Association of MMP-9, ADMA, and sCD40L with ischemic stroke and correlations with stroke severity: A case-control study

In the present study, we aimed to examine the correlation of serum levels of MMP-9, ADMA, and sCD40L with the occurrence and severity of acute ischemic stroke. Methods All routines analyses were performed using a Cobas platform. MMP-9, ADMA, and sCD40L were measured using ELISA kit. Results Compared with the controls, the stroke group showed higher MMP-9,higher sCD40L and higher ADMA. There were signicant positive correlations between the NIHSS scores and MMP-9, sCD40L and ADMA. Our study indicated that serum levels of MMP-9, sCD40L, and ADMA are associated with ischemic stroke and are correlated with the NIHSS scores at admission. Regression analysis was performed using the natural logarithm transformation of MMP-9, SCD40L, and ADMA.

Matrix metalloproteinase-9 (MMP-9) can degrade the extracellular matrix to weaken the brous cap of atherosclerosis plaques and damage the blood-brain barrier, thus causing brain edema and cerebral hemorrhage transformation [12,13]. Thus, high MMP-9 levels appear to be associated with the occurrence and progression of ischemic stroke and prognosis [12,13]. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (NOS) and is associated with endothelial damage and cardiovascular diseases [14][15][16]. Soluble CD40 ligand (sCD40L) is generated by the cleavage of CD40L. High levels of sCD40L can promote platelet activation and aggregation and play an important role in promoting thrombosis and in ammatory reaction [17]. Recent studies have shown a signi cant increase in plasma sCD40L in acute cerebral infarction patients [18,19].
Still, the association of these non-classical factors with stroke remains controversial, and it is poorly known whether they are independent of each other.
Therefore, this study aimed to examine the correlation of serum levels of MMP-9, ADMA, and sCD40L with the occurrence and severity of acute ischemic stroke. The results could help determine the value of these biomarkers for the occurrence of stroke. This study was approved by the Ethics Committee of the A liated Hospital of Chengde Medical College and all methods were performed in accordance with the Helsinki Declaration and relevant guidelines. The requirement for informed consent was waived because of the retrospective nature of the study.

Study design and patients
The inclusion criteria were 1) diagnosis of stroke in accordance with the Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke (2014) (i. acute onset; ii. focal neurological impairment; iii. imaging lesions or symptoms and signs lasting > 24 h; iv. non-vascular causes were excluded; v. cerebral hemorrhage was excluded by brain CT or MRI) and 2) acute ischemic stroke. The exclusion criteria were 1) cerebral hemorrhage, 2) history of previous stroke, 3) complicated with a serious injury to the heart, liver, kidney, or other organs, 4) chronic infectious diseases, 5) malignant tumors, 6) long-term use of immunosuppressant drugs or hormones before admission, or 7) severe mental disorders.
Control subjects were selected among the patients who visited the A liated Hospital of Chengde Medical College for routine health checkups during the same period. The exclusion criteria were 1) history of cerebrovascular disease, 2) complicated with serious damage to the heart, liver, kidney, or other organs, 3) chronic infectious diseases, 4) malignant tumors; 5) long-term use of immunosuppressants or hormones, or 6) severe mental disorders.

Data collection
Age, sex, smoking history, drinking history, history of hypertension, diabetes, and coronary heart disease, and general laboratory test results at admission (triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)) were collected from the medical charts.

Laboratory
All laboratory measurements were made on fasting peripheral venous blood samples (5 ml) taken within 24 h of admission, using EDTA tubes. Within 30 min after collection, the plasma was centrifuged for 15 min (3000 rpm) in a high-speed centrifuge, and the plasma was separated. The samples were stored at -80℃. All routines analyses were performed using a Cobas platform (Roche Diagnostics, Basel, Switzerland). MMP-9, ADMA, and sCD40L were measured using ELISA kit (Mlbio, Shanghai, China).

Stroke assessment
All patients admitted for stroke during the study period underwent a routine National Institutes of Health Stroke Scale (NIHSS) scoring.
Statistical analysis SPSS 26.0 (IBM, Armonk, NY, USA) was used for statistical analysis. The continuous data were tested for normal distribution using the Kolmogorov Smirnov test. The data conforming to the normal distribution are expressed as means ± standard deviations and were tested using the independent sample t-test. Data that did not conform to the normal distribution are presented using median (interquartile range) and were compared using the Mann-Whitney U-test. Categorical data are presented as n (%) and were tested using the chi-square test. The Spearman correlation analysis was used to analyze the correlations between the indicators. A multivariable logistic regression analysis was used to identify the factors associated with stroke. Two-sided P-values < 0.05 were considered statistically signi cant.

Results
Characteristics of the subjects A total of 197 subjects who visited the hospital between April 2015 and November 2015 were included.

