patients with a poor functional outcome, plasma S100A12 levels at day 90 were significantly lower than those measured on admission (day 0). The age- and sex-adjusted ORs for a poor functional outcome substantially increased as plasma S100A12 levels on admission became

Table 2 Baseline characteristics of patients according to a functional outcome. Functional outcome at day 90

Age, y Male Hypertension Dyslipidemia Diabetes mellitus Atrial fibrillation Cardioembolic infarction Body mass index, kg/m2 Systolic blood pressure, mm Hg NIHSS Thrombolytic therapy Edaravone therapy Casual blood glucose, mmol/L Leukocyte, ×109/L C-reactive protein, mg/L Fibrinogen, μg/mL S100A12, ng/mL

Favorable [mRS 0–1] (n = 97)

Poor [mRS 2–6] (n = 74)

66.7 ± 9.1 69 (71.1) 72 (74.2) 55 (56.7) 26 (26.8) 29 (29.9) 23 (23.7) 23.6 ± 3.6 155.4 ± 27.0 3 [1–4] 10 (10.3) 91 (93.8) 7.3 ± 2.9 7.1 ± 2.3 1.0 [0.4–2.0] 3.6 ± 1.1 1.1 [0.5–2.0]

70.5 ± 10.8 46 (63.0) 60 (81.8) 45 (60.8) 28 (37.8) 30 (42.5) 26 (35.1) 23.7 ± 4.0 168.7 ± 30.2 6 [4–13] 17 (23.0) 71 (96.0) 8.1 ± 3.7 7.7 ± 2.1 1.7 [0.7–5.0] 4.1 ± 1.3 2.1 [1.2–5.1]

higher (p for trend b0.001) (Table 3). Multivariate analysis adjusted for sex and age by forced entry, and for covariates with a marginal statistical significance (p b 0.1) for a functional outcome in the univariate analysis (baseline NIHSS, systolic blood pressure on admission, leukocyte count, casual blood glucose, C-reactive protein, fibrinogen, and thrombolytic therapy) (model 1) showed that patients with higher plasma S100A12 levels on admission (Q3 and Q4) showed a poor functional outcome at day 90, with significantly higher ORs than those with the lowest plasma S100A12 levels on admission (Q1) (p for trend = 0.005) (Table 3). These findings were observed even after adjustment for other known variables for stroke outcome regardless of their statistical significance in the univariate analysis (stroke subtypes [non-cardioembolic or cardioembolic], hypertension, dyslipidemia, diabetes mellitus, atrial fibrillation, body mass index, and edaravone [free radical scavenger] therapy), along with for covariates included in model 1 (p for trend = 0.006) (model 2). In these multivariate models, other possible markers for inflammation on admission, such as leukocyte count (model 1: OR 1.00, 95% CI 1.00–1.01, p = 0.99; model 2: OR

p

0.01 0.26 0.29 0.59 0.12 0.15 0.10 0.85 0.003 b0.001 0.02 0.54 0.09 0.06 0.003 0.004 b0.001

mRS: modified Rankin Scale. NIHSS: National Institute of Health Stroke Scale. Baseline data were obtained on admission. Edaravone: a free radical scavenger approved by the Japanese health authorities as a neuroprotective agent for the treatment of acute ischemic stroke. Values are expressed as mean ± SD, number of patients (%), or median [interquartile].

Fig. 1. Plasma S100A12 levels in ischemic stroke patients according to a functional outcome. Plasma S100A12 levels in patients with a poor functional outcome (P: mRS 2–6) were significantly higher than in those with a favorable outcome (F: mRS 0–1) from days 0 to 14. The line in each box indicates the median, the upper and lower lines of the boxes represent the upper and lower quartiles, and the whiskers mark 1.5-times the interquartile range. mRS: modified Rankin Scale. *p b 0.001. †p b 0.05 versus S100A12 levels on admission (day 0) in patients with a poor functional outcome.

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Table 3 Odds ratios of S100A12 for a poor functional outcome. Plasma S100A12 levels

Q1 Q2 Q3 Q4 p for trend

Patients with poor functional outcome n (%)

Age- and sex-adjusted OR

95% CI

11 (26.2) 13 (31.0) 23 (52.3) 27 (62.8)

1.00 1.17 2.97 4.24

Reference 0.45–3.12 1.20–7.66 1.67–11.35

p

0.74 0.02 0.002 b0.001

Multivariate-adjusted (model 1)

Multivariate-adjusted (model 2)

