Introduction

Atherosclerosis is a chronic inflammatory disorder characterized through activation of endothelial cell (EC), infiltration of monocytes and differentiation into macrophages and foam cell formation that cause endothelial dysfunction and proliferation of smooth muscle cell (SMC) [1]. Actually, the atherosclerosis pathogenesis consists of several processes with immune and non-immune cells involvement. Investigations through decades have revealed that various genes and signaling pathways are involved in the beginning and development of atherosclerosis [2].

For the first time, microRNAs (miRNAs) were introduced in C. elegans [3]. miRNAs are small (18–22 nucleotides), evolutionarily conserved, and single-stranded non-coding RNAs, which bind to the 3′-untranslated regions (3′-UTRs) of the target mRNA sequences and regulate gene expression. miRNAs through two important mechanisms causes gene suppression; first mechanism is inhibition of the mRNA translation and the next is degradation of the mRNA [4,5,6,7,8,9] (Fig. 1). It has been proposed that more than 60% of protein coding genes are regulated through miRNA [10]. In addition, a specific miRNA can bind and regulate several genes and, on the other hand, a specific gene can be regulated by many miRNAs. miRNAs are implicated in controlling of gene expression in different pathophysiological process, such as atherosclerosis development [2].

Fig. 1
figure 1

Biogenesis of miRNAs. miRNAs are single stranded non-coding RNAs with approximately about 22 nucleotides in length. At first, RNA polymerase II transcribes miRNA genes to generate primary miRNAs (pri-miRNAs). Afterwards, Drosha, which is an endonuclease, catalyze the pri-miRNA to generate precursor miRNAs (pre-miRNAs). Then, the pre-miRNA is transported to cytoplasm through exportin-5 located on the nucleus membrane. In the cytoplasm, Dicer, which is an endoribonuclease, cleaves the pre-miRNA to produce aseymetric duplexes of miRNA containing 19–22 nucleotides. The active strand of miRNA is located in the ribonucleoprotein (RNP) complex, which is named as RNA-induced silencing complexes (RISCs). In order to function, mature miRNAs within the RISC binds to 3′-untranslated region (UTR) of a given mRNA that culminates in to gene suppression. miRNA guide the RISC complex to a particular mRNA then the core component of RISC complex (Argonaute) directly implements the gene silencing [95] (license acquired from RightsLink, License Number: 4818641467208, [96])

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Recent studies in the field of microRNA (miRNA) have resulted in a big achievement in recognizing the cell types and diseases [11]. miRNAs are considered as important regulators of cellular migration, differentiation, proliferation, lipid uptake and efflux, and cytokine production. miRNAs have provided new molecular visions about atherosclerosis and been presented as a novel therapeutic approach. miRNAs can be evaluated in the body fluids and this feature confers them the potential as biomarkers for prognosis, diagnosis, and even follow up of patients. Recently, bioinformatic evaluations have provide promising insights on the biogenesis, function, and basic mechanisms of miRNAs in the etiology and pathogenesis of atherosclerosis [2].

This review article intends to review recent findings on the dysregulation of miRNAs in atherosclerosis cells/tissues and incorporate them with the previous knowledge, in order to get an insight on the potential of miRNAs to be used as prognostic marker in atherosclerosis. Ultimately, the implications for miRNA-based therapy will be clarified in the context of the disease.

