2021 Volume 85 Issue 2 Pages 213-219
Background: This study explored the value of cystatin C (CysC) in predicting stroke recurrence in patients with acute ischemic stroke.
Methods and Results: This was a post hoc analysis of the China Antihypertensive Trial in Acute Ischemic Stroke (CATIS) on 3,474 acute ischemic stroke patients with documented serum CysC and high-sensitivity C-reactive protein (hsCRP) concentrations. Study outcomes included stroke recurrence and combined vascular events within 2 years after stroke. In stroke patients with higher (i.e., ≥4.8ng/mL), but not lower, hsCRP concentrations, a higher CysC concentration (i.e., ≥0.78 mg/L) was associated with a 2.48-fold increase in the risk of recurrent stroke (95% confidence interval [CI] 1.37–4.51; P=0.003) and a 2.04-fold increase in the risk of vascular events (95% CI 1.27–3.28; P=0.003). Serum hsCRP concentrations significantly modified the association of serum CysC with recurrent stroke (Pinteraction=0.001) and vascular events (Pinteraction=0.007). Moreover, CysC may improve reclassification of stroke recurrence (net reclassification improvement [NRI] 42.9%, P=0.001; integrated discrimination improvement [IDI] 1.2%, P=0.001) and vascular events (NRI 35.8%, P=0.001; IDI 1.1%, P=0.004).
Conclusions: In ischemic stroke patients with high hsCRP concentrations, higher CysC concentrations increased the risk of stroke recurrence and vascular events. This indicates that the predictive value of CysC on stroke recurrence may depend on the inflammation status of patients.
Stroke is the second leading cause of death worldwide,1 but the first leading cause of death and adult disability in China.2,3 Despite advances in therapies and risk factor modification, stroke recurrence is common.4,5 Inflammation plays a pivotal role in the risk of incident stroke and stroke recurrence. The underlying mechanism includes atherosclerosis, plaque rupture, thrombosis, and subsequent vascular events. Cystatin C (CysC), a 13-kDa endogenous cysteine protease inhibitor, is a sensitive and accurate surrogate for kidney function independent of muscle mass, age, and other potential confounders.6,7 Previous studies indicated that CysC is a useful alternative to creatinine for detecting the risk of incident stroke8 and has potential prognostic value to determine mortality or poor functional outcome after stroke.9,10 However, it is unclear whether CysC can predict stroke recurrence, as in predicting the risk of coronary artery disease (CAD).11
Circulating CysC concentrations are closely correlated with several inflammatory biomarkers.12,13 The predictive value of CysC on CAD may depend on C-reactive protein (CRP) concentrations,14 and the results of studies are inconclusive.15,16 Niccoli et al hypothesized that the prognostic value of CysC was related to the atherosclerotic plaque burden.15 Because the role of CysC in predicting stroke recurrence and its interaction with high CRP concentrations are unclear, this study explored the relationship between CysC and stroke recurrence according to inflammatory status in patients with stroke based on data from the China Antihypertensive Trial in Acute Ischemic Stroke (CATIS).
This study was a prospective study using data from CATIS. Details of CATIS (i.e., rationale, design, and major results) have been published elsewhere.16 Briefly, CATIS was a multicenter, single-blind, blinded endpoints randomized clinical trial conducted in 26 hospitals across China. In all, 4,071 eligible acute ischemic patients with elevated systolic blood pressure (SBP; between 140 and <220 mmHg) and within 48 h of symptom onset were enrolled.
CATIS has been registered with Clinicaltrials.gov (ID: NCT01840072). This study was approved by the institutional review boards at Soochow University in China and Tulane University in the US, as well as ethics committee at each participating center. All procedures in this study were performed in accordance with the Declaration of Helsinki and the ethical standards of the responsible committee on human experimentation (institutional or regional). All participants or their legal representatives provided written informed consent.
Data CollectionPatient demographic information, clinical features, and medical and medication history were collected at the time of enrollment. Clinical features included time from symptoms onset to hospitalization, baseline SBP and diastolic blood pressure (DBP), body mass index (BMI), and triglycerides.
