Abstract and Figures

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. This review summarizes recent findings on the miRNAs in the regulation of VSMC proliferation, including miRNAs that exhibit the inhibition or promotion of VSMC proliferation, and their targets mediating the regulation of VSMC proliferation. Up to now, most of the studies were performed only in cultured VSMC. While the modulation of miRNAs is emerging as a promising strategy for the regulation of VSMC proliferation, most of the effects of miRNAs and their targets in vivo require further investigation.
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Int. J. Mol. Sci. 2019, 20, 324; doi:10.3390/ijms20020324 www.mdpi.com/journal/ijms
Review
The microRNAs Regulating Vascular Smooth Muscle
Cell Proliferation: A Minireview
Dongdong Wang 1,2,3,* and Atanas G. Atanasov 1,2,4,*
1 Department of Molecular Biology, Institute of Genetics and Animal Breeding of the Polish Academy of
Sciences, 05-552 Jastrzębiec, Poland
2 Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria
3 Institute of Clinical Chemistry, University Hospital Zurich, 8952 Schlieren, Switzerland
4 GLOBE Program Association (GLOBE-PA), 49418 Grandville, MI, USA
* Correspondence: dongdong.wang@univie.ac.at (D.W.); atanas.atanasov@univie.ac.at (A.G.A.)
Received: 5 December 2018; Accepted: 2 January 2019; Published: 14 January 2019
Abstract: 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. This
review summarizes recent findings on the miRNAs in the regulation of VSMC proliferation,
including miRNAs that exhibit the inhibition or promotion of VSMC proliferation, and their targets
mediating the regulation of VSMC proliferation. Up to now, most of the studies were performed
only in cultured VSMC. While the modulation of miRNAs is emerging as a promising strategy for
the regulation of VSMC proliferation, most of the effects of miRNAs and their targets in vivo require
further investigation.
Keywords: 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]. 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]. A plaque is made up of cholesterol, other lipids, and diverse cell types [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]. The monocytes locally differentiate into macrophages, which
scavenge the oxidized low-density lipoprotein (oxLDL) to become lipid-laden macrophage foam cells
[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]. The plaque is covered by a fibrous cap composed of VSMCs and a
VSMC-derived extracellular matrix [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
Int. J. Mol. Sci. 2019, 20, 324 2 of 15
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].
Recent studies have demonstrated that microRNAs (miRNAs) are expressed in the vascular
system and are involved in the control of VSMC proliferation [15,16]. Each miRNA is able to regulate
the expression of multiple target genes, frequently involved in the same cellular pathway.
Conversely, target genes may be affected by more than one miRNA. It is presumed that thousands
of human genes are targeted by miRNAs [16,17]. Mature miRNAs are short, noncoding ribonucleic
acid molecules, typically 22 nucleotides long, which bind to complementary sequences in the 3’-
untranslated regions (3’-UTR) of target mRNA transcripts. miRNA biosynthesis is initiated by the
transcription of a long capped and poly-adenylated primary miRNA (pri-miRNA) transcript [18,19].
The RNase III enzyme Drosha cleaves the pri-miRNAs into the precursor-miRNAs (pre-miRNAs)
[19]. Pre-miRNAs are exported from the nucleus to the cytoplasm, where they are processed by an
RNase II enzyme, Dicer. This cleavage event gives rise to a double-stranded product composed of the
mature miRNA guide strand and the miRNA passenger strand [20]. Mature miRNAs are then bound
to the miRNA-induced silencing complex, which contains two key proteins, argonaute 2 and
transactivation-responsive RNA binding protein [16,21]. The mature miRNA and miRNA-induced
silencing complex binds to complementary sites in the targeted mRNA transcripts and negatively
regulate gene expression. miRNA binding leads to mRNA degradation, inhibition of translation, or
both [22,23].
Some miRNAs have been implicated in regulating important VSMC nodal regulators such as
cytokines/growth factors and their receptors (e.g., platelet-derived growth factor (PDGF), insulin-like
growth factor-1 (IGF-1)), regulators of cell cycle progression (e.g., cyclins, cyclin-dependent kinase
inhibitors (CKI), a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1)),
signaling cascades (e.g., angiotensin II (Ang II)-mediated VSMC signaling pathways, extracellular
signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinases (MAPK), TGF-β signaling
cascades (Smad), signal transducer and activator of transcription (STAT)), transcription factors (e.g.,
serum-response factor (SRF), Krüppel-like factor-4 (KLF4)), nuclear receptors (e.g., estrogen receptor
α (ERα), neuron-derived orphan receptor 1 (NOR1)), and others (e.g., proviral integration site (Pim-
1), mammalian target of rapamycin (mTOR)). This minireview briefly summarizes recent findings on
the regulatory effects of miRNAs on VSMC proliferation, including both miRNAs that are associated
with inhibition or promotion of VSMC proliferation, see Table 1 and Figure 1.
Table 1. miRNAs implied in the regulation of VSMC proliferation.