Correlation analysis between the NIHSS score on admission and various indicators
The correlation results between the NIHSS score on admission and various indicators were shown in Table 3. The NIHSS scores at admission were not correlated with age, sex, hypertension, coronary heart disease, diabetes mellitus, smoking, drinking, triglycerides, TC, HDL-C, LDL-C, MMP-9, sCD40L, and ADMA.
On the other hand, there were signi cant positive correlations between the NIHSS scores and MMP-9 (r = 0.23, P = 0.007), sCD40L (r = 0.37, P < 0.001), and ADMA (r = 0.26, P = 0.003) ( Table 3 and Fig. 1). Discussion MMP-9, ADMA, and sCD40L are possibly associated with stroke. This study aimed to examine the correlation of serum levels of MMP-9, ADMA, and sCD40L with the occurrence and severity of acute ischemic stroke. The results showed that the serum levels of MMP-9, sCD40L, and ADMA are associated with ischemic stroke and are correlated with the NIHSS scores at admission.
MMP-9 is involved in the remodeling of the atherosclerosis plaques by digesting the extracellular matrix that constitutes the brous cap, making it more prone to rupture, in which case a thrombus will form when the highly thrombotic necrotic core is exposed to the blood [20]. MMP-9 is also involved in the destabilization of the blood-brain barrier, brain edema, and cerebral hemorrhage transformation, which can further deteriorate the condition of patients with stroke [12,13,21]. Thus, high MMP-9 levels appear to be associated with the occurrence and progression of ischemic stroke and prognosis [12,13]. A metaanalysis of case-control studies revealed that the MMP-9 levels are directly correlated with stroke volume, stroke severity, and functional outcome [22]. Although stroke volume and functional outcomes were not assessed in the present study, MMP-9 was independently associated with stroke and positively correlated with the NIHSS scores.
NO is a potent vasodilator and antioxidant necessary to the normal functions of the vascular endothelium [23,24]. Low levels of NO are associated with endothelial dysfunction, which is in itself a risk factor for the initiation and progression of atherosclerosis [25,26]. NO is produced by the NOS enzymes, and the endothelial NOS (eNOS) is directly involved in normal endothelial dysfunction [27,28].
Besides the numerous factors involved in the transcription levels of eNOS [27,28], ADMA is an endogenous inhibitor of eNOS, and high levels of ADMA are associated with endothelial damage and cardiovascular diseases [14][15][16]. Chen et al. [29] showed that ADMA is both a marker and a mediator of stroke. Janes et al. [30] showed that ADMA levels are associated with cerebral small vessel disease in asymptomatic patients. A recent review showed that high ADMA levels were associated with carotid atherosclerosis, which is a risk factor for ischemic stroke [31]. Still, some studies suggest that ADMA levels are not associated with stroke [32][33][34]. In the present study, ADMA levels were independently associated with stroke and correlated with the NIHSS. The discrepancies among studies could be due to several factors, including sample size, selection of the patients and controls, and genetics.
sCD40L is involved in platelet activation and aggregation, which is involved in ischemic stroke pathogenesis when the plaque necrotic core is exposed [17,35]. High levels of sCD40L are also involved in systemic in ammation, which is involved in pro-atherogenic mechanisms [17]. Plasma sCD40L levels are elevated in patients with acute ischemic stroke, but the causality relationship is unknown [18,19]. A study revealed that sCD40L levels were associated with in ammatory markers levels but not with stroke itself [36]. Since the in ammatory markers could not be analyzed in the present study because they were inconsistently measured among the patients, the present study could not determine whether the independent association of sCD40L with stroke was a direct independent association or through in ammation. Still, a study showed that sCD40L levels were associated with stroke severity and outcomes [18], supporting the correlation between sCD40L and the NIHSS score.
Still, the association of these non-classical factors with stroke remains controversial, and it is poorly known whether they are independent of each other. Indeed, several studies examined different panels of biomarkers for their association with stroke, but few studies examined MMP-9, ADMA, and sCD40L within the same study. In the present study, all three markers were each independently associated with stroke. Future studies should examine whether predictive models could be built using these markers.
The NIHSS is a common neurological function score used to determine the prognosis of patients with cerebral infarction at admission [37][38][39]. The NIHSS can be used to objectively re ect the severity of cerebral infarction and determine patient prognosis [37][38][39]. Still, it has limitations [40] that could not be controlled for in the present study. These limitations could explain, at least in part, why the correlations were weak (all r < 0.4). Another explanation could also be that MMP-9, sCD40L, and ADMA all contribute together to stroke. In addition, other factors could be involved but were not quanti ed in this study.
This study has limitations. It was a retrospective study limited to the data available in the charts. The sample size was small and did not allow deeper analyses such as receiver operating characteristics analyses and nomograms. Only the NIHSS score could be used to grade the stroke because it is the grading system used in all patients. Many factors that might be associated with stroke were not evaluated in this study.

Conclusions
In conclusion, the serum levels of MMP-9, sCD40L, and ADMA in patients with ischemic stroke are signi cantly higher than those in the control group, suggesting that the serum levels of MMP-9, sCD40L, and ADMA are correlated with the occurrence of acute ischemic stroke. In addition, MMP-9, sCD40L, and ADMA are correlated with the NIHSS score. Future studies should examine whether these markers could be used as predictors of the occurrence of acute ischemic stroke.

Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests Funding None.
Authors' contributions ML carried out the studies, participated in collecting data, and drafted the manuscript. HW performed the statistical analysis and participated in its design. YG participated in acquisition, analysis, interpretation of data, and drafted the manuscript. All authors have read and approved the nal manuscript.