MicroRNA regulation of phenotypic transformations in vascular smooth muscle: relevance to vascular remodeling

Abstract

Alterations in the vascular smooth muscle cells (VSMC) phenotype play a critical role in the pathogenesis of several cardiovascular diseases, including hypertension, atherosclerosis, and restenosis after angioplasty. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs (approximately 19–25 nucleotides in length) that function as regulators in various physiological and pathophysiological events. Recent studies have suggested that aberrant miRNAs’ expression might underlie VSMC phenotypic transformation, appearing to regulate the phenotypic transformations of VSMCs by targeting specific genes that either participate in the maintenance of the contractile phenotype or contribute to the transformation to alternate phenotypes, and affecting atherosclerosis, hypertension, and coronary artery disease by altering VSMC proliferation, migration, differentiation, inflammation, calcification, oxidative stress, and apoptosis, suggesting an important regulatory role in vascular remodeling for maintaining vascular homeostasis. This review outlines recent progress in the discovery of miRNAs and elucidation of their mechanisms of action and functions in VSMC phenotypic regulation. Importantly, as the literature supports roles for miRNAs in modulating vascular remodeling and for maintaining vascular homeostasis, this area of research will likely provide new insights into clinical diagnosis and prognosis and ultimately facilitate the identification of novel therapeutic targets.

Introduction

Blood vessels play a crucial role in maintaining the normal physiological functions of an organism and that regulates vascular remodeling, which alters the vascular configuration. Vascular activities are involved in communication between vascular smooth muscle cells (VSMCs) and other vascular cells. Upon exposure to pathophysiological stimuli, VSMC proliferation and migration are core processes in vascular remodeling and influenced by growth factors and signaling networks [1]. Vascular remodeling is characterized by the aggregation of VSMCs in intima and can change their phenotype in response to environmental cues. VSMC within the walls of blood vessels exhibit marked phenotypic plasticity characterized by transformation between differentiated (contractile) and proliferative (synthetic) phenotypes [2, 3]. Herein, we realize that the word synthetic is outdated, but that they mean by this term all VSMC phenotypes that are non-contractile phenotypes, which VSMC can acquire upon different stimuli during physiological or pathological processes. Such transformations occur in response to both the physiological stimuli and the pathological processes that underlie a number of cardiovascular diseases (CVD), including hypertension, atherosclerosis, restenosis following angioplasty, aneurysm formation, and chronic obstructive pulmonary disease [4–6]. Phenotypic modulation is a prerequisite for the proliferation and migration of VSMC from the tunica media to the tunica intima of the arterial wall. The main manifestations of this process include the transformation of cells from a contractile to a synthetic phenotype, which is characterized by altered proliferation, migration, and synthetic capabilities and by the secretion of large quantities of extracellular matrix (ECM) components [7–10]. Studies have also shown that diabetic and cardiovascular disorders induce phenotypic transformation of VSMC changing from contractile/differentiated to synthetic/proliferative phenotypes and thus contributing to vascular remodeling. During this course, SMC-specific contractile genes including Smooth Muscle α-actin (SMA), Smooth muscle22alpha (SM22α), Smooth muscle-myosin heavy chain (SM-MHC), and Smoothelin-B levels are decreased and the expression levels of SMC-specific synthetic genes, including nonmuscle myosin heavy chain B (SMemb), CCND1 (cyclin D1), and CCND2 (cyclin D2) are increased [11, 12]. Synthetic VSMC display alterations in increased proliferation and migration, and VSMC dedifferentiation impairs proliferative and migratory capacity. Additionally, VSMC undergo the phenotypic changes from contractile to synthetic state possibly enabling the mobilization and proliferation during arteriogenesis, and are capable to differentiate into pericytes to coat around capillaries during angiogenesis, which is a key phenomenon that influences vascular disease with the expansion of new vascular networks from the existing vascular system in a budding or non-budding form. Moreover, the ability of vascular endothelial cell (VEC) to proliferate and to secrete growth factors, such as vascular endothelial-derived growth factor (VEGF) and platelet-derived growth factor (PDGF), is crucial for the new vascular network development and subsequent arterialization via recruitment of mural cells.

MicroRNAs (miRNAs) are endogenous, short (approximately 19–25 base pairs in length) noncoding RNAs that act as negative regulators of eukaryotic gene expression and can inhibit the translation of target mRNA molecules by binding to the 3′ untranslated regions (3′-UTRs) of target gene mRNAs via complementarity [13, 14]. Thus, miRNAs have been shown to play important roles in multiple physiological processes, such as developmental timing, apoptosis, fat metabolism, neuronal development, cell differentiation, hormone secretion, and the occurrence and development of multiple diseases [15–17]. Of direct relevance to this review, the previous studies have demonstrated that the phenotypic transformation of VSMC is accompanied by tissue-specific expression of a number of miRNAs [18–20]. Such miRNAs appear to regulate the phenotypic transformations of VSMC by targeting specific genes that either participate in the maintenance of the contractile phenotype or contribute to the transformation to alternate phenotypes and thereby affect cell proliferation, migration, hypertrophy, and differentiation. Several studies have confirmed that various biological factors, e.g., growth factors and transcription factors, including those that are specific targets of certain miRNAs, can affect the phenotypic transformation of VSMC [21–23]. The additional functional miRNAs pathways involved in miRNA-mediated VSMC phenotypic transformations and the mechanisms regulating gene expression have recently become an active focus of research. This review summarizes miRNAs that have been identified to be involved in the phenotypic transformation of VSMC and recent progress toward understanding the mechanisms and functions underlying this process. Specifically, we emphasize the gene expression regulatory networks through which miRNAs regulate the phenotypic transformation of VSMC and the mechanisms through which miRNAs act on target genes. Furthermore, the molecular regulatory mechanisms through which miRNAs mediate the phenotypic modulation of VSMC and the broad clinical applications of miRNAs in the phenotypic transformation of VSMC, such as novel therapeutic targets and pathways that could be used for the clinical diagnosis and treatment of CVD, are discussed.

miRNAs implicated in VSMC phenotypic transformation

Since the first miRNA (miR-21) regulating VSMC proliferation was identified by Ji and colleagues [24], numerous studies have provided evidence for the role of more than 20 additional functional miRNAs, including miR-1/133, miR-22, miR-221, miR-143/145, miR-195, miR-424/322, and miR-638. It has been demonstrated that the high level of miR-21 expression in mouse carotid artery wall is determined by balloon injury and that miR-21 inhibition reverses the mice vascular remodeling induced by balloon injury [24]. More concretely, miR-21 directly targets phosphatase and tensin homologs (PTEN) and B-cell lymphoma 2 (Bcl-2), thereby regulating the proliferation and apoptosis of VSMC [25–27]. In addition, miR-21 expression was found to be regulated by the bone morphogenetic protein 4 (BMP4), while miR-21 inhibits the proliferation and migration of VSMC and stimulates the contraction by targeting and regulating programmed cell death protein 4 (PDCD4) and cytoplasmic division factor (DOCK) proteins [28–30]. However, in those initial studies, the miR-21-mediated regulation of the phenotypic transformation of VSMC was not investigated in depth. Interestingly, in a similar model, miR-145 was revealed to be highly expressed in VSMC and to control the formation of vascular neointimal lesions by targeting the Kruppel-like factor 5 (KLF5) gene to regulate the phenotypic transformation of VSMC [31]. Notably, as the scope of the study expanded, the functions of more miRNAs were gradually revealed. For example, PDGF-induced high expression of miR-221 significantly promoted VSMC proliferation and migration, partly by downregulating the expression of target genes c-KIT and p27 (kip1) to promote a synthetic phenotype of VSMC [32, 33] These findings demonstrate that miRNAs have important regulatory roles in VSMC phenotype and function, which may provide guidance in treatment of vascular diseases.

Fundamentally, since Dicer is the key enzyme responsible for most miRNA maturation, alteration of Dicer leads to a complete loss of miRNAs. The role of Dicer-dependent miRNAs in VSMC development and function in vivo was investigated using SM22Cre Dicer^flox (SMDicer-KO) mice. Dicer deletion was confirmed to increase embryonic lethality with abnormal vascular architecture and loss of VSMC contractile function [34]. Specific inactivation of Dicer significantly caused renal vascular abnormalities and striated fibrosis as well as type B aortic arch artery disruption [35, 36]. These results indicate that Dicer-generated miRNAs are essential for vascular remodeling and normal VSMC development, differentiation, and contractile function. Overall, miRNAs regulate the phenotypic transformation of VSMC and thereby play important regulatory roles in processes such as VSMC differentiation, contractile or synthetic function, and in vivo angiogenesis. Such miRNAs further influence the transformation of VSMC between contractile and synthetic phenotypes by regulating their proliferation, migration, and differentiation, as shown in Table 1. In the following sections, to illustrate how miRNAs contribute to VSMC phenotypic transformation, we explore specific miRNAs that have been reported to exert mechanistic roles in VSMC.

