ABC | Volume 114, Nº3, March 2020

Original Article Leocádio et al. Netrin-1 and IL-1 β : prognosis in ACS Arq Bras Cardiol. 2020; 114(3):507-514 Methods Study design The patients were participants of the “ERICO” (Strategy of Registry of Acute Coronary Syndrome) study, described in detail in previous reports. 14,15 Briefly, it is a prospective cohort study that included individuals admitted to treatment for ACS at the University of São Paulo Hospital (HU-USP), a teaching community hospital with 260 beds located in the District of Butantan, São Paulo, Brazil, from February 2009 to December 2013. The study protocol was in accordance with the Declaration of Helsinki. This study was approved by the Research Ethics Committee (CEP-HU/USP 866/08), and all patients signed the Informed Consent Form. Acute myocardial infarction (AMI) was defined by the presence of symptoms consistent with myocardial ischemia within 24 hours of hospital admission and troponin I level above the 99th percentile value with a coefficient of variation <10%. ST-segment elevation myocardial infarction (STEMI) was defined by the criteria for AMI, in addition to (a) the presence of persistent ST-segment elevation ≥1 mm in two contiguous electrocardiographic leads (lead ECG) or (b) new (or supposedly new) left bundle branch block (LBBB). Non‑ST‑segment elevation myocardial infarction (NSTEMI) was defined by the criteria for AMI plus the absence of persistent ST-segment elevation ≥1 mm in two contiguous ECG leads and of new or supposedly new LBBB. Unstable angina (UA) was defined as the presence of symptoms compatible with myocardial ischemia in the last 24 hours, absence of AMI diagnosis and at least one of the following five criteria: (a) history of previous coronary artery disease; (b) positive stratification of invasive or non-invasive ischemic heart disease; (c) dynamic or evolutionary ECGchanges; (D) troponin I >0.4 ng/mL (ensuring troponin I levels above the 99th percentile regardless of the utilized kit) or (e) agreement on UA diagnosis between two independent physicians. Data collection and outcomes After 6 months and annually for 2 years after the hospital admission, all individuals were contacted by telephone to update vital status information, including fatal and nonfatal cardiovascular outcomes. Whenever a participant reported a potential new MI event, new investigation procedures were initiated to confirm the event. Study outcomes were all-cause mortality, cardiovascular mortality, and the combined outcome (fatal AMI and new non-fatal AMI). The strategy for collecting and classifying mortality data, including searching for official death records, was detailed in a previous report. 15 In cases where it was not possible to determine the cause of death, the data were censored for all outcomes, except for death from all causes. During the hospital phase, trained interviewers collected data related to sociodemographic characteristics, cardiovascular risk factors, and medication, as previously described. 15 Blood samples were collected within 24 hours of admission. Analyses of plasma glucose, triglycerides, and total and HDL cholesterol were performed at HU-USP. LDL cholesterol was calculated using the Friedewald equation. 16 Concentrations of Netrin-1 and IL-1 β on admission were evaluated by Enzyme‑Linked Immunosorbent Assay (ELISA), following the kit instructions (Netrin-1: SEB827HU; USCN Life Science Inc., Wuhan, China and IL-1 β : 88-7010-88 eBioscience Inc., San Diego, CA, USA). Patients were classified according to Netrin-1 and IL-1 β concentrations in “low” and “high” groups if their concentration were below or above the median. Statistical analysis Da t a we r e a s s e s s ed f o r no rma l i t y us i ng t he Kolmogorov‑Smirnov test. The chi-square and Mann-Whitney (all continuous variables presented nonparametric distribution) tests were used to compare groups. Values were expressed as median (interquartile interval) or n (%). Kaplan-Meier curves were used, and the log-rank test was used to evaluate the difference between low and high groups. Risk estimates (hazard ratios with their respective 95% confidence intervals) for the events were calculated using Cox regression. In addition to Netrin-1 and IL-1 β , the following variables were used to construct models: age, type of ACS, diabetes, hypertension, and dyslipidemia. A two-tailed p-value < 0.05 was considered significant. The software programs SPSS (IBM SPSS Statistics for Windows, version 22.0, Armonk, NY: IBMCorp.) and GraphPad Prism (version 5.01 for Windows, San Diego, California: GraphPad Software) were used to carry out the analyses. Results A total of 803 patients were included in this study, including 333 women and 470 men. Comparing the main characteristics of male and female groups, we observed that women were older and had higher HDL-c concentration than men. Women were also more frequently affected by hypertension, diabetes, and dyslipidemia (Table 1). The most frequent type of ACS in male and female groups was NSTEMI (about 40% of cases) followed by UA and STEMI that had a similar frequency (about 30% each). During the 2 years follow up, there were 115 deaths from all causes (65 men and 50 women) including 78 deaths (67.8%) due to cardiovascular causes. We also identified 67 cases of AMI (fatal or non-fatal) in this same follow-up. Since age is an important factor involved in the mortality rate, we analyzed separately in women andmen younger and older than 60 years. To evaluate a possible role of Netrin-1 and IL-1 β as prognostic markers, we compared the frequency of ACS in the patients with levels of Netrin-1 and IL-1 β levels above and below the respective median. There were no associations between levels of Netrin-1 and IL-1 β and all-cause mortality, cardiovascular mortality and fatal or new non-fatal MI outcomes for males independently of their age (data not shown). For this reason, we focused our investigation on the female group (333 patients). The main characteristics of the female group (younger and older than 60 years) are shown in Table 2. At admission, women presented similar values of BMI, serum concentrations of glucose, triacylglycerol, and HDL cholesterol regardless in both age groups. The frequency of important risk factors such as hypertension, dyslipidemia and diabetes were higher in older women. However, levels of LDL cholesterol were lower in older ones. Current smokers 508