Trained neurologists assessed stroke severity upon admission using the National Institutes of Health Stroke Scale (NIHSS).17 Ischemic stroke patients recruited to the study were classified into 3 stroke subtypes (large artery atherosclerosis, embolic, and lacunar infarctions) according to the subtype classifications of acute ischemic stroke in Trial of Org 10172 in Acute Stroke Treatment (TOAST).18
Three blood pressure measurements were obtained at baseline by trained nurses using a standard mercury sphygmomanometer according to the study protocol. In addition, routine laboratory examinations, such as blood triglycerides, creatinine, and glucose, were performed for all patients at participating centers according to the study protocol. Estimated glomerular filtration rate (eGFR) was calculated by using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation with an adjusted coefficient of 1.1 for the Asian population.19,20
Serum CysC and hsCRP MeasurementsThe fasting blood samples were collected within 24 h after admission. All blood samples were sent to Soochow University through cold chain transportation and were frozen at −80℃. Serum CysC concentrations were determined using a CysC assay kit and the latex enhanced immunoturbidimetric method (Sichuan Maker Biotechnology, China). The primary reference material ERM-DA471/IFCC was used to calibrate the CysC assays. Serum hsCRP concentrations were measured using a commercially available immunoassay (R&D Systems, Minneapolis, MN, USA). Biomarker concentrations were measured by experienced technicians blinded to the baseline characteristics and clinical outcomes of the study participants. A high inflammation status was defined as an hsCRP concentration ≥4.8 ng/mL (75th percentile).
Outcome AssessmentStudy participants were followed-up in person at 3, 12, and 24 months after stroke by trained neurologists who were blinded to treatment assignment and baseline information. The outcome included stroke recurrence (fatal and non-fatal recurrent stroke) and vascular events (vascular deaths, non-fatal stroke, non-fatal myocardial infarction, hospitalized and treated angina, hospitalized and treated congestive heart failure, and hospitalized and treated peripheral arterial disease) within 24 months after stroke.
Statistical AnalysisIn this analysis, participants were first divided into 2 groups according to optimal cut-off points of CysC (0.78 mg/L) identified by receiver operating characteristic (ROC) curves. Baseline continuous variables are presented as the mean±SD or median with interquartile range (IQR) and were compared using Student’s t-test or the Wilcoxon rank test between patients with higher (≥0.78 mg/L) and lower CysC (<0.78 mg/L) concentrations. Categorical variables are presented as frequencies (%) and were compared using χ2 tests between the 2 CysC groups.
Kaplan-Meier analysis was used to estimate cumulative incidence rates of stroke recurrence and vascular events according to CysC levels at baseline, with comparisons between groups made using the log-rank test. We further evaluated the association between serum CysC concentrations and stroke recurrence or vascular events using Cox regression models (3 models); hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated for all 3 models. Model 1 was an unadjusted Cox model. Model 2 was adjusted for age, sex, current smoking status, alcohol consumption, time from onset to randomization, SBP, plasma glucose, triglycerides, ischemic stroke subtype, baseline NIHSS scores, hypertension, CAD, use of antihypertensive medications before stroke onset, family history of stroke, and randomized treatment. Model 3 was further adjusted for eGFR and hsCRP. The likelihood ratio test was used to examine the modified effect of inflammation on the predictive value of CysC for stroke recurrence and vascular events after adjustment for the covariates in Model 3.
Furthermore, the net reclassification index (NRI) and integrated discrimination improvement (IDI) were calculated to assess the reclassification performance by adding CysC or creatinine concentrations to a basic model with several important prognostic factors (age, SBP, baseline NIHSS scores, hypertension, and ischemic stroke subtype) in different hsCRP concentration groups. All P values are 2-tailed, and significance was set at P<0.05. Statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).