miRNA abnormalities in different cells/tissues of atherosclerosis

miRNA and endothelial cell

Recent knowledge about miRNAs introduced them as a novel class of inter- and intra-cellular molecules that affect endothelial cells (ECs) and change their profile. Investigations on miRNA signature illustrated that there are associations between miRNAs and pathogenesis of atherosclerosis [11]. Endothelial cells are the first cells that participate in the pathogenesis of atherosclerosis. Under the biochemical stimuli, ECs undergo a series of cellular and molecular changes that initiate the atherosclerotic plaque formation. For instance, expression of adhesion molecules like intracellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, and E-selectin facilitates the recruitment and migration of leukocyte to the marginal area of vessel that is the first stage of plaque formation [12]. Various miRNAs, including miR-17-3p, miR-31, and miR-126 modulate inflammation through regulation of the adhesion molecules, such as ICAM-1, E-selectin, and VCAM-1 [13]. It has been documented that miR-146a plays an important role in plaque destabilization and also modulates inflammation in atherosclerosis. Part of miR-146a functions originates from its effect on activation of nuclear factor (NF)-κB signal-transduction pathway [14]. In addition, miR-10 influences the NF-κB signaling pathway, hence plays a crucial role in atherosclerosis regulation [15]. On the other hand, miR-146 inhibits the Mitogen-activated protein (MAP) kinase and NF-κB pathways. Furthermore, it has been illustrated that miR-146 target HuR, a RNA binding protein, that leads to activation of ECs through inhibition of endothelial nitric oxide synthase (eNOS or NOS2) [16]. miR-126-5p in endothelial cells plays a protective role in the formation of atherosclerotic lesion through inhibition of Notch1 inhibitor delta-like 1 homolog (Dlk1) [17]. miR-126 has an important role in the angiogenesis through proliferation of ECs by inhibition of suppressors of the phosphatidylinositol kinase (PI3K) pathway [18]. miR-223 is involved in inhibition of cholesterol biosynthesis and controlling of high-density lipoprotein-cholesterol (HDL-C) uptake. In addition, this miRNA plays roles in cholesterol metabolism [19]. miR-26a has a therapeutic role and has been linked to cell death in atherosclerosis [20]. Thus, multiple miRNAs are involved in the modulation of ECs and have been implicated in pathogenesis of atherosclerosis.

miRNAs and monocyte/macrophage

The second stage of atherosclerosis is leukocyte recruitment and migration from blood flow to the artery wall in the areas of lipoprotein retention and endothelial dysfunction. One of important leukocyte which migrates to the artery wall is monocyte [2]. Monocytes are the precursors of myeloid-derived dendritic cells and tissue resident macrophages, which are differentiated into foam cells and cause plaque development [21]. Actually, these cells eventually differentiate into macrophages. These cells play a highlighted role in the pathophysiology of atherosclerosis through two main mechanisms; production of inflammatory mediators and modulation of lipid homeostasis. Lipoprotein uptake by macrophages leads to differentiation of macrophages to foam cell, which is a hallmark of atherosclerosis [22]. These foam cells stay in the location of injury of artery wall and promote the inflammatory immune responses and promote the plaque formation. These cells are the main producers of chemokines and cytokines, which involved in recruitment, migration, differentiation and activation of immune cells, thereby maintain and exacerbate the chronic inflammation [2]. Furthermore, these foam cells are implicated in the destabilization and rupture of plaques, which make these cells the most important cells in atherogenesis. Since doam cells are so important in atherosclerosis, researchers believe that they can modulate the pathological changes in the injury site by manipulating these cells [23]. One of the molecules that affects the infiltration of monocytes is miRNAs. For example, miR-124a, through regulating C-|C motif chemokine ligand 2 (CCL2) expression, promotes the rolling and migration of monocytes into the vessel wall [24]. Different miRNAs, such as miR-106a, miR-20a, and miR-17 control the infiltration of macrophages through direct inhibition of signal-regulatory protein-α (SIRPα) [25]. High expression of miR-145 by VSMCs blocks the macrophage infiltration and it could be a potential target for atherosclerosis management [26]. miR-223 directly inhibits several chemo-attractants, including CCL3 and chemokine C-X-C motif ligand 2 (CXCL2), thereby control infiltration of different myeloid cells [27]. High expression of nitro-oxidative stress through atherosclerotic lesion formation may be one of the functions of miR-342-5p, which is produced by macrophages and results in the atherosclerosis progression. Microarray analysis showed that miR-365, miR-145, miR-143, and miR-155 were downregulated, while miR-352, miR-214, miR-146, and miR-21 were upregulated in the neointimal formation models [28]. miR-342-5p by its effects on Akt pathway facilitates the activation of inflammatory macrophages during atherosclerosis. Since by targeting miR-342-5p in macrophages, the cascade of molecular events which proceed to form an inflammatory macrophage is prevented, it could be a promising therapeutic strategy [29].