Table 1.

Related miRNAs affect and regulate vascular remodeling for maintaining vascular homeostasis

miRNAs	Validated targets	Cellular functions	Physiological function and pathological changes	Inducer and regulator	References
miR-223	Downregulate PDGFRβ	Promote VSMC differentiation	Increase medial thickening, fragmentation of medial elastic fibers	imatinib	[19, 22]
miR-21	Decrease PTEN	Induce cell proliferation and decrease apoptosis	Promote abdominal aortic aneurysm development	/	[26, 27]
miR-124	Suppress SP1	Inhibit proliferation and migration	AmeliorateAtherosclerosis	PDGF-BB	[37]
miR-29	Upregulate ECM related proteins	Increase matrix synthesis	Promote the expansion of mouse aorta	Angiotensin II/Fibulin-4	[38]
miR-29a	Downregulate Fbw7/CDC4	Enhance VSMC proliferation	Promote atherosclerosis	oxLDL	[39]
miR-29b	Downregulate MMP2/elastin	Cause extracellular matrix deficiencies	Promote the early aneurysm development	/	[40]
miR-26b	Upregulate HMGA2	Promote cell viability and proliferation	Regulate Stanford type A aortic dissection	/	[41]
miR-9	Upregulate HIF-1α	Promote proliferative phenotype	Improve development of hypoxic pulmonary hypertension	Hypoxia 1α	[42]
miR-132	Upregulate PTEN	Antagonize the differentiation-promoting ability of cilostazol	Influence on the repair of carotid artery in rats with balloon injury	Cilostazol	[43]
miR-138	Decrease SIRT1	Enhance VSMC proliferation and migration	Improve diabetes blood vessels	High glucose	[44]
miR-146a	Decrease KLF4	Promote VSMC proliferation	Increase neointimal hyperplasia	KLF5	[45]
miR-214	Upregulate NCKAP1	Decrease proliferation and migration	Prevent neointimal SMC proliferation after injury	Serum-starved	[46]
miR-1260b	Upregulate GDF11	Promote Proliferation	Improve hypoxia-stimulated environmental stress	Hypoxia	[47]
miR-155-5p	Downregulate PKG1	Impair NO/cGMP-mediated maintenance of contractile phenotype and vasodilation	Improve inflammatory vascular disease	TNFα	[48]
miR-135a	Downregulate FOXO1	Attenuate pro-inflammatory responses	improve diabetes	/	[49]
miR-133a	Reduce LDLRAP1, oxLDL	Reduce lipid accumulation	Improve the formation of atherosclerosis	IL-19	[50]
miR-574	Inhibit ZDHHC14	Promote cell proliferation and inhibit apoptosis	Improve coronary artery disease	/	[51]
miR-31-5p	Increase myocardin	Attenuate contraction gene	Inhibit ablated pathological VSMC phenotypical switch	ALDH2, Max	[52]
miR-128	Downregulate KLF4	Promote contraction	Prevent endometrial hyperplasia	/	[53]
miR-501-5p	Decreases α-SMA, calponin and Smad3	Promotes VSMC proliferation and migration	Attenuates carotid balloon injury in rats	/	[54]
miR-214	Reduce Smad7 levels and increase Smad3 phosphorylation	Promotes VSMC proliferation and migration	Increase medial thickness and blood vessels areas	Ang II	[55]

miRNAs contribute to the phenotypic transformation of VSMC

Recent studies have used miRNA microarrays to identify differentially expressed miRNAs in the cardiovascular system and identify functional miRNAs that play key regulatory roles in VSMC phenotypic transformation. It was confirmed that some miRNAs are involved in regulating the proliferation, migration, and differentiation of VSMC and the remodeling of the cytoskeleton [52, 56]. To date, however, only the functions of a few miRNAs have been associated with the phenotypic transformation of VSMC. In this section, the role of miRNAs in the progression of vascular phenotypic transformation is discussed.

miRNAs regulate the transformation of VSMC to the synthetic phenotype

Reversing or inhibiting the transformation from a contractile to a synthetic VSMC phenotype or enhancing the stability of the contractile VSMC phenotype plays a key role in the prognosis of CVD, such as atherosclerosis [57–59]. The significant differential expression of a number of miRNAs in cardiovascular disease suggests that some miRNAs might regulate the transformation of VSMC to the synthetic phenotype. For example, Chan et al. [60] found that PDGF promotes the expression of miR-24, which targets and inhibits tribbles homologue-3 (TRIB3) and thereby disrupts the TRIB3-mediated degradation of ubiquitinated Smad ligase. This disruption leads to increased levels of Surf1 and subsequent reductions in Smad1/2/3 and Ras homologue family member A (RhoA) expression, which results in inhibition of the BMP-induced transformation of VSMC to the contractile phenotype and stabilization of the synthetic phenotype of VSMC. Additionally, PDGF receptor β (PDGFRB) levels can significantly affect VSMC phenotype. It was shown that myocardin reduced VSMC proliferation and migration by downregulating PDGFRB expression, and antagonizing miR-24 and miR-29a restored PDGFRB-induced VSMC migration and showed an augmented neointima formation [61]. Furthermore, miR-26a was significantly increased in PDGF-stimulated cultured VSMC and arteries with neointimal lesion formation by targeting Smad1, a key mediator of BMP pro-contractile signaling, to promote VSMC differentiation for the intervention of proliferative vascular disease [62]. Likewise, miR-31 plays a pivotal role in the synthetic phenotype of VSMC. Specifically, miR-31 levels in VSMC were significantly induced by PDGF, after which miR-31 promoted VSMC proliferation by targeting large tumor suppressor 2 (LATS2) levels and enhancing proliferating cell nuclear antigen (PCNA) expression [63]. Subsequently, miR-31 can also inhibit the expression of cellular repressor of E1A-stimulated genes (CREG) and genes involved in VSMC differentiation to promote their transformation to the synthetic phenotype [64]. In addition, miR-96 expression was regulated by BMP-4, further targeting and repressing the expression of tribbles homolog 3 (TRB3) to promote the transformation of VSMC to an undifferentiated phenotype [65]. These events trigger the transformation of VSMC to the synthetic phenotype.

Besides, Dong and colleagues further revealed that miR-146a is highly expressed in proliferating VSMC and that the knockout of miR-146a can inhibit the proliferation and migration of VSMC and stimulate VSMC apoptosis, which suggests that miR-146a can promote the transformation of VSMC to a synthetic phenotype [66]. Admittedly, inhibition of the contractile phenotype of VSMC in pulmonary hypertension is mediated by miR-214 through inhibition of the MEF2C–MYOCD–leiomodin1 (LMOD1) signaling axis, providing a theoretical basis for the treatment of vascular hyperproliferative diseases [67]. Collectively, these findings have concluded that the mechanisms of miR-24, miR-29a, miR-31, miR-96, miR-214, and multiple other miRNAs, including miR-138, miR-9, and miR-29b [44, 68, 69] contribute to the transformation of VSMC to a synthetic phenotype. Figure 1 shows the network of important miRNAs regulating a synthetic phenotype of VSMC.

Fig. 1.

MicroRNAs regulate synthetic phenotypic transformations in VSMC. Considerable amounts of miRNA, such as miR-221/222, miR-138, miR-24, and miR-146 through modulation of direct targets or in the presence of external stimuli can promote the conversion of VSMC to a synthetic phenotype. Growth factors, such as α-SMA, SM22α, and SMMHC, were significantly overexpressed during this process. The synthetic phenotype is characterized by increased proliferation and migration to maintain the stability of the VSMC synthetic phenotype against conversion to a contractile phenotype

Recently, Kruppel-like factor 4 (KLF4), an evolutionarily conserved zinc finger-containing transcription factor involved in various cell growth, differentiation, and proliferation, has been proved to cause substantial impacts on regulating CVD [70, 71]. Several miRNAs are closely associated with KLF4 in VSMC regulatory network. For example, miR-146a represses KLF4 expression by targeting its 3′-untranslated region (3′-UTR). Mechanistically, KLF4 competes with KLF5 to bind to and regulate the miR-146a promoter, and that KLF4 and KLF5 exert opposing effects on the miR-146a promoter[45, 72]. MiR-146a and KLF4 form a feedback loop to directly regulate the expression of each other, which provides new insights into the significant reduction of neointimal hyperplasia via targeting miR-146a and KLF4 axial in carotid arteries. Similarly, KLF4 mRNA-binding motif (CACCC) (or the reverse orientation sequence, GGGTG) binds to and regulate the miR-220c promoter, and represses its transcriptional level to reduce VSMC proliferation[73], potentially representing a novel approach to prevent restenosis. These results suggest that some miRNAs that form a negative feedback regulatory mechanism with KLF4 play an important regulatory role in the conversion of VSMC to a synthetic phenotype.

