Cystatin C as a predictor for carotid artery steno-occlusive disease in non-cardioembolic ischemic stroke

Abstract

Background

Stroke is the third leading cause of death in developed nations. Ultrasonography (US) of the carotid arteries is the modality of choice for diagnosis and monitoring of cases of atheromatous disease. Cystatin C (CysC) is a risk factor for cerebrovascular and cardiovascular disease.

Objectives

To detect the association of serum CysC levels with acute ischemic stroke, the correlation of its levels with stroke severity, and the association between CysC levels with extra- and intracranial stenosis.

Methods

Case-control study was conducted on 66 subjects, 33 patients within the first week of stroke onset with age range from 40 to 75 years, and 33 control healthy subjects with matched age and sex. All subjects underwent complete neurological examination, Extra- and transcranial duplex, CT brain, routine laboratory work-up, and serum CysC.

Results

Serum CysC levels were significantly raised in patients with acute ischemic stroke compared to controls. CysC levels were higher in patients with extracranial stenosis than in those with intracranial stenosis. Moreover, extracranial stenosis > 50% has higher levels of CysC. Serum CysC level showed a significant correlation with National Institutes of Health Stroke Scale (NIHSS) and intima-media thickness (IMT).

Conclusion

Serum CysC can be used as a marker for acute atherosclerotic ischemic stroke and stroke severity. High-grade extracranial stenosis has the highest level of CysC.

Introduction

About 25–30% of all cerebral ischemic events are caused by large vessel atherosclerosis. CysC may contribute to the process of carotid atherosclerosis via chronic inflammation [1]. Ultrasonography (US) of the carotid arteries is the modality of choice for diagnosis and monitoring of cases of atheromatous disease [2].

The aims of our work are to evaluate the association of serum CysC level within the first week of the stroke and to correlate its level with the severity of the stroke according to NIHSS and to determine whether CysC levels were associated with extra and transcranial large artery stenosis (LAS).

Patients and methods

In this case-control study, we chose 66 subjects who were divided into group A (study group) and group B (control group). Group A includes 33 patients within 1 week from the onset of ischemic stroke, 11 (33.3%) patients have extracranial stenosis, 11 patients with intracranial stenosis, and 11 patients with mixed intra- and extracranial stenosis, and they were recruited from the neurology in-patient stroke unit of Kasr El-Aini Teaching Hospital. Group B includes 33 healthy subjects with matched age and sex. The study was performed between November 2017 and May 2018. Inclusion criteria were as follows: age range 40–75 years old, both sex, intracranial and or extracranial stenosis by the duplex US, and we excluded patients < 40 years or > 75 years, patients with intracranial hemorrhage, potential cardiac embolic sources, malignancy, creatinine > 1.5 mg/dl, hypothyroidism, and chronic inflammatory diseases. All patients were subjected to full history, general, and neurological examination including NIHSS, extra-, and transcranial neurosonological assessments using Philips IU22 duplex machine software, version 2.0.13, USA 2012, and routine laboratory investigations and serum level of CysC using Siemens N Latex Cystatin C kit on BN ProSpec Nephelometer used in BN ProSpec Company from Siemens Healthcare Diagnostics, product Gmbh 35041, Marburg/Germany. Brain computed tomography (CT) with GE multislice 64, 32 detector*2, USA, 2015, and echocardiography with Samsung Medison HS60, Korea.

Statistical analysis

Data were coded and entered using the statistical package SPSS, version 25; SPSS Inc., Chicago, IL, USA. Data was summarized using mean and standard deviation for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons between groups were done using analysis of variance (ANOVA) with multiple comparisons post hoc test or unpaired test in normally distributed quantitative variables while non-parametric Kruskal-Wallis test and Mann-Whitney test were used for non-normally distributed quantitative variables [3]. For comparing categorical data, chi-square (χ2) test was performed. Exact test was used instead when the expected frequency is less than 5 [4]. Correlations between quantitative variables were done using Spearman correlation coefficient [5]. A multi-variate linear regression model was conducted. P values less than 0.05 were considered as statistically significant.

Results

Demographic and clinical data

Mean age of group A was (56.76 ± 11.12), and out of 33 patients, 20 were males (60.6%) and 13 were females (39.4%). The mean age of group B was (47.33 ± 5.19) with 25 males (75.8%) and 8 were females (24.2%).