The balance among the endogenous synthesis, uptake, efflux, hydrolysis and esterification of cholesterol leads to macrophage cholesterol homeostasis. A number of miRNAs has been identified that are involved in the cholesterol metabolism of macrophages. miR-27a/b through targeting genes implicated in efflux (ABCA1), uptake (CD36, LDL) and cholesterol esterification (ACAT1) may regulate cholesterol homeostasis of macrophages [30]. miR-146a and miR-125a-5p decrease cytokine release and lipid uptake in the oxidized-LDL (ox-LDL)-stimulated macrophages trough targeting toll-like receptor (TLR) 4 and oxysterol binding protein-like 9 (OSBPL9) genes, respectively [31, 32]. miR-155, through targeting HMG box-transcriptional protein 1 (HBP1), controls the development of foam cells. The transcriptional repressor HBP1 negatively regulates macrophage inhibitory factor (MIF), the protein which elevates the uptake of ox-LDL by macrophage [33]. Various miRNAs such as miR-758 [34], miR-302a [35], miR-301b [36], miR-148a [36], miR-130b [36], miR-128-1 [36], miR-26 [37], miR-106 [38], miR-33 [39,40,41], and miR-144 [42, 43] regulate the cholesterol efflux in macrophage through ABCA1 and, thereby, promote formation of macrophage foam cells.

Different environmental factors promote the development of two distinguish class of macrophages; the M1 that is named as classical or proinflammatory macrophage and the M2 that is called as alternative or anti-inflammatory macrophage. Different stages of atherosclerosis are associated with different kinds of macrophage profiles; the M1 phenotype is the predominance kind of macrophage involved in the progression stage and the M2 phenotype is the most frequent type of macrophage playing a role in the plaque regression [22]. There are an increasing list of miRNAs which are involved in regulating the balance between the M1 and M2 macrophages, including miR-223 [44], miR-155 [45], miR-19a [46], miR-33 [47], miR-let7a [48], miR-125a [49], miR-21 [50], miR-214 [51], miR-27a [52], miR-146a [53], and miR-124 [54]. miR-33 plays an important role in controlling efflux of cholesterol, as well as cellular metabolism of macrophages to modify their inflammatory profiles. On the one hand, miR-33 elevates aerobic glycolysis that maintains the M1-like macrophage phenotype, while decreases the oxidation of fatty acid that is critical for M2 macrophages development; however, the ultimate outcome is the predominance of M1 phenotype [47]. miR-33 inhibition metabolically resulted in the M2 predominance that led to tissue repair, resolving inflammation, and finally high level of atheroprotective regulatory T (Treg) cells [47]. miR-155, although with a controversial role in the atherosclerosis, can differentiate M1 from M2 macrophages and results in the elevated number of M1 phenotype [45]. This miRNA is induced by ox-LDL and there is an increased level of miR-155 expression in CD14+ monocytes of coronary artery disease (CAD) patients in comparison to healthy controls [33]. miR-155 inhibits the negative regulators of inflammatory cytokines signaling, including B cell lymphoma 6 (BCL6), Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1), and suppressor of cytokine signaling 1 (SOCS1), thereby promotes the production of proinflammatory cytokines [17, 55,56,57]. On the other hand, miR-223, by targeting Pknox1, can promote the M2 phenotype and also regulates some of lipid metabolism related genes [19, 44]. miR-27a has also been implicated in the formation of foam cell, upregulation of M2 markers like Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, also known as CD209) and CD206, as well as interleukin (IL)-10 production [52].