Meanwhile, to explore the linkage between miRNAs subgroups that regulate angiogenic phenotypic target genes, we performed enrichment analysis of known effector proteins and found the closest linkage between TP53, MYC, PTEN, STAT3, CCND1, and CTNNB1 proteins (Fig. 2A). Gene Ontology (GO) analysis revealed that target proteins, mainly represented by protein kinase complexes and serine/threonine protein kinase complexes, can significantly affect the regulation of angiogenesis and vasculature development, further affecting numerous DNA, RNA, and kinase activities, thus regulating the vascular synthesis phenotype (Fig. 2B). In addition, the major signaling pathways clustered in the PI3K–AKT signaling pathway and miRNAs in cancer (Fig. 2C). Interestingly, overexpression of miR-126 prevents vascular restenosis by downregulating PI3K/AKT signaling, thus activating MEK/ERK signaling pathway to enhance vascular remodeling and promoting VSMC migration and proliferation to reduce zone differentiation[74]. In conclusion, miRNAs are important for the regulation of synthetic phenotypes in vascular remodeling in a variety of vascular diseases.

Fig. 2.

Merged enrichment map of target proteins of synthetic phenotype regulated by miRNAs obtained from KEGG and GO. A Linkages between target proteins associated with miRNA-influenced synthesis phenotypes were analyzed by R language; B histograms of three typical enrichment items for molecular function (red), cellular composition (blue), and biological processes (green), and the top 10 GO items in each category are plotted according to p value. C KEGG enrichment analysis network based on the association of target genes; the most significant one is marked in red

miRNAs regulate the transformation of VSMC to the contractile phenotype

Under physiological conditions, the VSMC contractile phenotype is key to the maintenance of normal vascular function, including the regulation of vascular resistance, blood pressure, and contractile ability. Some miRNAs have been demonstrated to be specifically expressed in normal VSMC at high levels and to show differential downregulation after vascular injury, which suggests that these miRNAs might specifically regulate VSMC transformation to the contractile phenotype. For example, miR-1 overexpression induced by myocardin yielded the targeted negative regulation of the serine/threonine protein kinase Pim-1, which subsequently resulted in significant inhibition of Pim-1-mediated VSMC proliferation and indirect promotion of the transformation of VSMC to the contractile phenotype [75]. Moreover, miR-133 can promote the transformation of VSMC to the contractile phenotype via the PDGF/MAPK-ERK1/2/miR-133/Sp-1/KLF4/myocardin signaling pathway [76]. Specifically, exogenous PDGF can inhibit high miR-133 expression, and this inhibition can be alleviated by suppressing the MAPK/ERK1/2 signaling pathway. However, miR-133 overexpression can inhibit specificity protein-1 (Sp-1) expression to induce the downregulation of the transcription factor KLF4 and increase the expression of the downstream molecule myocardin, and these effects inhibit VSMC proliferation and migration and promote VSMC differentiation. Correspondingly, Tang et al. [37] revealed that miR-124 is expressed at low levels in proliferating VSMC, whereas upregulation of miR-124 expression significantly enhances the expression of VSMC differentiation-related protein genes, such as SMA, SM22α, and calponin. Their results further revealed that miR-124 can inhibit the expression of the downstream factor Sp-1 to prevent VSMC proliferation and thus indirectly promote the transformation of VSMC to the contractile phenotype.

Extensive studies have confirmed that miR-145 can be used as a potential marker for VSMC phenotypic transformation. A study has shown that lentivirus delivery micR-145 can significantly increase the area of fibrous cap, reduce the area of necrotic core and increase the content of plaque collagen in the aorta of apolipoprotein E^(−/−) mice by reducing KLF4 and increasing the expression of myocardial protein, so as to limit the morphology and cellular composition of atherosclerotic plaque and balance the stability of atherosclerotic plaque and plaque rupture [77, 78]. Interestingly, in a VSMC dedifferentiation model using balloon injury or PDGF induction, Cheng and colleagues found that promoting miR-145 expression reduced VSMC dedifferentiation, while inhibiting miR-145 expression led to VSMC dedifferentiation [79]. Mechanistically, miR-145 restores balloon-injured arterial neointima growth by targeting inhibition of KLF5 and its downstream signaling molecule myocardin. Meanwhile, miR-145 significantly reversed the suppression of atherosclerosis-induced VSMC differentiation marker genes, such as SMα actin, calmodulin, and MYOCD [79, 80]. These new findings may have broad significance for the diagnosis and treatment of a variety of proliferative vascular diseases. Venous grafts are commonly used as autografts in ischemic disease revascularization procedures, but long-term patency is poor, because exposure to arterial blood pressure accelerates VSMC hyperplasia. Japanese researchers reported that transduction of miR-145 in venous grafts attenuated venous intimal hyperplasia by maintaining more SM22α mature VSMC and fewer Ki-67-positive proliferating cells, allowing VSMC to shift from a proliferative to a contractile state [81, 82]. Further atherosclerosis was treated with nanoparticle micelles packed with miR-145, and miR-145 micelles were found to reverse the atherosclerosis-induced decrease in VSMC protective contractile markers, such as cardiac myosin, α-SMA, calmodulin, ACTA2, and MYH11 [83, 84]. Notably, miR-145 has been studied quite extensively in VSMC. Either exogenous overexpression of miR-145 effectively inhibits high glucose-induced VSMC over-proliferation and migration [85]; or induces VSMC initiation and development via CD40 and further relies on Jag-1 activation of Notch receptors leading to CBF1-dependent upregulation of miR-145, increasing differentiation and decreasing proliferation [86, 87]. These demonstrate that miR-145 is highly plastic in resident VSMC and is capable of converting VSMC from a migratory proliferative synthetic phenotype to a protective contractile phenotype, expanding the path for subsequent vasodilator drug development.

Analogically, high expression levels of miR-15b/16 are closely related to the contractile phenotype in VSMC, and overexpression of miR-15b/16 can promote the expression of genes encoding smooth muscle contractile-related proteins and inhibit the proliferation and migration of VSMC [88]. Studies have shown that miR-15b/16 can promote the conversion of VSMC to a contractile phenotype by targeting the downstream gene yes-associated protein 1 (YAP) [89]. Furthermore, Li et al. [90] found that the overexpression of miR-663 increases the expression of VSMC differentiation markers, such as SM-22α and calmodulin, and inhibits PDGF-induced VSMC proliferation and migration. Mechanistically, miR-663 promotes the transformation of VSMC to the differentiated phenotype through the targeted inhibition of JunB and its downstream molecules, such as myosin light chain 9 (MYL-9) and matrix metallopeptidase 9 (MMP-9).

Several studies have detailed other miRNAs and their impact on the VSMC contractile phenotype. For example, miR-424/322 directly targets and regulates Cyclin D1 and calumenin, indirectly targets stromal interaction molecule 1 (STIM1), and inhibits VSMC proliferation and migration, suggesting that miR-424/322 indirectly stimulates the transformation of VSMC to the contractile phenotype [91]. Furthermore, miR-638, which is highly expressed in differentiated VSMC, exhibits extremely low expression in dedifferentiated VSMC and can inhibit the expression of orphan nuclear receptor (NOR1), which leads to reduced expression of the downstream factor cyclin D1 and decreased VSMC proliferation and migration [92]. These events limit the conversion of VSMC to synthetic phenotypes by reducing the proliferation and migration of VSMC. In addition, miR-34c was significantly upregulated in a rat carotid artery injury model and increased the levels of p21, p27, and Bax through targeted suppression of stem cell factor (SCF), leading to the transformation of VSMC to a differentiated phenotype [93]. Furthermore, overexpression of miR-206 inhibited proliferation and migration of human pulmonary artery smooth muscle cells (HPASMC) by downregulating Notch-3 expression and further increased the levels of VSMC differentiation markers α-smooth muscle actin and calmodulin to promote VSMC differentiation, reflecting the importance of miR-206 in the contractile phenotype [94]. Accordingly, VSMC phenotypic transformation can modulate atherosclerosis progression by affecting cell proliferation and migration through the myocardial-miR-206-Cx43 loop [95]. In short, various miRNAs have a non-negligible role in VSMC phenotypic transformation and can provide good predictions for the progression of multiple diseases by facilitating the transformation to a contracted phenotype and serve as diagnostic markers and new targets for therapeutic intervention.

