microRNAs Associated with Carotid Plaque Development and Vulnerability: The Clinician’s Perspective

Ischemic stroke (IS) related to atherosclerosis of large arteries is one of the leading causes of mortality and disability in developed countries. Atherosclerotic internal carotid artery stenosis (ICAS) contributes to 20% of all cerebral ischemia cases. Nowadays, atherosclerosis prevention and treatment measures aim at controlling the atherosclerosis risk factors, or at the interventional (surgical or endovascular) management of mature occlusive lesions. There is a definite lack of the established circulating biomarkers which, once modulated, could prevent development of atherosclerosis, and consequently prevent the carotid-artery-related IS. Recent studies emphasize that microRNA (miRNA) are the emerging particles that could potentially play a pivotal role in this approach. There are some research studies on the association between the expression of small non-coding microRNAs with a carotid plaque development and vulnerability. However, the data remain inconsistent. In addition, all major studies on carotid atherosclerotic plaque were conducted on cell culture or animal models; very few were conducted on humans, whereas the accumulating evidence demonstrates that it cannot be automatically extrapolated to processes in humans. Therefore, this paper aims to review the current knowledge on how miRNA participate in the process of carotid plaque formation and rupture, as well as stroke occurrence. We discuss potential target miRNA that could be used as a prognostic or therapeutic tool.


Introduction
Ischemic stroke (IS) is one of the leading causes of mortality and disability in developed countries [1]. Atherosclerotic internal carotid artery stenosis (ICAS) accounts for about 20% cases of cerebral ischemia [2]. The present diagnostic tools for carotid artery assessment are based on imaging studies, including carotid Doppler ultrasonography, computed tomography, magnetic resonance, or conventional invasive angiography with a use of intravascular ultrasound (IVUS), and optical coherence tomography (OCT) [3][4][5]. They display the degree of ICAS, as well as carotid plaque morphology [6].
The current guidelines position carotid endarterectomy (CEA) and carotid artery stenting (CS) as the established treatment methods for ICAS [7]. In addition to invasive treatment, the optimal medical approach, including cardiovascular risk factor-control, as well as pharmacotherapy (i.e., antiplatelet and antidiabetic agents, lipid and blood pressure lowering medication), should be introduced in order to reduce IS risk [8,9]. The optimal timing for the intervention on carotid artery is controversial [10,11]. According to guidelines, CEA or CS is recommended in recently symptomatic ICAS with stenosis severity above 50% lumen reduction [10,11], whereas the intervention on asymptomatic ICAS is recommended in high-grade stenosis, or in carotid plaques exceeding 60% lumen reduction when features of high-risk plaque for cerebral ischemia are present [10]. As IS can result from a fragmented plaque debris release with a subsequent embolization of cerebral arteries, plaque rupture followed by local carotid artery thrombosis, or hypoperfusion of cerebral structures, the mechanism of cerebral ischemia is complex [12][13][14][15]. Thus, as evidenced, plaque morphology and structure, in addition to the degree of carotid stenosis, play the pivotal role in the IS risk assessment and decision on the intervention [16].
The serious drawback of the aforementioned imaging tools is that they do not allow for the assessment of early stages of atherosclerosis, i.e., those that precede intima-media complex thickening and early fatty lesions incidences [17]. Unfortunately, current guidelines miss laboratory biomarkers which could predict the incidence of IS and thus target the high-risk group of patients with preemptive treatment, whereas early intervention upon the initiation of atherosclerosis seems very attractive [18]. Data show the important roles of pro-atherothrombotic and pro-inflammatory biomarkers, including cytokines (IL-1β, IL-6, TNFα), platelets, and macrophages activity [19][20][21].
Recent studies emphasize that microRNA (miRNA) are the emerging particles that could potentially play a pivotal role in this approach [22]. miRNAs are small, non-coding RNA nucleotides, having a length that is typically between 18 and 27 nucleotides that regulate post-transcriptional gene expression, by binding to the 3 -(more often), or to 5 -untranslated regions of mRNA, or exons [23]. The role of the miRNA has already been confirmed in the broad range of both physiological and pathological processes [24]. They are responsible for target gene expression regulation after the transcription process, either by inhibiting the translation or mRNA degradation [25]. The diagnostic and prognostic role of circulating miRNAs in ICAS leading to IS has been studied, however the conclusions remain inconsistent. This paper aims to review the current knowledge and assess the newest studies on how the miRNA participate in the process of carotid plaque formation and rupture, as well as stroke occurrence.

