16 Attenuates Vascular Neointima Formation by Promoting the Contractile Phenotype of Vascular Smooth Muscle Through Targeting YAP.

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Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2016 October 01. Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 October ; 35(10): 2145–2152. doi:10.1161/ATVBAHA.115.305748.

MicroRNA-15b/16 attenuates vascular neointima formation by promoting the contractile phenotype of vascular smooth muscle through targeting YAP Fei Xu1, Abu Shufian Ishtiaq Ahmed2, Xiuhua Kang1,2, Guoqing Hu2, Fang Liu2, Wei Zhang1, and Jiliang Zhou2,#

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1Department

of Respiratory Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, China 2Department

of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912

Abstract Objective—To investigate the functional role of the miR-15b/16 in vascular smooth muscle phenotypic modulation.

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Approach and Results—We found that miR-15b/16 is the one of most abundant microRNAs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs convert to a synthetic phenotype miR-15b/16 expression is significantly reduced. Knocking-down endogenous miR-15b/16 in VSMCs attenuates smooth muscle-specific gene expression but promotes VSMC proliferation and migration. Conversely, over-expression of miR-15b/16 promotes smooth muscle contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, over-expression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced smooth muscle de-differentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP’s 3′-untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in up-regulation of YAP expression.

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Conclusions—These results indicate that miR-15b/16 plays a critical role in smooth muscle phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases. Keywords microRNA; YAP; smooth muscle; phenotypic modulation; neointima formation

#

Corresponding author: Jiliang Zhou, MD/PhD, Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, CB-3628 (Office); CB-3606 (Lab), 1459 Laney Walker Blvd, Augusta, GA 30912, Phone: 706-721-7582 (Office); 706-721-7583 (Lab); Fax: 706-721-2347, [email protected]. Disclosures None.

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Introduction

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Vascular smooth muscle cells (VSMCs) comprise the majority of the wall of blood vessels. The main function of VSMCs is to regulate the caliber of blood vessels by involuntary vasoconstriction or vasodilation thereby regulating blood flow and pressure. Mature VSMCs are quiescent and are geared for contraction through expressing a unique set of contractile proteins including smooth muscle α-actin, smooth muscle myosin heavy chain, 130-kDa myosin light chain kinase, Hic-5 1, calponin and SM22α 2. However, unlike striated muscles, VSMCs exhibit an extraordinary phenotype plasticity where, in response to diverse environmental cues, the cells may switch from a contractile phenotype to a “synthetic” phenotype that is characterized by increased proliferation and motility and decreased expression of the contractile proteins 2. Several serious pathological conditions in humans, such as intimal hyperplasia associated with restenosis are largely dependent upon VSMC phenotype modulation contributing to progression of intimal lesions that lead to vessel occlusion 2. Although a number of signaling pathways or factors have been reported to play roles in smooth muscle phenotypic switching, the mechanisms that control VSMC plasticity are still not fully understood.

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MicroRNAs (miRs) are a class of small non-coding RNAs composed of 18–25 bp nucleotides 3. Using the “seed” binding sequence, miRs usually bind to the complementary 3′ untranslated regions (3′-UTRs) of the messenger RNA (mRNA) transcripts, thereby inhibiting gene expression through inducing translational repression or mRNA degradation3. The “canonical” miR-16 family shares the same “seed” binding sequence AGCAGC therefore the members of miR-16 family likely have functional redundancy 4. As defined by the sequence similarity, members of the miR-16 family include miR-15 (a and b), miR-16 (1 and 2), miR-195, miR-424, and miR-497 4. Among them, miR-15 and miR-16 are cotranscribed from one of two different intragenic loci, miR-15a/-16-1 and miR-15b/-16-2 that are highly conserved among mammalian species 5. Although previous studies have demonstrated that miR-15/16 clusters act as tumor suppressors 6 and play a critical role in the control of cardiomyocyte proliferation and heart regeneration 7, 8, the functional role of miR-15/16 in VSMCs is unknown.

