Association of microRNAs with cerebral atherosclerosis progression: the role of endovascular interventions
AbstractBackground. Atherosclerosis of the brachiocephalic arteries is one of the leading causes of ischemic cerebrovascular disorders, which is based on a complex, multi–stage process regulated by epigenetic mechanisms. The latter, in particular, include changes in the expression of molecules such as microRNAs – small (on average, 22 nucleotides) non-coding RNA sequences that are heavily involved in most physiological and pathological processes.
Objective. The aim of the study was to evaluate the expression of miR–126-5p/-3p, miR-21-5p/-3p, miR-33a-5p/-3p, miR-29a-5p/-3p in relation to association with clinical cerebrovascular events and to identify differences in the expression pattern depending on the performed transluminal balloon angioplasty with carotid artery stenting for hemodynamically significant carotid stenosis.
Patients and methods. Our prospective study included 80 patients [average age – 66 years, men – 44 (55%)] with cerebral atherosclerosis of varying severity who were admitted to the vascular departments of the “Research Center of Neurology” (Moscow). Transluminal balloon angioplasty with stenting (TBAS) of the carotid artery was performed in 33 patients [in 14 (42%) patients – for symptomatic stenosis]. All patients underwent thorough clinical and neurological examination, laboratory and instrumental research methods, including isolation and analysis of microRNA expression (miR-126-5p/-3p, miR-21-5p/-3p, miR-33a-5p/-3p, miR-29a-5p/-3p).
Results. The microRNA expression pattern was significantly different in the patients who underwent TBAS: the levels of miR-126-5p/-3p and miR-29a-5p were statistically significantly lower, and those of miR-33a-5p/-3p higher compared with the patients who did not undergo angiosurgery. A multivariate linear regression model that included the expression of all the microRNAs studied identified miR-126-5p and miR-21-5p as statistically significant independent predictors of the degree of carotid artery stenosis.
Conclusion. The expression of a number of atherogenic microRNAs in patients who underwent transluminal angioplasty with carotid artery stenting appeared to be of a differentiated nature. The most significant biomarker was the expression level of miR-126-5p, which is associated, among other things, with the degree of carotid stenosis.
Funding. The study was financially supported by the grant of the Ministry of Science and Higher Education of the Russian Federation for large scientific projects on priority trends of scientific and technological development, agreement No. 075-15-2024-638.
Conflict of interest. The authors have no conflicts of interest to declare.
Authors’ contribution. Study conception and design – Tanashyan M.M., Raskurazhev A.A.; laboratory stage – Shabalina A.A.; data collection and handling – Annushkin V.A., Mazur A.S., Kuznetsova P.I..; statistical processing – Raskurazhev A.A.; draft manuscript preparation – Raskurazhev A.A., Tanashyan M.M., Mazur A.S.; manuscript revision – Tanashyan M.M.
For citation: Tanashyan M.M., Raskurazhev A.A., Shabalina A.A., Mazur A.S., Annushkin V.A., Kuznetsova P.I. Association of microRNAs with cerebral atherosclerosis progression: the role of endovascular interventions. Angiology and Vascular Surgery. Journal named after Academician A.V. Pokrovsky. 2025; 31 (1): 41–7. DOI: https://doi.org/10.33029/1027-6661-2025-31-1-41-47
Keywords: atherosclerosis; microRNA; cerebrovascular pathology; biomarkers
Introduction
Atherosclerosis of the brachiocephalic arteries is one of the leading causes of ischemic stroke [1]. Atherosclerosis today is considered to be a systemic chronic inflammatory disease of large and medium-sized arteries associated with subendothelial lipid penetration. Atherosclerotic plaque formation is a complex, multi-staged process, and according to modern concepts, epigenetic mechanisms play an important role in its regulation. The latter include alterations in the expression of certain molecules such as microRNA – small (on average 22 nucleotides) non-coding RNA sequences closely involved in most physiological and pathological processes [2].