OR

95% CI

OR

95% CI

1.00 1.08 3.70 3.77

Reference 0.32–3.70 1.16–12.81 1.13–13.48

1.00 1.09 3.61 4.01

Reference 0.30–4.04 1.02–13.85 1.09–16.10

p

0.90 0.03 0.03 0.005

p

0.89 0.04 0.03 0.006

The plasma S100A12 levels on admission were divided into quartile: Q1, 0.11–0.75 ng/mL; Q2, 0.76–1.59 ng/mL; Q3, 1.60–3.49 ng/mL; and Q4, 3.50–46.6 ng/mL. OR: odds ratio, CI: confidence interval. Model 1 was adjusted for sex and age by forced entry, and for covariates with a p value of b0.1 in the univariate analysis listed in Table 2 (baseline NIHSS, systolic blood pressure on admission, leukocyte count, casual blood glucose, C-reactive protein, fibrinogen, and thrombolytic therapy). Model 2 was adjusted for covariates included in model 1, along with other known variables for stroke outcome (stroke subtypes [non-cardioembolic or cardioembolic], hypertension, dyslipidemia, diabetes mellitus, atrial fibrillation, body mass index, and edaravone [free radical scavenger] therapy).

1.00, 95% CI 0.99–1.01, p = 0.85), C-reactive protein (model 1: OR 0.89, 95% CI 0.59–1.55, p = 0.62; model 2: OR 0.99, 95% CI 0.94–1.05, p = 0.59), and fibrinogen (model 1: OR 1.32, 95% CI 0.94–1.88, p = 0.11; model 2: OR 1.30, 95% CI 0.91–1.88, p = 0.15) were not associated with a poor functional outcome at day 90.

4. Discussion In this study, we found that plasma S100A12 levels on admission within 24 h after ischemic stroke onset were significantly higher in patients with a poor functional outcome than in those with a favorable outcome. Furthermore, multivariate analysis showed that patients with higher plasma S100A12 levels on admission had significantly higher ORs for a poor functional outcome at 90 days after stroke onset. These results suggest that plasma S100A12 on admission is an independent factor associated with the prognosis of patients with acute ischemic stroke. S100A12 is constitutively expressed and functions as a calciumbinding protein in granulocytes, but is secreted extracellularly in response to several cell stresses [4–6]. Interaction of secreted S100A12 with RAGE expressed in endothelial cells, macrophages, monocytes, microglia, and lymphocytes leads to inflammatory reactions by inducing the secretion of pro-inflammatory cytokines from these cells in various disease states [6]. Several clinical studies have shown that concentrations of S100A12 in peripheral blood correlate well with the activity of inflammatory diseases, suggesting that S100A12 is a sensitive marker reflecting the extent of localized inflammatory responses [6]. Inflammation plays a major role in adverse events in cardiovascular diseases [17]. An inflammatory response is also associated with a poor outcome in ischemic stroke. Post-ischemic inflammation exaggerates brain edema or directly promotes the cellular death in the penumbra, resulting in enlargement of the infarct volume and poorer functional recovery [1–3]. Some studies have suggested that S100A12 could be a marker for predicting poor prognosis in cardiovascular diseases. High plasma S100A12 levels are associated with major adverse cardiovascular events in patients with coronary artery disease [9]. Increased plasma S100A12 levels are also associated with all-cause and cardiovascular diseaserelated mortality in hemodialysis patients [18,19]. In this study, plasma S100A12 levels within 24 h after stroke onset were significantly associated with a poor functional outcome at 3 months, while there was no association between plasma S100A12 levels on admission and an initial stroke severity. We also found that plasma S100A12 levels in patients with a poor functional outcome were increased and sustained over, at least, 14 days after stroke onset. Our results were in line with previous study showing that treatment with neural cultures with RAGEmediated inflammatory insult for 48 h, but not for 24 h, was associated with neural death, and that RAGE-mediated inflammatory response contributes to a poor outcome after ischemic stroke [20,21].

