2.1 Study participants
The present study was performed based on the data from the China Antihypertensive Trial in Acute Ischemic Stroke (CATIS), a multicenter, single-blind, blinded end points, randomized clinical trial conducted in 26 hospitals across China to test whether moderate lowering of blood pressure (BP) within the first 48 hours after acute ischemic stroke onset would reduce death and major disability at 14 days or hospital discharge. Details about the CATIS design, methods and main results have been previously reported(13). Briefly, a total of 4071 ischemic stroke patients aged 22 years or older confirmed by CT or MRI of the brain within 48 hours of symptom onset with an elevated systolic BP between 140 mmHg and less than 220 mmHg were recruited in this trial. Patients with a BP ≥220/120 mm Hg, severe heart failure, acute myocardial infarction or unstable angina, atrial fibrillation, aortic dissection, cerebrovascular stenosis, or resistant hypertension, in a deep coma, or those treated with IV thrombolytic therapy were excluded from the CATIS trial. In the present study, 692 participants were further excluded because they did not offer blood samples, or some collected samples were hemolyzed in collections of blood samples, or we failed to determine plasma ANGPTL-4 concentrations.
2.2 Data collection
Baseline data including demographic characteristics, clinical characteristics, medical histories, and lifestyles were collected at the time of enrollment using a standard questionnaire. Stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS) by trained neurologists at baseline(14). Three BP measurements were obtained at baseline by trained nurses according to a common protocol adapted from procedures recommended by the American Heart Association(15). BP was measured with the participant in a supine position using a standard mercury sphygmomanometer based on arm circumference of participant. The mean of three BP measurements was used in analyses. Fasting blood samples were collected within 24 hours of hospital admission after at last 8 hour of fasting. Routine laboratory analyses (fasting blood glucose, blood lipids, blood creatinine, etc.) were performed for all enrolled patients, in each participating hospital at admission. All plasma and serum samples were frozen at -80°C in the Central Laboratory of School of Public Health in Soochow University until laboratory testing.
2.3 ANGPTL-4 detection
Plasma ANGPTL-4 concentrations were measured centrally at Soochow University with a commercially available DuoSet ELISA kit (R&D Systems, Minneapolis, MN). Intra-assay and interassay coefficients of variation were 3.3% and 3.9%, respectively. Laboratory technicians who performed these measurements were blind to the clinical characteristics and outcomes of the study participants.
2.4 Study outcomes
Participants were followed up in person at 3 months after ischemic stroke by trained neurologists. The primary outcome of present study was a combination of death and major disability (modified Rankin Scale [mRS] score ≥3). Secondary outcomes were separately those of death and major disability (modified Rankin scale score of 6 and 3–5, respectively), stroke recurrence and vascular disease events (i.e., recurrent nonfatal stroke, nonfatal myocardial infarction, hospitalized and treated angina, hospitalized and treated congestive heart failure, and hospitalized and treated peripheral arterial disease).
2.5 Statistical analysis
All participants were divided into 3 groups according to tertiles of plasma ANGPTL-4 concentrations and baseline characteristics were compared across 3 groups. The generalized linear regression analysis was used to test for trend across the tertiles of ANGPTL-4 for continuous variables, and the Cochran-Armitage trend χ2 test was used for categorical variables. Multivariate logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs) of primary and second outcomes for the highest tertile of ANGPTL-4 concentrations compared to the lowest tertile and for 1-SD increment of log-transformed ANGPTL-4 concentrations (0.44 ng/ml). Important conventional covariates like age, sex, antihypertensive treatment, time from onset to hospitalization, current smoking, current alcohol drinking, body mass index, dyslipidemia, heart disease, high-density lipoprotein cholesterol, fasting plasma glucose, history of hypertension, history of diabetes mellitus, family history of stroke, SBP at baseline, ischemic stroke subtypes and baseline NIHSS score were included in the multivariable models. Restricted cubic splines were performed to explore the shapes of the associations between plasma ANGPTL-4 concentrations and adverse clinical outcomes with 4 knots (at the 5th, 35th, 65th, and 95th percentiles). Furthermore, C statistics, net reclassification index (NRI), and integrated discrimination improvement (IDI) were used to evaluate the incremental prognostic value of plasma ANGPTL-4 concentrations beyond conventional risk factors (covariates in multivariable models)(16, 17).
Given that circulating ANGPTL-4 concentrations might be associated with stroke-related risk factors (i.e., obesity, metabolic syndrome, hypertension and diabetes mellitus). Thus, we performed subgroup analyses to determine the potential effect modification stratified by sex, age, baseline systolic BP, body mass index, dyslipidemia, current smoking, alcohol consumption, fasting plasma glucose and receiving immediate BP reduction in multivariate adjusted logistic regression models. Interactions between circulating ANGPTL-4 concentrations and covariates on primary outcome were tested by the likelihood ratio test of models with interaction terms. Two-tailed p < 0.05 was considered to be statistically significant. All statistical analyses were conducted using SAS statistical software (version 9.4).