The targets and functions of miR-23b and miR-182 have been extensively characterized in vascular smooth muscle. Specifically, Iaconetti et al. [96] found that miR-23b is downregulated after vascular injury in a rat carotid artery balloon injury model and that overexpression of miR-23b significantly reduces neointimal hyperplasia in balloon-injured arteries. These results further demonstrated that miR-23b can significantly promote the expression of VSMC marker genes, such as ACTA2 and MYH11, and that miR-23b can target the transcription factor Forkhead box O4 (FoxO4) to inhibit downstream MMP-9 expression and TNFα-induced VSMC migration. In addition, miR-23b can inhibit urokinase-type plasminogen activator and Smad3, thus inhibiting cell proliferation and migration mediated by the above two factors [97, 98]. In general, miR-23b can be speculated to be able to positively regulate VSMC transformation to the contractile phenotype. Similarly, miR-182 expression is significantly altered during the phenotypic transformation of VSMC. For example, miR-182 upregulation increases the expression of SMC contractile-specific genes, including α-smooth muscle actin (SMA), SM-22α, and calponin, whereas downregulation of miR-182 expression induces dedifferentiation of VSMC from the contractile to the synthetic phenotype [99]. Furthermore, additional results indicated that miR-182 could target fibroblast growth factor 9 (FGF9) and myeloid-associated differentiation marker (MYADM) by mediating PDGFRβ or TGF-β signaling to prevent SMC dedifferentiation [99–101]. These events all point to the ability of miR-182 to significantly modulate the contractile phenotype of VSMC, providing a promising target for the treatment of vascular diseases. The major miRNA networks regulating the VSMC contractile phenotype are shown in Fig. 3.

Fig. 3.

MicroRNAs regulate contractile phenotype transformation in VSMC. Inhibition of VSMC proliferation and migration and acceleration of VSMC differentiation are direct evidence of promoted VSMC conversion to a contractile phenotype. Factors, such as miR-1, miR-145, miR-195, miR-124, miR-34c through modulation of direct targets, or external stimulating factors can slow cell proliferation and migration or maintain the contractile phenotype of VSMC by partially affecting apoptosis through miR-206. Moreover, miR-145 and miR-133 could directly improve the differentiation ability of VSMC. In this process, tropoelastin, osteopontin, thrombospondin, and other proteins characteristic of the VSMC contractile phenotype were highly expressed to prevent conversion to the synthetic phenotype

Meanwhile, to explore the linkage between target genes regulating vasoconstriction phenotypic miRNAs, we performed enrichment analysis of known effector proteins and identified the closest linkage between TP53, MYC, VEGRA, PTEN, STAT3, HRAS, and ESR1 proteins (Fig. 4A). GO analysis showed that multiple protein complexes can significantly influence biological processes such as epithelial cell proliferation and glandular development, modulating DNA/RNA polymerase activity and partial protease/phosphatase-binding capacity, thereby regulating the vasoconstriction phenotype (Fig. 4B). In addition, the major signaling pathways clustered in the PI3K–AKT signaling pathway and miRNAs in cancer (Fig. 4C). Fortunately, miR-145 was highly expressed in small arteries of hypertensive patients remodeled by exercise, significantly reduced AKT phosphorylation and IGF-1R mRNA, and contributed to significant upregulation of the VSMC contractile marker calponin, which induced VSMC to maintain a contractile phenotype [102]. In conclusion, the regulation of contractile phenotype in vascular remodeling by miRNAs is not negligible and provides a therapeutic basis for some vascular diseases.

Fig. 4.

Merged enrichment map of target proteins of contractile phenotype regulated by miRNAs obtained from KEGG and GO. A The linkage between miRNAs affecting contracted phenotype-associated target proteins was Analyzed by R language; B histograms of three typical enrichment items for molecular function (red), cellular composition (blue) and biological processes (green), and the top 10 GO items in each category were plotted according to p value. C KEGG enrichment analysis network based on the association of target genes, the most significant one is marked in red

Taken together, these studies clearly demonstrate that multiple miRNAs have strong regulatory roles in maintaining VSMC phenotypic transformation. However, the exact role of these miRNAs seems to depend on the microenvironment and the cells responsible for disease pathology, and further refinement and expansion of the profound role of miRNAs in the contractile and synthetic phenotypes of VSMC is still needed.

VSMC phenotypic miRNAs involved in the regulation of vascular remodeling and homeostasis

Vascular remodeling and homeostasis are key components of many vascular diseases. Vascular remodeling is both an adaptive physiological process that maintains vascular homeostasis and a key pathological link that is commonly found in a number of vascular pathologies [103, 104]. In this regard, cardiovascular and cerebrovascular diseases, such as coronary heart disease, hypertension, stroke, PAH, and organ injury, pose substantial threats to human health and well-being [105, 106]. The vascular remodeling underlying these disorders results from an imbalance between vascular function and injury repair. As small regulatory transcripts, numerous miRNA species are involved in VSMC phenotypic changes, which are characterized by VSMC proliferation, migration and differentiation, oxidative stress and apoptosis, ECM synthesis, inflammation, osteoblast differentiation, and calcification and contribute to arterial stiffness, hypertension, and coronary heart disease [107–109], thus playing an important regulatory role in vascular remodeling and homeostasis (Fig. 5).

Fig. 5.

miRNAs regulate VSMC phenotypic transformations to influence vascular remodeling and homeostasis by regulating different pathophysiological aspects, thus affecting various cardiovascular disease processes. Vascular remodeling and homeostasis are key in many vascular diseases. miRNAs, as a class of small regulatory transcripts, can modulate the phenotypic transformation of VSMC to repair the balance between vascular function and injury by regulating the proliferation, migration and differentiation, oxidative stress and apoptosis, extracellular matrix (ECM) synthesis, inflammation, and osteoblast differentiation and calcification of VSMVs. Therefore, miRNAs have considerable potential for targeted therapy and prevention in cardiovascular diseases, including thoracic aortic coarctation, atherosclerosis, aortic aneurysm, diabetes, vasculitis, osteoporosis, and coronary artery disease

miRNAs in VSMC proliferation, migration, and differentiation

Recent studies have demonstrated that miRNAs are essential regulators of VSMC regulation and further identified several target genes that are implicated in VSMC proliferation, migration, and differentiation. For example, overexpression of miR-22 significantly increased the expression of VSMC phenotypic marker genes and led to inhibition of the proliferation and migration of VSMC, whereas the opposite effect was observed when endogenous miR-22 was knocked down [107]. Similarly, miR-22 overexpression in damaged vessels significantly reduced the expression of its target gene, eosinophil integration site 1 protein homolog (EVI1), and inhibited neointima formation in wire-injured femoral arteries by reducing VSMC proliferation [107]. In addition, a study by Tang et al. [37] found that overexpressed miR-124 significantly attenuated PDGF-induced human arterial smooth-muscle cell (HASMC) proliferation and phenotypic transformation. Overexpression of Sp1, as the downstream target of miR-124, appeared to reverse the anti-proliferative effect of miR-124 in HASMC, suggesting that miR-124 is a key regulator of HASMC differentiation, proliferation, and phenotypic transformation. In addition, a similar experiment performed by Chen et al. [110] showed that increased miR-34a around the blood vessels significantly reduced the expression level of its target gene Notch1 and thus decreased VSMC proliferation and inhibited neointimal formation in wire-injured femoral arteries. Mimetically, miR-134-5p effectively inhibited PDGF-induced VSMC phenotypic transformation and migration, while ectopic expression of miR-134-5p obviously promoted VSMC differentiation and expression of contractile markers [111]. The study further determined that miR-134-5p could inhibit the phenotypic transformation and thoracic aortic dissection (TAD) of VSMC by targeting signal transducer and activator of transcription 5B (STAT5B) and integrin beta 1 (ITGB1), suggesting that miR-134-5p is a critical regulator of phenotypic transformation and migration in human HVSMC.

Abnormal phenotypic transformation of VSMC is a hallmark of vascular disorders, and evidence shows that miRNAs play key roles in VSMC functions. For example, miR-128-3p, which is considered to be associated with VSMC phenotypic transformation and a new key factor in regulating vascular disease, increases the methylation status of the VSMC gene myosin heavy chain 11 (Myh11) to affect VSMC proliferation, migration, differentiation, and contractility after stress due to stimulation [112]. In addition, miR-145 is a key factor in regulating VSMC plasticity, which can be reduced in vascular disease. It was shown that miR-145 regulates the essential role of VSMC phenotype by inhibiting ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) mRNA translation to reduce proliferation and dedifferentiation [113]. Moreover, miR-145 could inhibit VSMC proliferation, migration, and phenotypic transformation by preventing activation of the PI3K/AKT/mTOR signaling pathway [114]. Overall, abnormalities in the VSMC phenotype and its regulators are factors that may have therapeutic potential in vascular lesions.