Within group A, 19 patients were diabetic (57.6%), 22 patients were hypertensive (66.7%), and 14 patients were smokers (42.4%) compared to 3 smokers (9.1%) only in group B.

Patients NIHSS range from 4 to 20 with a mean of 10.52 ± 4.29.

Twenty-one out of 33 patients had branch MCA infarction (63.6%.) in CT brain, 8 have a total MCA infarction (24.2%), 3 have lacunar infarction (9.1%), and only 1 patient was had MCA and PCA infarction (3.0%). Among 33 patients in group A, 24 (72.7%) had increased IMT.

In the current study, 9 out of 11 patients with extracranial stenosis had stenosis > 50% while 6 out of 11 patients with intracranial stenosis had stenosis > 50%, and only 4 patients with mixed intra- and extracranial stenosis had stenosis > 50%.

In group A, 22 patients had high CysC level (66.7%), but in group B, all had normal CysC level sensitivity 66% and specificity 100%.

TLC was high in 2 patients in group A (6.1%), lipid profile was high in 12 (36.4%), and coagulation profile, renal, hepatic function, and electrolyte level were normal in both groups.

Comparative analysis

Stroke patient (group A) had a significant higher level of CysC than healthy persons (group B) (P value = < 0.001). Moreover, patients with increased IMT had a higher level of CysC in comparison with patients with normal IMT in group A and all subjects in group B (P value = 0.003). The highest level of CysC was in patients with extracranial stenosis with statistical significance P value = 0.001, and it was high in patients with extracranial stenosis compared to patients with combined intra- and extracranial stenosis with P value = 0.015 (Fig. 1). Patients with > 50% stenosis have high level of CysC compared to patients with < 50% stenosis with statistical significance P value = 0.004 (Fig. 2). Among 19 patients with > 50% stenosis, CysC level was highest in extracranial stenosis (P value = 0.006). Intracranial stenosis > 50% had a higher level of CysC than to patients with intracranial stenosis < 50% with (P value = 0.03) (Fig. 3). The higher the patient score on NIHSS, the higher the level of CysC with (P value = < 0.001) (Table 1). No statistically significant difference was found between the level of CysC and size of infarction (P value = 0.403) (Table 2). Also, no statistically significant difference was found between CysC level and age or gender of patients, the presence or absence of hypertension, diabetes, and smoking. Regarding lipid profile and other laboratory findings in comparison to CysC level, no statistical significance was detected.

Fig. 1

Cystatin C level (mg/l) between 3 groups of patients

Fig. 2

Cystatin C level (mg/L) according to the degree of stenosis

Fig. 3

Cystatin C level (mg/l) in patients with intracranial stenosis according to the degree of stenosis

Table 1 Level of CysC according to groups, IMT, site, and degree of stenosis

Table 2 Level of CysC according to size of infarction

Discussion

In this study, we compared the serum level of CysC between ischemic stroke patients and healthy individuals, and we observed that CysC level was higher in patients within the first week of stroke onset than in control subjects. These findings interpreted in the context that CysC as an inhibitor of cysteine protease and high level of CysC affects the process of vascular remodeling by breaking the balance of proteolytic and anti-proteolytic activities. Therefore, CysC acts as a good biomarker for acute ischemic stroke. Going with our result, Xu and colleagues [6] noticed that elevated CysC levels were strongly associated with stroke. Yang and colleagues [7] have similar results and hypothesized that Cyst C plays an important role in the pathogenesis of atherosclerosis.

We should address that our study is limited by the small sample size which makes sensitivity 66% and specificity 100%.

In the present study, we found that there is no relation between the size of infarction and level of CysC, and we explain that serum levels of CysC were not affected by the size of infarction. First, because of the small number of participants with total MCA and combined MCA PCA territory infarction in comparison to patients with lacunar and branch MCA infarction. Second, that size of actual infarction may be smaller in brain image, and it appears large due to the presence of penumbra and vesogenic edema. On the other hand, Xiao and colleagues found that larger cerebral infarction size is associated with higher serum CysC. Therefore, CysC may be a predictor for the severity of ischemia and cell damage [8].

