In total, 210 patients with ACS (male, 146; female, 54; age, 66.28±10.47) who received coronary stent implantation or medical treatment at the Second Hospital of Hebei Medical University (Shijiazhuang, China) between January 2016 and June 2020 were recruited for the present study. Recruited patients exhibited symptoms compatible with angina pectoris (dyspnea and chest pain) and fulfilled ≥ one of the following criteria (25): i) Rise in serial troponin levels (the normal range of troponin being 0-0.04 ng/ml). The troponin level was detected at the time of admission. It was checked every 4 h in the first 24 h, every 8 h in the second 24 h and once in the third 24 h); ii) persistent ST-segment depression or elevation, dynamic electrocardiogram (ECG) changes or T-inversion, new left bundle branch block; and iii) diameter of coronary artery luminal stenosis ≥50% as detected by coronary angiography. The exclusion criteria were the following: i) Presence of inflammation, infection, autoimmune diseases, progressive hepatic and renal insufficiency, and tumor history; ii) history of ACS; and iii) pregnancy. Based on their clinical diagnoses, 160 patients with ACS were diagnosed with UAP and 50 patients with AMI. UAP is usually triggered by physical exercise, emotional excitement or a cold environment. The attack time is short and frequent. Nitroglycerin treatment can significantly alleviate the symptoms. For AMI, there is no obvious trigger. The attack time is long and up to several hours. There is no remission after nitroglycerin treatment. Patients often have fever, increased leukocytes and rapid erythrocyte sedimentation rate. The patients display a necrotic Q wave in ECG (26).

In addition, 50 healthy individuals with a mean age of 64.56±9.61 years (male, 22; female, 28) who underwent physical examination at the Second Hospital of Hebei Medical University (Shijiazhuang, China) were recruited in the present study as the control group between January 2016 and June 2020. All healthy subjects had normal coronary arteries and history with normal ECG characteristics and/or ECG treadmill test results. Participants with severe concomitant diseases, including cardiomyopathy, congenital heart disease, cerebrovascular or peripheral vascular diseases, trauma or surgery within the last 3 months, chronic or acute infection, malignant tumors, immune diseases, hepatic or renal failure or a history of recent cardiopulmonary resuscitation were excluded from this study.

The present study was approved by the Institutional Ethics Committee of the Second Hospital of Hebei Medical University. Written informed consent was obtained from every participant in the present study. A total of 5 ml peripheral blood samples were obtained from patients with ACS at the time of diagnosis and healthy individuals. Cell-free plasma was isolated within 6 h after collection by centrifugation at 4˚C (1,000 x g for 10 min), before being stored at -80˚C until further experimentation.

Exosomes were collected from the plasma by sequential ultracentrifugation. Plasma was centrifuged at 3,000 x g for 20 min at 4˚C, followed by centrifugation at 12,000 x g for 20 min at 4˚C. Subsequently, exosomes were collected by ultracentrifugation (100,000 x g for 70 min at 4˚C) in the pellet. After suspension in 20 ml PBS, the exosomes were collected again by ultracentrifugation (100,000 x g for 70 min at 4˚C) in the pellet. The morphological characteristics of the exosomes were verified by transmission electron microscopy. Nanoparticle tracking analysis (NTA) using a Malvern Zetasizer Nano ZS90 (Malvern Panalytical) was performed to measure the concentration and size of exosomes.

Freshly prepared exosomes ml) were diluted in PBS (20 ml) and fixed with 3.5% glutaraldehyde for 5 min at 4˚C. Subsequently, exosome preparation was allowed to adsorb in a mesh copper grid. The resulting grids were rinsed two times with wash buffer and contrasted by 2% uranyl-oxalate solution (pH 7.0) for 5 min at 25˚C. The visualization of exosomes morphology was performed using a H-9500 transmission electron microscope (Hitachi, Ltd.) at 300 kV, images were acquired using a F114 slow-scan CCD camera (Tietz Video and Image Processing Systems GmbH) and analyzed using EM-Menu v3.0 basic (Tietz Video and Image Processing Systems GmbH).