miR-21 and miR-147 decrease inflammation by attenuation the TLR-associated signaling of macrophages [58, 59]. Furthermore, miR-146a/b has been implicated in the inflammation resolution through attenuation of cytokine and TLR signaling in the macrophages. miR-146a expression in macrophages is induced by apolipoprotein E (apoE), an anti-atherosclerotic protein, that inhibits the inflammatory response of macrophages in vivo and in vitro [60]. Upregulation of miR-124a and miR-150 by the Krüppel-like factor 2 (KLF2) transcription factor, an anti-atherosclerosis factor, leads to low level of pro-atherosclerotic chemokines, such as CXCL1 and CCL2 [61]. miR-342-5p is one of the most important miRNAs that is induced during early stage of atherosclerosis in the macrophages located in the atherosclerotic lesions [29]. This miRNA, through inhibiting Akt1-mediated suppression of miR-155, increases the secretion of inflammatory mediators, such as IL-6 and inducible NOS (iNOS) from macrophages. Accordingly, miR-342-5p inhibition in Apoe−/− mice alleviates atherosclerotic lesions [29]. The bottom line is that miRNAs can influence on the atherosclerosis development through modulating different pathways in monocyte/macrophages.

miRNAs and vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are the cellular components of the normal blood vessel wall that maintains structural integrity and also, regulates the diameter of vessels. In the inflammatory condition, these VSMCs change their phenotypes and are converted into a synthetic phenotype that induces signals involved in the proliferation, migration, and finally inflammation [62]. Various miRNAs have been identified to be involved in the regulation of VSMC through different transcription factors, such as SMADs (involved in transforming growth factor (TGF)-β signaling), myocardin (a co-activator), Platelet-derived growth factor (PDGF; involved in the regulation of cytokines and growth factors), and serum-response factor (SRF/KLF4). Some of the important miRNAs implicated in the regulation of VSMCs and atherosclerosis development are presented in the next section [63].

miR-21 plays an important role in proliferation of VSMCs in response to injuries that culminated in the formation of atherosclerotic lesion. On the other side, inhibition of miR-21 in the mechanical balloon injury situation attenuates formation of neointimal lesion. miR-21 by directly targeting Phosphatase and tensin homolog (PTEN) and indirectly elevating the Bcl-2 expression level leads to high proliferation and low apoptosis of VSMCs [28]. Contractile phenotypes of VSMCs is induced by Bone morphogenetic proteins (BMPs) and TGF-β through miR-21. This miRNA inhibits the programmed cell death 4 (PDCD4), which acts as a negative regulator for genes that are involved in contractile form of VSMC. On the other hands, BMP and TGF-β signaling increase the expression of miR-21. This event mediated through Drosha complex by converting the pri-miR-21(primary transcripts of miR-21) into the pre-miR-21 (precursor miR-21) [64]. Further investigations will be required to evaluate the anti-proliferative effects of miR-21 inhibition and also its role in alleviate of atherosclerosis in non-mechanically injury of vessels.

Both miR-221 and miR-222 have an elevated level of expression in neointimal lesions. It has been documented that miR-221 and miR-222 are involved in VSMC proliferation. High expression of miR-221 and miR-222 in VSMCs, resulted in low expression of p27 (Kip1), c-Kit genes and also, some of genes related to SMC contractile [65]. Knockdown of miR-221 and miR-222 showed that proliferation of VSMC and also formation of neointimal lesion were reduced after mechanical injury by targeting p57(Kip2) and p27(Kip1) [66].

miR-143 and miR-145 are downregulated in vessel wall of atherosclerosis patients [67, 68]. Various loss-, and gain-of-function investigations proposed that miR-143 and miR-145 are two important microRNA with regulatory functions in VSMC contractile. Actually, mice with deficiency in miR-143 and miR-145 showed low level of expression in contractile marker and also their function in SMC, impairment in cytoskeletal dynamics and actin stress fibers and decreased medial thickness of vessel wall [67,68,69]. Furthermore, these mice showed a decreased blood pressure in response to vasopressor challenge and this effect attributed to low level of angiotensin converting enzyme (ACE) expression [70]. Conversely, high expression of miR-145 in ApoE−/− mice leads to a decreased size of atherosclerotic plaque and also, reduced macrophage accumulation and necrotic core area [26]. miR-143 and miR-145 play their roles in expression and function of contractile phenotype in VSMCs through low level of K Kruppel-like factor (KLF4) expression and high level of myocardin expression in the ApoE−/− vessel wall of mice [26]. miR-143 and miR-145 have other targets, such as KLF5 and ELK-1 transcriptional regulator which are implicated in differentiation of VSMC [67, 68, 71].