From Fatty Streaks and Foam Cells to Mature Plaque
Plaque formation initiates from stages that are not detectable by imaging tools [26]. First stages include endothelium dysfunction, accompanied by inflammation and modified low-density lipoprotein (LDL) retention in the intimal layer of the intima-media complex [27]. In the endothelium equilibrium, a great number of miRNAs are involved, including protective ones [28]. Their protective effect is achieved through many signaling pathways, however their major role is to prevent unfavorable lipid metabolism and reduce inflammation [28]. One of these miRNAs, miR-126, protects endothelial cells (ECs) through the suppression of NOTCH-1 inhibitor and activation of the vascular endothelial growth factor (VEGF) signaling (Table 1) [29,30]. At the beginning, miR-155 induces the downregulation of mitogen-activated protein 3 kinase 10 (MAP3K10), endothelin-1 (ET-1), and angiotensin II (ANG II) type I receptor [31,32]. The downregulation of ET-1 is important in many cardiovascular settings, as elevated levels of ET-1 are independently associated with increased cardiovascular mortality [33,34]. miR-146a and miR-125a decrease the lipid uptake in macrophages [35,36]. miR-146a also inhibits endothelial activation by increasing nitric oxide synthase (eNOS) expression [35]. miR-125 modulates extracellular vascular endothelial growth factor (VEGF) by manipulating macrophage soluble VEGF receptor-1 (sVEGFR1) production. This mechanism has a therapeutic potential in many diseases [36]. miR-206 and miR-223 regulate cholesterol synthesis through the reverse cholesterol transport from macrophages to the liver for excretion, attenuates pro-inflammatory cytokine production, and has a role in platelet activation [37][38][39][40][41]. T, inhibition with antago-miR-34a [48,49] Promotes cholesterol accumulation in macrophages, decreases reverse cholesterol transport miR-33a Targets hepatic ABCA1 inhibit efflux, increases macrophages ox-LDL uptake, foam cells accumulation T, inhibition of miR-33a facilitates atherosclerosis regression [50,51] Promotes atherosclerosis miR-155 repressing Bcl6 in macrophages, suppress eNOS increases pro-inflammatory NF-κB signaling, down-regulates the expression of eNOS and production of NO T, inhibition of miR-155 increased eNOS expression and NO production [32,52] Increases apoptosis in ECs miR-17-5p repression of ABCA1 expression through directly binding to its 3 -UTR rate of apoptosis in ECs T, inhibition of miR-17 suppresses apoptosis, hence decrease infarct size area, and improves microcirculation of the heart tissue, decreasing heart failure symptoms [53,54] Promotion of monocyte adhesion, proinflammatory Lipid metabolism miR-21 targets PPAR α targets TLR4 and NF-κB enhances the expression of VCAM-1 and MCP-1 and the adhesion of monocytes to ECs LPS-induced lipid accumulation and inflammatory response in macrophages Overexpression of miR-21 up-regulated AP-1 activation, which was attenuated by exogenous expression of PPARα overexpression of miR-21 significantly decreased the secretion of IL-6 and increased IL-10 levels [55,56]  Some miRs, such as miR-let-7g, also modulate ECs senescence by regulating anti-aging gene sirtuin 1 (SIRT1) and the insulin growth factor (IGF) 1 pathway [42]. In line with this, miR-143 is downregulated with advancing age and protects against vascular senescence [43,44]. On the contrary, miR-92a released from ECs stimulates macrophages to the pro-inflammatory responses and LDL uptake, which enhance atherosclerotic progression [85]. In mice, inhibition of miR-92a reduces endothelial inflammation and atheroma plaque size through the regulation of Kruppel-like factor 2 (KLF2) [47]. Similarly, miR-34a aggravates and accelerates vascular senescence through the downregulation of SIRT1 and AXL Receptor Tyrosine Kinase [48], whereas miR-34a inhibition by anti-miR-34a reduced vascular inflammation, senescence, and apoptosis [49,86]. In macrophages, ox-LDL increases miR-34a levels that target the cholesterol transporters' ATP-binding cassette subfamily A member 1 (ABCA1), and ATP-binding cassette subfamily G member 1 (ABCG1) [48]. This alters lipid metabolism, while miR-34a enhanced secretion of inflammatory cytokines promotes inflammation facilitating atherosclerotic plaque formation [48,49]. ECs dysregulation is enhanced by the lipid accumulation due to disturbed reverse cholesterol transport of cholesterol efflux from macrophages to the liver [50]. This results in lipid accumulation in macrophages with formation of foam cells [48]. miR-206 and miR-233 promote cholesterol efflux to the liver, whereas miR-33a inhibits reverse transport (Table 1) [39,40,51,87]. miR-33a/b have been shown to act as post-transcriptional regulators of a lipid metabolism, and their pharmacological inhibition diminished atherosclerosis by raising plasma high-density lipoprotein levels [87]. Nguyen et al. demonstrated that chitosan nanoparticles containing miRs can be delivered to macrophages [88]. In mice, macrophages treated with miR-33-loaded nanoparticles showed decreased reverse cholesterol transport [88]. In contrast, when efflux-promoting miRs were delivered the efflux was improved [88].
