Clinical studyStudy population

The present Biosignature study was a nationwide prospective cohort study that was carried out in nine centers across Taiwan from October 2012 to May 2015 to identify risk factors among stable CAD patients at baseline. Detailed protocols for this study have been published previously [29]. Briefly, the inclusion criteria were patients with significant CAD, as documented by a coronary angiogram, a history of myocardial infarction, as evidenced by a 12-lead electrocardiography (ECG) or hospitalization, or a history of angina with ischemic ECG changes or a positive response to stress test. Patients who received at least one successful PCI with either coronary stenting or balloon angiography and were considered stable on medical treatment for at least one month, were enrolled in the study. Patients were excluded if (i) they had been hospitalized for unstable angina, acute coronary syndrome, acute myocardial infarction, acute cerebrovascular events or other acute CV events within the 3 months prior to enrollment, (ii) they planned to receive further coronary revascularization or interventional procedures for other CV diseases during the following one year period, (iii) they had significant malignancies or tumors requiring advanced medical or surgical therapy or both in the following one year, or (iv) they had other major systemic diseases requiring hospitalization or operation in the following one year period. In addition, patients with a life expectancy < 6 months (e.g. malignant metastatic neoplasm), and those receiving treatment with immunosuppressive agents were also excluded. The study was approved by the Health Authority, Independent Ethics Committee, and Independent Review Board (IRB) at each hospital, as well as the Joint IRB Ethics Committee Review Board in Taiwan. All participants provided informed consent prior to their inclusion within the study.

Validation cohort

An independent validation cohort, consisting of 76 stable CAD patients with or without PCI enrolled, was also investigated for comparison with the study patients. All patients had confirmed CAD, defined as stenosis of ≥ 50% in any major epicardial vessel on an angiography at the Taipei Veterans General Hospital (Taipei, Taiwan), between November 2011 and December 2015. The exclusion criteria were the same as for the study patients, and they were given contemporary medical treatment. They were prospectively followed up until September 2019 or until the occurrence of an adverse CV event, such as CV death, fatal or non-fatal myocardial infarction, ischemic stroke, or target vessel revascularization for recurrent angina.

Biochemical measurements

When patients first visited the out-patient clinic after their hospital discharge, blood samples were drawn after a minimum 8 h fast. Total cholesterol and triglyceride were measured by the enzymatic calorimetric method. Plasma high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) cholesterol were measured using the selective detergent method. Plasma creatinine was measured via standard methods and the estimated glomerular filtration rate (eGFR) was calculated using the MDRD formula. Plasma hsCRP, tumor necrosis factor-α (TNF-α), N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) and endocan were detected using Milliplex MAP kit assays (Millipore Corporation, Darmstadt, Germany). In the validation cohort, plasma endocan was assessed using Sandwich ELISA kits (Abcam, Cambridge, MA, USA).

Clinical follow up for adverse events

All patients were regularly followed up and a trained study nurse performed data collection every 3 months for the first year and every 6 months from the second year onwards. The primary outcome was hard CV events, including CV mortality, non-fatal myocardial infarction and stroke. The secondary outcome was total CV events, including all-cause mortality, non-fatal myocardial infarction, stroke, hospitalization for coronary intervention, peripheral artery disease, heart failure and arrhythmia. Stroke was diagnosed if a focal deficit lasted for > 24 h. Maintenance therapy following PCI included aspirin (80–325 mg/day continuously) and clopidogrel (75 mg/day ≥ 6 months in patients with stents). Other medications were prescribed at the discretion of the attending physician.

In vitrostudyHuman EPC isolation and culture

The blood sample was collected from the peripheral veins of an independent group of 3 patients with stable CAD and 3 non-CAD subjects. The detailed methodology for the culture of EPCs has been mentioned in our previous study [22, 30,31,32]. In brief, after blood was collected, the total mononuclear cells were separated by Histopaque-1077 (Sigma-Aldrich, 10,771, Darmstadt, Germany) and centrifuged at 500 × g at room temperature for 30 min. The mononuclear cells were cultured in endothelial cell basal medium (Lonza, CC-3156, Basel, Switzerland), with supplements including hydrocortisone, human fibroblast growth factors, vascular endothelial growth factor (VEGF), R3-insulin-like growth factor-1, ascorbic acid, human epidermal growth factor, gentamicin sulfate-amphotericin and 20% fetal bovine serum on fibronectin-coated 6-well plates. After culture for 4 ~ 5 days, the nonadherent cells were removed and the attached early EPCs appeared. At 2–4 weeks, some early EPCs continued to grow into colonies of late (out-growth) EPCs, usually with the monolayer cobblestone-like shape in a pattern typical of mature endothelial cells at confluence. Various immunofluroscence stainings may be used to characterize the late EPCs as indicated. Only the late EPCs under passage 3 were used for consequent EPC study.