Several recent studies have indicated that type 2 diabetes mellitus (T2DM) can influence the proliferation, migration, and phenotypic transformation of VSMC and thus contribute to atherosclerosis and diabetes-induced vascular remodeling [74, 115]. Moreover, during the course of T2DM, the expression level of VSMC-specific miRNAs that are closely related to contractile or synthetic phenotypes will be differentially expressed. For example, using right carotid artery experimental angioplasty and RNA sequencing, Torella et al. [116] revealed that miR-29c and miR-204 were most significantly dysregulated in atherosclerotic plaques from patients with T2DM. miR-29c overexpression and miR-204 inhibition could enhance additive reductions in VSMC proliferation and thus attenuate VSMC phenotypic transformation in DM by targeting epithelial membrane protein 2 (Emp2) and caveolin-1 (Cav1), respectively. Another recent study demonstrated that miR-504 could promote diabetic VSMC dysfunction by targeting signaling adaptor growth factor receptor-bound protein 10 (Grb10) and transcription factor early growth response 2 (EGR2), thereby being involved in the metabolic memory of vascular complications [117]. Likewise, miR-504 Overexpression suppressed contractile genes and increased the migration and proliferation of VSMC from diabetic mice, indicating that miR-504 can promote VSMC dysfunction.

Collectively, these results appear to indicate that several miRNAs have been extensively characterized as key regulators of VSMC proliferation, migration, differentiation, and phenotypic transformation, and play important roles in the pathogenesis of many aspects of vascular disease.

miRNAs in VSMC apoptosis and oxidative stress

VSMC apoptosis plays an important role in vascular remodeling and in the pathophysiology of vascular diseases and atherosclerosis. Apoptosis of VSMC is considered to be triggered by oxidative stress present in the diseased vascular milieu. Identifying the regulatory mechanisms of oxidative stress-induced VSMC apoptosis potentially may reveal novel therapeutic approaches for vascular remodeling and atherosclerotic plaque instability. miRNAs have been experimentally validated to regulate apoptotic factors and oxidative stress, which is beneficial to the dissolution of atherosclerotic plaques, the proliferation of VSMC, and the formation of new intima. For example, Yang et al. [118] found that mammalian sterile 20-like kinase 2 (MST2) regulated miR-155-promoted inflammatory and oxidative stress responses by altering the interaction of MEK with Raf-1 and MST2 in response to vascular injury, indicating that suppression of endogenous miR-155 is a potential therapeutic strategy for vascular injury and remodeling. In addition, loss of VSMC through apoptotic cell death can cause fibrous cap thinning, necrotic core formation, and calcification that may destabilize plaques. miR-25 blocks corticosterone-induced VSMC apoptosis by targeting modulator of apoptosis 1 (MOAP1) and the p70S6k pathway [119]. A study by Peng et al. [120] elucidated the role of miR-26a in VSMC apoptosis and indicated that miR-26a protected VSMC against H₂O₂-induced injury through activation of the PTEN/AKT/mTOR pathway and may be considered a potential prognostic biomarker and therapeutic target in the treatment of abdominal aortic aneurysm (AAA). Similarly, miR-26 attenuates vascular smooth muscle maturation and indirectly promotes a differentiated smooth muscle phenotype and apoptosis through BMP signaling modulation in endothelial cells by targeting Smad1, which is a BMP signaling effector [121]. These results clearly reveal that miRNAs broadly regulate VSMC apoptosis to influence vascular remodeling and homeostasis, providing a reference for vascular disease treatment.

The cellular metabolism of all aerobic organisms produces reactive oxygen species (ROS), which have been associated with the physiopathology of several diseases and can be neutralized by nonenzymatic defenses and the activity of antioxidant enzyme. Notably, mammals have the ability to scavenge excess free radicals, which depends on the endogenous free radical scavenging system, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH), as well as nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade signal pathway [122–125]. Antioxidant enzymes can change the reactive oxygen free radicals in the body into substances with low activity, thus weakening their attack on the body [126, 127]. Although evidence for direct targeting of miRNAs to regulate oxidation-related enzymes has not emerged, however, some miRNAs can indirectly alter the activity of various oxidative enzymes to exert in vivo and cellular scavenging of free radicals for maintaining oxidative homeostasis in and around blood vessels [128, 129]. A recent study has shown that miR-135a-5p reversed ox-LDL-induced alterations in SOD and MDA activities in VSMC by increasing XBP1 expression, suggesting a potential therapeutic strategy for miRNAs in atherosclerosis treatment [130]. Moreover, miR-143-3p, which directly targets the inhibition of a disintegrin and metalloproteinase 10 (ADAM10), significantly affected the levels of superoxide dismutase and malondialdehyde, ameliorating the cytotoxicity triggered by angiotensin II in VSMC [131]. In addition, the VSMC isolated from the thoracic aorta of diabetes rats were transfected with miR-24, and it was found that the oxidative stress of VSMC was inhibited and the expression level of Nrf2/HO-1 was increased, and the degree of recovery was related to SOD and GSH-px [132]. The above results suggest that miRNAs regulate oxidative stress levels in VSMC by affecting related oxidative enzymes and proteins. Further exploration of their direct mechanisms and targets may provide more therapeutic prospects for the prevention and treatment of vascular diseases.

Moreover, upregulation of miR-21 and miR-221/222 in the area damaged by experimental arterial balloon injury promotes intimal hyperplasia and induces subsequent stenosis. In contrast, downregulation of miR-221/222 expression significantly inhibits VSMC proliferation and improves local vascular patency after angioplasty [29, 133]. Additional studies have proposed that a miR-143/145 cluster acts as a “switch” that regulates the phenotypic transformation of VSMC during vascular remodeling. Thus, the miR-143/145 cluster is elevated by dedifferentiation of the VSMC phenotype but decreased in lesions and atherosclerotic vessels. miR-143/145 expression during arterial repair postinjury inhibits VSMC proliferation and intimal growth. However, miR-143/145-knockout mice are also resistant to pathological vascular remodeling due to experimental injury, which suggests that the miR-143/145 clusters are both positive and negative regulators of VSMC differentiation and proliferation [81, 87]. Furthermore, their dual targeted vascular stress response mode can maintain the phenotypic plasticity of VSMC and promote appropriate responses to pathological injury. Overall, modulating vascular oxidative stress and affecting vascular apoptosis by altering the levels of miRNAs in vascular cells and blood circulation are a potentially effective means of treating some cardiovascular conditions.

miRNAs in VSMC ECM synthesis

VSMC are surrounded by ECM [134]_, which is a fundamental component of multicellular organisms that not only provides essential physical scaffolding for cellular constituents but also initiates crucial biochemical and biomechanical cues that are needed for vessel wall stability. Moreover, the ECM is a complex meshwork of both structural and functional proteins assembled in unique tissue-specific architectures that play important roles in guiding VSMC function [135]. Several studies have shown that miRNAs have emerged as key modulators of ECM homeostasis and thereby have been proposed to explore the role of miRNAs deeply in vascular remodeling. For example, Wang et al. [136] demonstrated that VSMC exhibit remarkable plasticity to undergo phenotypic modulation, in which the expression of VSMC markers is markedly attenuated, while conversely, ECM expression is dramatically increased. Myocardin not only activates smooth muscle-specific genes but also regulates ECM expression through induction of transcription of miR-143 during smooth muscle differentiation. Moreover, miR-126-5p promotes contractile transformation of VSMC by targeting VEPH1 and alleviates Ang II-induced abdominal aortic aneurysm in mice [137]. Ang II-induced upregulation of MMP9 and MMP2, two key proteases responsible for ECM degradation, in mouse aortas and human aortic smooth muscle cells (HAOSMC) was also reduced by miR-126-5p overexpression. The results reveal a novel role of miR-126-5p in inhibiting AAA development-associated aortic VSMC dedifferentiation.

