**3. Vascular smooth muscle cells**

The VSMCs of blood vessels in adult generally show a low proliferation rate and wellmaintained homeostasis [38]. However, upon external stimuli, such as cytokines, blood flow rates, shear stress, hormones, injury, and inflammation, provoke the phenotypic switch of VSMCs from contractile to synthetic [2, 39]. The contractile or differentiated VSMCs express contractile markers, smooth muscle myosin heavy chain (SM-MHC), alpha smooth muscle actin (SM α-actin), and calponin; whereas the synthetic or dedifferentiated VSMCs show an increase of cell proliferation, migration, upregulation of extracellular matrix proteins, such as collagen, elastin, and proteoglycans, and a decrease in the expression of contrac‐ tile markers [16, 40]. The plasticity of VSMCs plays important roles in the vascular development and pathological/physiological vascular remodeling. The contractile pheno‐ type is required to maintain the normal arterial wall structure and function, while the synthetic phenotype is involved in the development of atherosclerosis, restenosis after angioplasty, and hypertension [1, 39, 41].

The recent studies show that the regulation of VSMC phenotypic change is associated with the alteration of local environment as well as the modulation of gene expressions by miRNAs. Albinsson et al. reported that VSMC-specific deletion of Dicer-induced embryonic lethality at embryonic days 16 to 17 due to the thin and hypotensive vessel walls, impaired contractility, and hemorrhage [42]. In addition, the loss of Dicer in VSMCs of adult mice also showed a dramatic decrease in blood pressure, the impairment of contractile function, morphology, and phenotypic modulation [43]. These results strongly suggest that miRNAs are essential to vascular development and keep a balance of VSMC phenotype (Figure 2).

#### **3.1. miRNAs in the contractile phenotype of VSMCs**

#### *3.1.1. miR-143 and miR-145*

MiR-143 and miR-145 are the most well-known and enriched miRNAs in VSMCs and are in a bicistronic miRNA cluster on human chromosome 5 [44]. The significant role of miR-143 and miR-145 is to drive VSMC development and differentiation from human embryonic stem cells via targeting the stem cell pluripotency factors OCT4 (octamer-binding transcription factor 4), SOX2 [SRY (sex-determining region Y)-box 2], and KLF4 (Krüppel-like factor 4) [45]. After VSMC development, miR-143 and miR-145 are required for maintaining homeostasis of VSMCs through the suppression of multiple target proteins, such as KLF4, KLF5, E twentysix (ETS)-like transcription factor 1 (ELK1), versican, several actin remodeling proteins, and angiotensin-converting enzyme. The transcriptional activation of miR-143 and miR-145 also leads to the expression of contractile genes [41, 46–48].

The maintenance of the VSMC contractile phenotype is modulated by several signaling pathways including serum response factor (SRF)/myocardin, transforming growth factor β (TGF-β), bone morphogenetic proteins 4 (BMP4), and Jagged-1 (Jag-1)/Notch signaling, which regulates the expression of miR-143 and miR-145 (Figure 3) [46]. The promoter region of miR-143 and miR-145 represents highly conserved *cis* elements and potential binding sites containing CArG box of SRF and Nkx2.5. In addition, myocardin and myocardin-related transcription factor A (MRTF-A), cofactors of SRF, synergistically and strongly activate the miR-143 and miR-145 transcription [49, 50]. The upregulation of miR-143 and miR-145 by TGFβ and BMP4 mediates the induction of SRF cofactors: TGF-β upregulates myocardin via the activation of parallel pathways of p38MAPK and SMAD dependent [51], whereas BMP4 stimulates the nuclear translocation of MRTF-A [52]. The Jag-1-mediated activation of Notch receptors is also activating miR-143 and miR-145 transcription [46]. Activation of Notch receptor by Jag-1 results in proteolytic cleavage and translocation to the nucleus of the Notch intracellular domain (NICD). In the nucleus, NICD forms complex with C promoter binding factor 1 (CBF1), which is binding to miR-143 and miR-145 promoter and increases in miR-145 and miR-145 expression [46].

