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].

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].

miR-10a, miR-31, and miR-17-3p regulate inflammation modulating the expression of adhesion molecules in ECs, while miR-155 and miR-331 through the down-regulation of the anti-inflammatory suppressor of cytokine signaling 1 (SOCS1) protein [53,54,73,89,90]. During atherosclerosis development, miR-155 begins to stimulate atherosclerosis progression through repressing Bcl6 in macrophages, suppressing the expression of eNOS, and increasing pro-inflammatory NF-κB signaling [52].

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 VSMCs-enriched 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].

Some miRNAs were investigated in the context of carotid plaques, including miR-520, miR-455, and miR-105. Some are common for many arterial territories, including miR-21, miR-27, miR-100, and miR-122 (Table 1) [45,55,56,57,58,61,62,63,64,65,66,67,68,69,70,71,72,74,75,76,77,78,79,80,81,82,83,84,96,97,98,99,100,101,102].

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 H₂O₂-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.