Prognostic value of ST2 for MACEs and all-cause mortality in patients with coronary artery disease during a long-term follow up

Purpose ST2 has been proved the prognostic value in acute coronary syndrome (ACS), its prognostic value to predict cardiac events in established coronary artery disease (CAD) patients is unknown. The study ought to investigate the prognostic value of ST2 in patients with established coronary artery disease. Methods A total of 3650 consecutive patients were included in the study. The primary end point was major adverse cardiovascular events (MACEs). The secondary end point was all-cause death. To explore competing risks, cause-specic hazard ratios were obtained using Cox regression models. Results Kaplan–Meier indicated that the with had a signicantly increased risk of MACEs (log-rank p<0.001)and all-cause death(log-rank p<0.001). After adjustment for potential confounders, multiple COX regression models showed that higher level of ST2 was an independent predictor in developing MACEs(HR 1.31; 95% CI: 1.13–1.52; p<0.001) and all-cause death(HR 1.78; 95% CI: 1.38–2.30; p<0.001). We saw a signicant increase of AUC in ROC curve after addition of GDF-15 to a clinical model 0.586 vs 0.619 For MACEs (p<0.001).For long-term all-cause death the increase of AUC 0.766 vs 0.642 (95% CI 0.787–0.846(p<0.001).


Introduction
Coronary artery disease (CAD) remains the leading cause of death of the world [1]. Patients with previous coronary heart disease have a high probability of major adverse cardiac events(MACEs).This underscore the need for development of reliable prognostic and outcome biomarker that would be of vital importance in established CAD patients. Suppression of tumorigenesis-2 (ST2) is an interleukin-1 (IL-1) receptor family member. It exists in two isoforms: membrane-bound and soluble isoforms [2]. Previous studies has been suggested that IL-33 acts as an "alarm" to alert potential tissue stress or damage.IL-33/ST2 signaling protects the myocardium against hypertrophy and cardiac brosis following pressure overload [3,4]. An IL-1-related protein, called interleukin (IL)-33 was identi ed as a functional ligand for ST2 [5].Soluble ST2 is identi ed as a novel biomarker in in ammatory conditions and cardiovascular disease.
It has been widely reported that ST2 is a strong predictor of all-cause and cardiovascular mortality in patients with heart failure (HF) [6] [7] [8]. In ischemic heart disease, ST2 levels are elevated in non-ST segment elevation Acute Coronary Syndrome(NEST-ACS) and predict 1-year mortality [9]. In ST elevation myocardial infarction, ST2 levels in the upper quartile observed in ischemic heart disease(IHD) independently predict cardiovascular death and heart failure with an approximate doubling of risk [10].However, in long-term follow-up of CAD, whether ST2 is predictive of MACEs and all-cause remains inconclusive. We thus performed a large prospective large scale study. The aim of the present study is to evaluate the prognostic value of ST2 on MACEs and all-cause mortality in CAD during a long-term follow up.

2.1The study population
This study was an observational prospective study enrolling patients admitted as CAD in Chinese PLA general hospital between 2011 and 2015.A total of 4087 patients with an indication for diagnostic coronary angiography (CAG) and/or percutaneous coronary intervention (PCI) due to stable angina pectoris (SAP) or acute coronary syndrome (ACS) at our hospital were included.
All included patients were 18 years or older. Then 84 patients with the detailed data lost and 112 patients without angiographically determined CAD were excluded. 57 patients with one of the following diseases (severe heart failure, atrial brillation, aortic dissection, active infective disease, history of malignancy, end stage of renal disease, those in a deep coma) were excluded. 184 patients lost to follow-up were also excluded. As described in the owchart, the nal study group include 3650 patients. (Fig. 1).
Patients were considered to be hypertension with BP > 140/90 mmHg or under anti-hypertensive medication. Hyperlipidemia was de ned as known but untreated dyslipidemia or current treatment with lipid-lowering medications. Diabetes mellitus(DM) was de ned as the presence of symptoms of diabetes and a resting plasma glucose concentration ≥ 200 mg/dL, a fasting plasma glucose concentration ≥ 126 mg/dL, a 2-h plasma glucose concentration ≥ 200 mg/dL in a 75 g oral glucose tolerance test, or taking hypoglycemic agent or other medications for DM. Current smoking was de ned smoking if they reported any tobacco use in the last 30 days.