**Figure 2.** miRNAs in phenotypic switch of VSMCs.

such as collagen, elastin, and proteoglycans, and a decrease in the expression of contrac‐ tile markers [16, 40]. The plasticity of VSMCs plays important roles in the vascular development and pathological/physiological vascular remodeling. The contractile pheno‐ type is required to maintain the normal arterial wall structure and function, while the synthetic phenotype is involved in the development of atherosclerosis, restenosis after

The recent studies show that the regulation of VSMC phenotypic change is associated with the alteration of local environment as well as the modulation of gene expressions by miRNAs. Albinsson et al. reported that VSMC-specific deletion of Dicer-induced embryonic lethality at embryonic days 16 to 17 due to the thin and hypotensive vessel walls, impaired contractility, and hemorrhage [42]. In addition, the loss of Dicer in VSMCs of adult mice also showed a dramatic decrease in blood pressure, the impairment of contractile function, morphology, and phenotypic modulation [43]. These results strongly suggest that miRNAs are essential to

MiR-143 and miR-145 are the most well-known and enriched miRNAs in VSMCs and are in a bicistronic miRNA cluster on human chromosome 5 [44]. The significant role of miR-143 and miR-145 is to drive VSMC development and differentiation from human embryonic stem cells via targeting the stem cell pluripotency factors OCT4 (octamer-binding transcription factor 4), SOX2 [SRY (sex-determining region Y)-box 2], and KLF4 (Krüppel-like factor 4) [45]. After VSMC development, miR-143 and miR-145 are required for maintaining homeostasis of VSMCs through the suppression of multiple target proteins, such as KLF4, KLF5, E twentysix (ETS)-like transcription factor 1 (ELK1), versican, several actin remodeling proteins, and angiotensin-converting enzyme. The transcriptional activation of miR-143 and miR-145 also

The maintenance of the VSMC contractile phenotype is modulated by several signaling pathways including serum response factor (SRF)/myocardin, transforming growth factor β (TGF-β), bone morphogenetic proteins 4 (BMP4), and Jagged-1 (Jag-1)/Notch signaling, which regulates the expression of miR-143 and miR-145 (Figure 3) [46]. The promoter region of miR-143 and miR-145 represents highly conserved *cis* elements and potential binding sites containing CArG box of SRF and Nkx2.5. In addition, myocardin and myocardin-related transcription factor A (MRTF-A), cofactors of SRF, synergistically and strongly activate the miR-143 and miR-145 transcription [49, 50]. The upregulation of miR-143 and miR-145 by TGFβ and BMP4 mediates the induction of SRF cofactors: TGF-β upregulates myocardin via the activation of parallel pathways of p38MAPK and SMAD dependent [51], whereas BMP4 stimulates the nuclear translocation of MRTF-A [52]. The Jag-1-mediated activation of Notch receptors is also activating miR-143 and miR-145 transcription [46]. Activation of Notch receptor by Jag-1 results in proteolytic cleavage and translocation to the nucleus of the Notch

vascular development and keep a balance of VSMC phenotype (Figure 2).

**3.1. miRNAs in the contractile phenotype of VSMCs**

leads to the expression of contractile genes [41, 46–48].

*3.1.1. miR-143 and miR-145*

58 Muscle Cell and Tissue

angioplasty, and hypertension [1, 39, 41].

The expression levels of miR-143 and miR-145 significantly decrease in animal vascular diseases models including apolipoprotein E knockout mice (ApoE–/–), carotid artery ligation injury models, carotid balloon injury models, and in aortas from patients with aortic aneurism [53, 54]. MiR-143 and miR-145 knockout murine VSMCs showed a shift from contractile to synthetic state and the development of neointimal lesions [55], whereas the VSMC-specific overexpression of miR-143 and 145 increased the plaque stability and the expression of contractile proteins, and decreased macrophage infiltration, the expression of KLF4 and KLF5, and neointimal lesion formation [54, 56]. Altogether, these data indicate that miR-143 and miR-145 play an important role in the VSMC homeostasis and strengthen the VSMC contractile phenotype.

**Figure 3.** The signaling pathway of miR-143 and miR-145 expression.

#### *3.1.2. miR-1 and miR-133*

Similar to miR-143 and miR-145, miR-1 and miR-133 are also in bicistronic miRNA clusters and are induced by myocardin in VSMCs [57]. During differentiation of mouse ESC to VSMCs, the expression level of miR-1 is gradually increased, which results in the upregulation of VSMC-specific contractile proteins by repressing KLF4 [58]. Chen et al. demonstrated that overexpression of myocardin in VSMCs increased miR-1 expression and inhibited VSMC proliferation, which mediated the repression of Pim-1, a serine/threonine kinase [59].