Exosomes and vascular smooth muscle cells (VSMCs) were lysed using RIPA buffer containing protease inhibitors (Beyotime Institute of Biotechnology). Each sample was quantified by BCA protein quantitative kit (Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, 50 µg of protein per lane were separated by 10% SDS-PAGE and transferred onto 0.22-µm PVDF membranes. The membranes were then blocked with 5% non-fat milk for 60 min at 25˚C. The membranes were subsequently incubated with primary antibodies against CD9 (dilution 1:1,000; cat. no. ab223052; Abcam), CD63 (dilution 1:100; cat. no. ab216130; Abcam), flotillin-1 (dilution 1:500; cat. no. ab13493; Abcam), Cyr61 (dilution 1:1,500; cat. no. ab228592; Abcam) and tumor susceptibility 101 (TSG101; dilution 1:1,000; cat. no. ab30871; Abcam) at 4˚C for 12 h, followed by incubation with an HRP-conjugated secondary antibody (dilution 1:10,000; cat. no. ab205718; Abcam) for 120 min at 25˚C. Bound antibodies were detected using BM Chemiluminescence Western Blotting kit (cat. no. 11520709001; MilliporeSigma) and ImageJ v1.8 software (National Institutes of Health).

Exosomes were lysed using RIPA buffer containing protease inhibitors (Beyotime Institute of Biotechnology). Cyr61 levels were then measured using the CYR61 ELISA kit (cat. no. SBJ-H0152; Nanjing SenBeiJia Biological Technology Co., Ltd.) according to the manufacturer's instructions. Calibration and standardization of the assay was performed according to the manufacturer's protocol.

Human VSMCs from the National Infrastructure of Cell Line Resource (cat. no. 4201HUM-CCTCC00632) were cultured in DMEM (Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.) and incubated at 37˚C with 5% CO₂. To construct the atherosclerosis model in vitro, VSMCs were treated with ox-LDL (Shanghai Yeasen Biotechnology Co., Ltd.) at different concentrations (25, 50 and 100 mg/ml) for 24 h at 37˚C. In addition, VSMCs were treated with ox-LDL (50 mg/ml) for different times (12, 24 and 48 h) at 37˚C.

Small interference RNA (siRNA) and negative control (NC) for Cyr61 were synthesized by Sangon Biotech Co., Ltd. VSMCs (1x10⁶ cells/ml) were cultured in six-well plates. According to the manufacturer's instructions, Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) and siRNA (50 nmol/well) in serum-free DMEM medium were added to cultured cells for 4 h at 37˚C. The medium was then replaced with DMEM containing 10% FBS for 48 h at 37˚C. The siRNA sequences used were as follows: NC sense, 5'-UUCUCCGAACGUGUCACGCTT-3' and antisense, 5'-ACGUGACACGUUCGGAGAATT-3'; and siCyr61 sense, 5'-CAACGAGGACUGCAGCAAATT-3' and antisense, 5'-UUUGCUGCAGUCCUCGUUGAG-3'.

siCyr61-transfected VSMCs were treated with ox-LDL (50 mg/ml). Subsequently, VSMCs (3,000 cells/well) were seeded into 96-well plates in triplicate wells. After 24 h at 37˚C, 10 µl CCK-8 solution (Beijing Biosynthesis Biotechnology Co., Ltd.) was added and the VSMCs were cultured for 2 h at 37˚C. The optical density value at 450 nm was detected in each well using a microplate reader (Molecular Devices, LLC).

siCyr61-transfected VSMCs were treated with ox-LDL (50 mg/ml). Subsequently, VSMCs were incubated in binding buffer (1x10⁶ cells/ml), followed by staining with Annexin V-FITC (200 µl) and propidium iodide (PI) solutions (10 µl) at 25˚C for 15 min using the Annexin V-FITC/PI Apoptosis Detection kit (cat. no. A211-01; Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. The apoptotic rate was detected using a flow cytometer (FACSCanto III; BD Biosciences) and FlowJo v10.4 software (BD Biosciences).