There is a continuous crosstalk between ECs and vascular smooth muscle cells (VSMCs) [91]. ECs-derived miRNAs, like miR-126, miR-92a exert action on VSMCs, resulting in the VSMCsenriched miRNAs release, that often have reciprocal unfavorable effects on ECs [92,93]. On atherosclerosis initiation, VSCMs migrates from the medial arterial wall into the intimal space, resulting in promotion of plaque formation [91]. VSCMs migration and proliferation is one of key stages in early atherosclerosis. miR-520 regulates VSMCs function by targeting RelA/p65. This way, decreasing cells migration and proliferation exerts a protective role in atherosclerosis [45]. Moreover, miR-520c-3p mimics may act as a promising therapeutic strategy for atherosclerosis [45]. The VSMCs equilibrium plays a great role in the inhibition of atherosclerosis. The maintenance of contractile phenotype prevents atherosclerosis [45,59,60,[91][92][93][94]. Of the miRs capable of the contractile function recovery in VSMCs, miR-22 and miR143/145 are probably the most investigated ones, and are potential therapeutic targets [59,60,91,94]. Intravenous delivery of miR-143/145 extracellular vesicles blocked atherosclerotic lesion progression and exerted protective effects on intima-media complex [59,60], while miR-22 restores contractile phenotype of VSMCs without a negative impact on EC's function [95]. In addition, miR-22 inhibits intima-media hyperplasia, which is important both for inhibition of atherosclerosis plaque growth, as well as in the restenosis following stent implantation [46,95].
When the anti-atherothrombotic miRNAs are overbalanced by the pro-atherothrombotic miRNAs, we can steadily observe carotid intima-media complex thickening (CIMT), followed by the occurrence of focal non-calcified lesions [102]. Then, a formation of mature plaques composed of lipid and necrotic cores, fibrotic matrix, and calcifications are observed, accompanied by inflammation and angiogenesis. The first caution that should attract the attention of clinicians is CIMT [103][104][105]. CIMT, in ranges above the 75th percentile for age and gender, can even be observed in teenagers and young adults, particularly if accompanied by atherosclerosis risk factors, such as diabetes or familial hypercholesterolemia [106,107]. This parameter is well correlated with risk of cardiovascular events, such as cardiovascular death (CVD), IS, and myocardial infarction (MI) [103][104][105]. It has also a good predictive value for a presence of significant atherosclerosis in the other territories, e.g., coronary arteries [108,109]. Several miRNAs are associated with CIMT, including miR-22, miR-29a, miR-143/145, and miR-92a [46,70,110,111]. With increasing CIMT, atherosclerotic process accelerates. There is a huge role for metalloproteinases (MMP), such as MMP-2 and MMP-9, as they are associated with a promotion of plaque growth and CIMT increase, rather than a decrease in VSMCs contractility [111]. Interestingly, in advanced carotid plaques, migration and proliferation of VSMCs is beneficial, promoting the stability of the fibrous cap and prevention of plaque rupture [91,102]. This process in stimulated by the expression of miR-145 and miR-210 that drive the increase in plaque collagen content and a fibrous cap area, while at the same time reducing the necrotic core area [46,65,95].