miRNA Implied Targets References
miRNAs which inhibit VSMC proliferation
miRNA-1 Proviral integration site (Pim-1) [20,24]
miRNA-9 Platelet-derived growth factor receptor (PDGFR) and further downstream signaling
cascades [25]
miRNA-15a Krüppel-like factor-4 (KLF4) [26]
miRNA-15b/16 The potent oncoprotein yes-associated protein (YAP) and the pathways extracellular
signal-regulated kinase (ERK)1/2 and p38MAPK (mitogen-activated protein kinases) [27,28]
miRNA-22 Ecotropic virus integration site 1 protein homolog (EVI-1) [29]
miRNA-22-3p High mobility group box-1 (HMGB1) [30]
miRNA-23b The transcription factor forkhead box O4 (FoxO4) [31]
miRNA-24 The wingless-type Mouse Mammary Tumor Virus (MMTV) integration site family
member 4 (Wnt4)/disheveled-1 (Dvl-1)/β-catenin signaling pathway [32]
miRNA-29c N.A. [33]
miRNA-34a Neurogenic locus notch homolog protein-1 (Notch1) [34]
miRNA-34c Stem cell factor (SCF) [35]
miRNA-124 The 3′-UTR of the specificity protein-1 (Sp-1) gene or S100 calcium-binding protein A4
(S100A4) [36,37]
miR-125b Serum-response factor (SRF) [38]
Int. J. Mol. Sci. 2019, 20, 324 3 of 15
miRNA-126 Low-density lipoprotein receptor-related protein 6 (LRP6) [39]
miR-141 Pregnancy-associated plasma protein A (PAPP-A) [40]
miRNA-
143/145 KLF4, myocardin, ELK-1, and cluster of differentiation 40 (CD40) [41–43]
miRNA-145-5p
Smad4 and the TGF-β signaling cascades, including Smad2, Smad3 and TGF-β
[44]
miRNA-152 DNA methyltransferase 1 (DNMT1) and the methylation of ERα gene promoter region [45]
miR-155 20-like kinase 2 (MST2), the ERK pathway, or endothelial nitric oxide synthase (eNOS) [46,47]
miRNA-195 The Cdc42, cyclin D1, and fibroblast growth factor 1 (FGF1) genes [48]
miRNA-206 3′-UTR of the gap junction protein connexin 43 (Cx43) [49]
miRNA-214 NCK associated protein 1 (NCKAP1) [50]
miRNA-362-3p A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) [51]
miRNA-365 Cyclin D1 [52]
miR-379 3′-UTR of insulin-like growth factor-1 (IGF-1) [53]
miRNA-
442/322 Cyclin D1 and calumenin [54]
miRNA-490-3p PAPP-A [55]
miRNA-503 Insulin receptor (INSR). [56]
miRNA-542-3p Spleen tyrosine kinase (Syk)/signal transducer and activator of transcription (STAT)3-5
axis [57]
miRNA-612 AKT2 protein [58]
miRNA-638 Neuron-derived orphan receptor 1 (NOR1) [59]
miRNA-663 JunB/myosin light chain 9 [60]
miRNA-761 Mammalian target of rapamycin (mTOR) [61]
let-7d KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) [62]
let-7g Lectin-like oxidized-low-density lipoprotein receptor-1 (LOX-1), and PDGF/mitogen-
activated protein kinase kinase kinase 1 (MEKK1)/ ERK/KLF4 signaling [63]
miRNAs which promote VSMC proliferation
miRNA-17
Retinoblastoma (RB) protein mRNA-3′-UTR
[64]
miRNA-25 Cyclin-dependent kinase 6 (CDK6) [65]
miRNA-26a Smad1 and Smad4 [66]
miRNA-29a 3’-UTR of Fbw7/CDC4, KLF [67]
miRNA-29b SIRT1 [68]
miRNA-31 Cellular repressor of E1A-stimulated genes (CREG) expression. [69]
miRNA-130 The tumor suppressor p21 (CDKN1A) [70]
miRNA-130a CDKN1A, and growth arrest-specific homeobox (GAX). [70]
miRNA-133 CDKN1A [70]
miRNA-138 SIRT1 [71]
miRNA-146a KLF4 [72]
miRNA-146b-
5p The response to PDGF [73]
miRNA-155-5p N.A. [74]
miRNA-200c Ubc9 and KLF4 [75]
miRNA-204 N.A. [33]
miRNA-208 p21 [76]
miRNA-221 PDGFR [77]
miRNA-222 p27Kip1 and tissue inhibitor of metalloproteinase 3 (TIMP3) [78]
miRNA-574-5p
ZDHHC14
(Zinc Finger DHHC-Type Containing 14) gene [79]
miRNA-675
PTEN
[80]
miRNA which promotes and inhibits VSMC proliferation
miRNA-21 Promotion of VSMC proliferation: phosphatase and tensin homolog (PTEN), B-cell
lymphoma 2 (Bcl-2). [72,81]
Inhibition of VSMC proliferation: Programmed cell death 4, a tumor suppressor protein. [82]
N.A. = not available.
Int. J. Mol. Sci. 2019, 20, 324 4 of 15
Figure 1. The role of miRNAs in the regulation of vascular smooth muscle cell (VSMC) proliferation.
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 [25].
Furthermore, a small molecule that increased miRNA-9 expression also inhibited neointima
formation following balloon injury in vivo [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 [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 [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 [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 [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 [49]. It was reported that miRNA-379 inhibited
cell proliferation by targeting 3’-UTR of the IGF-1 gene [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 [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 [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 [83]. Furthermore, it inhibited neointima formation in wire-injured
femoral arteries [29]. Overexpression of miRNA-24 could attenuate VSMC proliferation and
Int. J. Mol. Sci. 2019, 20, 324 5 of 15
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 [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) [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 [48]. The administration of
miRNA-195 has been shown to substantially reduce neointima formation in vivo [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 [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 [51]. In addition, miR-365 was reported to inhibit VSMC proliferation partly
via modulating the expression of cyclin D1 [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 [54]. There are two restriction points in G1/S and G2/M interphases, which ensure correct
cell cycle progression [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 [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 [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 [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 [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 [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 [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) [46]. Another study reported that
miRNA-155 modulated the proliferation of VSMC by targeting endothelial nitric oxide synthase
(eNOS) [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 [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 [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 [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 [58].
The overexpression of let-7d in VSMCs reduced VSMC proliferation by targeting KRAS, which is a
Int. J. Mol. Sci. 2019, 20, 324 6 of 15
member of the small GTPase superfamily and regulates the pathway involved in proliferation [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)
[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 [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 [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 [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 [89], in human arterial smooth muscle cells
(HASMCs) [30]. Furthermore, miRNA-22-3p expression was downregulated and negatively
correlated with HMGB1 expression in arteriosclerosis obliterans tissue specimens [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 [31]. The
overexpression of miRNA-23b in balloon-injured arteries by Ad-miRNA-23b markedly decreased
neointimal hyperplasia [31]. Further study validated the transcription factor FoxO4 as a direct target
of miRNA-23b in VSMCs [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 [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 [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 [37]. Additionally, the expression of miRNA-125b was decreased in the arteries with
arteriosclerosis obliterans and PDGF-BB-stimulated VSMCs [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 [38].
Furthermore, exogenous miRNA-125b expression inhibited vascular neointimal formation in
balloon-injured rat carotid arteries [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) [41,42].
Deficiency of miRNA-145/143 promoted the synthetic phenotype of VSMCs [90,91]. Adenoviral-
mediated gene transfer of miRNA-145/143, which was downregulated after injury [81], inhibited
neointimal lesion formation in injured rat carotid arteries [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) [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 [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 [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 [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.
Int. J. Mol. Sci. 2019, 20, 324 7 of 15
2.5. miRNAs Regulating Nuclear Receptors in VSMCs
Some miRNAs have been implicated in regulating nuclear receptors in VSMCs. It was reported
that the overexpression of miRNA-152 decreased cell proliferation in LPS-treated VSMCs by
downregulating DNA methyltransferase 1 (DNMT1) and subsequently decreasing the methylation
of a nuclear receptor ERα gene promoter region [45]. Additionally, miRNA-638 was shown to inhibit
PDGF-BB-induced VSMC proliferation and migration by targeting a nuclear receptor NOR1 [59],
which is a critical regulator implicated in proliferative vascular diseases [93]. The effect of
miRNA-152 on VSMC proliferation in vivo still remains to be further studied.
