The study was a prospective cohort analysis of consecutive subjects who were admitted to our hospital from March 2023 to December 2023. Eligible participants had a documented history of at least one risk factor for atherosclerosis, which included hypertension, hyperlipidemia, diabetes, current smoking, obesity [body mass index (BMI) ≥28 kg/m²] or a family history of early-onset ischemic heart disease. All patients who underwent carotid artery and peripheral vascular ultrasound, coronary computed tomography angiography (CTA) or CT angiography examination were divided into two groups: the ASCVD group, which included patients with ASCVD, and the control group, which included patients without ASCVD. The specific diagnostic criteria for ASCVD (25) include the following: (i) coronary atherosclerotic heart disease (CAD), in which the level of stenosis in at least one coronary artery, as verified by CTA or coronary angiography (CAG), is 50% or greater; (ii) cerebrovascular disease, in which carotid and/or intracranial artery stenosis of 50% or more is verified through vascular ultrasound, head and neck CT angiography, or angiography (26); and (iii) peripheral blood vessel blockage of 50% or more, as determined by CT angiography or peripheral vascular ultrasound.

The exclusion criteria for the current study were as follows: severe liver or renal organ dysfunction, death during hospitalization, end-stage malignant tumors, connective tissue disorders, severe acute infections, and patients receiving anti-inflammatory or immunosuppressive medications.

The research was carried out following the guidelines of the Declaration of Helsinki and received approval from the Ethics Committee of the Peking University First Hospital (approval number 2023-132-002).

The morning after admission, fasting venous blood samples were collected in tubes containing disodium ethylenediaminetetraacetic tetraacetic acid (1 mg/ml) and aprotinin (500 KIU/ml; Sigma, St. Louis, MO, USA) and then promptly centrifuged at 3,500 rpm for 10 min at 4°C. The resulting plasma was divided into several aliquots and stored at −80°C until it was analyzed in the laboratory. The plasma levels of NRG-1 were measured using a specific and quantitative ELISA kit (Jiangsu Meibiao Biotechnology Co., Ltd.) according to the manufacturer's protocol.

Briefly, 50 μl of standards or prediluted samples (1:4 in sample diluent) were added to the appropriate wells, which had been precoatd with an anti-NRG-1 mimonoclonal antibody. After incubation at 37°C for 30 min, add 50 μl biotinylated antibody to each well. After incubation at 37°C for 30 min, the plates were washed by wash buffer. Add 50 μl of substrate A followed by 50 μl of substrate B to each well, incubate at 37°C in the dark for 10 min, and then add 50 μl stop solution. Finally, the plates were read at 450 nm using a microplate reader. The detection range was from 3 to 150 pg/ml. All plasma samples were assayed in duplicate and mean values were calculated.

Demographic and clinical parameters were collected for all of the subjects at baseline. All participants were asked about their smoking status, family history of disease, use of medications, and medical history. Clinical parameters, including age, sex, BMI, blood pressure, heart rate (HR), and left ventricular ejection fraction (LVEF), were collected from medical records. Laboratory data, including liver and kidney function, uric acid, blood lipid, fasting blood glucose, and glycosylated hemoglobin data, were collected from the laboratory. The follow-up information for ASCVD patients was gathered by reviewing our medical records or by contacting the patients or their family members via phone every three months with a period of 14–16 months. The primary endpoint was the occurrence of major adverse cardiovascular and cerebrovascular events (MACCEs), including angina pectoris, nonfatal MI, nonfatal stroke, new HF symptoms, and CVD-related death. If the patient died, the cause of death was determined by taking a history from family members and reviewing medical records.