Despite common fundamental mechanisms underlying atherogenesis the localization of the pathological process (e.g., coronary, iliac, carotid arteries, etc.) significantly affects its course and spread. The feasibility of translating scientific data regarding coronary atherosclerosis to carotid atherosclerosis (and vice versa) without first reproducing these studies remains an open question in case of microRNAs, which are subtle molecular genetic superstructures. This underscores the clinical and fundamental relevance of microRNA assessment in clear, clinically defined cohorts of patients – with the identification of those molecules that may have diagnostic and prognostic significance as biomarkers.
Earlier, in the framework of several pilot studies, we identified a number of atherosclerosis-associated microRNAs [3], the change in expression of which could serve as a marker of carotid atherosclerosis (CA) progression: miR-126-5p/-3p, miR-21-5p/-3p, miR-33a-5p/-3p, miR-29a-5p/-3p. The aim of this work was to study their expression in association with clinical cerebrovascular events (i.e. stroke development), as well as to identify differences in the expression pattern depending on the transluminal balloon angioplasty with stenting of the carotid artery (carotid artery stenting, CAS) performed for hemodynamically significant carotid stenosis.
Material and methods
This prospective study included 80 patients (44 men [55%], average age 66 years) with cerebral atherosclerosis of varying severity, from vascular departments of the Research Center of Neurology (Moscow, Russia). Inclusion criteria were: ultrasound signs of extracranial atherosclerosis of the brachiocephalic arteries (carotid and/or vertebral), signed informed consent to participate in the study. Exclusion criteria were: acute cardiovascular events (stroke/myocardial infarction/etc.) within the previous 3 months, decompensation of somatic pathology (renal/liver/respiratory failure), history of oncological diseases, refusal to participate in the study.
All patients underwent clinical and neurological examination, including evaluation of smoking status, concomitant cardiovascular pathology, antithrombotic and lipid-lowering therapy intake; ultrasound examination of the brachiocephalic arteries (duplex scanning, including determination of the degree of stenosis according to NASCET); blood tests (general and biochemical blood tests with determination of the lipid profile – the level of total cholesterol [TC], high-density lipoprotein cholesterol [HDL] and low-density lipoprotein [LDL], triglycerides [TG]) were made.
According to indications, transluminal balloon angioplasty with stenting of the CAS was performed in thirty-three patients [in 14 (42%) patients – due to symptomatic stenosis] to symptomatic stenosis) as part of the provision of high-tech medical care. Isolation and expression analysis of microRNA (miR-126-5p/-3p, miR-21-5p/-3p, miR-33a-5p/-3p, miR-29a-5p/-3p) were performed according to the previously described method [4]. Briefly: in the first stage, microRNA was isolated from leukocytes; in the second stage, the number of microRNA copies was determined on an amplifier using the reverse transcription method (the level of microRNA expression is presented as ×106 copies).
Ethical review
All patients signed informed consent to participate in the study. The work was approved by the local ethics committee of the Research Center of Neurology (protocol No. 11-4/19 dated November 20, 2019).
Statistical analysis
Statistical data processing was performed using the R programming language (version 4.4.1) in the RStudio software shell (version 2024.04.2), with the following plugins: ‘tidyverse’, ‘gtsummary’, ‘randomForest’, ‘stargazer’, ‘Hmisc’, ‘corrplot’. Methods of descriptive statistics (median and interquartile range, mean, proportion/frequency) and comparative statistics were used, including comparison of two independent samples using the Wilcoxon–Mann–Whitney test (for continuous variables), Pearson’s Chi-square tests and Fisher’s exact test (for categorical variables). Logistic regression was performed using the random trees method (package ‘randomForest’, ntree=1000), the average decrease in accuracy and the decrease in the Gini coefficient (both parameters reflect the relative decrease in the accuracy of the model when excluding a given variable) were used as a visualization of the accuracy of the model (Fig. 1). Correlation analysis was performed using the Pearson criterion. The significance level was 0.05, all tests were two-sided.