Collagen is the most abundant protein in animals, accounting for more than 30% of the total protein in the human body, is distributed in various organs and tissues, and can be synthesized and secreted by many kinds of cells constituting the framework structures in the ECM [138]. Collagen synthesis in VSMC is very important in atherosclerosis, as it affects plaque stability [139]. A study showed that miR-124-3p inhibited VSMC collagen synthesis and restrained protein levels of type I and type III collagen in aortas and atherosclerotic plaques by directly targeting prolyl 4-hydroxylase subunit alpha 1 (P4HA1), which might decrease atherosclerotic plaque stability [140]. Bekelis et al. [141] indicated that miR-21, miR-143, and miR-145 post-transcriptionally regulated the expression of multiple targets with a function in the ECM, such as collagen (COL1A1, COL5A1, and COL5A2) and MMP-13, which are associated with collagen formation, VSMC phenotypic modification, extracellular matrix remodeling, inflammation signaling, and lipid accumulation. These results indicate that microRNAs, as an important regulator of VSMC function, play a significant regulatory role in the formation and stability of vascular scaffolds, providing a theoretical basis for angiogenesis.

miRNAs in VSMC inflammation

Since VSMC proliferation and endothelial cell (EC) dysfunction are crucial in the pathogenesis of diabetic atherosclerosis and inflammatory symptoms, it is increasingly valuable to explore strategies for their prevention and treatment [142, 143]. Meanwhile, increasing evidence suggests that miRNAs and their target genes may be involved in VSMC inflammatory processes [144, 145]. For example, a study performed by Brennan et al. [146] showed that both PDGF- and TNF-α-induced VSMC and EC activation were associated with reduced let-7 miRNA expression via LIN28b, a negative regulator of let-7 biogenesis. Ectopic overexpression of let-7 in VSMC inhibited inflammatory responses, including proliferation, migration, monocyte adhesion, and nuclear factor-κB activation. The results suggested that restoration of let-7 expression in VSMC could provide a novel target for an anti-inflammatory approach in diabetic vascular disease. Furthermore, another study demonstrated that perivascular adipose tissue (PVAT)-derived extracellular vesicles (EVs) and their encapsulated miR-221-3p could be taken up into VSMC [147]. Transfer and direct overexpression of miR-221-3p dramatically enhanced VSMC proliferation and migration and caused vascular dysfunction by suppressing contractile genes peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) in the arterial wall. The results provided an EV-miR-221-3p-mediated mechanism by which PVAT triggers early stage vascular remodeling in the context of obesity-associated inflammation.

Adhesion molecules expressed by activated EC play a key role in regulating leukocyte trafficking to sites of inflammation. Resting endothelial cells normally do not express adhesion molecules, but cytokines activate endothelial cells to express adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), which mediate leukocyte adherence to endothelial cells. For example, Tamia A. Harris et al. [148] found that EC expressed miR-126, which inhibited VCAM-1 expression. Decreasing endogenous miR-126 levels increases leukocyte adherence to EC. These data suggest that miRNAs can regulate adhesion molecule expression and may provide additional control of vascular inflammation. In addition, inflammation and excessive proliferation of VSMC have key roles in various vascular disorders. However, how miRNAs mediate the regulation of VSMC proliferation during inflammation through NF-kappa B (NF-κB) activation remains unknown. Notably, the results from Yang et al. [149] indicated that VSMC proliferation under inflammation is regulated by NF-κB p65/miR-17/RB pathway activation, providing a mechanism for the excessive proliferation of VSMC under inflammation during vascular disorders, which may help identify novel targets for the treatment of vascular diseases.

miRNAs in VSMC osteoblast differentiation and vascular calcification

Vascular calcification is characterized by pathological deposition of calcium and phosphate in the arteries through an active and regulated biological process in which VSMC transform into osteoblast-like cells and is a key pathologic component of vascular diseases, such as atherosclerosis, coronary artery disease, and peripheral vascular disease. Several studies have demonstrated that miRNAs regulate osteoblast differentiation and thus modulate VSMC-mediated arterial calcification (Fig. 6). For example, miR-133a overexpression inhibits VSMC transdifferentiation into osteoblast-like cells by targeting the amounts of bone runt-related transcription factor 2 (Runx2) to inhibit alkaline phosphatase activity, osteocalcin secretion, and mineralized nodule formation [150]. The results suggested that miR-133a is a key negative regulator of the osteogenic differentiation of VSMC. However, another study performed by Zhao et al. [151] revealed that reticulocalbin-2 (RCN2) promoted VSMC osteogenic differentiation and calcification by inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation. Furthermore, inhibition of STAT3 activation promoted miR-155-5p expression in VSMC and inhibited RCN2 expression. Finally, RCN2 overexpression partially offset the miR-155-5p-mediated inhibition of VSMC calcification, acting as a positive feedback loop.

Fig. 6.

miRNAs act on the calcification signaling pathway to regulate VSMC phenotypic transformation. Calcification is one of the key regulatory signals in VSMC phenotypic transformation. Activation of VSMC calcification was promoted by miR-324-3p via enhancement of PI3K3A and MAP2K1 or miR-762/714 via activation of NCKX4 and PMCA1 or miR-32 and miR-34b/c targeting RUNX2. Subsequently, Ca²⁺ can promote increased ERK1/2 expression through activation of RAS, which further regulates downstream key factors regulating cell proliferation, migration, and transcription. In addition, the key protein ERK1/2 can activate miR-31 or be regulated by miR-181b to affect VSMC performance transformation

It is well known that vascular calcification significantly increases the risk of cardiovascular mortality. Several studies have shown that the use and accumulation of inorganic phosphate (Pi) raises the likelihood of vascular calcification and is one of the important causes of cardiovascular disease prevention and treatment that cannot be ignored. For example, microRNA miR-134-5p enhances inorganic phosphate (Pi)-induced calcium deposition in VSMC by inhibiting the expression of histone deacetylase 5 (HDAC5) and osteoprotegerin (OPG) and increasing Runx2 protein and bone morphogenetic protein 2 (BMP2) mRNA [152]. Similarly, miR-221 and miR-222 mimics caused increased VSMC calcium deposition by inducing significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit-1 expression, which increased cellular inorganic phosphate and pyrophosphate levels [153]. In addition to these, increased expression of miR-135a*, miR-762, miR-714, and miR-712* in VSMC may be involved in Pi-induced VSMC calcification by disrupting Ca(²⁺) efflux proteins by regulating the Ca²⁺ efflux proteins NCX1, PMCA1, and NCKX4 [154, 155]. These results suggest that some miRNAs have the potential to act as markers of vascular calcification, offering the possibility of clinical identification for early calcification in cardiovascular disease.

Arterial calcification is a common macroangiopathy that initiates from a VSMC-regulated process of osteoblastic differentiation. Accumulating evidence has demonstrated that epigenomic regulation by specific miRNAs might play an important role in VSMC calcification. For example, Cui et al. [156] have indicated that miR-204 is an endogenous attenuator of Runx2 in VSMC calcification. Downregulation of miR-204 may contribute to β-glycerophosphate-induced VSMC calcification by regulating Runx2, indicating that miR-204 is a potential novel therapeutic target in medial artery calcification. Interestingly, miR-204 overexpression attenuated osteoblastic differentiation of VSMC by targeting DNA methyltransferases 3A (DNMT3A). Moreover, when DNMT3A was knocked down, the methylation ratio of miR-204 was decreased significantly, and miR-204 expression resulted in abrogation of the effect of high-phosphate concentrations on VSMC calcification [157]. Furthermore, overexpression of miR-125b inhibited β-glycerophosphoric acid-induced osteogenic marker expression and calcification of VSMC, whereas knockdown of miR-125b promoted the phenotypic transformation of VSMC and calcification by regulating Ets1. Additionally, both miR-2861 and miR-3960 levels were increased during β-glycerophosphate-induced osteogenic transdifferentiation of VSMC, and miR-2861 and miR-3960 promoted osteogenic transdifferentiation of VSMC by targeting histone deacetylase 5 and homeobox A2, respectively, resulting in increased Runx2 protein production [158]. The above results suggest that the induction of osteogenic differentiation of VSMC by β-glycerophosphate to cause vascular calcification can be interfered by some miRNAs, thus reducing the possibility of large vessel disease development.

Vascular calcification is an actively regulated process similar to osteogenesis and osteoporosis. Increasing evidence suggests that miRNAs contribute to the development of VSMC calcification. For example, miR-34b/c, which is downregulated during VSMC calcification, exacerbated aldosterone-induced VSMC calcification by regulating the SATB2/Runx2 pathway, and overexpression of miR-34b/c attenuated aldosterone-induced VSMC calcification [159]. Conversely, miR-32 regulates the process of vascular calcification by activating phosphatidylinositol 3-kinase (PI3K) signaling and increasing RUNX2 expression and phosphorylation by targeting the 3′ untranslated region of the phosphatase and tensin homolog (PTEN) in mouse VSMC [160]. Homoplastically, miR-324-3p significantly increased PIK3CA and MAP2K1 expression to promote vascular calcification [161]. In addition, exosomes are stably present in circulation, which protects the functional miRNA from degradation and renders competence for the induction of VSMC calcification [162, 163]. Therefore, by exploring the evidence of circulating miRNA transduction to VSMC, it will facilitate the development of diagnostic, disease progression, and/or miRNA-derived therapeutic approaches in cardiovascular disease research.

