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INTRODUCTION
Atherosclerotic cardiovascular disease (CVD) stands as a significant contributor to global morbidity and mortality, imposing substantial burdens on health-care systems worldwide1. While conventionally understood to originate from atherosclerotic plaque rupture culminating in obstructive thrombus formation, contemporary perspectives propose novel mechanisms underlying CVD pathogenesis, extending beyond traditional cardiovascular risk factors2,3. In this scenario, inflammation has emerged as a pivotal component in the pathophysiological cascades driving acute coronary syndromes (ACS), with various inflammatory markers integrated into prognostic stratification algorithms and offering promising therapeutic targets4,5.
Gender exerts a notable influence on inflammatory responses, with women often exhibiting more pronounced reactions than men6,7. While the precise mechanisms underlying these gender disparities remain incompletely understood, recent investigations have centered on epigenetic regulators, particularly miRNAs, for their roles in modulating inflammatory processes and the progression of CVD8. Among these miRNAs, miR-155 has emerged as a potential mediator in the pathophysiology of ST-elevation myocardial infarction (STEMI). Derived from macrophages and other pro-inflammatory cells, miR-155 participates in numerous inflammatory and fibrotic processes, primarily through its interaction with the suppressor of cytokine signaling 19,10. Notably, clinical studies have demonstrated the diagnostic utility of measuring miR-155 in the emergency department for discerning the ischemic etiology of chest pain, aiding in prognosis and risk stratification1.
This study aims to investigate the association between miR-155 levels and inflammation, atherosclerotic burden, and major adverse cardiovascular events (MACE), with an emphasis on male patients to mitigate potential sex-related confounding factors11,12.
METHODS
Study design
An observational study was conducted at a specialized university clinical center dedicated to CVDs. The study recruited consecutive adult male patients admitted to the coronary care unit within 24 h of experiencing an episode of STEMI. The diagnosis of STEMI was established based on the presence of anginal symptoms coupled with evidence of myocardial injury, as indicated by elevated cardiac troponin T (cTnT) or creatine kinase MB (CK-MB) isoenzyme levels, with at least one measurement exceeding the 99th percentile upper reference limit. In addition, ST-segment elevation in at least two contiguous leads was required, defined as ≥ 2.5 mm in men under 40 years, ≥ 2 mm in men 40 years and older, or ≥ 1.5 mm in women in leads V2-V3, and/or ≥ 1 mm in other leads, in the absence of left ventricular hypertrophy or left bundle branch block on a 12-lead electrocardiogram. Exclusion criteria encompassed patients with prior reperfusion therapy, concurrent infections, neoplastic diseases, chronic heart failure, autoimmune disorders, or those using glucocorticoids or immunosuppressants. A reference group of young men without CVD or significant comorbidities was included for comparison of miR-155 levels.
Approval for the study protocol was obtained from the local ethics committee (number 21-1273), and adherence to the principles outlined in the Declaration of Helsinki and local regulations was ensured. Informed consent was obtained from all participants, granting permission for the use of clinical data and blood samples for research purposes. Medical interventions were administered solely at the discretion of treating physicians, and the study did not influence patient treatment or clinical decisions.
Upon hospital admission, clinical and demographic information was collected, and laboratory data were obtained directly from medical records. In addition, 4 mL of venous blood were drawn by peripheral puncture in a tube with activating gel, followed by centrifugation and storage of serum aliquots at −70°C until use. Coronary angiographies, conducted in the hemodynamics laboratory, were analyzed by interventional cardiologists from our institution. Significant coronary artery disease (CAD) was defined as a luminal diameter narrowing of > 50% in each major coronary artery. The atherosclerotic burden was assessed by determining the number of coronary vessels with > 50% occlusion. Patients were followed daily until discharge, and a composite outcome of MACE was scored, including acute heart failure, pulmonary edema, recurrent myocardial infarction, cardiogenic shock, or death.
Laboratory assays
Serum samples were thawed under standard conditions, and the levels of interleukin (IL)-1β and IL-6, and tumor necrosis factor (TNF) were quantified using multiplex assays with the Milliplex MAP panel (Millipore; Burlington, MA, USA).
To isolate RNA, serum samples underwent centrifugation at 10,000 g for 30 min at 4°C, followed by RNA extraction using the Qiagen serum/plasma mini kit (Qiagen; Hilden, Germany). During the RNA purification process, an equal amount of cel-miR-39 was added as a control. The presence of miR-155 (Assay ID: 002623) was identified using a two-step reverse transcription (RT)-quantitative polymerase chain reaction method involving specific RT primer assays and TaqMan probes (Applied Biosystems; Foster City, CA, USA). Normalization of miRNA concentrations was conducted using cel-miR-39 (Assay ID: 000200), and calculations were performed utilizing the formula 2ΔΔCt, with resulting values multiplied by 1000 and reported as arbitrary units (a.u.).
