Evaluation of neutrophil extracellular traps as the circulating marker for patients with acute coronary syndrome and acute ischemic stroke.

Abstract Introduction Neutrophil extracellular traps (NETs) are known to be induced by various factors. In this study, we tried to identify circulating levels of NETs in patients with acute coronary syndrome (ACS) and acute ischemic stroke (AIS) and to confirm its suitability as a new circulating marker in their detection. Methods We prospectively enrolled 95 patients with a diagnosis of ACS (N = 37) or AIS (N = 58) in Dong‐A University Hospital, Busan, Korea. The control group was selected from healthy adults (N = 25) who visited the hospital for health screening. Circulating levels of NETs were evaluated by measuring plasma concentrations of double‐stranded DNA (dsDNA) and DNA‐histone complex. Results The concentrations of dsDNA were statistically higher in patients with ACS or AIS than those in the control group (both P < .001). In the univariable and multivariable analyses, statistically significant risk factors were troponin I (TnI) level and dsDNA concentration in the ACS group (P = .046 and P = .015, respectively) and only dsDNA concentration in the AIS group (P = .002). In the receiver operating characteristic curve analyses, the area under the curve values for TnI level and dsDNA concentration in the ACS group were 0.878 and 0.968, respectively, and the value for dsDNA concentration in the AIS group was 0.859. Conclusions In this study, it was confirmed that the circulating level of NETs was increased in patients with ACS and AIS at initial presentation. Findings in this study show that NETs could be used as a new circulating marker for the initial diagnosis of ACS or AIS.


| INTRODUC TI ON
The number of Korean patients with acute coronary syndrome (ACS) and acute ischemic stroke (AIS) increases every year due to an aging population. As a result, patients with these diseases face a rapidly growing economic burden. 1 As a common mechanism of ACS, the rupture of the atherosclerotic plaque causes partial or complete occlusion of the coronary artery. When the collagen of the endothelium is exposed by the collapse of the plaque, coagulation cascades start with platelet activation, leading to thrombus formation. 2 In addition, it has been shown that excessive hyperactivity of platelets increases the risk of thromboembolism, leading to excessive formation of abnormal thrombosis together with atherosclerotic lesions, and is also a major factor in causing AIS. 3 The interaction between platelets and vascular endothelial cells could induce local inflammatory conditions in the blood vessels, leading to microcirculatory disturbances that promote the gradual progression to atherosclerosis. [4][5][6][7] Meanwhile, the neutrophil is the most abundant cell type in the leukocyte and plays a crucial role in the innate immune system, which serves as the first line barrier against microorganisms. 8,9 In 2004, Brinkmann et al found that activated neutrophils released histones, protein granules, and double-stranded DNA (dsDNA) to form fiber-like structures, termed neutrophil extracellular traps (NETs). 10 These NETs are known to be induced by various factors such as microorganisms, antibodies, activated platelets, and reactive oxygen species. 11 Although NETs have been studied mainly in terms of host defense mechanisms, there has also been interest in NETs for pathogenetic aspects of certain noninfectious diseases. For instance, the involvement of NETs in thrombosis formation has been demonstrated. 12 In addition to their immunological role, NETs provide a scaffold for red blood cells and platelets, contributing to coagulation cascades and formation of arterial and venous thromboses. 13,14 However, few analyses on NETs in patients with cardiocerebrovascular diseases exist. Therefore, there are limitations for their application to clinical fields.
In this study, we attempted to identify circulating levels of NETs using samples from patients with ACS and AIS and to confirm its suitability as a new circulating marker in detecting them.
The circulating levels of NETs were evaluated by measuring concentrations of dsDNA. To clarify the conclusions, we performed an additional assay measuring the DNA-histone complex, which was selected as the second marker. This selection was based on the observation that when neutrophils are activated by other stimuli, even in patients with ACS and AIS, they release DNA, which binds to histones and other cytoplasmic components. 10 As well, the DNA-histone complex is a relatively easy-to-measure test item in the laboratory. The results were analyzed in comparison with other clinical and laboratory markers.

| Blood samples and assays
Blood samples were collected in sodium citrate tubes. Within 1 hour after collection, the blood samples were centrifuged at 1600 × g for 15 minutes. The prothrombin time (PT) and activated partial thromboplastin time (aPTT) were tested using a CS-5100 coagulation analyzer (Sysmex).
After these laboratory tests, leftover plasma samples with 1800 μL or more were selected for this study and stored at −80°C until measure-

| Statistical analyses
Statistical analyses were performed using MedCalc for Windows, version 18.10 (MedCalc Software). A P-value <.05 was considered to be significant. All data were expressed as numbers with percentages or mean ± standard deviation. To compare continuous variables with the control group, the independent t test was used for variables with para-

| Baseline characteristics of the study population
At baseline, there were no statistically significant differences between the ACS and the control groups, and between the AIS and the control groups for the following variables: age, gender, underlying diseases (diabetes mellitus [DM], hypertension, and dyslipidemia), social histories (alcohol intake and current smoking), and laboratory data (PT and aPTT).
Neutrophil count, C-reactive protein, troponin I (TnI), and creatinine kinase MB (CK-MB) levels were significantly higher in both ACS and AIS groups than in the control group, and platelet count in both ACS and AIS groups was significantly lower than in the control group. The baseline characteristics of the study population are summarized in Table 1.

| Comparison of the circulating levels of NETs between the ACS, AIS, and control group
The circulating levels of NETs evaluated via measuring dsDNA concentrations were significantly higher in the ACS (743.28 ± 323.10 pg/ μL) and AIS (524.22 ± 370.06 pg/μL) groups than in the control group   Table 2.