Transfection of siRNA

EPCs were transfected with endocan siRNA (Santa Cruz Biotechnologies, sc-40543, Dallas, TX, USA) using Lipofectamine 2000 (Invitrogen, 12,252,011, Waltham, MA, USA) in culture medium.

Migration assay

The migratory function of EPCs was evaluated by a Boyden chamber assay (Transwell, Coster, San Diego, CA, USA). Briefly, the cells (1 × 104 cells) were seeded on in the upper chambers of 24-well Transwell plates with a polycarbonate membrane (8-mm pores). Then, cells migrated toward the lower chamber containing 600 μL cultured medium with FBS at 37 °C in 5% CO2. After 18 h, migrated cells were fixed in 4% paraformaldehyde and stained with hematoxylin solution. Images were captured by a high-power (× 100) microscope.

Tube formation assay

The cells (1 × 104 cells) were seeded into ECMatrix gel (Invitrogen, Carlsbad, CA, USA) in 96-well plates in 100 μL cultured medium with 10% FBS for 16 h at 37 °C in 5% CO2. Images were captured by high-power (× 40) microscope. The numbers of formed tubes of cells were calculated using Image-Pro Plus (Media Cybernetics, Inc. Rockville, MD, USA). The number of tube formation was evaluated by counting the total area of complete tubes formed by the cells in 6 representative fields.

Adhesion assay

THP-1 is a human monocytic cell line labeled with 10 Μm BCECF-AM (Invitrogen, B1170, Waltham, MA, USA) at 37 °C for 1 h in RPMI-1640 medium (Corning, Manassas, VA, USA). Confluent EPCs were incubated with THP-1 cells (5 × 105 cells/mL) at 37 °C for 1 h. Nonadherent THP-1 cells were removed, and plates were gently washed with PBS. The number of adherent THP-1 cells was counted under a 200 × high-power field well using a fluorescent microscope (Zeiss, Axiovert 200 M; White Plains, NY, USA). Six randomly chosen high-power fields were counted per well.

Western blot analysis

Total cell or tissue lysates were extracted using lysis buffer, and proteins were separated in 8–12% (v/v) SDS-PAGE gels. After electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA), the proteins were transferred onto PVDF membranes (Millipore, Darmstadt, Germany), and the membranes were incubated with anti-endocan (R&D Systems, AF1810, Minneapolis, MN, USA), VEGF (Santa Cruz Biotechnologies, sc-7269, Dallas, TX, USA), stromal cell-derived factor (SDF)-1 (Cell Signaling, Danvers, #3740, MA, USA), vascular cell adhesion molecule-1 (VCAM-1; Cell Signaling, #13,662, Danvers, MA, USA), intercellular adhesion molecule-1 (ICAM-1; Cell Signaling, #67,836, Danvers, MA, USA), E-selectin (Santa Cruz Biotechnologies, sc-137054, Dallas, TX, USA), anti-actin (Merck, 3,423,208, Darmstadt, Germany) at 4 ℃ overnight. After washing three times, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the membranes were visualized using the ECL kit.

Statistical analysis

Statistical analysis was performed using the Statistical Product and Service Solutions software, version 18.0. Descriptive statistics were expressed as the mean ± standard deviation for continuous variables, and as the number of cases and percentage (%) for categorical variables. Analyses of differences between groups were performed using the Pearson Chi Squared, Student’s t-test or one-way analysis of variance (ANOVA). The Cox proportional hazard model was used to assess the association between endocan and cardiovascular events, while adjusting for baseline cardiovascular risk factors and clinical variables. A two-tailed P-value of < 0.05 was considered statistically significant.