All the statistical analyses were performed using SPSS Statistics 26.0 software and R software version 4.3.1. Continuous variables are expressed as the mean ± standard deviation (SD) or error (SE), and categorical variables are presented as frequencies (percentages). Normally distributed quantitative data were analyzed via independent-samples two-tailed Student't-tests. For nonparametric data, the Wilcoxon matched-pair signed-rank (two samples) test was used. Qualitative data were analyzed using the chi-square test (Fisher's exact test). Pearson/Spearman correlations and regression analyses were applied to assess the relation between NGR-1 and the inflammatory mediators. Binary logistic regression was used to determine whether NRG-1 was an independent predictor of ASCVD risk, considering various relevant covariates. These included the baseline characteristics of all participants (age, sex, BMI, and medical history) as well as risk factors for atherosclerosis [such as HbA1c, blood lipids, fasting blood glucose, creatinine (Cr), serum uric acid, and some inflammatory factors]. Mediation analysis was performed using R, along with Zstats v1.0 (https://www.zstats.net) to evaluate the proportional contributions of inflammatory factors (MCP-1, MPO, VCAM-1, and hs-CRP) to the associations of the NRG-1 with ASCVD risk.

Cardiovascular and cerebrovascular event rates were determined using the Kaplan‒Meier method and compared via the log-rank test. To determine the associations between baseline NRG-1 levels and the risk of MACCEs, cox proportional hazards models were used. Furthermore, to identify potential related changes, a variety of subgroup analyses and interaction analyses were performed. Patients with ASCVD were stratified into diverse subgroups, according to age (<60 vs. ≥60 years), sex, smoking status, and the presence of hypertension, dyslipidemia, diabetes, and hyperuricemia. A two-tailed P < 0.05 was considered to indicate statistical significance.

A total of 690 subjects had at least one risk factor for atherosclerosis and were hospitalized during the study period; 229 were excluded based on the basis of the exclusion criteria (Figure 1). According to the inclusion and exclusion criteria, a total of 461 patients were selected and divided into the ASCVD group (n = 220) and the control group (n = 241) on the basis of the grouping criteria (Supplementary Table S1). Propensity score matching (PSM) was performed on a 1:1 scale on the basis of sex and age. In the final study analysis, 370 patients were divided into two groups: the ASCVD group, which included 185 patients with ASCVD (160 men; mean age 74.54 ± 12.15 years old), and the control group, which included 185 subjects without ASCVD (153 men; mean age 74.66 ± 11.91 years old). Among the ASCVD group, there were 161 cases of CAD, 1 case of ischemic stroke, 8 cases of peripheral artery stenosis, 9 cases of carotid artery stenosis, 4 cases of CAD combined with ischemic cerebrovascular disease, and 2 cases of carotid artery combined with peripheral artery stenosis.

The two groups had similar demographic characteristics, distributions of pubertal stages and anthropometric measurements (Table 1). However, there were notable differences in the levels of Cr (P = 0.006), total cholesterol (TC, P < 0.001), low-density lipoprotein cholesterol (LDL-c, P < 0.001), and LVEF (P = 0.035) between ASCVD patients and controls. ASCVD patients presented lower concentrations of TC and LDL-c, which may be associated with the use of lipid-lowering drugs.

NRG-1 levels were significantly lower in ASCVD patients than in the controls (123.45 ± 0.87 pg/ml vs. 139.76 ± 0.83 pg/ml, P < 0.001), whereas the concentrations of proinflammatory mediators such as MCP-1, MPO, VCAM-1, and hs-CRP were higher in ASCVD patients than in the controls (Figure 2, Supplementary Table S2).

Spearman's correlation analysis revealed that human circulating NRG-1 levels were negatively associated with MCP-1 (−0.278, P < 0.001), MPO (−0.171, P = 0.001) and VCAM-1 (−0.351, P < 0.001) levels. However, there was no significant correlation between NRG-1 and CRP (Supplementary Table S3).

Logistic regression was used to determine whether high levels of NRG-1 are independently protective against ASCVD development. After adjustment for other covariables (age, sex, BMI, blood pressure, heart rate, smoking status, family history of early-onset CVD, hypertension, dyslipidemia, diabetes, hyperuricemia, use of lipid-lowering drugs, glycosylated hemoglobin, creatinine, eGFR, uric acid, glucose, TG, TCHO, HDL-c, LDL-c, LVEF, MCP-1, MPO and VCAM-1), a binary analysis revealed that among the set of influencing variables for ASCVD, NRG-1 remained an independent protective factor (OR = 0.855, 95% CI = 0.814–0.899, P < 0.001; Table 2). However, the OR for TCHO is below 1, which could be linked to the fact that a significant portion (338/360, 93.89%) of the participants in our study were using lipid-lowering drugs. The use of lipid-lowering drugs conferred cardiovascular protection (OR = 0.051, 95% CI = 0.008–0.316, P < 0.001).