Blood samples
Blood samples were collected in the early morning. On the basis of protocol, the blood were obtained by the EDTA-anticoagulated plastic tubes. All the blood samples were centrifuged at 1000 g for 10 min and serum samples were stored at 80 ℃. The plasma levels of total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C) and triglycerides (TG) levels were measured using commercial reagents following standard procedures. The ST2 levels were determined in serum in single measurements by using a quantitative sandwich monoclonal enzyme-linked immunosorbent assay (Presage ST2 Assay, Critical Diagnostics, Inc., San Diego, California).

Follow up and outcomes
Patients were followed up until February 2020 or until the occurrence of cardiovascular event. All participants were followed up by analyses of clinical materials and telephone contact quarterly. The primary endpoint was MACEs, the second endpoint was all-cause death. MACEs was de ned as cardiac death, unstable angina, myocardial infarction and unplanned revascularization. All deaths were considered cardiac unless a de nitive non cardiac cause was established. Myocardial infarction was de ned as the rise of cardiac biomarkers with evidence of myocardial ischemia. Unstable angina pectoris was de ned as new or accelerating symptoms of myocardial ischemia accompanied by new ischemic ST-T changes. Coronary revascularization was diagnosed if the patient underwent percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) with evidence of myocardial ischemia. We obtained follow-up for all patients until the primary outcome or date of censoring. All-cause death was de ned as death from any cause. Written informed content was obtained from all study participants, and the study was approved by the ethics committee of Chinses PLA General Hospital.

Statistical analysis
Variables with a normal distribution are presented as mean ± SD, whereas in case of nonnormality the medians are presented. Categorical data are presented as counts or percentages. Patients were divided into two groups according to the median level of ST2 and differences in baseline characteristics between the two groups were evaluated by chi-square tests (categorical variables), analysis of variance as appropriate. Spearman correlation coe cient was used to assess the correlation between ST2 values and other continuous variables. Kaplan-Meier curves was used to estimate the cumulative incidence risks of outcomes across baseline ST2 levels and compared by log-rank tests. Cox proportional hazards models were used to evaluate the association of baseline ST2 levels with the study endpoints. The results are presented as the hazard ratios (HRs) and 95% con dence intervals (CIs) according to levels of ST2.We tted two multivariate proportional hazards models. Model 1 was adjusted for clinical variables include age, sex, BMI, current smokers, hypertension, hyperlipidemia, diabetes mellitus, previous myocardial infarction(MI), previous PCI/CABG,TC,TG,HDL-C,LDL-C. Model 2 was adjusted for ST2 level and Moedel1.The relation of ST2 levels with outcomes is presented with COX proportional hazard models both with ST2 as a continuous variable and with ST2 as a categorical variable. Area under ROC curve was used to compare the predictive ability of the parameters of interest. SPSS version 21.0 software (IBM Corp., Armonk, New York) was used for descriptive data analysis. All statistical tests were 2-tailed, and p values < 0.05 were considered statistically signi cant.

Baseline characteristics of CAD patients.
Baseline measurements of ST2 were available in 3650 patients. The median concentration of ST2 was 19 ng/ml. The baseline characteristics of the consecutive CAD patients are shown in Table 1. we divided the patients into two groups based on the median concentrations of ST2.Patients with ST2 ≥ 19 ng/ml was older, more often men, higher rate of previous PCI/CABG. They also had a higher level of TG. (Table 1).

