Longitudinal change of serum exosomal miR-186-5p estimates major adverse cardiac events in acute myocardial infarction patients receiving percutaneous coronary intervention

Objective Our recently published study discovers that exosomal microRNA (miR)-186-5p promotes vascular smooth muscle cell viability and invasion to facilitate atherosclerosis. This research aimed to explore the prognostic implication of serum exosomal miR-186-5p in acute myocardial infarction (AMI) patients receiving percutaneous coronary intervention (PCI). Methods One hundred and fifty AMI patients receiving PCI and 50 healthy controls (HCs) were screened. Serum exosomal miR-186-5p was detected by reverse transcriptase-quantitative polymerase chain reaction assay in AMI patients at admission and after PCI, as well as in HCs after enrollment. Major adverse cardiac events (MACE) were recorded during follow-up in AMI patients receiving PCI. Results Serum exosomal miR-186-5p was raised in AMI patients vs. HCs (P < 0.001). Besides, serum exosomal miR-186-5p was positively linked to body mass index (P = 0.048), serum creatinine (P = 0.021), total cholesterol (P = 0.029), and C-reactive protein (P = 0.018); while it was reversely linked with estimated glomerular filtration rate (P = 0.023) in AMI patients. Interestingly, serum exosomal miR-186-5p was correlated with the diagnosis of ST-segment elevation myocardial infarction (P = 0.034). Notably, serum exosomal miR-186-5p was decreased after PCI vs. at admission (P < 0.001). The 6-, 12-, 18-, and 24-month accumulating MACE rates were 4.5%, 8.9%, 14.8%, and 14.8% in AMI patients. Furthermore, serum exosomal miR-186-5p ≥3.39 (maximum value in HCs) after PCI (P = 0.021) and its decrement percentage <median (35%) decrement (P = 0.044) estimated elevated MACE in AMI patients. Conclusion Serum exosomal miR-186-5p is reduced after PCI, and its post-PCI high level or minor decrease estimates increased MACE risk in AMI patients.


Introduction
Acute myocardial infarction (AMI) is the most fatal type of coronary artery disease, which is sorted into ST-segment elevation myocardial infarction (STEMI) and non-STEMI (NSTEMI) (1).Percutaneous coronary intervention (PCI) is a fundamental therapy for AMI patients, which has enhanced the clinical outcomes of these patients (2).Unfortunately, major adverse cardiac events (MACE), such as cardiac death, cardiac arrest, recurrent myocardial infarction, and repeat revascularization, occur even after successful PCI, which severely affects the clinical outcomes of AMI patients (3,4).It is estimated that the incidence of 2-year MACE after PCI is approximately 16%, and the incidence of 3-year MACE after PCI increases to nearly 20% in AMI patients (5)(6)(7)(8).Therefore, seeking potential markers that predict MACE may be vital to enhance the management of AMI patients receiving PCI.
Accordingly, the current research intended to investigate the relationship of serum exosomal miR-186-5p with MACE in AMI patients receiving PCI.

Study population
A total of 150 AMI patients receiving PCI treatment between January 2021 and May 2023 were continuously included in this study.The inclusion criteria were: (i) diagnosed as AMI per guideline from the American Heart Association/American College of Cardiology Joint Committee (18); (ii) ≥18 years old; (iii) treated with PCI; (iv) willing to cooperate with blood sample collection.The exclusion criteria were: (i) with malignant cancers or hematological malignancies; (ii) with severe liver or kidney dysfunction; (iii) with acute or chronic infectious diseases; (iv) with surgical operation in the past 6 months; (v) pregnant women or lactating mothers.Additionally, a total of 50 healthy people were screened as healthy controls (HCs), whose age and sex were matched with AMI patients.The eligible criteria for HCs were: (i) without abnormalities in recent physics examination; (ii) ≥18 years old; (iii) willing to cooperate with this study.The exclusion criteria for AMI patients were suitable for the HCs as well.The Ethics committee of the Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology gave approval to this study.The informed consent was obtained from all AMI patients or their families, and HCs.