Given that the protective effect of NRG-1 may be associated with inflammatory factors, we conducted a mediation analysis to evaluate the proportional contributions of inflammatory factors (MCP-1, MPO, VCAM-1, and hs-CRP) to the associations of the NRG-1 with ASCVD risk. According to the results of the mediation analyses (Table 3), MCP-1, MPO, and VCAM-1 accounted for 20.2%, 8.8%, and 30.1%, respectively, of the associations of NRG-1 with ASCVD. However, there were no significant mediating effects observed for hs-CRP.

The follow-up period ranged from 14 to 16 months (mean 13.8 ± 1.7 months), during which 33 patients experienced MACCEs. This included 8 patients who were hospitalized for HF, 22 patients with ACS, 1 patient with malignant arrhythmia, 1 patient with stroke, and 1 patient with sudden cardiac death. The initial baseline characteristics and biomarker levels of the patients with ASCVD are presented in Supplementary Table S4 according to the occurrence of MACCEs during follow-up. The results revealed that the mean NRG-1 concentration in patients with MACCEs was 112.04 ± 1.24 pg/ml, which was lower than that in patients without MACCEs (125.93 ± 0.90 pg/ml, P < 0.001). However, no significant intergroup differences were detected in MCP-1 (294.89 ± 4.19 ng/L vs. 296.63 ± 1.89 ng/L, P = 0.707), MPO (88.74 ± 1.14 ng/ml vs. 89.95 ± 0.54 ng/ml, P = 0.338) and VCAM-1 levels (553.34 ± 8.52 ng/ml vs. 548.85 ± 3.80 ng/ml, P = 0.633).

On the basis of the observed relationship between NRG-1 levels and the occurrence of MACCEs, we further evaluated the prognostic value of NRG-1 for predicting the occurrence of MACCEs. Patients with ASCVD were divided into two groups on the basis of their median NRG-1 level (122.5 pg/ml): the lower group (n = 92) and the higher group (n = 93). In the lower group (NRG-1 < 122.5 pg/ml), MACCEs were reported in 30 patients, which included 7 individuals hospitalized due to HF, 20 patients with ACS, 1 patient with severe arrhythmia, 1 patient with stroke, and 1 patient who experienced sudden cardiac death. In the higher group (NRG-1 ≥ 122.5 pg/ml), 3 patients were admitted to the hospital because of ACS recurrence or HF (Table 4). The Kaplan‒Meier survival curves suggested that patients with NRG-1 levels <122.5 pg/ml tended to have lower survival rates than did those with higher NRG-1 levels (Figure 3, P < 0.001).

According to the cox models, the unadjusted hazard ratio was 0.892 (95% CI, 0.852–0.934). After adjusting for demographics, this association between NRG-1 and MACCEs remained significant (Table 5, Model 1). With the addition of covariates associated with NRG-1 from our cross-sectional analyses (Model 2), there were minimal changes in the relative hazard (HR 0.860, 0.816–0.907, P < 0.001). Furthermore, in the fully adjusted model (Model 3), which included additional potential confounders (LVEF and the levels of proinflammatory mediators), significance was maintained (HR 0.857, 0.809–0.908, P < 0.001). No single covariate changed the univariate HR between NRG-1 and MACCEs by more than 10%, and the backward elimination method did not reveal further evidence of confounding factors. Thus, circulating NRG-1 was an independent predictor of MACCEs in patients with ASCVD.

To further investigate the relationship between the NRG-1 and MACCEs in patients with ASCVD, a series of subgroup analyses were conducted. As shown in Figure 4, none of the subgroups, including age, smoking history, family history of early-onset cardiovascular disease, and prevalence of hypertension, diabetes mellitus or hyperuricemia, profoundly changed the relationship between NRG-1 and MACCEs (all P values for interaction >0.05).