Statistical analysis
Data distribution was assessed using the D'Agostino-Pearson test. Patients were categorized into tertiles based on miR-155 levels for analysis. Categorical data were presented as percentages and analyzed using the χ2 test, whereas continuous variables were described using the median with minimum-maximum range and analyzed using the Mann-Whitney U test for two groups or the Kruskal-Wallis test for three groups. Correlations were examined using Spearman's rho (ρ) coefficient with 95% confidence intervals. Statistical analyses were two-tailed, with a significance threshold set at p < 0.05. Calculations and data visualization were performed using the Social Science Statistics website (http://socscistatistics.com) and GraphPad Prism version 9.5.1 (GraphPad Software; La Jolla, CA, USA).
RESULTS
A total of 85 male subjects participated in the study, comprising 69 patients diagnosed with STEMI and 16 asymptomatic individuals serving as the reference group. Patients with STEMI had a median age of 60 (28-45) years, significantly higher than the median age of 36 (32-38) years observed in healthy controls. Significantly lower levels of miR-155 (Fig. 1A) were found in STEMI patients compared to healthy individuals (54.2, 10.1-549.9 a.u. vs. 152.8, 30.6-999.1 a.u.; p = 0.003).
Figure 1. Results A: the relative expression levels of miR-155 were significantly lower in men with ST-elevation myocardial infarction (STEMI) (orange circles) compared to the control group (olive triangles). B: inflammatory marker levels were similar among STEMI patients stratified by tertiles of miR-155 expression: lower (green circles), medium (blue circles), and higher (red circles). Horizontal lines represent the median values. CRP: C-reactive protein; IL-1β: interleukin 1-β; IL-6: interleukin-6; NLR: neutrophil-to-lymphocyte ratio; PLR: platelet-to-lymphocyte ratio; TNF: tumor necrosis factor.
STEMI patients were further grouped into tertiles based on miR-155 levels. The relative expression of miR-155 in the first, second, and third tertiles was 25.4 a.u. (10.1-40.9), 54.2 a.u. (44.0-68.8), and 110.8 a.u. (70.1-549.9), respectively, with corresponding median ages of 58 years (41-77), 62 years (39-79), and 60 years (33-80). Traditional CVD risk factors were prevalent in all patient groups, with no significant differences in clinical or demographic characteristics, including STEMI severity (Table 1). Laboratory data indicated trends toward higher leukocyte and neutrophil levels in patients within the third tertile, although statistical significance was not reached. Likewise, cardiac biomarkers showed elevated trends in the second tertile for cTnT, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and CK-MB, but no significant differences were found between the groups (Table 1).
Table 1. Clinical data at admission and in-hospital outcomes of STEMI patients
| Variable | Distribution according to
miR-155 levels |
p |
||
|---|---|---|---|---|
| 1st tertile | 2nd tertile | 3rd tertile | ||
| Age, years | 58 (41-77) | 62 (39-79) | 60 (33-80) | 0.898 |
| Systolic blood pressure, mmHg | 133 (86-190) | 129 (106-177) | 140 (118-210) | 0.105 |
| Diastolic blood pressure, mmHg | 80 (59-120) | 80 (60-110) | 81 (60-120) | 0.541 |
| Body mass index, kg/m2 | 27.4 (21.0-37.9) | 25.5 (22.5-35.2) | 27.7 (19.4-36.1) | 0.189 |
| Hypertension, n (%) | 14 (60) | 8 (34) | 13 (56) | 0.165 |
| Diabetes mellitus, n (%) | 6 (26) | 11 (47) | 10 (43) | 0.278 |
| Dyslipidemia, n (%) | 7 (30) | 8 (34) | 7 (30) | 0.935 |
| Active smoker, n (%) | 7 (30) | 9 (39) | 11 (47) | 0.481 |
| Killip classification | 2 (1-4) | 1 (1-3) | 1 (1-3) | 0.075 |
| GRACE risk score | 125 (70-168) | 111 (72-198) | 111 (62-186) | 0.753 |