To sum up, these findings suggest a potential role of miRNAs, which determine VSMC fate and reduce neointima formation following arterial injury, thereby acting as therapeutic targets for restenosis and vascular remodeling after angioplasty. Increasing discoveries are being made about miRNAs and their targets affecting multiple CVD by regulating VSMC phenotypic transformation (Table 2), and we need to identify specific miRNAs in specific CVD and interfere with them for therapeutic purposes. In addition, deeply systematic and functional analysis of miRNAs in vascular smooth muscles is required before a clear set of restricted patterns of tissue and principles will become more apparent.

Table 2.

Characterization of VSMC phenotypic transformations regulated by miRNAs in vascular remodeling for maintaining vascular homeostasis

Characteristics affecting phenotypic transformation	ncRNAs	Target gene	Regulation category	Physiological function	Disease type	References
Proliferation, Migration, and Differentiation	miR-124	Sp1	Attenuat VSMC proliferation and phenotypic transformation	Reduce aortic dissection	Aortic dissection	[37]
	miR-128	KLF4/ Myh11	Reduce VSMC proliferation and migration	Reduce proliferation and migration, improve differentiation and contractility	Carotid restenosis	[53]
	miR-22	EVI1	Reduce VSMC proliferation	Inhibit neointima formation in wire-injured femoral arteries	Vascular injury	[107]
	miR-34a	Notch1	Reduce VSMC proliferation and migration	Inhibit neointima formation	Vascular injury	[110]
	miR-134-5p	STAT5B/ ITGB1	Inhibit VSMC phenotypic transformation and migration	Suppress the aorta dilatation and vascular media degeneration	Thoracic aortic dissection	[111]
	miR-145	UHRF1	Reduce VSMC proliferation and migration	Prevent intimal hyperplasia in mouse carotid artery	Intimal hyperplasia/ restenosis	[113]
	miR-504	Grb10/Egr2	Enhance VSMC proliferation and migration	Improve VSMC dysfunction	Diabetes mellitus	[117]
	miR-93	Mfn2	Promote VSMC proliferation and migration	Promote neointimal formation	Atherosclerosis and restenosis	[164]
	miR-101	DOCK4	Reduce VSMC proliferation and migration	/	/	[165]
	212-5p	PAFAH1B2	Reduce VSMC proliferation and migration	Promote contraction	Hypertension	[166]
	miR-4463	Bfgf	Reduces VSMC proliferation and intimal hyperplasia	Reverse the injury-induced dedifferentiation	Arterial injury	[167]
Apoptosis and Oxidative stress	MiR-25	MOAP1	Inhibit apoptosis	Reduce the formation of unstable plaque	Atherosclerosis	[119]
	miR-26	PTEN	Enhance cell viability	Protecte VSMCs against H2O2-induced injury	Abdominal aortic aneurysm	[120]
	miR-24	Nrf2/Ho-1	Suppress HG-induced oxidative stress	Promotes re-endothelialization in balloon-injured diabetic rats	Balloon Injury	[132]
	miR-31-5p	FNDC5	Attenuate oxidative stress	Promote VSMC migration	Spontaneously hypertensive	[168]
	miR-4787-5p	PC1	Promote apoptosis	Induced polycystic kidney disease	Aortic dissection	[169]
ECM synthesis	miR-143	Versican	Attenuate ECM versican protein expression	Inhibit migration and proliferation	/	[136]
	miR-126-5p	VEPH1	Attenuat aortic dilation and elastin degradation	Restore hAoSMCs differentiation	Abdominal aortic aneurysm	[137]
	miR-124-3p	P4HA1	Decrease type I and type III collagen	Decrease content of plaques	Atherosclerosis	[140]
	miR-106a	TIMP-2	Accelerate ECM degradation	Inhibit cell viability and promote apoptosis	Abdominal aortic aneurysm	[170]
Inflammation	let-7	Lin28b	Inhibit inflammatory responses	Reduce proliferation, migration and monocyte adhesion	Atherosclerosis	[146]
	miR-221-3p	PGC-1α	Promote inflammatory macrophage infiltration	Induce fat cell hypertrophy and increase lipogenesis	Adiposity	[147]
	miR-17	RB	Improve inflammatory reaction	Stimulate VSMCsproliferation, enhance G1/S transition	/	[149]
	miR-712-5p	/	Promote TNF-α-induced VSMC inflammation	Reduce carotid artery ligation-induced intimal hyperplasia	Proliferative vascular diseases	[171]
	miR-376b-3p	KLF15	Promote inflammatory	Induce expression of inflammatory cytokines IL-1β and TNF-α	Diabetes and hyperglycemia	[172]
	miR-378a	PGC-1α	Reduce inflammation	Inhibit free fatty acid-induced cell proliferation and migration	Atherogenesis	[173]
Osteoblast differentiation and Vascular calcification	miR-133a/204	Runx2	Inhibit VSMCs osteogenic differentiation	Inhibit calcification	Atherogenesis	[150]
	miR-155-5p	RCN2	Reduce SMC osteogenic differentiation and calcification	Inhibit calcification	Peripheral arterial disease	[151]
	miR-125b	Ets1	Inhibit osteogenic markers expression	Inhibit VSMCs calcification	Chronic kidney disease	[157]
	miR-2861/3960	HDAC5/ HOXA2	Promote VSMCs osteogenic transdifferentiation	Promote vascular calcification	/	[158]
	miR-34b/c	SATB2	Alleviate VSMC calcification	Antagonize the effects of aldosterone	Vascular disease	[159]
	miR-32	PTEN	Promote osteogenic protein expression	Promote vascular calcification	Coronary artery calcification	[160]
	miR-204	DNMT3A	Alleviate VSMCs osteoblastic differentiation	Reduce arterial calcification	Uremia	[174]

Furthermore, other non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), have been reported to play important roles in the phenotypic transformation of VSMC. Here, we summarized the role of lncRNAs and circRNAs in regulating VSMC phenotypic transformation, as shown in Table 3, which indicates that other ncRNAs also have great potential in the treatment of CVD, including coronary artery disease, hypertension, and aortic coarctation. However, given the limited data reported, this does not provide definitive corroboration and needs to be further explored.

Table 3.

Characterization of VSMC phenotypic transformations regulated by lncRNAs and circRNAs in vascular remodeling for maintaining vascular homeostasis

ncRNAs	Target gene	Regulation category	Physiological function	Disease type	References
lncRNA-PEBP1P2	CDK9	Repress VSMC proliferation, migration, and dedifferentiation	Suppress neointima formation	Coronary heart disease and carotid atherosclerotic	[175]
lncRNA-CARMN	Myocardin	Maintain contractile phenotype of VSMC	Suppress neointima formation	Carotid artery injury	[176]
lncRNA-PSR	YBX1	Promote VSMC proliferation and migration	Promote neointima formation	Spontaneous hypertension	[177]
lncRNA-MYOSLID	MKL1	Promote VSMC differentiation and inhibit proliferation	Amplify VSMC differentiation program	/	[178]
lncRNA-AK098656	MYH11/FN1	IncreaseVSMC proliferation and migration, elevate extracellular matrix proteins	Narrow resistant arteries	Spontaneous hypertension	[179]
LncRNA- MRAK048635_P1	/	Inhibit VSMC proliferation and migration and promote apoptosis	Reduce vascular damage in hypertension	Essential hypertension	[180]
lncRNA-NEAT1	WDR5	Promote VSMC proliferation and migration	Promote neointima formation	Occlusive vascular diseases	[181]
lncRNA-FOXC2_AS1	Akt/mTOR	Promote VSMC proliferation, migration, and dedifferentiation	Promote neointima formation	/	[182]
lncRNA-SENCR	Myocardin	Inhibit VSMC proliferation, migration and synthetic phenotype-related gene expression	Suppress rupture of mice aortic media	Aortic dissection	[183]
circRNA-SATB2	SM22-α/STIM1	Promote VSMC proliferation and migration	Promote neointima formation	Coronary heart disease	[184]
circRNA-ZXDC	ABCC6	Increase VSMC proliferation and migration activity	Increase MMD vessels intima thickness	Moyamoya disease	[185]
circ-COL1A1	miR-30a-5p/SMAD30	Promote VSMC proliferation and migration	Promote human saphenous vein tissue growth	Atherosclerosis	[186]

Outlook

The identification of molecular mechanisms related to the miRNA-mediated phenotypic transformation of VSMC and the rapid progress in relevant research have enabled screening and identification of miRNAs that are differentially expressed during the phenotypic transformation of VSMC. These miRNAs can act as potential markers of the phenotypic transformation of VSMC that can be used for early clinical diagnosis, prognosis, and efficacy evaluations of diseases, such as atherosclerosis and restenosis after angioplasty, as shown in Table 1. Research on miRNAs that regulate vascular remodeling and homeostasis in vascular lesions has become an area of intense research interest. Previous studies have shown that some miRNAs involved in the phenotypic transformation of VSMC play an important role in the regulation of vascular remodeling and homeostasis. Therefore, the key goal of the current research is to conduct a more comprehensive and thorough investigation of the upstream regulatory factors and downstream target factors of miRNAs involved in the phenotypic transformation of VSMC, which would allow a more detailed understanding of the overall regulatory network of miRNA-mediated VSMC phenotypic transformation. Based on the current research results, the present review identifies potential gene expression regulatory pathways of miRNAs involved in the phenotypic transformation of VSMC.