Data documentation
Characteristics of AMI patients were collected for analyses, which included age, sex, body mass index (BMI), marital status, education level, location, smoke, hypertension, hyperlipidemia, diabetes, diagnosis, culprit lesion, multivessel disease, blood biochemical indexes, symptom-to-balloon time, thrombus aspiration, number of implanted stents, stent diameter, total stent length, and infarct size.

Sample collection and detection
Peripheral blood samples from AMI patients were collected at admission and after PCI (at the time of discharge); while samples from HCs were collected at the time of enrollment.After sample collection, the serum was immediately isolated.Then, the exosomes were separated from the serum through Total Exosome Isolation (from serum) (No.Cat.4478360, Invitrogen TM , Waltham, USA).All test procedures were performed in strict accordance with the instructions.
Serum exosomal miR-186-5p was detected using a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay.The U6 was used as the internal reference, and the final results were calculated using the 2 −ΔΔCt method.The primer sequences of serum exosomal miR-186-5p and U6 were the same as in our previous study (17).

Follow-up and prognosis analyses
AMI patients underwent routine follow-ups until July 2023.Follow-up visits were conducted once per month in the first three months, and then every three months thereafter.During the follow-up, MACE was recorded, which contained recurrent thrombosis in stents, recurrent myocardial infarction, revascularization, malignant arrhythmia, cerebral infarction, and cardiac death (19).Besides, the accumulating MACE rates were calculated for prognosis analyses.In this study, the serum exosomal miR-186-5p at admission and after PCI of AMI patients was divided into high and low levels, utilizing the maximum value of 3.39 in HCs.The decrement percentage of serum exosomal miR-186-5p was divided into <median (35%) decrement and ≥median (35%) decrement.

Statistical analyses
SPSS version 26.0 (IBM, USA) was used for the data process.The Wilcoxon rank sum test was used to compare serum exosomal miR-186-5p between AMI patients and HCs, as well as compare serum exosomal miR-186-5p between AMI patients with different characteristics (except for education level using Spearman test and culprit lesion using Kruskal-Wallis H-test).The receiver operating characteristic (ROC) curve was used to show the distinguishing ability of serum exosomal miR-186-5p to separate HCs from AMI patients.The Spearman test was performed to analyze the correlation of serum exosomal miR-186-5p with continuous variables in AMI patients.The Wilcoxon signed-rank test was used for comparing serum exosomal miR-186-5p at admission and after PCI.The Kaplan-Meier curves were displayed to show the accumulating MACE rates.The Log-rank test was used to compare the accumulating MACE rates between AMI patients with different levels of serum exosomal miR-186-5p.A P-value less than 0.05 indicated statistical significance.