2.6. Others
Some observations suggest that the induction of miRNA-1 by myocardin led to an inhibition of
VSMC proliferation by downregulation of Pim-1, which is also named as serine/threonine-protein
kinase Pim-1 [20,24]. It was shown that miRNA-761 inhibited Ang-II-induced VSMC proliferation
and migration by targeting mTOR, which is a serine/threonine-specific protein kinase [61]. miRNA-
29c activation in diabetes mellitus arterial tissues is necessary and sufficient to prevent the
exaggerated VSMC growth upon injury [33]. MiRNA-29c overexpression in the injured artery
robustly reduced arterial stenosis in diabetes mellitus rats [33]. The targets of miRNA-29c in VSMCs
remain to be further investigated.
3. miRNAs Which Promote VSMC Proliferation
3.1. miRNAs Influencing Growth Factors/Cytokines, Growth Factor Receptors, and Other Membrane
Receptors in VSMCs
It has been demonstrated that miRNA-26a promoted VSMC proliferation by directly targeting
Smad1 and Smad4, which are two TGF-β- and BMP-related pro-differentiation factors [66].
VSMCdeficient in miRNA-26a shows a significant reduction in proliferation [66]. Some researchers
also found miRNA-146b-5p was upregulated in PDGF-BB treated VSMCs. Inhibition of miRNA-146b-
5p reduced VSMC proliferation and migration by blocking the VSMC response to PDGF [73]. The
detailed molecular mechanisms by which the miRNA-146b-5p regulates the PDGF signaling pathway
is so far not clear.
3.2. miRNAs Influencing Regulators of Cell Cycle Progression in VSMCs
It was reported that miRNA-17 stimulated the proliferation of VSMCs, enhanced cell cycle G1/S
transition, and increased levels of proliferating cell nuclear antigen and E2F1 by directly targeting the
retinoblastoma (RB) protein mRNA-3′-UTR and then suppressed the expression of RB [64]. In
addition, activation of NF-κB p65 resulted in increased miRNA-17 expression in VSMCs, whereas
inactivation of NF-κB p65 resulted in decreased expression of miRNA-17 in VSMCs [64]. It was
shown that miRNA-25 promoted VSMC proliferation by directly targeting cyclin-dependent kinase
6 (CDK6) [65]. Transfection of VSMC with miRNA-130 decreased the expression of CDKN1A (cyclin-
dependent kinase inhibitor 1A) and, in turn, significantly increased smooth muscle proliferation.
Conversely, inhibition of miRNA-130 by anti-miRNAs and seed blockers increased the expression of
CDKN1A and inhibited VSMC proliferation [70]. The hypoxia-induced miRNA-130a controlled
pulmonary SMC proliferation by directly targeting the tumor suppressor p21 (CDKN1A) [70], and
growth-arrest-specific homeobox (GAX) [94]. One study indicated that miRNA-208 promoted
insulin-induced VSMC proliferation through the downregulation of its potential target p21.
However, a miRNA-208 inhibitor alone had no effect on VSMC proliferation [76]. Expression of
miRNA-221 and miRNA-222 can be transcriptionally induced by PDGF [77]. The proliferative effect
of miRNA-221 and miRNA-222 on VSMC was mediated through silencing of their target proteins,
CKI p27Kip1, p57Kip2, and c-kit [77]. Reduction of miRNA-221 blocked the effects of PDGF on the
proliferation of VSMCs. In the same line, knockdown of miRNA-221 or miRNA-222 inhibited VSMC
proliferation and neointimal formation in rat carotid artery after injury [77]. MiRNA-222 also could
Int. J. Mol. Sci. 2019, 20, 324 8 of 15
promote pulmonary arterial smooth muscle cells (PASMC) proliferation at least partially through
targeting p27Kip1 and the tissue inhibitor of metalloproteinase 3 (TIMP3) [78]. Furthermore, miRNA-
221 sponge therapy significantly reduced miRNA-221 activity and inhibited neointimal hyperplasia
in vein grafts, possibly by targeting p27Kip1 [95]. It is shown that overexpression of miRNA-675
promoted VSMC proliferation in vitro by targeting phosphatase and the tensin homolog (PTEN),
which is involved in the regulation of the cell cycle and aggravates restenosis in vivo [80]. So far, only
miRNA-221 has been verified to exhibit proliferation-regulating effects on VSMCs in vivo. The effects
of other promising miRNAs on VSMC proliferation in vivo need further investigation.
3.3. miRNAs Regulating Signaling Cascades in VSMCs
It has been shown that overexpression of miRNA-133 inhibited VSMC proliferation in vitro
through an ERK1/2 kinase-dependent pathway and increased expression of Sp-1 [96]. Furthermore,
adenoviral overexpression of miRNA-133 in the balloon-injured rat carotid significantly reduced
neointimal formation [96]. Key signaling cascades involved in VSMC proliferation include
Ras/MAPK cascades, Src-activated signal transduction, PI3K/Akt, PLC-γ, phosphatase and Jak/STAT
signaling pathways [88]. The influence of the miRNAs regulating these signaling cascades on VSMC
proliferation remains to be further studied in vitro and in vivo.
3.4. miRNAs Regulating Transcription Factors in VSMCs
A study suggested that the increased amount of miRNA-29a enhanced VSMC proliferation and
promoted atherogenesis, probably through downregulation of Fbw7 (F-box and WD repeat domain-
containing 7)/CDC4 (cell division control protein 4) expression by targeting the 3’-UTR of
Fbw7/CDC4, subsequently increasing a transcription factor KLF5 stability by reducing the
Fbw7/CDC4-dependent ubiquitination of KLF5 [67]. In addition, overexpression of miRNA-146a
increased VSMC proliferation by directly targeting KLF4 which could upregulate p21 [72].
Knockdown of miRNA-146a attenuated PDGF-mediated increase of VSMC proliferation [97].
Treatment of balloon-injured rat carotid arteries with antisense oligonucleotides against miRNA-146a
resulted in reduced neointima formation and VSMC proliferation in vivo [97]. The reduction in
miRNA-200c levels led to increased target gene expression (e.g., Ubc9 and KLF4), which further
repressed miRNA-200c levels and accelerated VSMC proliferation [75]. All these miRNAs (miRNA-
29a, miRNA-146a, miRNA-200c) targeted a common transcription factor, KLF4, which indicates that
KLF4 might be a key target in miRNA-mediated regulation of VSMC proliferation.
