This prospective cohort study was conducted in the Department of Cardiology and Clinical Pharmacology, SRM MCH and RC, Kattankulathur, between January 2016 and March 2018. The study was approved by the SRM Institutional Ethics Committee (IEC), India (664/IEC/2014), SRM Medical College Hospital and Research Centre, Kancheepuram and all study procedures were performed in accordance with the provisions of the Declaration of Helsinki. Written Informed consent was obtained from all the patients who participated in the study.

Study population: The study group comprised of consecutive patients who presented with ACS and were hospitalised in the Department of Cardiology between January and August 2016. Patients above 18 years of age, of both gender and willing to give informed consent and comply with all study-related procedures, were included in the study. ACS patients comprised of both STEMI and NSTEMI/UA. The condition STEMI was defined as a clinical syndrome with the presence of clinical symptoms associated with myocardial ischaemia, a constant ST elevation and release of biomarkers associated with necrosis [12]. NSTEMI/UA was defined as a clinical syndrome where there is a persistent absence of ST elevation and the release of necrotic markers [13]. Patients with other inflammatory diseases such as infections, cancer, Heart failure (HF), Chronic Stable Angina (CSA) and autoimmune diseases were excluded from the study [Table/Fig-1].

[Table/Fig-1]:

Flow chart illustrating the recruitment process.

Laboratory investigations: From each patient, 4 mL of peripheral blood was collected from the vein in the forearm prior to CAG in an Ethylenediaminetetraacetic Acid (EDTA) coated vacutainer tubes within 48 hours of admission in the cardiac intensive care unit. After adequate centrifugation at 2500 revolutions per minute (rpm) for 10 minutes, the serum samples were extracted and were stored at -80°C in deep freezer. The level of Serum ANGPTL-4 was estimated using ELISA with a commercially available (Ray Biotech Human ANGPTL-4 ELISA, GA, USA) kit. The inter and intra assay coefficient of variation was 8.8% and 8% respectively. The laboratory measurements were performed in a blinded manner and the investigators performing the assay were unaware of the patient diagnosis.

Follow-up: The patients were followed up through a telephonic interview to record MACE. MACE was defined as cardiovascular mortality, re-hospitalisation, recurrent MI, cerebrovascular diseases and CABG [14].

The normality of data for continuous variables was checked using Q-Q plot in SPSS. Continuous variables were summarised as the mean±standard deviation (SD) or median (IQR) and categorical data were expressed as frequency with percentages. Differences in the categorical variables between groups were evaluated using the chi-square test. Parametric or non parametric tests were used based on the distribution of data. The differences in continuous variables between groups were analysed with Independent samples t-test based on the normality of data. The optimal cut-off level of ANGPTL-4 to predict MACE in ACS patients was evaluated using the area under the ROC curve. Kaplan-Meier survival analysis was performed and the survival rate between those with high and low ANGPTL-4 levels was compared using the log-rank test. All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) software version 16.0 (SPSS Inc., Chicago, IL). All p-values less than 0.05 were considered statistically significant.

Baseline characteristics of study patients: The study sample comprised of ACS patients namely NSTEMI/UA (n=63) and STEMI (n=57). The study patients were compared for characteristics such as age, gender, diabetes mellitus, hypertension, smoking habits, dyslipidaemia, and family history of coronary artery disease, renal disorders and sedentary lifestyle [Table/Fig-2]. In comparison, the study showed no significant differences in study characteristics like age, laboratory parameters and clinical risk factors.

Follow-up: During the median follow up of 24 (22-25.75) months, MACE had occurred among 14 patients. The total number of events occurring among NSTEMI/UA and STEMI patients were 9 (7.5%) and 5 (4.16%) respectively. Among the STEMI patients, five patients died due to cardiovascular causes and among the NSTEMI/UA patients, five died due to cardiovascular causes, three were re-hospitalised and one underwent CABG. Ten patients were lost to follow-up. Moreover, there were no significant differences in the demographic, clinical and laboratory study characteristics among the study patients based on the status of MACE. However, the presence of diabetes (p=0.002) and hypertension (p=0.013) were significantly higher among patients with MACE than among patients without MACE [Table/Fig-3]. ROC was plotted to predict the ability of ANGPTL-4 to predict MACE among ACS patients. However, ANGPTL-4 failed to predict MACE among ACS patients {AUC=0.493, p=0.935; (95% CI 0.321-0.665)}. Kaplan Meier survival curve was plotted for patients with low and high ANGPTL-4 levels and the log-rank test failed to show a significant difference in the survival rate between the two groups (χ² = 0.100, p=0.75) [Table/Fig-4].

[Table/Fig-4]:

Comparision of survival curves between patients with high and low angiopoietin-like 4 (ANGPTL-4).