In all, 3,474 patients were included in this analysis. The mean age of patients was 61.9±10.8 years and 1,256 (36.2%) were female. Of the patients in this study, 1,528 had high CysC concentrations (≥0.78 mg/L) and 1,946 had low CysC concentrations (<0.78 mg/L). The baseline characteristics of the study participants are given in Table 1. Compared with patients with lower CysC concentrations, those with higher CysC concentrations were more likely to be elderly and male and have higher SBP, creatinine, and serum hsCRP concentrations. In addition a greater proportion of those with higher CysC concentrations had hypertension, CAD, embolic stroke, and a high inflammation status, and more used antihypertensive medications. However, patients with higher CysC concentrations had lower DBP, fasting plasma glucose, and eGFR, and a lower proportion of alcohol consumption, family history of stroke, and thrombotic stroke, than those with lower CysC concentrations.
| High CysC | Low CysC | P value | |
|---|---|---|---|
| No. patients | 1,528 | 1,946 | |
| Demographic data | |||
| Age (years) | 64.8±11.0 | 59.6±10.1 | <0.001 |
| Male sex | 1,022 (66.9) | 1,196 (61.5) | 0.001 |
| Current smoker | 571 (37.4) | 706 (36.3) | 0.51 |
| Current alcohol drinker | 443 (29.0) | 632 (32.5) | 0.03 |
| Clinical features | |||
| Time from onset to hospitalization (h) | 10.3 [4.3–24.0] | 10.0 [4.5–24.0] | 0.99 |
| Baseline SBP (mmHg) | 167.9±17.1 | 165.3±16.9 | <0.001 |
| Baseline DBP (mmHg) | 96.2±11.3 | 97.0±10.9 | 0.03 |
| BMI (kg/m2) | 24.9±3.2 | 24.9±3.1 | 0.78 |
| Triglycerides (mmol/L) | 1.5 [1.0–2.2] | 1.4 [1.0–2.2] | 0.21 |
| Plasma glucose (mmol/L) | 5.7 [5.0–7.0] | 5.8 [5.1–7.4] | 0.001 |
| CR (μmol/L) | 77.0 [66.0–91.3] | 63.9 [53.2–74.0] | <0.001 |
| eGFR (mL/min/1.73 m2) | 96.8 [80.4–107.4] | 110.4 [101.3–119.2] | <0.001 |
| Admission NIHSS score | 5.0 [3.0–8.0] | 4.0 [2.0–8.0] | 0.21 |
| Medical history | |||
| History of hypertension | 1,246 (81.5) | 1,490 (76.6) | <0.001 |
| History of hyperlipidemia | 96 (6.3) | 149 (7.7) | 0.12 |
| History of diabetes | 260 (17.0) | 349 (17.9) | 0.48 |
| History of CAD | 195 (12.8) | 161 (8.3) | <0.001 |
| Family history of stroke | 247 (16.2) | 394 (20.3) | 0.002 |
| Medication history | |||
| Use of antihypertensive medications | 822 (53.8) | 884 (45.4) | <0.001 |
| Use of lipid-lowering medications | 47 (3.1) | 69 (3.6) | 0.44 |
| Ischemic stroke subtype | |||
| Thrombotic | 1,140 (74.6) | 1,515 (77.9) | 0.03 |
| Embolic | 96 (6.3) | 86 (4.4) | 0.01 |
| Lacunar | 325 (21.3) | 399 (20.5) | 0.58 |
| Randomized treatment | 770 (50.4) | 965 (49.6) | 0.64 |
| Inflammation status | |||
| hsCRP (ng/mL) | 2.5 [0.9–6.7] | 1.5 [0.6–3.8] | <0.001 |
| High hsCRP (≥4.8 ng/mL) | 490 (32.1) | 386 (19.4) | <0.001 |
Data are given as the mean±SD, median [interquartile range], or n (%). AA cut-off point of 0.78 mg/L cystatin C (CysC) was obtained from receiver operating characteristic curves. BMI, body mass index; BP, blood pressure; CAD, coronary artery disease; CR, creatinine; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; hsCRP, high-sensitivity C-reactive protein; NIHSS, National Institutes of Health Stroke Scale; SBP, systolic blood pressure.