siCyr61-transfected VSMCs were treated with ox-LDL (50 mg/ml). Subsequently, VSMCs resuspended in FBS-free DMEM were added to the upper chamber of Transwell plates (Corning, Inc.) with a cell density of 1x10⁵ cell/ml. Subsequently, the bottom chamber was filled with DMEM supplemented with 30% FBS. After a 48-h culture at 37˚C, VSMCs that failed to migrate to the lower chamber were removed. Next, the lower membrane was fixed with 4% paraformaldehyde for 30 min at 25˚C and the migratory cells were stained with 0.1% crystal violet for 20 min at 25˚C. Migratory cells from five randomly chosen fields (magnification, x40) from each chamber were photographed using light microscopy and counted using ImageJ v1.8 (National Institutes of Health).

Statistical analyses in the present study were conducted using SPSS 22.0 software (IBM Corp.). χ² test, Fisher's exact test and t-test were performed for comparisons between two groups. Differences among > two groups were evaluated by one-way analysis of variance followed by Tukey's test. The ROC curves were constructed to evaluate the diagnostic accuracy of exosomal Cyr61. A multivariate analysis was applied to identify independent predictors of the existence of ACS. A two-tailed P<0.05 was considered to indicate a statistically significant difference.

The biochemical and clinical parameters of the 160 patients with UAP, 50 patients with AMI and 50 healthy individuals are presented in Table I. No significant difference was observed in the distribution of age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP) or history of hypertension, diabetes, dyslipidemia or smoking. However, compared with those in healthy subjects, the male sex was significantly more prevalent among patients with ACS. Furthermore, between healthy UAP patients and AMI patients, there were significant differences in ejection fraction (EF), hemoglobin A1c (HbA1c), creatinine (CR), glucose and high-sensitivity C-reactive protein (hs-CRP; all P<0.05; Table I).

Firstly, plasma exosomes were isolated from blood samples collected from healthy individuals, patients with UAP and patients with AMI. Transmission electron microscopy and nanoparticle tracking analysis revealed that the plasma exosomes displayed a round-shaped morphology with diameters ranging from 60 to 110 nm (Fig. 1A). The size distribution of the exosomes isolated from healthy individuals, UAP, and AMI plasma samples was 85.73±10.13, 89.7±10.81 and 87.56±13.92 nm, respectively (Fig. 1B). Furthermore, the expression of exosome markers CD9, CD63, flotillin and TSG101 were examined by western blotting. Exosomes isolated from healthy individuals, patients with UAP and patients with AMI were tested positive for CD9 expression in addition to CD63, flotillin and TSG101 (Fig. 1C). To assess the expression pattern of Cyr61 derived from the exosomes, ELISA was performed. As shown in Fig. 1D, the level of Cyr61 in exosomes isolated from patients with ACS was significantly higher compared with that in healthy individuals (P<0.001). Subsequently, changes in exosomal Cyr61 levels among the three ACS subtypes were evaluated. Compared with those in healthy individuals, exosome-derived Cyr61 levels in the UAP subgroup (P<0.001; Fig. 1D) or AMI subgroup (P<0.01) were also significantly higher. However, there was no significant difference in exosomal Cyr61 levels between those in the UAP and AMI subgroups (Fig. 1D).

Based on the median value of exosomal Cyr61 levels obtained from ELISA experiments, the 210 patients with ACS were equally divided into two groups, the high Cyr61 group (exosomal Cyr61 >1.545 pg/ml) and the low Cyr61 group (exosomal Cyr61 <1.545 pg/ml). Association analysis of exosomal Cyr61 levels with the clinical characteristics of the 210 patients with ACS were then performed. As shown in Table II, high exosomal Cyr61 levels were significantly associated with sex (P=0.016), family history of ACS (P=0.002) and glucose levels (P=0.001). By contrast, there is no significant association between exosomal Cyr61 levels and other parameters, namely age, BMI, SBP, DBP, left ventricular internal diameter (LVID) at diastole, LVID at systole, left atrial, fractional shortening, EF, HbA1c, CR, estimated glomerular filtration rate, TG, TC, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and hs-CRP (Table II).