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Results
The main results of the study are presented in Table 1. The following were chosen as two main criteria for searching for relevant clinical and epigenetic markers: a) history of ipsilateral ischemic stroke (IS); b) carotid angioplasty with stenting (CAS). When comparing the patient groups according to stroke status, the following anticipated statistically significant differences were identified: higher incidence of type 2 diabetes mellitus (67% vs 30%, p=0.001), predominance of male patients (73% vs 44%, p=0.011), higher degree of carotid artery stenosis (60% vs 50%, p=0.006), and higher triglyceride levels (1.87 vs 1.30 mmol/L, p=0.005) in patients with IS. Among microRNAs, miR-126-3p and miR-29a-3p should be emphasized, whose expression was statistically significantly lower in the IS group.
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Other statistically significant differences were found in the group of patients who underwent CAS: for example, percutaneous interventions were performed more often in patients with other cardiovascular diseases (in particular, every second patient had a history of diagnosed coronary heart disease [55% vs 21%, p=0.002]). The microRNA expression pattern was also significantly different in patients with prior CAS: miR-126-5p/-3p and miR-29a-5p levels were statistically significantly lower and miR-33a-5p/-3p levels were higher compared to the group that did not undergo angiosurgery.
Of note is a rather high adherence to treatment in the studied cohort of patients: more than 80% of patients took the prescribed antiplatelet (in particular, acetylsalicylic acid) and hypolipidemic (statins) therapy.
In a multiple analysis of factors potentially associated with previous CAS, a significant contribution of the expression of a number of microRNAs acting as independent predictors was identified (Fig. 1) – in particular miR-126 and miR-29a.
In contrast, a multiple linear regression model that included the expression of all microRNAs examined identified miR-126-5p and miR-21-5p as statistically significant independent predictors of the degree of carotid artery stenosis (Table 2); and decreased expression of both microRNAs was associated with higher grades of ICA stenosis.
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Correlation analysis also demonstrated certain associations in CAS patients (Fig. 2).
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Noteworthy is the presence of statistically significant correlations of all microRNA expression with the degree of carotid stenosis (positive – for miR-33a-5p/-3p, negative – for miR-126-5p/-3p, miR-29a-5p/-3p, miR-21-5p/-3p) in the group of patients who did not undergo CAS. Only correlations of miR-126-5p (r=-0.44) and miR-29a-5p (r=0.40) with the degree of carotid stenosis remain in the CAS group. In addition, a cluster of unidirectional, statistically significant correlations of miR-33a with lipid profile parameters, which had not been observed before, was revealed in this cohort of patients.
Discussion
For the first time in a cohort of patients with cerebral atherosclerosis, we demonstrated a differentiated pattern of expression of atherogenic microRNAs depending on prior carotid artery stenting (CAS), as well as the association of a number of epigenetic markers with ischemic stroke (IS). The choice of the studied microRNAs was made on the basis of earlier works, which demonstrated their significant alterations depending on the severity of carotid stenosis [5].
One of the main microRNAs whose expression was statistically significantly lower in the CAS group was miR-126, an endothelium-specific microRNA closely associated with the initiation and progression of atherosclerosis. Special attention has been paid to miR-126 because of its ability to maintain the functional integrity of the endothelium by stimulating reparative processes, inhibiting vascular inflammation, and promoting neointima formation [6]. Many authors consider miR-126 as “mechanosensitive”, i.e. its expression is highest in areas of turbulent blood flow (e.g., the bifurcation of the common carotid artery) [7].
In our work, the lowest values of miR-126 expression were observed in the group of patients who underwent CAS – ×106 vs 9×106, respectively (p<0.05). There can be several explanations for this phenomenon: firstly, CAS implies changes in the morphostructure of atherosclerotic plaque in the carotid artery due to the physical impact of the stent, which can lead to changes in epigenetic regulation at this level and, accordingly, alterations in the miRNA expression pattern. Taking into account that miR-126 is most significant exactly for those parts of carotid arteries in which atherogenesis is active, the decrease in its expression after CAS may indicate a decrease in pathologic activation of endothelial cells. On the other hand, if we consider high expression of miR-126 as an atheroprotective factor, its decrease may not be directly related to CAS, but indirectly – because a priori these are patients with a higher degree of carotid stenosis. This can be indirectly confirmed by the multiple linear regression model presented in the article, according to which decreased expression of miR-126-5p (along with miR-21-3p) is a significant independent predictor of a higher degree of carotid artery stenosis.