The expression level of miR-133 is downregulated in proliferating VSMCs and after vascular injury. Overexpression of miR-133 reduces VSMC growth, but the downregulation of miR-133 induces VSMC proliferation *in vitro* [57]. Both the transcription factor Sp-1 (specificity protein 1) which regulates VSMC phenotypic switch and the actin-binding protein moesin that modulates VSMC migration are direct targets of miR-133. Accordingly, miR-133 overexpres‐ sion in the rat carotid artery reduced neointimal hyperplasia, whereas anti-miR-133 increased VSMC proliferation and neointimal formation after balloon injury [57].

#### *3.1.3. miR-21*

The expression of miR-21 mediates TGF-β and BMP4 signaling pathway and promotes the contractile phenotype of VSMCs via downregulating programmed cell death 4 (PDCD4), a negative regulator of smooth muscle contractile genes [60]. Another target of miR-21 is the family of the dedicator of cytokinesis (DOCK) proteins, which promote VSMC migration by modulating the activity of small Rac1 GTPase [61]. On the other hand, Horita et al. reported that Fos-related antigen (FRA)-1, a direct target of miR-143, was a positive regulator of increased miR-21 expression [62]. The upregulation of miR-143 by SRF repressed FRA-1 expression, which led to the decrease of VSMC proliferation via the repression of miR-21 and subsequent increase of phosphatase and tensin homolog (PTEN), one of the target proteins of miR-21 [62]. The dual function of miR-21 in promoting both contractile and synthetic pheno‐ type of VSMCs may suggest that diverse targets of miR-21 are involved in different biological processes depending on the cellular environments or context.

#### *3.1.4. Other miRNAs*

**Figure 3.** The signaling pathway of miR-143 and miR-145 expression.

Similar to miR-143 and miR-145, miR-1 and miR-133 are also in bicistronic miRNA clusters and are induced by myocardin in VSMCs [57]. During differentiation of mouse ESC to VSMCs, the expression level of miR-1 is gradually increased, which results in the upregulation of VSMC-specific contractile proteins by repressing KLF4 [58]. Chen et al. demonstrated that overexpression of myocardin in VSMCs increased miR-1 expression and inhibited VSMC

The expression level of miR-133 is downregulated in proliferating VSMCs and after vascular injury. Overexpression of miR-133 reduces VSMC growth, but the downregulation of miR-133 induces VSMC proliferation *in vitro* [57]. Both the transcription factor Sp-1 (specificity protein 1) which regulates VSMC phenotypic switch and the actin-binding protein moesin that modulates VSMC migration are direct targets of miR-133. Accordingly, miR-133 overexpres‐

proliferation, which mediated the repression of Pim-1, a serine/threonine kinase [59].

*3.1.2. miR-1 and miR-133*

60 Muscle Cell and Tissue

Our group has reported that miR-29b and miR-365 inhibit the proliferation and migration of VSMCs [63, 64]. The interleukin-3 (IL-3) is known to stimulate proliferation and migration in vascular diseases, and it downregulates miR-29b expression. Mir-29b significantly decreases the proliferation and migration of VSMCs through the inhibition of the signaling pathway related to Mcl-1 (myeloid cell leukemia 1) and MMP2 (matrix metallopeptidase 2). Consistent with the declined miR-29b expression in balloon-injured rat carotid arteries, overexpression of miR-29b by local oligonucleotide delivery can inhibit neointimal formation [64]. The proliferation of VSMCs by various stimuli, including platelet-derived growth factor (PDGF)- BB, angiotensin II (Ang II), and serum, led to the downregulation of miR-365 expression levels. The cell-cycle-specific cyclin D1 was found to be a potential target of miR-365; thus exogenous miR-365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, blocking transition of G1/S [63, 65].

Li et al. reported that miR-663 is related to human VSMC phenotypic switch and the devel‐ opment of neointimal formation [66]. According to this particular study, overexpression of miR-663 upregulated VSMC differentiation marker genes and inhibited PDGF-induced VSMC proliferation and migration via targeting the transcription factor JunB and myosin light chain 9 (MYL9). Overexpression of miR-663 also dramatically attenuated the neointimal lesion formation in mice after carotid artery ligation [66]. In addition, the same group identified that miR-638 also is markedly downregulated by PDGF-stimulated human VSMCs, whereas upregulated in human VSMCs cultured in differentiation medium, a condition that inhibits proliferation [67]. Furthermore, the orphan nuclear receptor NOR1 (neuron-derived orphan receptor 1) was identified as a target of miR-638 and downregulation of NOR1 was implicated in the miR-638-mediated inhibitory effects on cyclin D1 expression, cell growth, and migration of PDGF-induced VSMCs.