3.5. Others
In VSMCs cultured in the presence of the proliferation stimulator PDGF-BB, miRNA-29b was
upregulated significantly [68]. miRNA-29b is demonstrated as being an important regulator in the
PDGF-BB-mediated VSMC phenotypic transition by targeting SIRT1 (gene encoded Sirtuin-1, also
known as NAD-dependent deacetylase sirtuin-1) [68]. The same target was also described for another
miRNA. An in vivo study shows that miRNA-138 promoted VSMC proliferation and migration in
db/db mice through downregulation of SIRT1 [71]. Additionally, the inhibition of miRNA-204 in
diabetes mellitus arterial tissues has been shown to prevent the exaggerated VSMC growth upon
injury [33]. Moreover, miRNA-204 inhibition in the injured artery significantly reduced arterial
stenosis in diabetes mellitus rats [33]. Other studies indicated that miRNA-204 targets Sirtuin-1 in
endothelial cells [98,99]. However, the targets of miRNA-204 in VSMCs remain to be further studied.
miRNA-31 has been proven to promote the VSMC contractile phenotype by repressing the cellular
repressor of E1A-stimulated genes (CREG) expression, a secreted glycoprotein that inhibits cell
growth [69]. It was reported that miRNA-574-5p expression was elevated in the sera and VSMCs of
patients with CAD [79]. Additionally, miRNA-574-5p overexpression promoted cell proliferation and
inhibited apoptosis in VSMCs by directly targeting ZDHHC14 (Zinc Finger DHHC-Type Containing
14) gene [79]. Overexpression or transfection of miRNA-155-5p mimic elevated the proliferation and
migration of VSMCs, which was blocked by treatment with an inhibitor of miRNA-155-5p [74]. The
Int. J. Mol. Sci. 2019, 20, 324 9 of 15
targets of miRNA-155-5p in VSMCs require further investigation. Both miRNA-29b and miRNA-138
targeted SIRT1 to regulate VSMC proliferation, which suggests that SIRT1 might be a key target
mediating the miRNA-modulatory effects on VSMC proliferation.
4. miRNA-21 Which Promotes and Inhibits VSMC Proliferation
Some studies suggest that the function of miRNA-21 is likely to be complex and highly context-
dependent. In addition to promoting contractile gene expression, miRNA-21 has been found to
promote VSMC proliferation [81]. MiRNA-21 is demonstrated to promote VSMC proliferation by
silencing PTEN, a tumor suppressor protein, and increasing B-cell lymphoma 2 (Bcl-2), which
increased VSMC proliferation and survival [72,81]. In the same line, a depletion of miRNA-21 caused
decreased VSMC proliferation. Local delivery of an antisense oligonucleotide to knockdown miRNA-
21 inhibited neointima formation in a rat carotid artery after angioplasty [72,81]. The decrease of the
expression of miRNA-21 by adenovirus-mediated miRNA-21 sponge gene therapy significantly
reduced the proliferation in cultured VSMCs and the proliferation rates in grafts compared with
controls at 28 days after bypass surgery [100]. miRNA-21 sponge gene transfer therapy also reduced
the intimal/media area ratio in vein grafts compared with the controls and improved vein graft
hemodynamics probably by targeting PTEN in vein grafts [100]. On the other hand, in some studies,
the increased miRNA-21 is shown to promote VSMC differentiation and inhibit VSMC proliferation
by upregulating VSMC-restricted contractile proteins by silencing programmed cell death protein 4
(PDCD4), a tumor suppressor protein [82]. To understand the complicated role of miRNA-21 in the
regulation of VSMC proliferation, further studies need to be performed.
5. Conclusions
Extensive research during the last decade investigated the association of miRNAs with VSMC
proliferation. In this minireview, we briefly summarize the miRNAs that regulate VSMC
proliferation. These miRNAs exert their regulation of VSMC proliferation by regulating a number of
target proteins and signaling cascades. The targets modulated by miRNA implied in the inhibition of
VSMC proliferation include PDGFR, KLF4, YAP, EVI-1, HMGB1, Cyclin D1, p21, Sp-1, S100A4, SRF,
LRP6, PAPP-A, myocardin, ELK-1, ERα gene, Cx43, NCKAP1, ADAMTS1, cyclin D1, IGF-1, AKT2,
Notch1 gene, PDGFR downstream signaling cascades, Ang-II-mediated VSMC signaling pathways,
TGF-β signaling cascade (Smad2, Smad3, Smad4, and TGF-β), ERK1/2, p38MAPK, Wnt4/Dvl-1/β-
catenin signaling pathway, Syk/ STAT3-5 axis, DNMT1, MST2, Raf-1, MEK, eNOS, Cdc42, cyclin D1,
FGF1, INSR, NOR1, Jun B/myosin light chain 9, mTOR, KRAS, LOX-1 and MEKK1/ERK/KLF4
signaling. The targets modulated by miRNAs implied in increased proliferation of VSMC include the
RB protein, NF-κB p65, CDK6, TGF-β- and BMP-related pro-differentiation factors (Smad1 and
Smad4), Fbw7/CDC4, KLF5, SIRT1, CREG, CDKN1A, GAX, Sp-1, ERK1/2 kinase-dependent
pathway, SIRT1, KLF4, p21, Ubc9, CKI p27Kip1, p57Kip2, and c-kit, ZDHHC14, PTEN, Bcl-2, and
PDCD4. The majority of the studies up to now were performed only in cultured VSMC. The effects
of these miRNAs and their targets in vascular muscle tissue in vivo need to be further investigated.
Furthermore, there is one open question on the specificity of miRNA action for VSMCs. To this end,
there are some tools (such as TissueAtlas [101] and IMOTA (https://ccb-web.cs.uni-
saarland.de/imota/)) that have been generated and can be used to check whether the miRNAs and
their target genes are expressed in specific tissues [102]. Additionally, endothelial barriers play a very
important role in atherosclerosis, restenosis, and other CVDs. Targeting of VSMC proliferation may
influence the endothelial cell layer by modulating the proliferation of endothelial cells. Therefore,
when investigating the effects of miRNAs on VSMC proliferation, it is also of relevance to examine
their effects on endothelial cell proliferation.
Author Contributions: conceptualization, D.W. and A.G.A.; writing—original draft preparation, D.W.;
writing—review and editing, D.W. and A.G.A.