Clinical information on AMI patients receiving PCI
The enrolled patients had a mean age of 61.4 ± 10.0 years, and there were 107 (71.3%) male patients.Meanwhile, 111 (74.0%) patients were diagnosed with STEMI, and 39 (26.0%) patients were diagnosed with NSTEMI.A total of 65 (43.3%)patients had multivessel disease.The median [interquartile range (IQR)] symptom-to-balloon time was 4.1 (2.5-7.9)hours.Additionally, the number of implanted stents was 1 in 117 (78.0%) patients, and it was 2 in 33 (22.0%) patients.Furthermore, the mean infarct size was 21.7 ± 8.1%.The mean left ventricular ejection fraction value was 43.5 ± 12.5%.The specific clinical features of AMI patients receiving PCI are listed in Table 1.
Regarding categorical variables, serum exosomal miR-186-5p was only linked with the diagnosis of STEMI (P = 0.034).But it was not linked to sex, marital status, educational level, location, smoking status, hypertension, hyperlipidemia, diabetes, diagnoses, culprit lesion, multivessel disease, thrombus aspiration, or number of implanted stents in AMI patients receiving PCI (all P > 0.05) (Table 3).
The current study further explored the relationship of serum exosomal miR-186-5p with clinical properties in AMI patients receiving PCI.Firstly, it was discovered that serum exosomal miR-186-5p was positively related to BMI and TC in AMI patients receiving PCI.A potential reason might be that exosomal miR-186-5p might facilitate lipid accumulation, which further led to a higher BMI and TC (15).Secondly, serum exosomal miR-186-5p was positively correlated with Scr and inversely linked with eGFR in AMI patients receiving PCI.It would be due to the fact that exosomal miR-186-5p might modulate Smad5 and toll-like receptor 7/8 axis to induce renal inflammation and fibrosis, which impaired renal function and resulted in a higher Scr and lower eGFR (24, 25).Thirdly, serum exosomal miR-186-5p was positively related to CRP in AMI patients receiving PCI.A reason behind this could be that exosomal miR-186-5p might regulate the PTEN/AKT pathway to facilitate the secretion of proinflammatory cytokines, which aggravated the inflammation, thereby leading to higher CRP (26).Fourthly, serum exosomal miR-186-5p was related to the diagnosis of STEMI in AMI patients receiving PCI.It was speculated that exosomal miR-186-5p could facilitate atherosclerosis to exacerbate the occlusion of blood vessels, leading to STEMI (17).
The change of miR-186-5p after PCI in patients with cardiovascular diseases has been reported by one previous study, which discloses that serum miR-186-5p declined after PCI in ACS patients (22).This previous study also indicates that higher serum miR-186-5p at admission estimates a higher MACE risk in ACS patients after PCI (22).In our study, we discovered that serum exosomal miR-186-5p was reduced after PCI in AMI patients.A probable explanation might be that exosomal miR-186-5p could facilitate the progression of AMI, while disease progression was attenuated after PCI, which contributed to a lower exosomal miR-186-5p (17).Apart from this finding, we also discovered that high level serum exosomal miR-186-5p after PCI and its decrement percentage <median (35%) decrement was related to increased MACE in AMI patients.The potential arguments would be that: (1) exosomal miR-186-5p might regulate C1q/TNF-related protein 3 and Yin Yang 1 pathway to aggravate myocardial ischemia/reperfusion-induced injury after PCI, which led to the occurrence of MACE (27,28).(2) as It should be clarified that immune cells play a fundamental role in the pathology and progression of AMI (29).Additionally, miR-186-5p shows the potential to regulate the inflammatory response, migration, proliferation, foaming, and polarization of immune cells    (15,27,30,31).Therefore, we speculated that miR-186-5p might also regulate the functionality of immune cells to engage in AMI pathology, which could be study direction for our subsequent studies.Moreover, in our previous study, we discover that macrophage-derived exosomal miR-186-5p is involved in the pathology and progression of AMI (17).In addition, macrophages play a fundamental role in regulating inflammation and atherogenesis, and one previous study indicates that miR-186-5p regulates LOX-1 to enhance macrophage foaming, thereby accelerating atherosclerosis (15,32).Therefore, it might be meaningful to explore the abundance of macrophages in AMI patients, and the correlation between exosomal miR-186-5p and the abundance of macrophages could also be explored by subsequent studies.Apart from macrophages, regulatory T cells (Tregs) regulates cardiac fibrosis, inflammation, and angiogenesis to engage in AMI (33).Hence, further studies should also explore the abundance of Tregs in AMI patients.Several limitations should be mentioned in the current study.(1) this was a single-center study; thus, selection bias would unavoidably exist.(2) the follow-up duration could be prolonged to explore the long-term prognostic implication of serum exosomal miR-186-5p in AMI patients receiving PCI.
(3) the number of HCs was small and unmatched to the number of AMI patients receiving PCI, which might affect the results.(4) immune cells played fundamental role in the pathology and progression of AMI; further studies should explore the longitudinal changes of distinct immune cells in AMI patients, and the effect of exosomal miR-186-5p on the functionality of distinct immune cells should be further investigated as well.
In conclusion, serum exosomal miR-186-5p is reduced after PCI; its post-PCI high level and minor reduction predicts increased MACE risk in AMI patients.

FIGURE 1
FIGURE 1Serum exosomal miR-186-5p was higher in AMI patients receiving PCI than HCs.Comparison (A) and discriminative ability (B) of serum exosomal miR-186-5p between AMI patients receiving PCI and HCs.

TABLE 1
Characteristics of AMI patients.
3.3 Relationship of serum exosomal miR-186-5p with clinical properties in AMI patients receiving PCI

TABLE 2
Correlation of serum exosomal miR-186-5p with continuous variables in AMI patients.

TABLE 3
Comparison of serum exosomal miR-186-5p in AMI patients with different categorical variables.