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The Hippo signaling is an evolutionally conversed pathway that controls organ size and tumorigenesis 9. YAP (yes-associated protein) is a major down-stream effector of this signaling pathway that is inactivated by phosphorylation by its upstream Lats (large tumor suppressor) kinases 9. YAP is over-expressed in a spectrum of human primary tumors and is a potent oncoprotein through its ability to induce expression of genes involved cell cycle progression 9. Recently, several studies showed that the Hippo-YAP pathway plays a critical role in mouse cardiovascular development 10, 11 and vascular disease 12, 13. In particular, our recent study showed that YAP expression is induced during smooth muscle phenotypic modulation and this increased expression of YAP inhibits smooth muscle-specific gene expression while promotes smooth muscle proliferation and migration in vitro and in vivo 13. However, the mechanisms through which YAP is up-regulated during smooth muscle phenotypic switching remains elusive.

Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2016 October 01.

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In this study, we demonstrate that miR-15b/16 promotes VSMC contractile phenotype, at least in part, by targeting YAP and therefore restoring expression of miR-15/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases.

Materials and Methods Rat aortic SMCs were prepared as previously reported 1. Rat carotid artery balloon injury was performed as described in our previous reports 1, 12–14. Full materials and methods are available in the online-only Data Supplement.

Results Expression of miR-15b/16 is down-regulated during smooth muscle phenotypic switching from a contractile to synthetic state in vitro and in vivo

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In order to explore the potential function of the miR-16 family miRs in VSMCs, we first examined their expression in vascular tissue and primary VSMCs. By qRT-PCR we found miR-16 highly expressed in the rat thoracic aorta and was approximately 20% of miR-145, the most abundant miR that was previously identified in the vascular tissue 15–17 (Online Figure IA). Moreover, in both rat primary aortic SMCs and human coronary artery SMCs (HCASMCs), miR-16 and its co-transcribed family member, miR-15b are the most abundant miRs among the miR-16 family (Online Figure I B and C). Compared to endothelial cells and macrophages in mouse, primary VSMCs have highest expression level of miR-15b and miR-16 (Online Figure I D). We therefore first examined the expression of miR-15b/16 during smooth muscle phenotypic switching in vitro and in vivo. In culture, rat aortic tissue undergoes a dramatic switch toward a synthetic proliferative phenotype 1, 18, that was associated with significantly decreased expression of miR-15b/16 along with smooth muscle contractile phenotype marker Hic-5 1 (Figure 1A). In response to PDGF-BB (plateletderived growth factor BB), a growth factor known to further inhibit smooth muscle contractile protein expression while promoting VSMC proliferation and migration 19, miR-15b/16 expression was significantly down-regulated, whereas miR-221 was significantly induced as previously reported 20 (Figure 1B). Consistent with the in vitro data we found that miR-15b/16 was significantly reduced in the balloon injured rat carotid arteries, a model resembling angioplasty in humans that promotes VSMC proliferation and migration while reducing expression of smooth muscle differentiation markers (Figure 1C) 21. Taken together, these data demonstrate that expression of miR-15b/16 is attenuated in phenotypically modulated synthetic VSMCs in vitro and in vivo. MiR-15b/16 attenuates VSMC proliferation and migration

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Proliferation and migration of VSMCs are key events for the development of neointima thickening. Since miR-15b/16 was reduced in phenotypically modulated VSMCs and these cells are known to be proliferative and migratory, we directly examined the effects of miR-15b/16 on VSMC proliferation and migration. Using WST-1 assays we found that over-expression of miR-15b/16 (approximately 15-fold, Online Figure IIA) significantly impaired VSMC proliferation in a variety of cell culture media (Figure 2A). Cell cycle analysis further demonstrated that miR-15b/16 over-expression resulted in an accumulation


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of cells in the G0/G1 phase and fewer cells in S phase (Figure 2B), indicating a cell cycle arrest in G0/G1. This cell cycle retardation resulted in a significant decrease in cell growth (Figure 2C) of rat VSMCs and HCASMCs (Figure 2D). To test the functional role of endogenous miR-15b/16 on VSMC proliferation, we utilized anti-sense oligonucleotides to knock-down endogenous miR-15b/16. qRT-PCR revealed that the endogenous miR-15b/16 was almost completely depleted after transfection with anti-sense oligonucleotide (Online Figure IIB). Silencing miR-15b/16 resulted in enhanced rates of VSMC proliferation and growth (Figure 2E and F). Moreover, over-expression of miR-15b/16 in rat aortic primary SMCs also significantly attenuated VSMC migration as assessed by Boyden chamber assays (Figure 2G). In contrast, silencing endogenous miR-15b/16 promoted VSMC migration (Figure 2H). Together, these data demonstrate that miR-15b/16 is a potent inhibitor for VSMC proliferation and migration.