Decreased miR-126 expression is also a good predictor of atherosclerotic plaque instability in carotid arteries [8]; in the same study, the cyclooxygenase type 2 gene (COX2) was identified as one of the target genes for miR-126, and its expression was inversely correlated with the level of circulating miR-126. In our work, the clinical equivalent of carotid plaque instability was a history of ipsilateral ischemic stroke – it appeared that lower miR-126-3p expression was associated with a cerebral atherothrombotic event. In a mouse experiment, administration of endothelial exosomes rich in miR-126 promoted neuroreparative effects in an ischemic stroke model [9].
Another microRNA with reduced expression in the CAS group was miR-29a-5p. Y. Cao et al. showed that miR-29a mediates the apoptotic effect of tumor necrosis factor-α on endothelium, thus playing a significant role in the progression of atherosclerosis [10]. Given the high representation of this microRNA in endothelial cells, the decrease in its expression after CAS is potentially a favorable factor reflecting the reduction in the area of atherogenic changes. On the other hand, studies on models of hyperlipidemia-induced vascular injury have shown a decrease in the level of circulating miR-29a [11], indicating its dual role in the processes of atherogenesis. In our study, decreased miR-29a-3p expression was statistically significantly associated with ischemic stroke.
miR-33a, a microRNA affecting lipid metabolism, showed opposite directional changes: its expression was higher in the group of CAS-treated patients than in the comparison group. We have previously demonstrated the association of changes in this microRNA with proatherogenic alterations of lipid profile [12]. Inhibition of miR-33a-5p in macrophages is atheroprotective by modulating ABCA1-dependent cholesterol efflux [13]. Some authors postulate, in particular, that targeted delivery of anti-miR-33a-5p therapies may be promising with respect to personalized treatment of atherosclerosis and its complications [14]. The reason for the higher expression of miR-33a in patients who underwent CAS remains not completely clear – it may be due to proinflammatory properties of miR-33a, which are activated in response to endothelial damage as a result of stenting.
It is interesting to note that one of the independent predictors of severe carotid stenosis was a decrease in miR-21-3p expression, which showed no significant association with either stroke or CAS. miR-21 is one of the important epigenetic regulators of cardiovascular pathology, and targeting it with various agents is considered as a promising avenue of treatment [15]; however, the results of studies regarding the role of miR-21 in atherogenesis are contradictory and do not provide a clear understanding of the significance of alterations in its expression. Since miR-21 is expressed in all major effector cells of the atherosclerotic process (macrophages, endotheliocytes, smooth muscle cells), it most likely mediates different mechanisms (proatherogenic and atheroprotective) depending on the stage of the disease.
In general, a superficial search of available literature sources revealed only a small number of publications directly or indirectly discussing the involvement of epigenetic mechanisms against the background of carotid artery stenting, which determines the relevance of the presented study. The demonstrated changes in the expression pattern of atherogenic microRNAs associated with clinically significant events (ischemic stroke, CAS), firstly, substantiate the relevance of microRNAs not only as diagnostic but also as predictor biomarkers (including differentiating the response to treatment); secondly, they emphasize the need for further investigation of their prognostic value in prospective studies; thirdly, they convincingly argue for the need to investigate microRNAs as therapeutic agents.
The limitations of this study include a relatively small sample size; potential selection bias, i.e. selection of patients on the basis of one clinic, which introduces a certain bias in the generalization of the results obtained; selection of a small number of microRNAs; non-prospective nature of the study.
Conclusion
Carotid atherosclerosis is a complex, heterogeneous pathologic process that forms the basis for a large number of cerebrovascular disorders. The study of epigenetic regulation (in particular, microRNAs) of the development and progression of this condition is promising not only from the fundamental point of view, but also in the aspect of searching for precision diagnostic and prognostic biomarkers. In our study in patients who underwent carotid artery stenting, we identified a differential expression pattern of a number of atherogenic microRNAs. The most significant biomarker in this respect was the level of miR-126-5p expression, which was associated, among other things, with the degree of carotid stenosis. Decreased expression of miR-126-3p and miR-29a-3p was associated with ischemic stroke, which emphasizes the need for their further study in prospective cohort studies.
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