In the current study, a positive correlation was found between CysC level and NIHSS scores with statistical significance, so we concluded that CysC is a prognostic tool for stroke severity as the higher the CysC level, the severer the stroke. Zhiqiang and colleagues [6] show a positive correlation between CysC and NIHSS. While Zeng and colleagues [9] observed that correlation between CysC levels and NIHSS was not significant, that was conversely, to our results due to our selection criteria as our patients were in the first week of stroke.

As regards CysC predictive value, in the present study, increased IMT had a higher level of CysC with positive statistical significance. Matching with our result, Zhu and colleagues [10] noticed that CysC was strongly associated with carotid thickening and plaque. Moreover, Kaneko and colleagues [11] noticed that CysC is strongly and independently associated with arterial wall elasticity, which reflects the degree of subclinical atherosclerosis. On the contrary, Bui and colleagues [12] found no association between CysC with internal and common carotid IMT and such differences are due to their participants that were four different self-reported ethnic groups (white, African American, Hispanic, and Chinese).

In our study, when we compared the level of CysC between patients with extracranial, transcranial, and combined stenosis, we noticed that the highest level of CysC present in patients with extracranial stenosis with statistical significance that can be explained by that CysC contributes to the process of carotid atherosclerosis. Extracranial arteries are elastic arteries while intracranial arteries are muscle arteries, and atherosclerosis and vascular calcification occur in extracranial vessels while oxidative stress which induces endothelial dysfunction occurs in intracranial vessels. Moreover, patients with stenosis > 50% had higher levels of CysC compared to patients with stenosis < 50% with positive statistical significance. Extracranial stenosis > 50% had the highest level of CysC with positive statistical significance. Xu and colleagues [13] noticed that serum CysC was highly associated with large cerebral artery stenosis but not the location of large cerebral artery stenosis whether extracranial or intracranial. Going with our result, Umemura and colleagues [14] suggested that higher levels of CysC were independently associated with symptomatic extracranial ICA stenosis, but not with intracranial ICA/MCA stenosis in patients with non-cardioembolic stroke.

Peliseket and colleagues [15] also suggested that an imbalance between cysteine proteinase and CysC could impact changes in vascular structure and stenotic lesions. While Huang and colleagues [16] observed serum CysC is high in stroke patients but were not correlated with the presence of intracranial arterial stenosis.

In the present study, patients’ age and gender had no correlation with CysC level, and our finding implies that CysC was included in the process of carotid atherosclerosis via chronic inflammation and not related to demographic risk factors. Matching with our result, Yang and colleagues [7] observed no statistical significance between CysC and age or gender of stroke patients. Although Kobayashi and colleagues [17] observed a higher level of CysC in males.

In the current study, no significant correlation was found between CysC level and hypertension, diabetes, or smoking. So CysC is strongly and independently associated with arterial wall atherosclerosis. Going with our finding, Xiao and colleagues [8] and Huang and colleagues [16] found that serum CysC concentrations in stroke patients were not correlated with hypertension or diabetes. On the other hand, Yang and colleagues [7] observed a significant association between CysC level and hypertension and between CysC and diabetes. Kobayashi and colleagues [17] also noticed statistical significance between CysC level and hypertension, DM, and smoking, taking into consideration that there were few participants with severe kidney disease in these studies.

In our study, no statistical significance was found in comparing cystatin c level and lipid profile. Matching with our study result, Xiao and colleagues [8] and Huang and colleagues [16] found that CysC levels were not correlated with serum total cholesterol, high-density lipoprotein, and low-density lipoprotein levels. While on the contrary, Xu and colleagues [6] observed a significant correlation with serum CysC levels and serum triglycerides and cholesterol.

In the present study, no statistical significance was found between CysC level and electrolyte. Going with our finding, Gharaibeh and colleagues [18] found that serum electrolyte levels did not show any significant changes in cases compared to controls.

Conclusion

From all previous results, it could be concluded that serum cystatin C can be used as a marker for acute atherosclerotic ischemic stroke and good indicator for stroke severity. Higher levels of CysC closely associated with extracranial stenosis than with transcranial stenosis. So, CysC is a good predictor, diagnostic, and prognostic biomarker in acute stroke.