### **3.2. miRNAs in the synthetic phenotype of VSMCs**

#### *3.2.1. miR-221 and miR-222*

Similar to TGF-β and BMP4 promote VSMC contractile phenotype, PDGF induces VSMC phenotype switch from contractile to synthetic type [41]. Mir-221 and miR-222 are well characterized in VSMC phenotype switch and clustered on the X chromosome, and have same seed sequence [68]. Mir-221 and miR-222 are transcriptionally induced by activating PDGF pathway, which reduces the expression of contractile genes and promotes VSMC proliferation and migration [69]. In addition, the expression of miR-221 and miR-222 are elevated in neointimal lesion from balloon-injured rat carotid arteries [70]. The targets of miR-221 and miR-22 include p27Kip1 and p57kip2, both of which are a negative regulator of VSMC proliferation [70, 71]. Interestingly, miR-221 decreased the expression of c-kit, and this reduced c-kit expression subsequently repressed the expression of a VSMC-specific nuclear coactivator myocardin [69]. As the PDGF-treatment stimulated phenotype change of VSMCs, overexpres‐ sion of miR-221 and miR-222 accelerated VSMC proliferation and migration, while downre‐ gulation of them attenuated VSMC proliferation and neointimal formation in rat carotid artery after angioplasty [70]. These data indicate that miR-221 and miR-222 have a crucial role in regulating VSMC phenotype change, and suggest their therapeutic potential in pathological vascular remodeling.

#### *3.2.2. miR-146a*

Recent study has shown that miR-146a is upregulated in rat balloon-injured arteries and serum-induced proliferative VSMCs [72, 73]. KLF4, a negative regulator of VSMC prolifera‐ tion, was downregulated by miR-146a, and in turn, KLF4 repressed miR-146a expression through binding to CACCC (or GGGTG) elements on the miR-146a promoter. This negative feedback regulatory network between miR-146a and KLF4 regulates transcription of each other, controlling VSMC proliferation *in vitro* and vascular neointimal hyperplasia *in vivo* [72]. Dong et al. demonstrated that miR-146a upregulation is related to the downregulation of critical transcriptional factors such as the protein expression of nuclear factor-κBp65 (NFκBp65) and PCNA. In contrast, miR-146a knockdown is linked to the increase in pro-apoptotic protein Bax expression [73]. Therefore, miR-146a is a novel regulator of VSMC proliferation in cardiovascular diseases.

#### *3.2.3. miR-24*

Both TGF-β and BMP4 signaling are important in maintaining the VSMC contractile pheno‐ type, whereas PDGF signaling pathways can promote the synthetic phenotype conversion of VSMCs. Although these signaling pathways play key roles in VSMC phenotypic changes, the crosstalk between these pathways is not elucidating until quite recently. Chan et al. reported that despite of BMP4 stimulation, the contractile genes are downregulated by co-treatment with PDGF in human primary pulmonary SMCs (PASMCs) [74]. Furthermore, PDGF stimu‐ lation decreases the expression of Tribbles-like protein-3 (Trb3), an important modulator of the BMP and TGF-β signaling pathway. Interestingly, the result of Trb3 promoter-luciferasereporter assay showed that the downregulation mechanism of Trb3 expression by PDGF is associated with the effect of PDGF-induced miR-24, directly targeting 3URT of Trb3 mRNA, rather than PDGF stimulation [74]. The knockout endogenous miR-24 by antisense oligonu‐ cleotides restored Trb3 expression as well as attenuates PDGF-mediated synthetic activity, suggesting that PDGF-induced miR-24 expression which downregulated Trb3, and this miR-24-mediated downregulation of Trb3 is sufficient to induce synthetic phenotypic changes of VSMCs [74].