MiRNA as diagnostic tool

Recent researches have illustrated that miRNAs could be applied as diagnostic and prognostic biomarkers for various disorders, such as cardiovascular diseases, diabetes, kidney diseases, rheumatoid arthritis, and cancer. Since the proteins-based biomarkers could not fulfill the diagnostic criteria for diagnosis of cardiac diseases, then diagnostic markers need an improvement. Investigations from clinical samples illustrated that miRNAs appear to be the most important biomarker for a proper diagnosis and even could be applied as a therapeutic agent for various cardiovascular diseases, including hypertension, stroke, atherosclerosis, heart failure, acute myocardial infarction, and even cancer [72, 73]. miR-423-5p showed an elevated level of expression in heart failure patients irrespective of gender and age and could be applied as a sensitive agent for heart disorders [74]. Conversely, miR-126 and miR-145 showed low level of expression in patients with coronary artery disease [75]. Tissue samples from myocardial infarction (MI) patients showed that miR-133a/b and miR-1 were downregulated and miR-208 was upregulated [76]. Therefore, miRNAs could be applied as an important biomarker for diagnosis, prognosis and, even identification of atherosclerosis.

Therapeutic opportunities and challenges

The miRNA ability to target genes opens a door to disease treatment through gene modulations. Since one miRNA could target many genes, there is also potential side effects of other genes targeting. Yet, this approach might be effective in complex disorders such as atherosclerosis which various pathways are implicated in disease pathogenesis [2].

In order to obtain an efficient miRNA-based therapy, it is critical to find important dysregulated genes and pathways which are implicated in atherosclerosis pathogenesis. Investigations on miRNAs illustrated that these molecules have an important role in atherosclerosis development and also its initiation (Table 1). Since miRNA mimics and inhibitors can target genes which are responsible for atherosclerosis pathogenesis, further researches are required to develop a novel miRNA based drug candidates [11].

Table 1 MicroRNA involvement in atherosclerosis pathogenesis through endothelial, monocyte, macrophage and Vascular Smooth Muscle Cells (VSMCs)
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Anti-sense oligonucleotides provide an opportunity to miRNA silencing to downregulate expression of miRNA or to fine tune special pathways which dysregulated by the disease. To chemically promote the anti-sense oligonucleotide approach, many methods have been used to enhance tissue uptake, stability of miRNAs and target affinity [77]. To minimize unanticipated toxicities and potential side effects, careful assessment of these chemical modifications will be necessary. Single stranded anti-miR oligonucleotides do not need lipid-based delivery systems and can be formulated in saline for intravenous or subcutaneous delivery. After systemic delivery of miRNAs, rapidly taken up by various organs such as liver, kidney, spleen, bone marrow and adipose tissue [78, 79]. After a cell taken up the anti-sense oligonucleotides, the anti-miR forms a high affinity and stable bond with the corresponding miRNA and inhibits the binding of the miRNA to its mRNA target. Preclinical investigations on non-human primates illustrated that using naked anti-miR oligonucleotides could be efficient in targeting of miRNAs (miR-33 and miR-122) especially in the liver [80, 81]. To elevate cellular uptake of anti-miRs, cholesterol analogs have been used and this increased their incorporation into LDL and high-density lipoprotein (HDL) [82,83,84]. miRNA decoy or sponge transcripts is another approach to inhibit miRNA. These transcripts act as competitive inhibitors of the target miRNA [85]. The miRNA sponges have various binding sites which have complementary sequences with the miRNA seed sequence and interfere with miRNA and finally inhibit its function. Viral vectors can be used to deliver miRNA sponges and their expression could be inducible in a specific stage or in a certain cell line through specific promoters. Studies illustrated that after cell transfection with viral vectors containing miRNA sponges, the corresponding miRNAs showed a decreased level of expression [86].