Over a 2-year follow-up, 261 (7.5%) and 315 (9.1%) patients experienced recurrent stroke and vascular events, whereas 156 (4.5%) were lost to follow-up. Cumulative incidence rates of stroke recurrence and vascular events are shown in the Figure. Among patients with a high inflammation status, those with high CysC concentrations had a significantly higher cumulative incidence of stroke recurrence (log-rank P=0.001) and vascular events (log-rank P=0.001) than those with low CysC concentrations. However, among patients with a low inflammation status, there was no significant difference in cumulative incidence rates of stroke recurrence and vascular events between patients with high and low CysC concentrations (log-rank P>0.1 for both).
Cumulative incidence of the rate of (A,B) stroke recurrence and (C,D) vascular events according to cystatin C levels at baseline in patients with high (A,C) and low (B,D) inflammation status, defined as high-sensitivity C-reactive protein (hsCRP) concentrations ≥4.8 and <4.8 ng/mL, respectively.
The associations between CysC levels and risk of stroke recurrence or vascular events are given in Table 2. Among patients with a high inflammation status, those with high CysC concentrations had increased incidence rates of recurrent stroke (10.2% vs. 4.2%) and vascular events (14.1% vs. 7.0%) compared with patients with low CysC concentrations. After adjustment for age, baseline NIHSS scores, medical histories, and other important covariates in Model 2, high CysC concentrations were associated with a 2.39-fold increased risk of stroke recurrence (HR 2.39; 95% CI 1.33–4.30; P=0.004) and 2.01-fold increased risk of vascular events (HR 2.01; 95% CI 1.26–3.21; P=0.003) in the group with high inflammation status. These significant associations remained after further adjustment for eGFR and hsCRP. However, in the group with low inflammation status, high CysC concentrations were not significantly associated with an increased risk of recurrent stroke (HR 0.89; 95% CI 0.66–1.21; P=0.45) or vascular events (HR 1.01; 95% CI 0.76–1.35; P=0.92). A significant interaction was found between serum CysC and hsCRP concentrations on the risk of stroke recurrence (Pinteraction=0.001 in Model 3) and vascular events (Pinteraction=0.007 in Model 3).
| High inflammation statusA | Low inflammation status | Pinteraction | |||||
|---|---|---|---|---|---|---|---|
| Low CysC | High CysC | P value | Low CysC | High CysC | P value | ||
| Stroke recurrence | |||||||
| No. patients (%) | 16 (4.2) | 50 (10.2) | 120 (7.7) | 75 (7.2) | |||
| Model 1 | 1.00 | 2.57 (1.47–4.52) | 0.001 | 1.00 | 0.94 (0.71–1.26) | 0.68 | 0.002 |
| Model 2 | 1.00 | 2.39 (1.33–4.30) | 0.004 | 1.00 | 0.88 (0.65–1.19) | 0.41 | 0.001 |
| Model 3 | 1.00 | 2.48 (1.37–4.51) | 0.003 | 1.00 | 0.89 (0.66–1.21) | 0.45 | 0.001 |
| Vascular events | |||||||
| No. patients (%) | 27 (7.0) | 69 (14.1) | 126 (8.1) | 93 (9.0) | |||
| Model 1 | 1.00 | 2.09 (1.34–3.26) | 0.001 | 1.00 | 1.09 (0.83–1.43) | 0.53 | 0.01 |
| Model 2 | 1.00 | 2.01 (1.26–3.21) | 0.003 | 1.00 | 1.02 (0.77–1.35) | 0.89 | 0.007 |
| Model 3 | 1.00 | 2.04 (1.27–3.28) | 0.003 | 1.00 | 1.01 (0.76–1.35) | 0.92 | 0.007 |
Unless indicated otherwise, data show hazard ratios with 95% confidence intervals in parentheses. AHigh inflammation status was defined as hsCRP ≥4.8 ng/mL. Model 1 was an unadjusted Cox model. Model 2 was adjusted for age, sex, current smoking, alcohol consumption, time from onset to randomization, SBP, plasma glucose, triglycerides, ischemic stroke subtype, baseline National Institutes of Health Stroke Scale scores, hypertension, CAD, the use of antihypertensive medications before stroke onset, a family history of stroke and randomized treatment. Model 3 was adjusted for all the factors in Model 2 and further adjusted for eGFR and hsCRP. Abbreviations as in Table 1.