The occurrence and development of cardiovascular disease is caused by the phenotypic transformation of VSMC, which is accompanied by the differential expression of a large number of specific markers, including SM22α, SMA, and CCND1. As a special marker, miR-21 has considerable potential for cardiovascular disease prediction. For example, miR-21 overexpression increased the spread cell area and proliferative capacity of saphenous VSMC and MMP-1 expression while reducing RECK protein, indicating a transformation to the synthetic phenotype [199]. Conversely, low miR-21 expression downregulated the mRNA and protein expression levels of α-SMA and AP-1, further inhibiting VSMC proliferation, invasion, and migration [200]. miR-21 has similar regulatory effects on TSP-1 and ERK 1/2 signaling [201]. Overall, inhibition of miR-21 expression significantly reduced VSMC proliferation and migration, which may provide new therapeutic targets for some human cardiovascular pathologies [202]. Thus, the development of safe and efficient miR-21 inhibitory drugs may be effective for controlling acute cardiovascular events by reducing VSMC proliferation and migration. Interestingly, if VSMC phenotypically transformed miRNA markers can specifically increase or inhibit the expression of VSMC-specific marker proteins in patients, miRNA gene therapy can be applied and intervene in the expression of target VSMC marker proteins in vivo, providing directions for future CVD or even vascular tumor diseases. Accumulating evidence has shown that miRNAs exhibit great potential in the diagnosis and prognosis of cardiovascular diseases with high sensitivity. For example, the high-level expression of miR-208, miR-134, miR-449, miR-21, miR-133a, miR-29b, miR-566, miR-7-1, miR-92a, miR-455-3p, miR-126, miR-636, miR-486, and miR-129 was significantly indicative of the occurrence of acute myocardial infarction [187–191]. Significant correlation with the development of heart failure was shown by detecting the levels of multiple miRNAs, including miR-320a, miR-22, miR-126, miR-132, and miR-103 [192–194]. Meanwhile, miR-126, miR-182, miR-197, and miR-223 were found to predict the ability of future adverse cardiovascular events in the general population and in patients with known CVD [195, 196]. These results suggest that some miRNAs are specific to CVD and may serve as important biomarkers with clinical applications for the diagnosis and therapy of disease activity. In addition, the major drawbacks of miRNA therapeutic regimens are off-target effects in organ systems. Thus, the implementation of local or cell-type-specific delivery mechanisms using nanoparticles on stents is highly desirable for application in miRNA therapeutics. For example, coating miR-145-containing nanoparticles on drug-eluting stents can further prevent restenosis by enabling VSMC growth around the stent [197]. Similarly, anti-miR-21 can reduce restenosis by reducing peri-coated stent intimal neogenesis [198]. Therefore, the specific role, contribution, and therapeutic potential of miRNA drugs have shown great potential to treat cardiovascular diseases.

Vascular remodeling is reportedly a key step in vascular injury repair that is accompanied by the phenotypic transformation of VSMC characterized by their proliferation [203–205]. Based on the discovery of some miRNA markers for VSMC phenotypic transformation, the in vivo expression of some functional miRNAs that regulate VSMC phenotypic transformation can be upregulated or downregulated to improve pathological vascular remodeling. The miR-143/145 cluster is both a positive and negative regulator of VSMC differentiation and antiproliferation [206]. Based on this dual targeted vascular stress response model, specific gene therapy drugs that target miR-145 can be developed to effectively maintain the phenotypic plasticity of VSMC, significantly inhibit reactive remodeling after vascular injury, and achieve effective prevention and treatment of restenosis, aneurysm, and other diseases after angioplasty [207, 208]. In addition, the high expression of miR-221/miR-222 after vascular injury plays a key role in the transformation of VSMC to the contractile phenotype. This finding suggests that clinical drugs developed to inhibit miR-221/miR-222 in vivo can effectively inhibit synthesis and improve pathological vascular remodeling, which would allow the effective prevention and treatment of vascular restenosis after stenting, coronary artery bypass grafting, heart transplantation, and other procedures [32, 33, 209].

However, several limitations complicate the current understanding of miRNA regulation of phenotypic transformations in vascular smooth muscle. For example, a given miRNA can regulate the expression of many different targets, and a single target can be regulated by multiple miRNAs, suggesting that the mechanisms of specific miR-mRNA interactions and their regulatory patterns during the phenotypic transformation of VSMC are elaborate and complex. Moreover, further investigations are necessary to determine whether other epigenetic conditions, such as DNA methylation and histone modifications, can influence the differential expression of VSMC phenotypic miRNAs, thereby promoting or inhibiting pathological smooth muscle remodeling.

The discovery of miRNA markers for the phenotypic transformation of VSMC and the clarification of their gene expression regulatory mechanisms provide a new platform for further investigation of the molecular mechanisms underlying the phenotypic transformation of VSMC. In-depth investigation of the comprehensive miRNA regulatory network of VSMC phenotypic transformation will contribute to the design of future studies investigating the complex molecular mechanisms of miRNA-mediated VSMC phenotypic transformation. Functional studies of miRNA markers involved in the phenotypic transformation of VSMC that could be used for early diagnosis, treatment, and prognosis and efficacy evaluations of CVD and in the development of gene therapies are likely to become future areas of intense clinical research.

Abbreviations

VSMC

Vascular smooth muscle cells

CVD

Cardiovascular diseases

ncRNA

Non-coding RNA

miRNA

MicroRNAs

lncRNAs

Long non-coding RNAs

circRNAs

Circular RNAs

PTEN

Phosphatase and tensin homologue

Bcl-2

B-cell lymphoma 2

KLF4

Kruppel-like factor 4

BMP-4

Bone morphogenic protein-4

PDCD4

Programmed cell death protein 4

DOCK

Dedicator of cytokinesis

VEGF

Vascular endothelial-derived growth factor

PDGF

Platelet-derived growth factor

TRIB3

Tribbles homologue-3

RhoA

Ras homologue family member A

MAPK

Mitogen-activated protein kinase

ERK

Extracellular signal-related kinase

PCNA

Proliferating cell nuclear antigen

LATS2

Large tumor suppressor 2

CREG

Cellular repressor of E1A-stimulated genes

TRB3

Tribbles homologue 3

Sp-1

Specificity protein-1

ELK1

ETS transcription factor ELK1

ACE

Angiotensin-converting enzyme

MYL9

Myosin light chain 9

MMP-9

Matrix metallopeptidase 9

STIM1

Stromal interaction molecule 1

NOR1

Orphan nuclear receptor

SCF

Stem cell factor

PAH

Pulmonary arterial hypertension

HPASMC

Human pulmonary arterial smooth muscle cells

FoxO4

Forkhead box O4

FGF9

Fibroblast growth factor 9

MYADM

Myeloid-associated differentiation

ECM

Extracellular matrix

EVI1

Eosinophil integration site 1 protein homologue

HASMC

Human arterial smooth-muscle cells

STAT5A

Signal transducer and activator of transcription 5A

ITGB1

Integrin beta 1

Myh11

Myosin heavy chain 11

T2DM

Type 2 diabetes mellitus

Emp2

Epithelial membrane protein 2

Cav1

Caveolin-1

Grb10

Growth factor receptor-bound protein 10

EGR2

Early growth response 2

MST2

Mammalian sterile 20-like kinase 2

MOAP1

Modulator of apoptosis 1

AAA

Abdominal aortic aneurysm

HAOSMC

Human aortic smooth muscle cells

P4HA1

Prolyl 4-hydroxylase subunit alpha 1

PVAT

Perivascular adipose tissue

EVs

Extracellular vesicles

EC

Endothelial cell

VCAM-1

Vascular cell adhesion molecule 1

NF-κB

NF-kappa B

RCN2

Reticulocalbin-2

STAT3

Signal transducer and activator of transcription 3

DNMT3A

DNA methyltransferases 3A

SMA

Smooth Muscle α-actin

SM22α

Smooth muscle22alpha

SM-MHC

Smooth muscle-myosin heavy chain

Author contribution

GW, YL, DF, and ML conceived the idea, analysis of literature, and writing of the manuscript; GW and FY read the literature and revised the article; XG, YL, and JW read through and corrected the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [Grant no. 81800434], and Grant of Sichuan Province Science and Technology Agency Grant [2022YFS0627].

Availability of data and materials

Not applicable.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest regarding the publication of this article.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.