In contrast, plaque instability is associated with increasing levels of MMP-2, MMP-9, the increasing size of plaque and the lipid and necrotic core, particularly when abundant in lipids [111][112][113][114]. MMP-9 is particularly important as it predicts future adverse cardiovascular events [71,[111][112][113]. It was observed that MMP-9 is regulated by several miRNAs, including miR-92a, which is a predictor of plaque instability [114]. However, miR-92a is not necessarily always negative [115,116]. The upregulation of MMP-9 and the downregulation of TIMP3 in H 2 O 2 -induced VSMCs were observed to be reversed by mimicking miR-92a in addition to SIRT1 and siRNA, which may prevent a phenotypic change of VSMCs [115,116]. Other miRNAs associated with serum concentration of MMP-9 include miR-100, miR-155, miR-133a, and miR-223 [111,114]. These miRNAs are also linked to plaque instability and might be used as biomarkers of plaque conversion from a stable state into a vulnerable state [114][115][116][117][118][119].
Thus, it is of the utmost importance to identify carotid plaques that are likely to undergo transformation from the stable ones to vulnerable ones, prone to rupture and cause symptoms of cerebral ischemia. The research is ongoing for the identification of specific microRNAs that could prevent IS through the manipulation of their expression levels.

Degree of Carotid Plaque, Patients with Asymptomatic and Symptomatic ICAS: Prognostic miRNAs
One of the key issues addressed in recent studies was the role of miRNAs in differentiating patients with stable asymptomatic ICAS from those who may develop symptomatic ICAS, thus the identification of patients characterized with high risk of ICAS-related IS [114,119]. Alas, the majority of studies on carotid atherosclerosis describe carotid lesions that are non-occlusive, whereas the occlusive carotid artery lesions regarding miRNA expression profile and levels are under-investigated. Notably, ICAS causes at least a 50% stenosis, which increases the risk of carotid lesion-related IS [120]. miRNA as potential markers of carotid plaque that is non-obstructive compared to plaques causing a significant carotid stenosis (equal or greater than 50% lumen reduction) were investigated by Raskurazhew et al. (Table 2) [100]. In this study, the levels of miR-126-3p, miR-126-5p, miR-21-3p, and miR-29a-3p were higher in non-obstructive carotid plaques, meanwhile they were low in carotid plaques which caused lumen reduction of 50% or greater [100]. In contrast, levels of miR-33a were lower in early carotid plaques, while higher in asymptomatic ICAS exceeding 50% [100].
In the study by Dolz et al., the authors focused on the progression of asymptomatic ICAS and the potential role of miRNAs as non-invasive biomarkers of carotid plaque instability and ICAS progression [121]. The analysis identified a different miRNA expression profile in RNA samples from patients with ICAS progression during the follow-up period, compared to the patients without ICAS progression. Progression of ICAS was associated with significantly higher expression levels of miR-199b-3p  [121]. However, in the multivariate analysis, only the overexpression of a proangiogenic miR-130a-3p was independently associated with the ICAS progression (ORs, 5.4; 95% CI, 1.03-28.2; p = 0.025) and IS risk [121].
In line with this, miR-637 was assessed as a potential biomarker of ICAS and IS occurrence [122]. The prospective analysis revealed that down-regulation of miR-637 was associated with the degree of ICAS, with a sensitivity of 85.6%, and a specificity of 83.3% for ICAS of at least 50% lumen narrowing [122]. During the 5 year follow-up, the Kaplan-Meier analysis showed that the lower miR-637 levels were associated with a higher incidence of ICAS-related IS (HR = 0.073, 95% CI = 0.017-0.313, p < 0.001), along with severe ICAS (HR = 0.144, 95% CI = 0.045-0.463, p = 0.001) [122]. The study proved that the miR-637 downregulation is responsible for cardiovascular events in patients with atherosclerosis [137] by promoting the proliferation and migration of VSMCs by regulating IGF-2 [138].
In another study by Zhu et al., the sensitivity and the specificity of miR-486 for a diagnosis of significant asymptomatic ICAS were 82.4% and 89.7%, respectively [123]. The results showed that HAEC apoptosis was weakened by increased miR-486-5p, reflecting its protective effect against vascular ECs injury [123]. Vascular ECs injury may promote the release of intracellular adhesion factors and then trigger the transformation of macrophages into foam cells. In vitro experimental results demonstrated that miR-486-5p can also inhibit the release of ICAM-1 in HAECs. Besides its regulatory role in the apoptosis of vascular ECs, the findings of the study also demonstrated the influence of miR-486-5p on inflammation and oxidative stress [139].