NRI and IDI values for serum CysC for stroke recurrence and vascular events are given in Table 3. In patients with high inflammation status, adding serum CysC to a basic model with important prognostic factors led to significant improvements in reclassifying patients at risk of recurrent stroke (NRI 42.9%, P=0.001; IDI 1.2%, P=0.001) and vascular events (NRI 35.8%, P=0.001; IDI 1.1%, P=0.004), whereas no such significant improvements were observed in patients with low inflammation status. In addition, adding creatinine to the basic model did not significantly improve the risk prediction of stroke recurrence and vascular events in either the high or low inflammation status groups.
| NRI (continuous) | IDI | |||
|---|---|---|---|---|
| Estimate (95% confidence interval), (%) |
P value | Estimate (95% confidence interval), (%) |
P value | |
| High inflammation statusA | ||||
| Stroke recurrence | ||||
| Conventional model | Reference | Reference | ||
| Conventional model + CysC | 42.9 (21.1, 64.7) | 0.001 | 1.2 (0.5, 1.8) | 0.001 |
| Conventional model + Cr | 9.9 (−14.7, 34.4) | 0.44 | 0.02 (−0.07, 0.1) | 0.61 |
| Vascular events | ||||
| Conventional model | Reference | Reference | ||
| Conventional model + CysC | 35.8 (16.5, 55.1) | 0.001 | 1.1 (0.4, 1.8) | 0.004 |
| Conventional model + Cr | 6.7 (−14.0, 27.5) | 0.53 | 0.008 (−0.03, 0.04) | 0.69 |
| Low inflammation status | ||||
| Stroke recurrence | ||||
| Conventional model | Reference | Reference | ||
| Conventional model + CysC | 3.4 (−10.8, 17.6) | 0.65 | 0.03 (−0.03, 0.1) | 0.29 |
| Conventional model + Cr | 1.0 (−13.5, 15.6) | 0.89 | 0.009 (−0.04, 0.05) | 0.71 |
| Vascular events | ||||
| Conventional model | Reference | Reference | ||
| Conventional model + CysC | 5.3 (−8.4, 19.0) | 0.45 | −0.001 (−0.02, 0.02) | 0.94 |
| Conventional model + Cr | 4.7 (−9.1, 18.5) | 0.50 | NA | 0.80 |
AHigh inflammation status was defined as hsCRP ≥4.8 ng/mL. The conventional model included age, SBP, baseline NIHSS, hypertension, and ischemic stroke subtype. IDI, integrated discrimination index; NA, not available; NRI, net reclassification improvement. Other abbreviations as in Table 1.
This study showed that higher CysC concentrations increased the risk of stroke recurrence and vascular events within 24 months in patients who had had a stroke and who had a higher inflammation status. No such association was found in those with lower hsCRP concentrations. Those patients with higher hsCRP and CysC concentrations had a higher cumulative risk and greater HRs of stroke recurrence and vascular events after adjustment for important confounding factors. Furthermore, adding serum CysC to a basic model with important prognostic factors, including age, SBP, baseline NIHSS scores, hypertension, and ischemic stroke subtype, improved the reclassification of the model for recurrent stroke and vascular events.
