Encyclopedia
Vascular smooth muscle cell (VSMC) proliferation plays a critical role in atherosclerosis. At the beginning of the pathologic process of atherosclerosis, irregular VSMC proliferation promotes plaque formation, but in advanced plaques VSMCs are beneficial, promoting the stability and preventing rupture of the fibrous cap. Recent studies have demonstrated that microRNAs (miRNAs) expressed in the vascular system are involved in the control of VSMC proliferation.
- miRNA
- VSMC
- proliferation
- atherosclerosis
- CVD
- restenosis
1. Introduction
Atherosclerosis is the main cause of cardiovascular diseases (CVDs), which are a leading cause of death worldwide [1][2][1,2]. Atherosclerosis is an inflammation-associated condition, in which arteries become narrowed and hardened due to an excessive buildup of plaque in the inner lining of the arteries [3][4][3,4]. A plaque is made up of cholesterol, other lipids, and diverse cell types [1][2][1,2]. At the beginning of the pathologic process of atherosclerosis, vascular smooth muscle cells (VSMCs) migrate from the media into the intima and proliferate within the lesions in response to mediators secreted by monocytes and lymphocytes [5][6][5,6]. The monocytes locally differentiate into macrophages, which scavenge the oxidized low-density lipoprotein (oxLDL) to become lipid-laden macrophage foam cells [7][8][7,8]. Over years, foam cells and extracellular lipid droplets form a core region of the plaque in the wall of the affected arteries [7][8][7,8]. The plaque is covered by a fibrous cap composed of VSMCs and a VSMC-derived extracellular matrix [1][2][1,2]. Inflammatory molecules and proteolytic enzymes can weaken the cap, transforming the stable plaque into a vulnerable structure that can rupture [8]. Plaque rupture induces thrombosis and can lead to life-threatening events such as heart attacks and strokes [9]. Therefore, at the beginning of the pathologic process of atherosclerosis, irregular VSMC proliferation promotes plaque formation, but in advanced plaques VSMCs are beneficial, promoting the stability and preventing rupture of the fibrous cap [10].
Aberrant VSMC proliferation represents a significant problem not only during initial plaque formation but also after surgical interventions like percutaneous transluminal coronary angioplasty or bypass surgery contributing to pathological re-narrowing (restenosis) of the affected blood vessel [11]. Restenosis occurring in the first six months after angioplasty has been reported in as many as 25–50% of the patients [12]. Currently, therapy aimed at the inhibition of VSMC proliferation is an established approach against restenosis. The most prominent drugs used in drug-eluting stents so far have been paclitaxel (a microtubule stabilizing agent) and sirolimus (an mTOR inhibitor) [13][14][13,14].
2. miRNAs Which Inhibit VSMC Proliferation
2.1. miRNAs Influencing Growth Factors/Cytokines, Growth Factor Receptors, and Other Membrane Receptors in VSMCs
It was shown that the increase of miRNA-9 inhibited the serum-induced proliferation of VSMCs by directly targeting the PDGF receptor (PDGFR) disrupting downstream signaling cascades [15][25]. Furthermore, a small molecule that increased miRNA-9 expression also inhibited neointima formation following balloon injury in vivo [15][25]. It was demonstrated that the overexpression of miRNA-34a in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miRNA-34a dramatically promoted VSMC proliferation by targeting neurogenic locus notch homolog protein-1 (Notch1), a single-pass transmembrane receptor [16][34]. The miRNA-34c inhibited VSMC proliferation and neointimal hyperplasia by targeting the stem cell factor (SCF), a cytokine that binds to the c-kit receptor [17][35]. miRNA-141 inhibited oxLDL-induced abnormal VSMC proliferation through targeting pregnancy-associated plasma protein A (PAPP-A), a secreted protease targeting IGF-1 binding proteins [18][40]. A similar effect was also described for another miRNA—miRNA-490-3p, which inhibited the proliferation of VSMCs induced by oxLDL by also targeting PAPP-A [19][55]. The overexpression of miRNA-206 inhibited VSMC proliferation by silencing the expression of the gap junction protein connexin 43 (Cx43), a component of gap junctions and intercellular channels, via targeting Cx43 3′-UTR [20][49]. It was reported that miRNA-379 inhibited cell proliferation by targeting 3’-UTR of the IGF-1 gene [21][53]. It was shown that miRNA-503 inhibited PDGF-induced human aortic VSMC proliferation and migration by targeting the insulin receptor (INSR), a transmembrane receptor that is activated by insulin, IGF-1 [22][56]. In addition to PDGFR and INSR, there are other growth factors and their respective receptors which also influence the VSMC proliferation, including fibroblast growth factor (FGF), and epidermal growth factor (EGF). It is very interesting to further investigate the effect of the miRNAs which influence these growth factors and their receptors on VSMC proliferation.