There is limited data on the miRNAs acting as triggers for symptom development in patients with a significant ICAS. In the study by Grosse et al., the levels of several popular circulating miRNAs (miR-92a, miR-126, miR-143, miR-145, miR-155, miR-210, miR-221, miR-222, and miR-342-3p) were analyzed in patients with symptomatic and asymptomatic ICAS that were referred to CEA [124]. The study has not found miRNAs diagnostic for symptomatic ICAS, except from the trend to difference for miR-92a in multivariate analysis [124]. The authors have not observed either the relationship between miRNAs expression levels and micro embolic signals that correspond to plaque fragility and distal embolization to cerebral arteries by debris released from the plaque [140].
The study by Luque et al. aimed to present the associations between atherosclerotic plaque instability and circulating miR-638 level [125]. The results showed significantly lower miR-638 levels in patients with symptomatic ICAS, as compared to asymptomatic ICAS [125]. The study demonstrated that miR-638 level was correlated with the IS incidence and IS recurrence, smoking habit, bilateral ICAS, coronary artery disease, and hypercholesterolemia [125]. The authors considered miR-638 as a potential marker associated with plaque vulnerability and IS.
In the study by Badacz et al., the differences in expression levels of circulating miR-133a, miR-124, and miR-134 were found in patients with symptomatic vs. asymptomatic ICAS [83]. The authors observed higher serum levels of miR-1 (p = 0.032) in a thrombotic plaque, as well as higher levels of miR-1 (p = 0.04) and miR-16-5p (p = 0.003) in patients with ulcerated plaques [83].
Similarly, in the study by Zhang et al., the expression levels of miR-106b-5p were analyzed in asymptomatic ICAS patients [126]. The expression of miR-106b-5p was associated with hypertension, dyslipidemia, and degree of ICAS. In ROC analysis, miR-106b-5p showed a good predictive value for significant ICAS compared to healthy individuals, with a sensitivity of 89.7% and a specificity of 83.6% [126]. Furthermore, patients with higher miR-106b-5p expression levels were more likely to suffer from IS during follow-up (log rank p = 0.020; HR 5.431, 95% CI: 1.592-18.520, p = 0.007).
The other study by Zhang et al. focused on miR-637 and its possible role as a biomarker for IS risk [122]. The multivariate analysis revealed that downregulation of miR-637 independently predicted future IS (HR = 0.073, 95%CI = 0.017-0.313, p < 0.001), thus making the analyzed miRs possible biomarkers in ICAS patients [122].
In line with this, Li et al. analyzed expression levels of miR-483-5p in patients with asymptomatic ICAS and evaluated its diagnostic and predictive value for IS [127]. The results revealed that patients with high miR-483-5p levels had higher a risk of IS (log-rank p = 0.011), as compared to patients with lower miR-483-5p expression levels (HR 2.670; 95% CI 1.099-6.484; p = 0.030) [127]. Similarly, miR-342-5p was proposed as an IS risk predictor at a 5 year follow-up by Zhou et al. [128].
Several more studies found possible predictive value of specific miRNAs for the onset of IS. Chen at al. stated that elevated miR-92a expression could differentiate patients with asymptomatic ICAS from healthy subjects, and was an independent predictive factor for the occurrence of IS (HR = 2.971, 95% CI = 1.230-7.173, p = 0.015) [129], while Zhou et al. observed miR-342-5p as a potential predictor of IS in asymptomatic patients with ICAS (HR = 5.512, 95% CI = 1.370-22.176, p = 0.016) [129].
Finally, the recent study by Badacz et al. analyzed cardiovascular outcomes in patients with diabetes who underwent intervention on ICAS [134]. During a follow-up period of 7 years, miR-134-5p occurred an independent risk factor of CVD/MI/recurrent IS (HR = 1.12; 95% CI = 1.05-1.21, p < 0.001) in diabetic patients, while miR-16 in patients without diabetes [134]. The authors suggested the diversity of miRNAs profile in patients with and without diabetes for cardiovascular outcomes. In line with this, Sardu et al. found miR-24 as a possible predictor of ischemic cardiovascular events and hospitalization for heart failure in pre-diabetic patients at a 2 year follow-up [141].