Application of miRNA mimic has two purposes; one is to reconstitute a downregulated miRNA and the second is to decrease expression of genes which are implicated in disease pathogenesis. For instance, downregulation of miR-181b in ECs of CAD patients could be restored with miRNA mimics [87]. Drug delivery vehicles such as lipoprotein-based drug, polymeric micelles, and liposomes carriers have been expanded to deliver these therapeutic oligonucleotides to targets. It is illustrated that to deliver miRNAs between cells, miRNAs are associated with HDL particles and this issue proposes that use of HDL infusion to deliver miRNAs could be efficient [88]. Two of important challenges which the scientists faced with are; specific delivery of miRNAs to a target or a cell, and the second is to achieve an optimal repression multiple doses of miRNA are required.

Since most of oligonucleotide inhibitors and miRNA mimics are mostly taken up by liver, enough amount of these oligonucleotide could not reach to the vessel wall and the success rate is lower than optimal. Studies showed that if miRNAs penetrated specifically to the peripheral blood mononuclear cells and vascular endothelium of the vessel wall the success rate of treatment would be better [87, 89]. In other word, tissue or cell specific delivery of miRNA mimics or inhibitors instead of systemic delivery proposes a novel opportunity to prevent atherosclerosis development and its progression. As a matter of fact, in order to get the highest efficiency with lowest doses and minimal side effects it is critical to find an appropriate translational dosing regimen.

Currently, various miRNA-based therapies are in preclinical stage and two of these therapies are in clinical trials. The first miRNA therapeutic in clinical trial is Miravirsen, a LNA against miR-122, which targets hepatitis C virus (HCV) RNA [90]. Investigations on non-human primates illustrated that inhibition of miR-122 leads to suppression of HCV viremia and considerable side effects and viral resistance has not been reported [91]. The second is the miRNA mimic of miR-34, which stimulating anti-tumor immune responses and inhibit various oncogenic pathways and thereby considered as a tumor suppressor molecule [92]. Investigations on MRX34 [93], a miR-34 mimic encapsulated in a liposomal nanoparticle formulation, in patients with hematological malignancies or advanced solid tumors showed promise results.

Several years of hard working resulted in significant progress in atherosclerosis treatment. Especially, statins (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors) showed promise to treats CAD patients. Although, statins significantly decrease LDL levels and resulted in cardiovascular improvement, but the disease burden even in CAD treated patients remained [2].

To treat disease such as atherosclerosis which complex signaling pathways are involved, novel and complementary therapeutic approaches are required. Since a miRNA may have various targets to suppress gene expression, treatment of patients with miRNAs may have side effects on cell function, biological pathways, and homeostasis in the periphery, liver, and vessel wall. Delivery sets of miRNA mimics or inhibitors could be considered as an attractive and applicable approach in atherosclerosis improvement and also management of its complications [2].

Future perspectives

Over the course of past few years, miRNAs have been evidenced to be involved in the etiology and pathogenesis of atherosclerosis, predominantly through modulating genes playing roles in the process of inflammation. That notwithstanding, it is still critical to further explore and identify the miRNAs with bona fide implication in the atherosclerosis initiation and perpetuation. Furthermore, it is important to clarify miRNA signature in the early and late stage of the disease, thereby facilitating the way toward devising diagnostic biomarkers of the atherosclerosis. On the other hand, miRNA-based therapy or mesenchymal stem cell-derived exosomes could be more efficient in treatment of atherosclerosis [94]. Research in field of miRNA of atherosclerosis is at its infancy, but confers a promising future, as application of miRNAs as a biomarker in diagnosis, prognosis, and even therapy is quiet exciting. Although animal researches showed promising results, still some practical difficulties and technical challenges need to be addressed before translation from researches into clinical practices. As soon as these challenges are resolved, the miRNA-based approaches will be a potential therapy to compete with other therapeutics.