2.2. miRNAs Influencing Regulators of Cell Cycle Progression in VSMCs
It was reported that miRNA-22 inhibited VSMC proliferation and migration through repressing ecotropic virus integration site 1 protein homolog (EVI-1) gene expression [23][29], which is a regulator of cell cycle progression by stabilizing the FBXO5 (F-Box Protein 5) protein and promoting cyclin-A accumulation during interphase [24][83]. Furthermore, it inhibited neointima formation in wire-injured femoral arteries [23][29]. Overexpression of miRNA-24 could attenuate VSMC proliferation and neointimal hyperplasia after vascular injuries in diabetic rats. This result is possibly related to the regulation of the expression of Cyclin D1 and CKI p21 through the wingless-type MMTV (Mouse Mammary Tumor Virus) integration site family member 4 (Wnt4)/disheveled-1 (Dvl-1)/β-catenin signaling pathway [25][32]. A different mechanism of proliferation suppression was demonstrated for miRNA-24, which inhibited high glucose-induced VSMC proliferation and migration by targeting high mobility group box-1 (HMGB1) [26][84]. A study indicated that miRNA-195 inhibited oxLDL-induced VSMC proliferation by repressing the expression of the cell division control protein 42 homolog (Cdc42), cyclin D1, and fibroblast growth factor 1 (FGF1) genes [27][48]. The administration of miRNA-195 has been shown to substantially reduce neointima formation in vivo [27][48]. The upregulation of miRNA-362-3p was demonstrated to inhibit VSMC proliferation and migration, and impede the G1/S cell cycle transition by binding to the 3′-UTR of ADAMTS1 and decreasing the levels of its mRNA and protein expression [28][51]. Further study revealed that significant downregulation of miRNA-362-3p was observed in 110 atherosclerotic coronary artery disease (CAD) patients and not in the 84 control subjects [28][51]. In addition, miR-365 was reported to inhibit VSMC proliferation partly via modulating the expression of cyclin D1 [29][52]. Increased levels of miRNA-424/322 inhibited VSMC proliferation in vitro and in injury-induced remodeling in vivo by directly targeting cyclin D1 and calumenin [30][54]. There are two restriction points in G1/S and G2/M interphases, which ensure correct cell cycle progression [31][85]. The cell cycle phases are strictly regulated by many regulatory mechanisms. Key regulatory proteins include the cyclins, cyclin-dependent kinases (CDK), CDK inhibitors (CKI), retinoblastoma protein (RB), and the tumor-suppressor gene product [32][86]. The degree to which the miRNAs influence these factors on VSMC proliferation remains to be examined.
2.3. miRNAs Regulating Signaling Cascades in VSMCs
There are some miRNAs regulating signaling cascades in VSMCs. It was shown that the overexpression of miRNA-15b/16 promoted SMC contractile gene expression while attenuating VSMC proliferation by repressing the potent oncoprotein Yes-associated protein (YAP). Knockdown of endogenous miRNA-15b/16 in VSMCs attenuated VSMC-specific gene expression and promoted VSMC proliferation and migration [33][27]. A different mechanism of proliferation suppression was demonstrated for miRNA-16, which was observed to be highly expressed in VSMCs and to be involved in the Ang-II-mediated VSMC signaling pathways [34][28]. Lentiviral vector-mediated miRNA-16 knockdown promoted Ang-II-induced cell proliferation and migration, which was associated with the pathways involving ERK1/2 and p38MAPK [34][28]. It was reported that the expression of miRNA-126 inhibited VSMC proliferation by targeting the low-density lipoprotein receptor-related protein 6 (LRP6) that is involved in a canonical Wnt pathway. Furthermore, it repressed neointima formation in vitro and in vivo [35][39]. It was reported that the overexpression of miRNA-145-5p inhibited PDGF-induced VSMC proliferation and migration by directly targeting Smad4 and dysregulating the TGF-β signaling cascades, including Smad2, Smad3, and TGF-β [36][44]. In addition, miRNA-155 was shown to inhibit VSMC proliferation though directly repressing 20-like kinase 2 (MST2) and thus activating the ERK pathway by promoting an interaction between Raf proto-oncogene serine/threonine-protein kinase (Raf-1) and mitogen-activated protein kinase kinase (MEK) [37][46]. Another study reported that miRNA-155 modulated the proliferation of VSMC by targeting endothelial nitric oxide synthase (eNOS) [38][47]. The overexpression of miRNA-214 in serum-starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miRNA-214 dramatically increased the proliferation and migration [39][50]. Further study indicated that NCK (non-catalytic region of tyrosine kinase adaptor protein 1) associated protein 1 (NCKAP1), involved in the transduction of signals from Ras to Rac, is the functional target of miRNA-214 in VSMCs [39][50]. Furthermore, upregulation of miRNA-542-3p in old VSMCs significantly inhibited VSMC proliferation, whereas downregulation of miRNA-542-3p in young VSMCs increased VSMC proliferation by targeting spleen tyrosine kinase (Syk)/STAT3-5 axis [40][57]. It has been shown that the overexpression of miRNA-612 significantly inhibited PDGF-BB-induced migration and invasion of VSMCs through inducing cell cycle arrest at the G1 stage by directly decreasing AKT2 (AKT serine/threonine kinase 2) protein expression [41][58]. The overexpression of let-7d in VSMCs reduced VSMC proliferation by targeting KRAS, which is a member of the small GTPase superfamily and regulates the pathway involved in proliferation [42][62]. The transfection of let-7g into VSMCs has also been shown to significantly inhibit VSMC proliferation induced by oxLDL through targeting lectin-like oxidized-low-density lipoprotein receptor-1 (LOX-1) [43][63]. Another study indicated that let-7g maintained VSMC in the contractile status by reducing the PDGF/mitogen-activated protein kinase kinase kinase 1 (MEKK1)/ERK/KLF4 signaling, which further reduced VSMC atherosclerotic change [44][87]. The signaling cascades in VSMCs are very complicated, including Ras/MAPK cascades, Src-activated signal transduction, phosphatidylinositol 3-kinase (PI3K)/AKT, phospholipase C-γ (PLC-γ), phosphatase and Janus kinase (Jak)/STAT signaling pathways [45][88]. The influence of the miRNAs regulating other signaling cascades on VSMC proliferation remains to be further investigated in vitro and in vivo.