#### *3.2.4. miR-26a*

**3.2. miRNAs in the synthetic phenotype of VSMCs**

Similar to TGF-β and BMP4 promote VSMC contractile phenotype, PDGF induces VSMC phenotype switch from contractile to synthetic type [41]. Mir-221 and miR-222 are well characterized in VSMC phenotype switch and clustered on the X chromosome, and have same seed sequence [68]. Mir-221 and miR-222 are transcriptionally induced by activating PDGF pathway, which reduces the expression of contractile genes and promotes VSMC proliferation and migration [69]. In addition, the expression of miR-221 and miR-222 are elevated in neointimal lesion from balloon-injured rat carotid arteries [70]. The targets of miR-221 and miR-22 include p27Kip1 and p57kip2, both of which are a negative regulator of VSMC proliferation [70, 71]. Interestingly, miR-221 decreased the expression of c-kit, and this reduced c-kit expression subsequently repressed the expression of a VSMC-specific nuclear coactivator myocardin [69]. As the PDGF-treatment stimulated phenotype change of VSMCs, overexpres‐ sion of miR-221 and miR-222 accelerated VSMC proliferation and migration, while downre‐ gulation of them attenuated VSMC proliferation and neointimal formation in rat carotid artery after angioplasty [70]. These data indicate that miR-221 and miR-222 have a crucial role in regulating VSMC phenotype change, and suggest their therapeutic potential in pathological

Recent study has shown that miR-146a is upregulated in rat balloon-injured arteries and serum-induced proliferative VSMCs [72, 73]. KLF4, a negative regulator of VSMC prolifera‐ tion, was downregulated by miR-146a, and in turn, KLF4 repressed miR-146a expression through binding to CACCC (or GGGTG) elements on the miR-146a promoter. This negative feedback regulatory network between miR-146a and KLF4 regulates transcription of each other, controlling VSMC proliferation *in vitro* and vascular neointimal hyperplasia *in vivo* [72]. Dong et al. demonstrated that miR-146a upregulation is related to the downregulation of critical transcriptional factors such as the protein expression of nuclear factor-κBp65 (NFκBp65) and PCNA. In contrast, miR-146a knockdown is linked to the increase in pro-apoptotic protein Bax expression [73]. Therefore, miR-146a is a novel regulator of VSMC proliferation in

Both TGF-β and BMP4 signaling are important in maintaining the VSMC contractile pheno‐ type, whereas PDGF signaling pathways can promote the synthetic phenotype conversion of VSMCs. Although these signaling pathways play key roles in VSMC phenotypic changes, the crosstalk between these pathways is not elucidating until quite recently. Chan et al. reported that despite of BMP4 stimulation, the contractile genes are downregulated by co-treatment with PDGF in human primary pulmonary SMCs (PASMCs) [74]. Furthermore, PDGF stimu‐ lation decreases the expression of Tribbles-like protein-3 (Trb3), an important modulator of the BMP and TGF-β signaling pathway. Interestingly, the result of Trb3 promoter-luciferase-

*3.2.1. miR-221 and miR-222*

62 Muscle Cell and Tissue

vascular remodeling.

cardiovascular diseases.

*3.2.3. miR-24*

*3.2.2. miR-146a*

Leeper et al. demonstrated that serum-starved human aortic SMCs upregulates the expression of contractile marker genes, and miR-26a is significantly downregulated during serumstarvation and in murine abdominal aortic aneurysm (AAA) development models [75]. Overexpression of miR-26a promoted VSMC proliferation and migration as well as inhibited apoptosis, whereas downregulation of miR-26a induced contractile gene expression and reduced proliferation and migration. The potential targets of miR-26a include SMAD1 and SMAD4, members of TGF-β and BMP superfamily downstream signaling cascade. Consis‐ tently, knockdown of miR-26a led to SMAD1 and Samd4 upregulation and activates TGF-β and BMP signaling pathway. Thus, miR-26a is expected to actively participate in the regulation of VSMCs as an enhancer of cell growth and migration, and an inhibitor of apoptosis, modu‐ lating TGF-β and BMP signaling [75].

#### *3.2.5. miR-31*

Although miR-31 plays an important role in cancer cell growth and proliferation [76–78], the biological function of miR-31 in VSMCs remains unclear. Recently, Liu et al. found that miR-31 was an abundant miRNA in VSMCs of vascular walls, and was significantly upregulated in proliferative VSMCs and rat carotid arteries with neointimal growth after balloon injury [79]. The serum- or PDGF-induced VSMC proliferation is inhibited by miR-31 inhibitor, 2′OMemiR-31 (the antisense oligonucleotide for miR-31 that is modified at each nucleotide by an Omethyl moiety at the 2′-ribose position). On the contrary, overexpression of miR-31 using the adenoviruses expressing rat miR-31 promotes VSMC proliferation [79]. The pro-proliferative effect of miR-31 is mediated by the direct inhibition of the large tumor suppressor homolog 2 (LATS2) expression, and is accelerated by the activation of mitogen-activated protein kinase/ extracellular regulated kinase (MAPK/ERK) [79].