High ANGPTL-4 was not a significant predictor of major adverse cardiac events in acute coronary syndrome patients (χ²=0.100, p=0.75)

Prediction of MACE in diabetic and hypertensive ACS patients: As the occurrence of MACE was higher among diabetic and hypertensive ACS patients, the Kaplan Meier survival curve was plotted for patients with low and high ANGPTL-4 levels for both the diabetic and hypertensive patients with ACS. On performing the analysis, the log-rank test failed to show any significant difference in the survival rate in both diabetic (χ²=0.002, p=0.97) and hypertensive (χ²=0.002, p=0.96) ACS patients. ROC was plotted to predict the occurrence of MACE among diabetic and hypertensive ACS patients. With regard to the diabetic {AUC=0.466, p=0.741; (95% CI 0.251-0.681)} and hypertensive {AUC=0.386, p=0.292; (95% CI 0.166-0.606)} ACS subjects, ANGPTL-4 failed to predict MACE among the study patients.

To the best of our knowledge, this is the first prospective cohort study conducted in the Indian population, where authors have investigated the prognostic ability of ANGPTL-4 for predicting MACE in ACS patients. However, the present work failed to demonstrate the prognostic ability of ANGPTL-4 in predicting MACE among ACS patients. In an earlier study reported by Muendlein A et al., among 490 Caucasian patients, no significant difference was observed in the levels of circulating ANGPTL-4 between patients with and without CAD [15]. In a prospective study, the plasma ANGPTL-4 and ANGPTL-4 variants predicted cardiovascular events independently from conventional cardiovascular risk factors. In another recent study performed among COPD patients, it was reported that ANGPTL-4 was a predictor of atherosclerosis and cardiac dysfunction in COPD patients independent of established risk factors such as high sensitivity C-reactive protein (hsCRP) [16]. The difference in the study population, sample size and follow-up period may have led to the different study findings. The study reported by Muendlein A et al., included a larger group of CAD patients and the follow-up period was much longer compared to the present study [15]. The study reported by Waschki B et al., included COPD patients, which is a population very diverse from present study. ANGPTL-4 encodes a glycosylated; secreted protein containing a C-terminal fibrinogen domain which is highly expressed in the liver, heart, muscle and adipose tissue. This encoded protein is induced by the peroxisome proliferator-activated receptors and acts as a serum hormone that is involved in the regulation of glucose homeostasis, lipid metabolism, and insulin sensitivity [16]. Furthermore, this protein is a strong LPL inhibitor and plays a significant role in modifying LPL activity in conditions of fasting and exercise [17,18].

In present study, the occurrence of MACE events was significantly higher among diabetic and hypertensive ACS patients. Therefore, authors investigated the prognostic ability of serum ANGPTL-4 for predicting the occurrence of MACE among both diabetic and hypertensive ACS patients, but the biomarker did not help in distinguishing patients who were likely to develop events in this subset. Diabetes and hypertension are well-known risk factors for the occurrence of cardiovascular disease [19]. However, not all patients with these risk factors have similar outcomes. Having a circulatory biomarker with prognostic ability will enable us to identify the subset of diabetic and HTN patients; who require more aggressive therapeutic measures. The protein marker ANGPTL-4 is being widely studied among diabetic and hypertensive subjects [20]. Previous genomic studies have reported that ANGPTL-4 variants E40K and T266M were associated with the cardiovascular disease risk among T2DM (type II diabetes mellitus) patients [21,22]. Furthermore, Smart-Halajko C et al., reported that ANGPTL-4 E40K and T266M variant were linked with lower triglyceride levels in T2DM patients and the ANGPTL-4 genotype position did not modify the triglyceride reaction even after the lifestyle alteration [23]. In one of the cross-sectional study, the plasma levels of ANGPTL4 were considerably elevated amongst hypertensive patients than the non-hypertensive. It was also observed that the plasma levels of ANGPTL-4 were elevated among subjects with T2DM and hypertension than in those with T2DM but not hypertension [24].

The sample size of present study population was relatively smaller, and hence this precluded us from getting a greater number of major adverse cardiac events. There was heterogeneity in the follow-up periods as they were performed at different intervals of time. It is not known if the patients who were lost to follow-up had any MACE. Moreover, authors could not collect information on the prior use of drugs which might have potentially influenced the level of ANGPTL-4 in the study population.

In conclusion, the present study failed to demonstrate that ANGPTL-4 could be a potential biomarker for the prediction of major adverse cardiac events among acute coronary syndrome patients. Serial measurement of ANGPTL-4 should be performed at different time intervals to understand the pathophysiology of ANGPTL-4 during acute coronary syndrome. However, larger studies are warranted to investigate the prognostic value of ANGPTL-4 among patients with acute coronary syndrome.