Funding: This work was supported by the HOMING programme of the Foundation for Polish Science co-
financed by the European Union under the European Regional Development Fund (Homing/2017-4/41), the
Int. J. Mol. Sci. 2019, 20, 324 10 of 15
Polish KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal-Safe Food” decision
of Ministry of Science and Higher Education No. 05-1/KNOW2/2015, and the Peter und Traudl Engelhorn
Foundation for the promotion of Life Sciences.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
VSMC Vascular smooth muscle cell
miRNAs microRNAs
CVDs Cardiovascular diseases
oxLDL Oxidized low-density lipoprotein
IGF-1 Insulin-like growth factor-1
PDGF Platelet-derived growth factor
CKI Cyclin-dependent kinase inhibitor
Ang II Angiotensin II
ADAMTS1 A disintegrin and metalloproteinase with thrombospondin motifs 1
ERK Extracellular signal-regulated kinase
STAT Signal transducer and activator of transcription
SRF Serum-response factor
KLF Krüppel-like factor
ERα Estrogen receptor α
NOR1 Neuron-derived orphan receptor 1
mTOR Mammalian target of rapamycin
PDGFR PDGF receptor
SCF Stem cell factor
Cx43 Connexin 43
HMGB1 High mobility group box-1
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... The original set of miRNA that was shown to modulate VSMC functions were called myomiRs and initially included miR-1, miR-133, miR-206 and miR-208. Later, myo-miRs were expanded to include many other miRNAs [65][66][67]. miRNAs are abundantly expressed in the vascular wall, where they contribute to numerous VSMC functions. For instance, miR-26, miR-29a, miR31, miR133, among several others, regulate VSMC proliferation [65,68]. ...
... miRNAs are abundantly expressed in the vascular wall, where they contribute to numerous VSMC functions. For instance, miR-26, miR-29a, miR31, miR133, among several others, regulate VSMC proliferation [65,68]. Emerging evidence shows an important contributory role of miRNAs in the pathogenesis of several CVDs including atherosclerosis. ...
... In addition, miRNAs have been found to regulate VSMC migration. For example, miR15b/16, miR22, miR24, miR34a, miR214, miR362-3p, miR379, miR638, and others were reported to regulate VSMC migration as well as proliferation by silencing the mRNA levels of an array of VSMC phenotype switching-related genes [65,70]. Importantly, miRNAs are involved in pathological VSMC migration and proliferation, such as in the case of atherosclerosis [70]. ...
Article
Cardiovascular disease (CVD) continues to be the primary cause of global mortality. Vascular smooth muscle cells (VSMCs) are integral components of vascular structure and function, evident by their vital roles in modulating blood flow and pressure. Such roles exist due to the differentiated contractile phenotype of VSMCs. However, VSMCs may switch to a dedifferentiated, proliferative synthetic phenotype in a phenomenon known as phenotypic switching. This switch involves dramatic changes in VSMC migration, proliferation, gene expression programs, differentiation, cellular stiffness and extracellular matrix (ECM) deposition. In this review, we explore the role of the small GTPase Rho and its effector, Rho-associated kinase (ROCK), in phenotypic switching as well as apoptotic pathways in VSMCs. We critically dissect how RhoA promotes cell migration and proliferation as well as its role in modulating the expression of a battery of VSMC marker proteins. We also discuss how RhoA modulates apoptosis, induces dedifferentiation, increases vascular stiffness, or modifies ECM accumulation. These alterations in VSMC phenotypes contribute to multiple vascular dysfunctions, including hypertension and atherosclerosis. Understanding the molecular underpinnings and the signaling pathways involved in these altered phenotypes may provide novel avenues of drug design and other therapeutic interventions for the management of CVDs.
... LncRNAs are potential regulators of the inflammatory response [155] and can regulate gene expression through epigenetic regulation, transcriptional regulation, post-transcriptional regulation, molecular sponge effects, and as molecular chaperones, thus playing a key role in the regulation of plaque stability [145,156]. MiRNAs are expressed in the vascular system, participate in vascular inflammation and smooth muscle cell proliferation, and are now recognized as main regulators in the signaling pathways leading to plaque instability [147,157]. Although lncRNAs transcripts are more numerous than protein-coding genes, their expression, function, and mechanism of action in the relevant cell types during development of the atherosclerotic plaque are unclear. ...
... However, there are still some studies on how the release of miRNAs in the plasma reflects the dynamics and regulates the instability of carotid plaque. Some tools such as Tissue Atlas [157] and IMOTA (https://ccb-web.cs.uni-saarland.de/ imota/) provide powerful means for the subsequent detection of the expression of miRNAs and their target genes in specific tissues. ...
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Cardiovascular diseases (CVDs) caused by arteriosclerosis are the leading cause of death and disability worldwide. In the late stages of atherosclerosis, the atherosclerotic plaque gradually expands in the blood vessels, resulting in vascular stenosis. When the unstable plaque ruptures and falls off, it blocks the vessel causing vascular thrombosis, leading to strokes, myocardial infarctions, and a series of other serious diseases that endanger people's lives. Therefore, regulating plaque stability is the main means used to address the high mortality associated with CVDs. The progression of the atherosclerotic plaque is a complex integration of vascular cell apoptosis, lipid metabolism disorders, inflammatory cell infiltration, vascular smooth muscle cell migration, and neovascular infiltration. More recently, emerging evidence has demonstrated that non-coding RNAs (ncRNAs) play a significant role in regulating the pathophysiological process of atherosclerotic plaque formation by affecting the biological functions of the vasculature and its associated cells. The purpose of this paper is to comprehensively review the regulatory mechanisms involved in the susceptibility of atherosclerotic plaque rupture, discuss the limitations of current approaches to treat plaque instability, and highlight the potential clinical value of ncRNAs as novel diagnostic biomarkers and potential therapeutic strategies to improve plaque stability and reduce the risk of major cardiovascular events.
... Therefore, inhibition of VSMCs proliferation and migration represents an effective therapeutic strategy for hypertension [6]. miRNA, or microRNAs, are small non-coding RNAs with about 19-23 nucleotides in length in a class of organisms [7]. It has been reported that miRNAs could be involved in multiple pathological processes, including VSMCs proliferation and migration, through targeting 3 ′ untranslated region (UTR) of mRNAs [7]. ...
... miRNA, or microRNAs, are small non-coding RNAs with about 19-23 nucleotides in length in a class of organisms [7]. It has been reported that miRNAs could be involved in multiple pathological processes, including VSMCs proliferation and migration, through targeting 3 ′ untranslated region (UTR) of mRNAs [7]. Moreover, miR-NAs are closely related to vascular remodeling and endothelial cell growth regulation [8]. ...
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MicroRNAs (miRNAs) have been regarded as modulators in vascular pathologies, including hypertension. Dysregulated proliferation and migration of VSMCs (vascular smooth muscle cells) contributes to vascular remodeling during hypertension. miR-634 was reported to be dysregulated in hypertensive patients. The involvement of miR-634 in hypertension and the role of miR-634 on VSMCs proliferation and migration were then evaluated. Firstly, HASMCs (human aortic smooth muscle cells) were incubated with 2 μM angiotensin (Ang) II for 12 hours to establish the cell model of Ang II-induced hypertension. Results showed that Ang II treatment promoted proliferation and migration of HASMCs. Secondly, miR-634 was down-regulated in the hypertensive patients, and reduced in Ang II-induced HASMCs in a time dependent manner. Functional assays revealed that Ang II promoted proliferation and migration of HASMCs were suppressed by miR-634 mimic. Lastly, miR-634 targeted 3' untranslated region (UTR) of Wnt4, and reduced Wnt4 expression in HASMCs. miR-634 inhibited β-catenin nuclear translocation. Over-expression of Wnt4 counteracted the suppressive effects of miR-634 on Ang II-induced proliferation and migration of HASMCs. In conclusion, miR-634 inhibited HASMCs proliferation and migration through inactivation of Wnt4/β-catenin pathway.