2.4. miRNAs Regulating Transcription Factors in VSMCs
Some miRNAs have been implicated in the regulation of transcription factors. It was demonstrated that miRNA-15a, which is upregulated in VSMCs treated with a transcription factor KLF4, can strongly inhibit the proliferation of VSMCs [46][26]. Some studies show that miRNA-22-3p overexpression had anti-proliferative and anti-migratory effects by directly targeting HMGB1, a co-factor for gene transcription and proinflammatory factor [47][89], in human arterial smooth muscle cells (HASMCs) [48][30]. Furthermore, miRNA-22-3p expression was downregulated and negatively correlated with HMGB1 expression in arteriosclerosis obliterans tissue specimens [48][30]. miRNA-23b was also shown to suppress the urokinase-type plasminogen activator, Smad3, and a transcription factor forkhead box O4 (FoxO4) expression in phenotypically modulated VSMCs [49][31]. The overexpression of miRNA-23b in balloon-injured arteries by Ad-miRNA-23b markedly decreased neointimal hyperplasia [49][31]. Further study validated the transcription factor FoxO4 as a direct target of miRNA-23b in VSMCs [49][31]. The overexpression of miRNA-124 significantly attenuated PDGF-BB-induced human aortic VSMC proliferation and phenotypic switch by directly targeting the 3′-UTR of the specificity protein-1 (Sp-1) gene and then repression of transcription factor Sp-1 expression [50][36]. MiRNA-124 was further shown to be dramatically downregulated in the aortic media of clinical specimens of the dissected aorta and correlated with molecular markers of the contractile VSMC phenotype [50][36]. Another study suggested that miRNA-124 inhibited VSMC proliferation by targeting the S100 calcium-binding protein A4 (S100A4), which prevented protein phosphatase 5 (PP5) activation [51][37]. Additionally, the expression of miRNA-125b was decreased in the arteries with arteriosclerosis obliterans and PDGF-BB-stimulated VSMCs [52][38]. miRNA-125b suppressed VSMC proliferation and migration but promoted VSMC apoptosis by directly targeting SRF, a member of the MADS (MCM1, Agamous, Deficiens, and SRF) box superfamily of transcription factors [52][38]. Furthermore, exogenous miRNA-125b expression inhibited vascular neointimal formation in balloon-injured rat carotid arteries [52][38]. Some studies indicated that the overexpression of miRNA-145 or miRNA-143 is sufficient to promote differentiation and inhibit proliferation of cultured VSMCs by targeting a network of transcription factors, including a transcription factor KLF4, a transcriptional coactivator myocardin, and a transcription activator ELK-1 (member of ETS oncogene family) [53][54][41,42]. Deficiency of miRNA-145/143 promoted the synthetic phenotype of VSMCs [55][56][90,91]. Adenoviral-mediated gene transfer of miRNA-145/143, which was downregulated after injury [57][81], inhibited neointimal lesion formation in injured rat carotid arteries [54][42]. A recent study suggested that miRNA-145 inhibited VSMC proliferation by targeting a member of the tumor necrosis factor (TNF)-receptor superfamily cluster of differentiation 40 (CD40) [58][43]. It has been demonstrated that overexpression of miRNA-663 potently inhibited PDGF-induced VSMC proliferation and migration. This most likely occurred by inhibition of the expression of a transcription factor Jun B/myosin light chain 9 [59][60]. Furthermore, adeno-miRNA-663 markedly suppressed the neointimal lesion formation in mice after vascular injury induced by carotid artery ligation, specifically via decreased Jun B expression [59][60]. Transcription factors are proteins that control the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence [60][92]. The miRNA-22-3p regulating HMGB1 might be a very promising target to treat diseases related to VSMC proliferation since its inhibitory effect on VSMCs has been verified in both animal models and humans.