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MiR-15b/16 promotes the smooth muscle contractile phenotype by targeting YAP


Since miR-15b/16 was down-regulated in tandem with the reduction of smooth musclespecific markers during smooth muscle phenotypic switching (Figure 1), we next determined the role of miR-15b/16 on smooth muscle differentiation. Over-expression of miR-15b/16 in rat primary aortic SMCs promoted the expression of smooth muscle contractile genes including SM22α, Hic-5, SM α-actin and calponin whereas expression of proliferative markers such as PCNA and cyclin D1 were decreased (Figure 3A). Similar results can be obtained in HCASMCs (Figure 3B). Recently we showed that the expression of YAP is induced in all of in vitro and in vivo smooth muscle phenotypic modulation models as described in figure 1 13. Furthermore, we found the induced expression of YAP plays an integrated role in inhibiting smooth muscle-specific gene expression while promoting VSMC migration and proliferation 13. Since the expression of miR-15b/16 and YAP is negatively correlated and their functions exert opposite effects in VSMCs, we postulated that YAP may be a potential target of miR-15b/16. Consistent with this idea, over-expression of miR-15b/16 attenuated YAP protein expression without affecting expression of the YAP associated protein, TEAD1 (Figure 3A). The inhibition of YAP by miR-15b/16 is through blocking YAP translation as YAP mRNA levels were unaltered (Figure 3C). In contrast, a known miR-16 target, cyclin D1 22 appeared to be regulated through altered mRNA stability (Figure 3A and B). Furthermore, silencing endogenous miR-15b/16 induced YAP expression, inhibited expression of smooth muscle-specific differentiation genes and up-regulated cycline D1 expression (Figure 3D). Data from these gain/loss-of-function assays suggest that YAP is a potential target of miR-15b/16. Targetscan.org and microRNA.org prediction algorithms indicated that the 3′-UTR of the human YAP gene harbors a putative consensus site for miR-15b/16 and this binding site is evolutionarily conserved among vertebrates (Figure 3E). To experimentally validate YAP as a miR-15b/16 target gene, we first generated luciferase reporters containing the YAP 3′UTR region harboring the wild-type or mutated putative miR-15b/16 binding site and then transient transfection experiments were performed. Data from these experiments revealed that luciferase activity of the wild-type YAP 3′-UTR luciferase reporter was significantly decreased in a dose-dependent manner in the presence of miR-15b/16, whereas luciferase activity of constructs in which the miR-15b/16 binding site was mutated were unaffected by miR-15b/16 over-expression (Figure 3F). To further determine the role of YAP in the


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miR-15b/16 mediated inhibition of VSMC growth, adenovirus encoding miR-15b/16 was co-transduced into rat primary aortic SMCs with or without YAP adenovirus which lacks its 3′-UTR, and then the SMC growth was evaluated by WST-1 assays and cell number counting. Data from these experiments revealed that in the presence of YAP lacking its 3′UTR, the miR-15b/16 mediated VSMC growth inhibition was significantly attenuated (Figure 3G and H), suggesting YAP, as a down-stream target of miR-15b/16 was involved in miR-15b/16-mediated effects on VSMC proliferation. Taken together, these data indicate that miR-15b/16 promotes smooth muscle contractile phenotype through directly targeting YAP. Restoring the expression of miR-15b/16 attenuates arterial injury-induced neointima formation in the rat carotid artery balloon injury model

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Given that miR-15b/16 strongly inhibits VSMC proliferation and migration while promoting SMC contractile gene expression through targeting YAP in vitro (Figure 2 and 3), we next tested miR-15b/16 function in vivo in the rat carotid artery balloon injury model. Following the protocol we recently established for the local viral infusion in the balloon injured carotid artery 13, we first confirmed that local delivery of miR-15b/16 adenovirus increased the expression of miR-15b/16 2–4 folds compared to the control RCA (right carotid artery) (Figure 4A). Moreover, over-expression of miR-15b/16 in vivo significantly inhibited neointima formation as measured by decreased neointima/media ratio and reduced neointima area as compared to control GFP virus transduced carotid arteries (Figure 4B, C and D). These data demonstrate that restoring the expression of miR-15b/16 attenuates arterial injury-induced neointima formation in the rat carotid artery balloon injury model. Over-expression of miR-15b/16 promotes VSMC contractile phenotype in vivo