... MiRNAs are receiving increasing attention for their roles in biological processes in human diseases [14]. Previous studies showed the involvement of miRNAs in VSMC dysregulation [15]. For example, miR-146b-3p restrained cell growth, migration, and phenotype transition in PDGF-BB-exposed VSMCs [16]. ...
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Background Human aortic vascular smooth muscle cells (HA-VSMCs) play vital roles in the pathogenesis of vascular diseases. Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs. In this study, the functions of circRNA pecanex homolog (circPCNX) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs were investigated. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circPCNX, DNA methyltransferase 1 (DNMT1), and microRNA-1278 (miR-1278). 5′-Ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry analysis, wound healing assay, and transwell assay were used to examine cell proliferation, cell cycle, and migration. Western blot assay was utilized to measure protein levels. RNA immunoprecipitation (RIP) assay, RNA pull down assay, and dual-luciferase reporter assay were adopted to analyze the relationships among circPCNX, miR-1278, and DNMT1. Results CircPCNX was upregulated in PDGF-BB-treated HA-VSMCs in a dose- or time-dependent manner. CircPCNX knockdown alleviated PDGF-BB-induced cell proliferation, cell cycle progression, and migration in HA-VSMCs. CircPCNX knockdown could reverse PDGF-BB-induced HA-VSMC progression by regulating DNMT1. Moreover, circPCNX was identified to regulate DNMT1 expression by sponging miR-1278. Inhibition of miR-1278 reversed circPCNX knockdown-mediated effects on cell proliferation and migration in PDGF-BB-induced HA-VSMCs. MiR-1278 overexpression suppressed PDGF-BB-stimulated HA-VSMC proliferation and migration by targeting DNMT1. Conclusion CircPCNX promoted PDGF-BB-induced HA-VSMC proliferation and migration by elevating DNMT1 expression through sponging miR-1278.
... MicroRNAs (miRNAs) are a kind of small (19-23 nucleotides) non-coding RNA molecules that function as endogenous suppressors of gene expression by binding to the 3'-untranslated region (3'-UTR) of target mRNAs to facilitate translational inhibition or mRNA cleavage [7]. Generally, miRNAs, as crucial posttranscriptional regulators, are involved in many physiological and pathological processes, including autophagy [8,9], cell differentiation [10], cell proliferation [11][12][13], DNA damage and repair [14], organ development [15,16] and tumorigenesis [17,18]. Numerous miRNAs are directly or indirectly correlated with oncogenes and function as oncomiRNAs or tumor suppressor miRNAs, especially in colorectal cancer [17,19,20]. ...
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Colorectal cancer (CRC) is the most common malignant tumor occurred in digestive system. However, the prognosis of CRC patients is poor. Therefore, it is urgent to illuminate the mechanism suppressing CRC and explore novel targets or therapies for CRC treatment. MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of 20–23 nucleotides encoded by endogenous genes, which are associated with the development of a variety of cancers, including CRC. Studies have shown that miR-19a is identified as oncogenic miRNA and promotes the proliferation, migration and invasion of CRC cells. However, the relationship between miR-19a and ferroptosis in CRC remains unknown. Here, we reported that iron-responsive element-binding protein 2 (IREB2), as an inducer of ferroptosis, was negatively regulated by miR-19a. IREB2 is a direct target of miR-19a. In addition, ferroptosis was suppressed by miR-19a through inhibiting IREB2. Thus, we proposed a novel mechanism of ferroptosis mediated by miR-19a in CRC cells, which could give rise to a new strategy for the therapy of CRC.
... miR-21 exerts its effect in the early stages of atherosclerosis, acting in particular on vascular smooth muscle cells (VSMCs) by increasing the proliferative capacity and the synthesizing phenotype. In addition to miR-21, the modulatory role of miR-1 on contractile proteins in VSMCs has also been demonstrated, leading to suppression of α-smooth muscle actin [86][87][88]. ...
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The atherosclerotic vascular disease is a cardiovascular continuum in which the main role is attributed to atherosclerosis, from its appearance to its associated complications. The increasing prevalence of cardiovascular risk factors, population ageing, and burden on both the economy and the healthcare system have led to the development of new diagnostic and therapeutic strategies in the field. The better understanding or discovery of new pathophysiological mechanisms and molecules modulating various signaling pathways involved in atherosclerosis have led to the development of potential new biomarkers, with key role in early, subclinical diagnosis. The evolution of technological processes in medicine has shifted the attention of researchers from the profiling of classical risk factors to the identification of new biomarkers such as midregional pro-adrenomedullin, midkine, stromelysin-2, pentraxin 3, inflammasomes, or endothelial cell-derived extracellular vesicles. These molecules are seen as future therapeutic targets associated with decreased morbidity and mortality through early diagnosis of atherosclerotic lesions and future research directions.
... However, the miR-146a expression is higher in coronary and carotid atherosclerotic plaques than in intact vessels [28,34,37]. The target genes and molecular mechanisms (proliferation, apoptosis and migration of SMCs, modulation of immune response) by which miR-146a acts in atherosclerosis-associated cells are being actively studied [104,105]. ...
... Conversely, disruption of these epigenetic and transcriptional complexes by factors such as KLF4, TCF21, histone deacetylases, and DNA methyltransferases leads to repression of the SMC contractile genes and the activation of the previously repressed genes associated with proliferation, migration, inflammation, and other processes involved in both SMC adaptive and maladaptive response (Nagao et al., 2020;Salmon et al., 2012;Shankman et al., 2015;Zhuang et al., 2017). In addition to these transcription and epigenetic mechanisms, noncoding RNAs such as miRNAs, long noncoding RNAs, and circular RNAs are directly involved in the posttranscriptional regulation of transcript abundance in SMC (Vacante et al., 2021;Wang and Atanasov, 2019;Zeng et al., 2021). ...