| Leukocytes ×103/mm3 | 11.8 (6.2-25.5) | 10.4 (6.7-18.8) | 12.4 (6.5-20.2) | 0.433 |
| Hemoglobin, g/dL | 16.1 (10.9-19.3) | 15.8 (12.0-17.3) | 15.3 (12.5-17.9) | 0.965 |
| Platelets ×103/mm3 | 222 (94-340) | 258 (130-594) | 232 (133-351) | 0.828 |
| Albumin, g/dL | 4.1 (3.3-5.0) | 4.2 (2.8-4.8) | 4.1 (3.3-4.6) | 0.973 |
| Creatinine, mg/dL | 1.11 (0.77-2.34) | 1.04 (0.66-3.29) | 1.10 (0.78-2.59) | 0.933 |
| cTnT, ng/mL | 5883 (59-100000) | 11158 (75-100000) | 10397 (171-100000) | 0.879 |
| CK-MB, U/L | 67 (3-300) | 108 (2-300) | 97 (6-300) | 0.618 |
| LDH, U/L | 308 (149-2125) | 315 (127-942) | 425 (146-2627) | 0.606 |
| NT-proBNP, ng/L | 596 (36-24211) | 934 (27-4640) | 871 (35-9708) | 0.909 |
| Pharmacological therapies, n (%) | ||||
| ASA | 23 (100) | 22 (95) | 22 (95) | 0.597 |
| P2Y12 inhibitors | 23 (100) | 23 (100) | 23 (100) | > 0.999 |
| Statins | 22 (95) | 22 (95) | 23 (100) | 0.597 |
| RAAS inhibitors | 22 (95) | 22 (95) | 20 (86) | 0.422 |
| Primary PCI, n (%) | 22 (95) | 21 (91) | 22 (95) | 0.766 |
| Number of occluded coronary arteries, n (%) | 0.007 | |||
| One or two vessels | 21 (91) | 20 (86) | 15 (65) | |
| Three vessels | 1 (4) | 3 (13) | 8 (34) | |
| Left main coronary artery, n (%) | 0 | 4 (17) | 1 (4) | 0.569 |
| Mechanical ventilation, n (%) | 0 | 0 | 1 (4) | 0.217 |
| Intra-aortic balloon pump, n (%) | 1 (4) | 4 (17) | 3 (13) | 0.357 |
| Major clinical outcomes, n (%) | 3 (13) | 4 (17) | 6 (26) | 0.258 |
| Acute heart failure | 2 (8) | 3 (13) | 6 (26) | 0.107 |
| Pulmonary edema | 0 | 0 | 3 (13) | 0.030 |
| Cardiogenic shock | 1 (4) | 1 (4) | 1 (4) | > 0.999 |
| Death | 0 | 1 (4) | 1 (4) | 0.379 |
| Days of hospital stay | 3 (1-15) | 4 (1-11) | 6 (2-18) | 0.256 |
Data are presented as median (minimum-maximum range) unless otherwise specified. Coronary artery involvement and the main clinical outcomes were analyzed using the trend principle (X2 for trends). Significant p-values are in bold. ASA: acetylsalicylic acid; CK: creatine kinase; CK-MB: MB isoenzyme of CK; cTnT: cardiac troponin T; GRACE: Global Registry of Acute Coronary Events risk score; LDH: lactic dehydrogenase; MI: myocardial infarction; NT-proBNP: N-terminal pro-B-type natriuretic peptide; PCI: percutaneous coronary intervention; P2Y12: platelet receptor for adenosine 5' diphosphate; RAAS: renin-angiotensin-aldosterone system.
Pharmacological therapies administered at admission and primary percutaneous coronary interventions did not differ between groups. However, assessment of atherosclerotic burden revealed a significantly higher prevalence of three-vessel occlusions in the third tertile compared to lower tertiles (34% vs. 13% vs. 4%; p = 0.007). In addition, the occurrence of MACE tended to be more common in higher tertiles, with pulmonary edema notably more frequent in the third tertile (13% vs. 0 vs. 0; p = 0.030). Mortality rates did not significantly differ between the groups (Table 1).
Regarding the association between miR-155 levels and inflammatory biomarkers (Fig. 1B), no differences were found in high-sensitivity C-reactive protein (hsCRP), IL-6, TNF, or IL-1β levels among STEMI patients stratified according to miR-155 expression levels. Neutrophil-to-lymphocyte ratios (NLR) and platelet-to-lymphocyte ratios (PLR) were similar between tertiles. Correlation analyses further indicated a lack of association between miR-155 levels and the aforementioned inflammatory biomarkers, including NLR (ρ = 0.05, −0.15-0.33), PLR (ρ = 0.15, −0.09-0.38), hsCRP (ρ = 0.17, −0.06-0.40), IL-6 (ρ = 0.14, −0.10-0.37), TNF (ρ = 0.01, −0.22-0.26), and IL-1β (ρ = −0.05, −0.29-0.18), as well as cardiac biomarkers such as cTnT (ρ = 0.09, −0.15-0.33), NT-proBNP (ρ = 0.08, −0.15-0.32), or CK-MB (ρ = 0.18, −0.06-0.40).