In addition to miRNA, there is probably a huge role for microparticles in vascular damage in diabetic patients [142]. Some of these macromolecules have angiogenetic properties or participate in modulation of vascular senescence or remodeling, taking part in the process of vascular aging [142]. The other miRNAs that could be potentially associated with plaque instability and cerebrovascular outcomes are: miR-21, miR-221, miR-133b, and miR-124 were independent predictors (Table 2) [135,136].

microRNA Expression Levels in Plaques Removed during CEA
Apart from circulating serum or plasma miRNAs, the carotid plaque can contain large amounts of miRNAs. These miRNAs often differ or have diverse expression levels in plaque, compared to systemic miRNAs [97]. Intra-plaque miRNAs play the role in the process of carotid plaque evolution and progression, leading to plaque rupture and IS, as well [97]. These miRNAs can be explored only when a biological material is available, e.g., after the excision of carotid plaque that takes place during CEA.
The study by Cipollone et al. covered the large-scale miRNA analysis of symptomatic and asymptomatic carotid plaques excised during CEA (Table 3) [97]. The results presented significant up-regulation of several miRNAs (out of 41 analyzed), miR-145 and miR-133a in particular, in symptomatic plaques as compared to asymptomatic ones [97]. In the study by Maitrias et al., seven miRNAs were dysregulated, including miR-100, miR-125a, miR-127, miR-133a, miR-145, and miR-221 in carotid plaques obtained during CEA in 30 symptomatic and asymptomatic patients [22]. The identified miRNA profile was corresponding to the inflammatory process leading to plaque destabilization. miR-100, miR-125a, and miR-127 were proven to modulate the vascular inflammation and angiogenesis, as well as oxidative stress [125,[146][147][148]. Finally, miR-133a, and miR-145 are considered to play the key role in regulating the vascular remodeling and inflammation especially by controlling VSMCs proliferation and differentiation [149,150]. In the study by Bazan et al., the miR-221 and miR-222 expressions were evaluated in carotid plaques obtained from CEA in three groups of patients: with asymptomatic ICAS, symptomatic ICAS (neurologic symptoms within 5 days), and patients who underwent neurologic event earlier [143]. Patients in acute phases of neurologic symptoms related to ICAS exhibited a significant decrease in miR-221 and miR-222 expression as compared to the other groups [143,150]. This confirms the direct association of miRNAs dysregulation in the mechanisms of plaque instability [143].
Another promising potential biomarker of plaque instability in carotid atherosclerosis is miR-200c [144]. The miR-200c plaque concentration was up-regulated in carotid plaques that presented unstable features on imaging studies. Additionally, miR-200c positively correlated with instability biomarkers, such as COX2, IL-6 or MMP-9 [144]. miR-146a and pro-inflammatory cytokines in patients with severe ICAS were addressed in the study by Huang et al. [151]. The expression levels of miR-146a, IL-6, and TNF-α in the ICAS group were higher than those in the control group and positively correlated with the degree of ICAS and plaque vulnerability [151]. In the study by Wei et al., upregulation of miR-330-5p level was identified in symptomatic carotid plaques [145].
Thus, the complex process of plaque destabilization is attributed to both intra-plaque action of miRNAs and is simultaneously regulated by various circulating miRNAs.

Conclusions
MicroRNA have become a point of interest in recent years of scientific research including broad range of pathophysiological issues. Multiple recent studies proved miRNAs to be the important factor of atherosclerotic process, including coronary, peripheral, or carotid artery disease. miRNA seem to be directly associated with a patient's atherosclerosis severity and functional prognosis, suggesting that miRNAs can be used as potential biomarkers for the diagnosis and prognosis evaluation of cardiovascular events. According to modern evidence, some miRNAs reveal a potential diagnostic or prognostic value in revealing the potential vulnerable plaque features or predicting future ischemic events.
Several miRNAs were proven to be a viable diagnostic tool in assessing the risk of carotid plaque destabilization, and as a result of plaque rupture, the risk of IS occurrence is high. Moreover, the studies have shown that different miRNAs were able to differentiate the symptomatic and asymptomatic course of ICAS.
However, the studies struggle with a wide range of miRNAs, missing identification of the narrow group of pivotal and key miRNAs responsible for the atherosclerosis processes, which may play the role of biomarkers in everyday routine practice. Therefore, dedicated study is necessary in order to establish which miRNAs could be used in carotid atherosclerosis.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Conflicts of Interest:
The authors declare that there are no conflict of interest regarding the publication of this article.