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miRNAs are versatile regulators of smooth muscle cell (SMC) fate and behavior in vascular development and disease. Targeted loss-of-function studies have established the relevance of specific miRNAs in controlling SMC differentiation or mediating phenotypic modulation. Our goal was to characterize SMC miRNAome and its contribution to transcriptome changes during phenotypic modulation. Small RNA sequencing revealed that dedifferentiation led to the differential expression of over 50 miRNAs in cultured SMC. miRNA/mRNA comparison that predicted that over a third of SMC transcript expression was regulated by differentially expressed miRNAs. Our screen identified the miR-200 cluster as highly downregulated during dedifferentiation. miR-200 maintains SMC quiescence and represses proliferation, migration, and neointima formation, in part by targeting Quaking, a central SMC phenotypic switching mediator. Our study unraveled the substantial contribution of miRNAs in regulating the SMC transcriptome and identified the miR-200 cluster as a pro-quiescence mechanism and a potential inhibitor of vascular restenosis.
... NFKB participates in many biological processes, such as inflammation, immunity, differentiation, cell growth, tumorigenesis, and apoptosis. The crosslink between NFKB and cardiovascular diseases has also been built [33]. Emerging evidence shows that intense inflammatory responses occur during AMI. ...
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Acute myocardial infarction (AMI), one of the most severe and fatal cardiovascular diseases, remains the main cause of mortality and morbidity worldwide. The objective of this study is to investigate the potential biomarkers for AMI based on bioinformatics analysis. A total of 2102 differentially expressed genes (DEGs) were screened out from the data obtained from the gene expression omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) explored the co-expression network of DEGs and determined the key module. The brown module was selected as the key one correlated with AMI. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses demonstrated that genes in the brown module were mainly enriched in ‘ribosomal subunit’ and ‘Ribosome’. Gene Set Enrichment Analysis revealed that ‘TNFA_SIGNALING_VIA_NFKB’ was remarkably enriched in AMI. Based on the protein–protein interaction network, ribosomal protein L9 (RPL9) and ribosomal protein L26 (RPL26) were identified as the hub genes. Additionally, the polymerase chain reaction (PCR) results indicated that the expression levels of RPL9 and RPL26 were both downregulated in AMI patients compared with controls, in accordance with the bioinformatics analysis. In summary, the identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of AMI.
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Cellular reprogramming through targeting microRNAs (miRNAs) holds promise for regenerative therapy due to their profound regulatory effects in proliferation, differentiation, and function. We hypothesized that transdifferentiation of vascular smooth muscle cells (SMCs) into endothelial cells (ECs) using a miRNA cassette may provide a novel approach for use in vascular disease states associated with endothelial injury or dysfunction. miRNA profiling of SMCs and ECs and iterative combinatorial miRNA transfections of human coronary SMCs revealed a 4‐miRNA cassette consisting of miR‐143‐3p and miR‐145‐5p inhibitors and miR‐146a‐5p and miR‐181b‐5p mimics that efficiently produced induced endothelial cells (iECs). Transcriptome profiling, protein expression, and functional studies demonstrated that iECs exhibit high similarity to ECs. Injected iECs restored blood flow recovery even faster than conventional ECs in a murine hindlimb ischemia model. This study demonstrates that a 4‐miRNA cassette is sufficient to reprogram SMCs into ECs and shows promise as a novel regenerative strategy for endothelial repair.
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MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.
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Background Phenotypic switch of vascular smooth muscle cells (VSMCs) participates in the etiology of various vascular diseases. It has been proved that microRNAs (miRNAs) serve as crucial regulators of functions of VSMCs. This study aimed to discover how miR-29b regulates the transformation of VSMCs phenotypes in mice. Material/Methods Primary VSMCs of aorta in mice were cultured in DMEM medium. A series of experiments involving transfection of oligonucleotides in cultured VSMCs, quantitative reverse transcription PCR (qRT-PCR), luciferase reporter assay, and Western blotting analysis were performed in this study. Results We found that in VSMCs cultured in presence of stimulator, platelet-derived growth factor-BB (PDGF-BB), miR-29b was upregulated significantly and expressions of VSMC-phenotype-related genes (α-SMA, calponin, and SM-MHC) were regulated by miR-29b. Moreover, through downregulation of sirtuin 1 (SIRT1), miR-29b affects phenotypic transformation of VSMCs. Luciferase report assay identified a significant increase of SIRT1 3′-UTR activity in treatment with miR-29b inhibitor, which, however, was reversed in the presence of miR-29b mimic. Suppression of miR-29b reversed the activation of NF-κB induced by PDGF-BB in VSMCs. Conclusions We concluded that miR-29b is an important regulator in the PDGF-BB-mediated VSMC phenotypic transition by targeting SIRT1. Interventions aimed at miR-29b may be promising in treating numerous proliferative vascular disorders.
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cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNF-α-dependent inflammatory vascular disease is unclear. Here, we found that TNF-α treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-untranslated region of PKG1 mRNA. TNF-α induced VSMC phenotypic switching from a contractile to a synthetic state through the downregulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNF-α. In addition, TNF-α-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNF-α-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNF-α impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by downregulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-kB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.
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Background There is accumulating evidence demonstrating that microRNAs (miRNA) play essential roles in proliferation, migration, and invasion of vascular smooth muscle cells (VSMCs). However, the exact function of these molecules and the mechanisms involved are not fully understood. In this study, we defined the role of miR-145-5p in VSMCs. Material/Methods This study used the PDGF-bb-induced VSMCs proliferation model. Expression of miR-145-5p and its target, Smad4, were detected and measured by real-time PCR and Western blot analysis. The luciferase reporter of miR-145-5p was used to elucidate miRNA-target interactions. The functions of miR-145-5p in proliferation and migration were detected by CCK-8 assay, Transwell assay, and scratch test. Results This study demonstrates that miR-145-5p is downregulated in PDGF-mediated VSMCs in both time- and dose-dependent manners. The in vitro results suggest that overexpression of miR-145-5p results in a reduction in SMAD4 and an increase in SMAD2, Smad3, and TGF-β at the mRNA and protein levels. Overexpression of miR-145-5p inhibited PDGF-induced VSMCs proliferation and migration. Moreover, SMAD4 was identified as a direct target of miR-145-5p and is involved in PDGF-mediated VSMC proliferation. Downstream factors such as Smad2, Smad3, and TGF-β were also influenced by miR-145-5p. Conclusions We identify miR-145-5p as a novel regulator of VSMC. Moreover, miR-145-5p inhibits VSMCs proliferation and migration by directly targeting Smad4 and dysregulating the transforming growth factor-β signaling cascade, including Smad2, Smad3, and TGF-β.