Precise mechanisms underlying the association between S100A12 and a poor functional outcome after ischemic stroke remain unclear in this study. Recently, toll-like receptor (TLR)-2 and TLR-4 signaling pathways have been reported to play a pivotal role for post-ischemic inflammation, subsequent ischemic damage, and a poor outcome after ischemic stroke [22]. Activation of TLR-2 and TLR-4 has been shown to enhance the expression of S100A12 on monocytes [8]. S100A12 has also been suggested to bind to TLRs to activate inflammatory responses [23]. Thus, it seems possible to suggest that S100A12 amplifies subsequence RAGE-mediated inflammatory response through positive feedback interaction with TLR-2 and TLR-4, thereby playing a crucial role for post-ischemic inflammation, and affecting functional outcome in patients with ischemic stroke, although S100A12 might be a surrogate marker of inflammatory responses after ischemic stroke. In contrast to plasma S100A12 levels, other well-known inflammation-related factors on admission, such as leukocyte counts, C-reactive protein, and fibrinogen, were not associated with poor functional outcome in our study. Our results are in accordance with a previous study, which showed that these factors were not associated with a poor functional outcome in patients with ischemic stroke when evaluated within 24 h after stroke onset [24]. Granulocytes begin to infiltrate into ischemic brain tissue within a few hours after stroke onset [25]. Protein expression of S100A12 is induced in granulocytes immediately after hypoxic insults [11], and gene expression of S100A12 is increased in peripheral leukocyte in patients with acute ischemic stroke [10]. RAGE-mediated inflammation increases permeability of blood–brain barrier [26]. Collectively, these results suggest that granulocytes infiltrating into ischemic lesions increase the expression of S100A12 and secrete it into the plasma immediately after the onset of ischemic stroke, although it was not clear whether S100A12 was increased in the brain in parallel with plasma levels. Infiltration of granulocytes peaks at around 3 days, and although reduced, continues through couples of weeks after ischemic stroke [25]. Macrophages are also recruited into ischemic brain following granulocyte infiltration, and remained in ischemic tissue for substantially long time periods [25]. Although S100A12 is constitutively expressed in granulocytes, S100A12 is also induced in and released from macrophages under inflammatory condition [27]. Therefore, macrophages recruited into ischemic brain would be another source of S100A12 in delayed phase of ischemic stroke. Edaravone, in addition with its antioxidant effect, has been shown to suppress proinflamamtory responses and to improve functional outcome after brain ischemia [16,28]. We found, however, no significant effect of edaravone on functional outcome in this study. The discrepancy between previous studies and ours is likely related to that most patients were administered with edaravone in this study, resulting in insufficient statistical power. There are several limitations in this study. First, we did not assess infarct size in this study. However, stroke volume correlates with neurological severity assessed by the NIHSS [29]. Because we included the

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NIHSS in our analyses, we expected that the NIHSS could be substituted for infarct size in the present study. Second, we do not have data on prestroke levels of plasma S100A12. We consider, however, that pre-stroke levels of plasma S100A12 were not different between patients with a favorable and a poor functional outcome, because plasma S100A12 levels in patients with a poor functional outcome were decreased to levels compatible with those in patients with a favorable outcome at day 90. Third, we do not have data on granulocyte counts, in which S100A12 is abundantly expressed. It will be a future study to elucidate the association between plasma S100A12 levels and granulocyte counts. Fourth, although we did not register patients with signs of infection on admission in this study, we may not completely exclude patients with infection from the study, which is a potential cofounder as infection is associated with both higher levels of inflammatory markers and with a poor outcome after stroke. Fifth, sample size was relatively small in this study. Further studies with a larger number of subjects are required to confirm the clinical significance of S100A12 in ischemic stroke. In conclusion, increased plasma S100A12 levels on admission within 24 h after the onset of stroke are significantly and independently associated with a higher risk for poor functional outcome after ischemic stroke. Plasma S100A12 levels in the acute phase of stroke could be a blood biomarker for predicting a functional outcome in patients with ischemic stroke. Conflict of interest All authors declare no conflicts of interest. Acknowledgments The authors wish to thank Associate Professor Hitoshi Inoue (Research Institute for Information Technology, Kyushu University) for his technical support for the secure FSR Data Collection System. We are grateful to all the clinical research coordinators (Hisayama Research Institute for Lifestyle Diseases) for their help in obtaining informed consent and collecting clinical data. References [1] Shichita T, Ago T, Kamouchi M, et al. Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke. J Neurochem 2012;123(Suppl. 2):29–38. [2] Smith CJ, Emsley HC, Gavin CM, et al. Peak plasma interleukin-6 and other peripheral markers of inflammation in the first week of ischemic stroke correlate with brain infarct volume, stroke severity and long-term outcome. BMC Neurol 2004;4:2. [3] Whitely W, Chong WL, Sengupta A, et al. Blood markers for prognosis of ischemic stroke: a systematic review. Stroke 2009;40:e380–9. [4] Foell D, Wittkowski H, Vogl T, et al. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol 2007;81: 28–37. [5] Hofmann MA, Drury S, Fu C, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 1999;97: 889–901.