Correlations of ST2 levels with other clinical biochemical factors
Patients with higher levels of ST2 at presentation were older, more males, higher rate of previous PCI/CABG, higher level of TG(    (Fig. 4b).
4 Discussion Our study established a higher level of ST2 was a signi cant and independent predictor of cardiovascular event. In this study, we found that higher concentrations of ST2(≥ 19 ng/ml) was associated with an increased risk of all-cause death in patients with coronary heart disease. Higher concentrations of ST2 remained an independent indicator of MACEs and all-cause mortality after adjustment for established risk factors for CV disease and other prognostic biomarkers. Furthermore, our study con rmed that the incremental prognostic value of ST2 for MACEs and all-cause mortality beyond the clinical model by ROC curve analysis .Our results provide updated information on the long-term prognostic role of ST2 in established CAD patients. Our results suggest that the addition of plasma ST2 measurements to established CV risk factors may further improve risk strati cation.
Biomarkers have become increasingly important tools helping to improve patient care over the past two decades. Numerous biomarkers have been identi ed in the diagnosis, prognosis and risk prediction of cardiovascular disease but few have made their way to clinical practice [11]. The most extensively used cardiovascular biomarkers are the natriuretic peptides in the diagnosis and prognosis of heart failure and cardiac troponins in the diagnosis of acute myocardial infarction. Deeper experimental studies of the pathophysiology of atherosclerosis have identi ed a large number of molecules as potential prognostic biomarkers in cardiovascular disease [12].Previous studies suggested that ST2 maybe a potential biological marker for mechanical overload in the heart.ST2 was markedly upregulated in mechanicallystimulated cardiomyocytes. Furthermore, ST2 has been proved to be a predictor of outcome in patients with HF [6] [13] [8] [14].Recent evidences suggest that ST2 may be predictive in patients with ACS [15] [16]. It has been shown to be a powerful independent prognosticator for patients with acute myocardial infarction (AMI) [6] [7].According to Eggers KM 's research, ST2 levels are elevated early in NSTE-ACS and predict 1-year mortality [9]. Wang YP's research showed that Serum levels of ST2, IL-33 and BNP were positively correlate with MACEs in patients with acute myocardial infarction (AMI) after percutaneous coronary intervention (PCI) [17].However, there was no to investigate the long-term value of ST2 in the prediction of MACEs or all cause death in patients with CAD in a large population.
The in ammatory hypothesis of atherosclerosis suggests that in ammatory cell signaling drives the formation, development, and eventual instability of atherosclerotic plaques [18]. Under this respect, the IL-33-ST2 pathway deserves consideration. In fact, ST2 are particularly expressed in arterial endothelial cells, involving in the progression of atherosclerosis [19] [20].IL-33 was originally reported as a modulator of in ammation, tipping the balance towards CD4 + T helper-cell type 2 mediated immune responses [21]. IL-33 may play a protective role in the development of atherosclerosis. The effect of IL-33 on the function of foam cells indicated the protect role of IL-33 in atherosclerosis [22]. ST2 acts as a decoy receptor for IL-33, thus blocking its protective effects. It has been reported that mice treated with soluble ST2 developed signi cantly larger atherosclerotic plaques in the aortic sinus of the ApoE(-/-) mice compared with the control mice [23].These results suggested that ST2 may be proposed as a marker of plaque burden and predictors of future cardiovascular event [24].Although the above data suggest that ST2 has a role in the prognosis of patients presenting with an acute coronary syndrome, whether ST2 contributes to cardiovascular risk prediction in a large scale CAD patients during a long-term follow up remains uncertain.
To evaluate the prognostic value of a biomarker in CVD, researchers must demonstrate the elevated risk of an cardiovascular events associated with higher levels of the new biomarker with adjustment for other established risk factors. The results should be presented as hazards ratios relative risk estimates from a Cox model and a probability value test of signi cance of the marker in the multivariable models [25].  [29].Our result demonstrated that in a median follow up of 6.4 years, higher level of ST2 is signi cantly associated with all-cause death, MACEs and provides incremental prognostic value beyond traditional risks factors.

Limitations
While the study provides a large, well characterized study sample with adjudicated outcomes, the research is limited to a single center, these data represent the results of an observational analysis in a clinical trial population. As in any observational study, we cannot exclude residual confounding. However, this is probably minimal because we used a comprehensive adjustment strategy to control for known variables that are commonly used to stratify the risk of CAD patients.

Conclusions
Higher values of ST2 confer a markedly adverse prognosis characterized by a large excess risk of MACEs and all-cause death over a long period of follow-up. Measurement of ST2 should be considered as part of approaches to risk strati cation in CAD patients during a long-term follow up. Availability of data and material

Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  Flowchart of the study Figure 3 Kaplan-Meier curves for prediction of MACEs(a) and all-cause death in patients with higher levels of ST2 (ST2≥19ng/ml) and lower levels of ST2 ST2 19ng/ml Figure 4 ROC curve analyses that relate ST2 levels to MACEs (a) and all-cause death (b).