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Background/aims: Increasing evidence links microRNAs to the pathogenesis of peripheral vascular disease. We recently found microRNA-125b (miR-125b) to be one of the most significantly down‑regulated microRNAs in human arteries with arteriosclerosis obliterans (ASO) of the lower extremities. However, its function in the process of ASO remains unclear. This study aimed to investigate the expression, regulatory mechanisms, and functions of miR-125b in the process of ASO. Methods: Using the tissue explants adherent method, vascular smooth muscle cells (VSMCs) were prepared for this study. A rat carotid artery balloon injury model was constructed to simulate the development of vascular neointima, and a lentiviral transduction system was used to overexpress serum response factor (SRF) or miR-125b. Quantitative real‑time PCR (qRT‑PCR) was used to detect the expression levels of miR‑125b and SRF mRNA. Western blotting was performed to determine the expression levels of SRF and Ki67. In situ hybridization analysis was used to analyze the location and expression levels of miR-125b. CCK-8 and EdU assays were used to assess cell proliferation, and transwell and wound closure assays were performed to measure cell migration. Flow cytometry was used to evaluate cell apoptosis, and a dual-luciferase reporter assay was conducted to examine the effects of miR‑125b on SRF. Immunohistochemistry and immunofluorescence analyses were performed to analyze the location and expression levels of SRF and Ki67. Results: miR-125b expression was decreased in ASO arteries and platelet-derived growth factor (PDGF)-BB-stimulated VSMCs. miR-125b suppressed VSMC proliferation and migration but promoted VSMC apoptosis. SRF was determined to be a direct target of miR-125b. Exogenous miR-125b expression modulated SRF expression and inhibited vascular neointimal formation in balloon-injured rat carotid arteries. Conclusions: These findings demonstrate a specific role of the miR-125b/SRF pathway in regulating VSMC function and suggest that modulating miR-125b levels might be a novel approach for treating ASO.
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miRNAs are a class of non-coding endogenous small RNAs that control gene expression at the posttranscriptional level and involved in cell proliferation, migration and differentiation. Dysregulation of miRNA expression is involved in a variety of human diseases including cardiovascular diseases. miRNAs have been shown to regulate vascular smooth muscle cell (VSMC) function and play vital roles in hypertension, restenosis and atherosclerosis. Here we reported that miR-16 as one of miRNAs in the miR-15 family was highly expressed in vascular smooth muscle cells (VSMCs) and involved in angiotensin II (Ang II) mediated VSMC signaling pathways. Ang II downregulated miR-16 expression in VSMCs. Lentiviral vector mediated miR-16 knockdown promoted Ang II-induced cell proliferation and migration. Moreover, silencing miR-16 enhanced Ang II induced cell cycle associated gene expression and promoted Ang II-activated cell proliferative pathways ERK1/2 and p38. Our finding demonstrated for the first time that miR-16 was a potential therapeutic target by participating in the Ang II-associated multiple signaling pathways in cardiovascular diseases.
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Harnessing the mechanisms underlying the exacerbated vascular remodelling in Diabetes Mellitus (DM) is pivotal to prevent the high toll of vascular diseases in DM patients. microRNAs (miRNA) regulate vascular smooth muscle cell (VSMC) phenotypic switch. However, miRNA modulation of the detrimental diabetic VSMC phenotype is underexplored. Streptozotocin-induced Type 1 Diabetes (T1DM) Wistar rats and T2DM Zucker rats underwent right carotid artery experimental angioplasty and global miRNA/mRNA expression profiling was obtained by RNA sequencing (RNA-Seq). 2 days after injury a set of 6 miRs were found to be uniquely down-regulated or up-regulated both in VSMCs in T1DM and T2DM. Among these, miR-29c and miR-204 were the most significantly mis-regulated in atherosclerotic plaques from DM patients. miR-29c overexpression and miR-204 inhibition per se attenuated VSMC phenotypic switch in DM. Concomitant miR-29c overexpression and miR-204 inhibition fostered an additive reduction in VSMC proliferation. Epithelial membrane protein 2 (Emp2) and Caveolin-1 (Cav1) mRNAs were identified as direct targets of miR-29c and miR-204, respectively. Importantly, contemporary miR-29c overexpression and miR-204 inhibition in the injured artery robustly reduced arterial stenosis in DM rats. Thus, contemporaneous miR-29c activation and miR-204 inhibition in DM arterial tissues is necessary and sufficient to prevent the exaggerated VSMC growth upon injury.
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Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the process underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.
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Many natural products have been so far tested regarding their potency to inhibit vascular smooth muscle cell proliferation, a process involved in atherosclerosis, pulmonary hypertension and restenosis. Compounds studied in vitro and in vivo as VSMC proliferation inhibitors include, for example indirubin-3'-monoxime, resveratrol, hyperoside, plumericin, pelargonidin, zerumbone and apamin. Moreover, taxol and rapamycin, the most prominent compounds applied in drug-eluting stents to counteract restenosis, are natural products. Numerous studies show that natural products have proven to yield effective inhibitors of vascular smooth muscle cell proliferation and ongoing research effort might result in the discovery of further clinically relevant compounds.
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Background: Vein graft failure due to neointimal hyperplasia remains an important and unresolved problem of cardiovascular surgery. MicroRNA-221 (miR-221) has been shown to play a major role in regulating vascular smooth muscle cell (VSMC) proliferation and phenotype transformation. Thus, the purpose of this study is to determine whether adenovirus mediated miR-221 sponge gene therapy could inhibit vein graft neointimal hyperplasia. Methods: Adenovirus encoding miR-221 sponge (Ad-miR-221-SP) was used to inhibit VSMC proliferation in vitro and neointimal formation in vivo. Expression of miRNA-221 was evaluated in cultured VSMC and in rat vein graft models following transduction with Ad-miR-221-SP, Ad-Control-SP (without miR-221 antisense binding sites), or Ad-GFP (control). To accelerate the transfer of miR-221 sponge gene to the vein grafts, 20% poloxamer F-127 gel was used to extend virus contact time and 0.25% trypsin to increase virus penetration. Results: miR-221 sponges can significantly decrease the expression of miR-221 and proliferation in cultured VSMC. Cellular proliferation rates were significantly reduced in miR-221 sponge treated grafts as compared with controls at 6weeks after bypass surgery (19.8% versus 43.6%, P=0.0028). miR-221 sponge gene transfer reduced the neointimal area (210.75±24.13 versus 67.01±12.02, P<0.0001), neointimal thickness (171.86±27.87 versus 64.13±16.23, P<0.0001) and neointima/media ratio (0.74±0.21 versus 1.95±0.25, P<0.0001) in vein grafts versus controls. miR-21 sponge treatment was also improved hemodynamics in vein grafts. We have further identified that p27 (Kip1) is a potential target gene of miR-221 in vein grafts. Conclusion: miR-221 sponge therapy can significantly reduce miR-221 activity and inhibit neointimal hyperplasia in vein grafts. Locally adventitial delivery of adenoviruses mediated miRNA sponges may be promising gene therapies to prevent vein graft failure.