Circulating CysC is one of the major biomarkers secreted by all nucleated cells and has a role in vascular remodeling and atherosclerosis. Moreover, CysC is a sensitive indicator of kidney function and is better than creatinine-derived eGFR in detecting a preclinical stage of renal disease. Thus, higher circulating CysC concentrations may be indicative of occult chronic kidney disease,21 which is closely related to stroke outcome. Epidemiological studies have shown that elevated circulating CysC concentrations are an independent risk factor for mortality and cardiovascular disease22 or stroke,23 with a nearly linear relationship. In addition, CysC increases the risk of poor outcomes after acute coronary syndrome.24,25 In stroke patients, associations were also observed between elevated CysC concentrations and the risks of poor outcome and mortality.9,10 However, the relationship between CysC and the risk of stroke recurrence has rarely been explored. Recently, a single-center study documented that higher CysC concentrations were associated with recurrent ischemic stroke over a 1-year follow-up.26 However, that study did not consider other potential confounding variables that could have affected the relationship between CysC and stroke outcomes, such as age, sex, and inflammation. Evidence suggests that inflammation not only plays a central role in the risk of stroke, but that it is also correlated with CysC concentrations.11,27,28 A cohort study found that CysC was significantly associated with the risk of CAD even after adjusting for traditional risk factors, but this association disappeared after adjustment for CRP, suggesting that the predictive value of CysC may be dependent on CRP.14 The present study supports the assumption that CysC has predictive value for stroke recurrence, but only in the setting of a higher hsCRP concentration. A significant interaction between CysC and hsCRP was also detected in this study. These findings suggest that inflammation modifies long-term prognostic values of baseline CysC on stroke recurrence.
The mechanisms underlying the prognostic role of CysC in stroke recurrence remains to be elucidated. Kidney dysfunction and atherogenesis are 2 proposed pathophysiological etiologies. CysC is involved in the progression of atherosclerosis. Previous studies showed that CysC was highly correlated with the extended burden of carotid atherosclerosis29 and coronary atherosclerosis.15 In addition, CysC is regarded as an indicator of endothelial dysfunction throughout the vascular system. High concentrations of CysC may indicate extended cerebrovascular damage and an increased the risk of stroke recurrence. Inflammation is also closely associated with the development of atherosclerosis and plays an important role in stroke recurrence.30 A high concentration of hsCRP may promote the detrimental effect of CysC and increase the risk of stroke recurrence. The present study showed that CysC increased the risk of stroke recurrence in patients with acute ischemic stroke, but this predictive value of CysC was affected by the inflammation status. This suggests that inflammatory status should be considered when focusing on the predictive value of CysC for stroke recurrence in patients with an acute ischemic stroke.
This study was conducted based on a large-scale, multicenter, randomized clinical trial with strict quality control for data collection and outcome assessment. Nearly all important confounders were adjusted for in the analysis. However, several limitations need to be considered. First, a selection bias was inevitable because patients with extremely high blood pressures (SBP/DBP ≥220/120 mmHg) were excluded. Nevertheless, the baseline features of participants in this study were basically similar to those from the China National Stroke Registry.31 Second, serum CysC was measured only once at baseline. Repeated measurements at different time points may provide information on dynamic changes between the acute and recovery stages of stroke. Third, the detailed subtypes of recurrent stroke (ischemic or hemorrhagic) were not recorded when data were collected, although ischemic stroke is well-known predominant type of stroke in China. Fourth, study participants were from China and so the findings of this study need to be verified in other populations.
Higher CysC increased the risk of stroke recurrence and vascular events in stroke patients with a high hsCRP concentration. This predictive value of CysC on stroke recurrence was related to inflammation status in stroke patients.
The authors thank the study participants, their relatives, and the clinical staff at all participating hospitals for their support and contribution to this project.
The authors declare no conflicts of interest.
This study was supported by the National Key Research and Development Program of China (Grant no. 2016YFC1307300), the National Natural Science Foundation of China (Grant no. 81673263, 81701149), and a Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions, China; Jiangsu Provincial Medical Key Discipline Project (ZDXKB 2016022).
This study was approved by the institutional review boards at Soochow University, China, and Tulane University, USA (Reference no. 2007IRB1).
The deidentified participant data will not be shared.