Previously we have shown that the expression of YAP was significantly induced in rat balloon injury model and blocking the induction of YAP significantly up-regulated smooth muscle marker expression and decreased SMC proliferation thereby attenuating neotima formation after arterial injury 13. Since over-expression of miR-15b/16 in vivo attenuated neointima formation following arterial injury (Figure 4) and YAP was identified as a direct target of miR-15b/16 (Figure 3), we sought to determine if the beneficial effects of miR-15b/16 were associated with decreased YAP expression in vivo. Western blotting of proteins isolated from either GFP control or miR-15b/16 virus treated vessels 14 days following balloon injury revealed that exogenous over-expression of miR-15/16 impeded the arterial injury-induced YAP expression and significantly alleviated injury-induced downregulation of smooth muscle-specific differentiation genes (Figure 5A and B). Furthermore, over-expression of miR-15b/16 significantly decreased neointima VSMC proliferation by 40% as indicated by Ki-67 staining (Figure 5C and D). Taken together, these data demonstrate that miR-15b/16 promotes the VSMC contractile phenotype by targeting YAP thereby attenuating arterial-injury induced neointima formation (Figure 5E).

Discussion This study provides the first evidence demonstrating a novel role for miR-15b/16 in targeting YAP to regulate the phenotype of VSMCs. We found that miR-15b/16 was highly


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expressed in contractile VSMCs and was down-regulated in phenotypically modified VSMCs concomitant with increased YAP expression (Online Figure 1 and Figure 1) 13. We identified YAP as a direct down-stream target of miR-15b/16 (Figure 3E and F). Because miR-15b and miR-16 share the identical seed sequence to bind to YAP 3′UTR, it is conceivable that individual miR-15b and miR-16 have similar effects on YAP 3′UTR reporter activity. Furthermore, we found that over-expression of miR-15/16 significantly represses YAP expression in vitro and in vivo (Figure 3A, B and Figure 5A). Conversely, depletion of endogenous miR-15b/16 resulted in an up-regulation of YAP expression and decreased expression of contractile proteins (Figure 3D). Therefore, our study suggests that the down-regulation of miR-15b/16 in response to vascular injury, alleviates its repression of YAP, in vitro and in vivo permitting YAP to drive VSMC from a contractile phenotype toward a synthetic proliferative phenotype. In further support of the negative relationship between miR-15b/16 and YAP expression, previous studies have shown that miR-16 family members are drastically up-regulated 8 whereas the expression of YAP is significantly down-regulated during the arrest of cardiomyocyte proliferation in the postnatal mouse heart 23. Similarly, the expression of a subset of miR-16 family miRs is strikingly upregulated during the maturation of mouse aorta when the VSMCs transit from a proliferative to a quiescent phenotype 24.


In addition to directly targeting YAP it is likely that miR-15b/16 modulates VSMC proliferation by also targeting YAP regulated genes that control cell cycle. The miR-16 family has been proposed to be a set of “master” inhibitors for cell proliferation by silencing many overlapping target genes involved in the cell cycle progression, especially G1-S transition, including cyclin D1, cyclin E1, and cdc25A 22, 25. Furthermore, several other cell cycle genes were revealed to be regulated by miR-16 family, including cyclin D2, cyclin D3, cyclin-dependent kinase 6, check point kinase 1, cdc2, Birc5, cdc42 4. YAP activates a spectrum of genes involving in cell cycle that significantly overlap with the miR-16 family targets including cyclin D1, cyclin E1, check point kinase 1, cdc2 and Birc5 8, 23, 26, 27. Among these, cyclin D1 was initially identified as a bona fide target gene of YAP 26 and can be targeted by miR-16 to both promote mRNA degradation and inhibit protein translation 22. Previous study has shown that increased cyclin D1 expression was sufficient to mediate the injury-induced neointima formation by promoting VSMC proliferation 28. Moreover, we have shown that YAP can induce cultured VSMC proliferation in vitro through induction of cyclin D1 13. Together, these findings suggest that the inhibitory effects of over-expressed miR-15b/16 on neotima formation in vivo likely result from a combination of its ability to target both YAP and cyclin D1. Therefore over-expression of miR-15b/16 would have more potent inhibitory effects on smooth muscle cell proliferation than silencing YAP alone since miR-15b/16 may target additional cell cycle regulators other than the shared target cyclin D1 with YAP. Although miR-195, another member of the miR-16 family has been reported to inhibit VSMC proliferation 29 we found that expression of miR-195 is very low compared to miR-15b/16 in VSMCs (Online Figure I). In this study we provide a novel mechanistic insight into the post-transcriptional regulation of YAP expression in VSMCs by miR-15b/16. Since YAP protein is over-expressed in an array of tumors in humans and in injured vessels, studying the regulation of endogenous