| ROC curve analyses for the ACS and AIS groups
The ROC curve analyses for the ACS and AIS groups were performed on the variables showing significant results in the Cox regression analyses ( Figure 2). In the AIS group, ROC curve analysis was performed only on dsDNA concentration, which showed a statistically significant result in the previous multivariable analyses. In the ACS group, the AUC values for TnI level and dsDNA concentration were 0.878 and 0.968, respectively, although there was no statistically significant difference in comparison of the two ROC curves (P = .060). The AUC value for dsDNA concentration in the AIS group was 0.859 (Table 3). For dsDNA concentrations in the ACS and AIS groups, cutoff values representing an optimal sensitivity/specificity pair were calculated as 400.69 pg/μL (sensitivity of 89.2% and specificity of 96.0%) and 281.28 pg/μL (sensitivity of 86.2% and specificity of 76.0%), respectively.  (Table 4), but there were no statistically significant differences between the subgroups of each group, the ACS and AIS (P = .522 and P = .243, respectively). The Kaplan-Meier event-free cumulative curves for dsDNA concentrations in patients with ACS and AIS are depicted in Figure 3. When comparing the event-free probability based on the median values of TnI, there were also no statistically significant differences in the ACS and AIS groups (P = .059 and P = .171, respectively, and data not shown).

| D ISCUSS I ON
In the present study, the circulating levels of NETs were evaluated by measuring dsDNA concentrations in patients with ACS and AIS.
After the Cox regression analyses were performed to determine significant variables in ACS and AIS, the ROC curve analyses were carried out to confirm whether dsDNA concentration could be a useful marker at the initial stages of ACS and AIS. We also investigated occurrences of MACEs during the follow-up period to check whether the elevated dsDNA concentration would be associated with poor prognosis in patients with ACS and AIS. P-values lower than 0.05 were considered to be significant and they were shown in bold. and it has been demonstrated that inflammatory responses involving neutrophil activation play a major role in atherosclerotic changes and progression. [22][23][24][25] According to a recent study, circulating extracellular DNA levels in AIS patients are associated with innate immune system activation. 26 Though clear mechanisms are yet to be explored, these processes could lead to the elevated level of NETs in the ACS and AIS groups in this study. In this respect, it is necessary to examine the possibility that the increased level of NETs is associated with the pathogenesis of ACS and AIS as one of many damage-associated molecular patterns.

F I G U R E 1
Though the presence of DM was not considered as a confounding factor in this study, a previous in vitro study discovered that production of NETs from isolated neutrophils was promoted by high glucose levels, and especially, type 2 DM patients with elevated glycated hemoglobin (HbA1c) had high levels of nucleosomes, neutrophil elastase, and other components of NETs. 27 Meanwhile, dyslipidemia could injure endothelial cells and induce neutrophilia, which is eventually associated with the promotion of lipid deposition and atherosclerotic plaque burden. 28 In addition, dyslipidemia could promote serum levels of C-X-C motif ligand (CXCL)-1, which enhances neutrophil mobilization. 29 In the present study, there was no statistical difference in the prevalence of DM, hypertension, and dyslipidemia of the ACS, AIS, and control groups, and this is probably because the control group was selected, in part, according to age. TnI and CK-MB were higher in AIS patients than those in the control group. In interpreting the cardiac Abbreviations: ACS, acute coronary syndrome; AIS, acute ischemic stroke; CI, confidence interval; CK-MB, creatinine kinase MB; CRP, C-reactive protein; dsDNA, double-stranded DNA; OR, odds ratio; TnI, troponin I.
P-values lower than 0.05 were considered to be significant and they were shown in bold.
markers, such as TnI and CK-MB in AIS patients, there are various hypotheses about how cerebrovascular events directly endanger the heart, but there is no convincing mechanistic explanation as of yet. [30][31][32][33][34][35] Based on the results of this study, it was confirmed that the circulating level of NETs increased in patients with ACS or AIS at initial presentation. In particular, the dsDNA concentrations in the ACS group were significantly higher, not only to those in the control group but also to those in the AIS group. This is, perhaps, due to the lack of consideration of disease severity in the AIS group at the time of admission. According to the previous study of Vallés et al, 2017, a positive correlation exists between NETs level and the National Institutes of Health Stroke Scale (NIHSS) score, reflecting stroke severity, and patients with a higher NIHSS score had a higher NETs level (P < .001). 36 Interestingly, the results of the ROC curve analyses for the ACS group showed that the dsDNA concentration would be an equivalent marker to TnI. Additionally, the ROC curve analysis for patients with UA, having suggestive symptoms of cardiac ischemia without elevated TnI levels, showed a slightly higher AUC value of 0.973 (P < .001, data not shown). These results suggest that measuring the dsDNA concentration could be a more useful marker than TnI for the initial diagnosis of UA.
During a relatively short period, we investigated the occur- On the other hand, there have been recent studies on chronic neurological diseases using cell-free DNA in cerebrospinal fluid (CSF) samples. [40][41][42] There was a study that analyzed serial nuclear and mitochondrial DNA levels in plasma and CSF for patients with subarachnoid hemorrhage. 43 It would be worthwhile to investigate whether the levels of NETs in CSF samples could be a superior marker for the diagnosis of AIS.
In summary, this study demonstrated that measuring the circulating levels of NETs has diagnostic power equivalent to TnI in ACS patients at initial presentation. Similarly, NETs analyses in patients with AIS showed potential for their use as novel circulating markers for the initial diagnosis of AIS.

E TH I C A L A PPROVA L
This study was approved by the institutional review board of Dong-A University Hospital. Written consents from the patients were waived because it was a study using residual samples without any additional blood collection.