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[6] Pietzsch J, Hoppmann S. Human S100A12. A novel key player in inflammation? Amino Acids 2009;36:381–9. [7] Goyette J, Geczy CL. Inflammation-associated S100 proteins: new mechanisms that regulate function. Amino Acids 2011;41:821–42. [8] Abbas A, Aukrust P, Dahl TB, et al. High levels of S100A12 are associated with recent plaque symptomatology in patients with carotid atherosclerosis. Stroke 2012;43: 1347–53. [9] Saito T, Hojo Y, Ogoyama Y, et al. S100A12 as a marker to predict cardiovascular events in patients with chronic coronary artery disease. Circ J 2012;76:2647–52. [10] Barr TL, Conley Y, Ding J, et al. Genomic biomarkers and cellular pathways of ischemic stroke by RNA gene expression profiling. Neurology 2010;75:1009–14. [11] Vince RV, Chrismas B, Midgley AW, et al. Hypoxia mediated release of endothelial microparticles and increased association of S100A12 with circulating neutrophils. Oxid Med Cell Longev 2009;2:2–6. [12] Matsuo R, Ago T, Kamouchi M, et al. Clinical significance of plasma VEGF value in ischemic stroke: Research for Biomarkers in Ischemic Stroke (REBIOS) study. BMC Neurol 2013;13:32. [13] Kamouchi M, Matsuki T, Hata J, et al. Prestroke glycemic control is associated with the functional outcome in acute ischemic stroke. Stroke 2011;42:2788–94. [14] Hata J, Doi Y, Ninomiya T, et al. Combined effects of smoking and hypercholesterolemia on the risk of stroke and coronary heart disease in Japanese: the Hisayama Study. Cerebrovasc Dis 2011;31:477–84. [15] Pellis L, van Erk MJ, van Ommen B, et al. Plasma metabolomics and proteomics profiling after a postprandial challenge reveal subtle diet effects on human metabolic status. Metabolomics 2012;8:347–59. [16] The Edaravone Acute Brain Infarction Study Group. Effect of a novel free radical scavenger, edaravone (MCI-186), on acute brain infarction: randomized, placebocontrolled, double-blind study at multicenters. Cerebrovasc Dis 2003;15:222–9. [17] Barron HV, Cannon CP, Murphy SA, et al. Association between white blood cell count, epicardial blood flow, myocardial perfusion, and clinical outcomes in the setting of acute myocardial infarction: a thrombolysis in myocardial infarction 10 substudy. Circulation 2000;102:2329–34. [18] Nakashima A, Carrero JJ, Qureshi AR, et al. Effects of circulating soluble receptor for advanced glycation end products (sRAGE) and the proinflammatory RAGE ligand (EN-RAGE, S100A12) on mortality in hemodialysis patients. Clin J Am Soc Nephrol 2010;12:2213–9. [19] Shiotsu Y, Mori Y, Nishimura M, et al. Prognostic utility of plasma S100A12 levels to establish a novel scoring system for predicting mortality in maintenance hemodialysis patients: a two-year prospective observational study in Japan. BMC Nephrol 2013;14:16. [20] Muhammad S, Barakat W, Stoyanov S, et al. The HMBG1 receptor RAGE mediates ischemic brain damage. J Neurosci 2008;28:12023–31. [21] Tang SC, Wang YC, Li YI, et al. Functional role of soluble receptor for advanced glycation end products in stroke. Arterioscler Thromb Vasc Biol 2013;33:585–94. [22] Brea D, Blanco M, Ramos-Cabrer P, et al. Toll-like receptors 2 and 4 in ischemic stroke: outcome and therapeutic values. J Cereb Blood Flow Metab 2011;31: 1424–31. [23] Foell D, Wittkowski H, Roth J. Mechanisms of disease: a ‘DAMP’ view of inflammatory arthritis. Nat Clin Pract Rheumatol 2007;3:382–90. [24] Whiteley W, Wardlaw J, Dennis M, et al. The use of blood biomarkers to predict poor outcome after acute transient ischemic attack or ischemic stroke. Stroke 2012;43: 86–91. [25] Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol 2010;87:779–89. [26] Okuma Y, Liu K, Wake H, et al. Anti-high mobility group box-1 antibody therapy for traumatic brain injury. Ann Neurol 2012;72:373–84. [27] Goyette J, Yan WX, Yamen E, et al. Pleiotrophic roles of S100A12 in coronary atherosclerotic plaque formation and rupture. J Immunol 2009;183:593–603. [28] Zhang N, Komine-Kobayashi M, Tanaka R, et al. Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 2005;36:2220–5. [29] Tong DC, Yenari MA, Albers GW, et al. Correlation of perfusion- and diffusionweighted MRI with NIHSS score in acute (b6.5 hours) ischemic stroke. Neurology 1998;50:864–70.

Plasma S100A12 is associated with functional outcome after ischemic stroke: Research for Biomarkers in Ischemic Stroke.

Ischemic stroke is accompanied by an inflammatory response, which exacerbates brain injury and deteriorates functional outcome. S100A12 is expressed a...
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