Given that Cyr61 levels were significantly higher in plasma exosomes of patients with ACS, the diagnostic accuracy of exosomal Cyr61 as a biomarker to discriminate patients with ACS from individuals was evaluated further. As shown in Fig. 2A and Table III, exosomal Cyr61 levels could differentiate between patients with ACS and healthy subjects with high accuracy [area under the curve (AUC), 0.763; 95% confidence interval (CI), 0.705-0.822; P<0.01]. At the cut-off value of 1.435 pg/ml for exosomal Cyr61, the optimal sensitivity and specificity were 55.2 and 92%, respectively. Subsequently, the efficiency of using exosomal Cyr61 as a diagnostic marker for identifying the ACS subgroups was analyzed.

ROC curve analyses (Fig. 2B and Table III) revealed that exosomal Cyr61 could discriminate patients with UAP from healthy individuals with AUC values of 0.790 (95% CI, 0.730-0.851; P<0.001). The optimal cut-off values of exosomal Cyr61 were 1.435 pg/ml (57.5% sensitivity and 92% specificity) to detect UAP. Furthermore, exosomal Cyr61 was also found to be an accurate marker for discriminating patients with AMI from healthy individuals, with AUC values of 0.678 (95% CI, 0.567-0.788; P=0.002). At the cut-off value of 1.485 pg/ml for exosomal Cyr61, the optimal sensitivity and specificity were 48 and 94%, respectively (Fig. 2C and Table III). However, the ability of exosomal Cyr61 in differentiating patients with UAP from patients with AMI was not satisfactory (P=0.136; Fig. 2D and Table III).

Multivariable logistic regression analyses were subsequently performed to identify potential independent contribution of each parameter to the presence of ACS. The results (Table IV) revealed that exosomal Cyr61 (P<0.001) and CR (P=0.001) were independently associated with the presence of ACS. However, EF, HbA1c, glucose and hs-CRP levels were not significantly associated with ACS (Table IV).

To investigate the role of Cyr61 in atherosclerosis, the expression levels of Cyr61 were measured in VSMCs after stimulation with ox-LDL. As shown in Fig. 3A and B, Cyr61 levels were significantly increased in VSMCs following a 24-h exposure to ox-LDL in a concentration-dependent manner. Ox-LDL treatment also induced a significant increase in Cyr61 expression in a time-dependent manner (Fig. 3C and D). Subsequently, the efficiency of Cyr61 knockdown was confirmed after using siRNAs in VSMCs: siCyr61 transfection significantly decreased the expression level of Cyr61 compared with the NC group (P<0.01; Fig. 3E). Moreover, the increased expression of Cyr61 induced by ox-LDL was partially inhibited by siCyr61 transfection (P<0.01; Fig. 3F and G). Cell viability analysis revealed that, when compared with that in the control group, exposure to ox-LDL (50 µg/ml) significantly enhanced cell viability (P<0.01), whilst knocking down Cyr6 expression significantly reduced cell viability in the presence of ox-LDL at 48 and 72 h, compared with the ox-LDL group (P<0.01; Fig. 3H). Furthermore, following treatment with 50 µg/ml ox-LDL for 24 h, the levels of cell apoptosis were significantly reduced (P<0.01; Fig. 3I and J). By contrast, Cyr61 knockdown significantly increased the apoptosis rate of VSMCs compared with that in the ox-LDL group (P<0.01; Fig. 3I and J). In addition, a significant increase in the number of migratory cells was observed in response to ox-LDL treatment, then was partially but significantly reversed by Cyr61 knockdown (P<0.01; Fig. 3K and L).