In the subanalysis based on coronary artery involvement, miR-155 levels in patients with left anterior descending (LAD) artery involvement (n = 50) were similar to those without LAD involvement (55.6, 10.1-549.9 a.u. vs. 48.8, 12.8-150.5 a.u.; p = 0.161). Similarly, no significant differences were found in cTnT (14239, 75-100000 ng/mL vs. 5616, 59-100000 ng/mL; p = 0.792), CK-MB (86.8, 2.7-300 U/L vs. 108.7, 3.8-300 U/L; p = 0.713), or hsCRP (5.1, 0.5-476.6 mg/L vs. 5.4, 0.6-196.0 mg/L; p = 0.802) between these groups.
DISCUSSION
In this study, we explored the participation of miR-155 levels in inflammation and myocardial necrosis among male patients experiencing STEMI. Our findings provided evidence of miR-155 dysregulation in STEMI, suggesting its potential role in the underlying atherosclerotic burden and the occurrence of MACE, particularly pulmonary edema.
The observed downregulation of circulating miR-155 in STEMI patients aligns with prior studies, reinforcing its potential as a biomarker of cardiovascular risk. Yao et al. and Fichtlscherer et al. reported decreased miR-155 levels in ACS and CAD patients, respectively, highlighting its inverse correlation with Th17 cell differentiation11,13. Similarly, Zidar et al. found a pronounced downregulation of miR-155 in myocardial infarction patients with ventricular rupture, suggesting a regulatory role in inflammation intensity14. The downregulation of miR-155 likely acts as a negative feedback loop controlling immune response upregulation. Activation of miR-155 can occur through conventional transcription factors such as nuclear factor κB and signaling cascades involving T and B cell receptors. MiR-155 is co-expressed with IL-17A and inversely correlates with the frequency of Th17 cells, regulating cell differentiation toward Th1 and secretion of proinflammatory cytokines such as TNF and IL-613,15. While our study did not find an association between miR-155 levels and inflammatory markers, it does explain why patients with the highest atherosclerotic burden exhibited the highest levels of miR-155, suggesting an attempt to attenuate atherogenesis. Once activated by oxidized lipoproteins and other atherogenic debris, cells involved in atherosclerotic plaque, including macrophages, dendritic cells, and CD4+ and CD8+ T lymphocytes, can increase their production of miR-15516,17. This enhanced miR-155 response would attempt to attenuate atherogenesis, improving cholesterol efflux by macrophages and leading to a shift toward an antiatherogenic lymphocyte profile18. Alternatively, the reduction in circulating miR-155 levels in ACS patients might result from the uptake of circulating miRNAs into atherosclerotic lesions. Experimental evidence suggests the release of miRNAs by apoptotic bodies and microparticles within atherosclerotic plaques19.
The potential role of miR-155 in STEMI severity extends beyond inflammation. Saadatian et al. reported peak miR-155 expression 2-7 days post-myocardial infarction onset, with lower expression in subjects with non-significant CAD compared to those with significant disease20. In addition, Guo et al. demonstrated that inhibiting miR-155 reduced cardiomyocyte apoptosis and improved cardiac function21. In our cohort, higher miR-155 levels were associated with increased atherosclerotic burden and a higher incidence of MACE, suggesting its role as both a mediator of vessel injury and a marker of disease severity.
From a translational perspective, these findings suggest the potential of miR-155 as a valuable biomarker in STEMI. If validated in future research, the measurement of miR-155 at hospital admission could inform clinical decisions, identifying patients at a higher risk of adverse outcomes. Furthermore, interventions aimed at modulating miR-155 expression represent promising avenues for therapeutic strategies. The strengths of our study lie in its homogeneous sex composition, which reduces confounding due to sex-based differences in cardiovascular outcomes, and the precise assessment of atherosclerotic burden using cardiac catheterization. However, the limitations of its observational design and small sample size preclude causal inferences. Further mechanistic studies are necessary to elucidate the specific role of miR-155 in regulating the interplay between immune activation, myocardial injury, and plaque instability.
In conclusion, this study revealed dysregulation of miR-155 levels in male patients with STEMI, suggesting its potential influence on disease severity (atherosclerotic burden) and the occurrence of MACE (pulmonary edema), regardless of conventional inflammation or myocardial markers.