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YAP expression is of great clinical importance. Initial studies have concentrated on investigating YAP phosphorylation, degradation, and nuclear localization 9. However, emerging evidences suggest that the expression of YAP can be regulated at both transcriptional and post-transcriptional levels. For example, c-Jun 30, β-catenin 31, CREB (cAMP response element-binding protein) 32 and the Ets family member GA-binding protein GABP 33 can regulate YAP gene transcription. Recently, the activating transcription factor, ATF-4, has also been shown to promote YAP transcriptional induction in the cells experiencing endoplasmic reticulum stress thereby promoting cell survival 34. YAP expression is known to be regulated post-transcriptionally by several miRs. During preparation of the manuscript, we noticed that miR-15a/16 targeted YAP to inhibit cell proliferation and migration in gastric adenocarcinoma cells 35. In addition to miR-16 family members, YAP can be regulated by miR-375 in liver 36, lung 37, pancreatic 38 and thyroid cancer cells 39. Additionally, miR-141, 200a and 506 have been implicated in regulating YAP expression in esophageal squamous cell carcinoma 40, breast 41 and hepatoma cancer cells 42, respectively. A recent report also demonstrated that YAP can be regulated at the epigenetic level 43. Together, these studies suggest that regulation of YAP gene expression is complex and is developmental stage, cell and tissue-context dependent. Interestingly, in this study we found over-expression of miR-15b/16 specifically down-regulates YAP protein expression without affecting YAP mRNA expression level (Figure 3A, B and C), suggesting the inhibitory effects of miR-15b/16 is through suppressing the translation of YAP. However, we previously reported that YAP protein and mRNA levels are both increased after vascular injury 13, suggesting that miR-15b/16 mediated decrease in YAP protein expression are only a part of mechanism accounting for increased YAP expression following vascular injury. Given that ATF-4 promotes YAP expression 34 and ATF-4 is induced in VSMCs after arterial injury to mediate neointima thickening 44, it is likely that ATF-4 plays a role in the transcriptional up-regulation of YAP in VSMCs following injury. Additional studies are need to confirm this possibility. Restoring expression of miR-15b/16 following balloon injury significantly attenuated neointima formation (Figure 4 and 5). This data together with strong effects of miR-15b/16 on promoting vascular SMC contractile phenotype suggest that over-expression of miR-15b/16 family may be an attractive therapeutic strategy for treating proliferative VSMC related diseases such as atherosclerosis and restenosis. However, the potent ability of miR-16 family to inhibit cell proliferation may be a “double-edged sword” since it also affects endothelial cell proliferation and migration that is involved in angiogenesis and reendothelialization 45, 46. Therefore, any therapies aimed at targeting miR-16 family in the cardiovascular system are required to be cell type-specifically targeted.

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In summary, this study identifies miR-15b/16 as a critical modulator in promoting VSMC contractile phenotype by targeting, at least in part, YAP. Therefore, restoring miR-15b/16 expression in the VSMCs would be an attractive therapeutic approach for treatment of VSMC-related occlusive vascular diseases.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.


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Acknowledgments We thank Dr. Paul Herring for a critical reading of the manuscript. Sources of funding The project described was supported by a grant (R01HL109605) from the National Heart, Lung, and Blood Institute, NIH to J. Z.. F. X., X. K. and W. Z. were supported by a fund from Chinese National Key Specialty Program of Respiratory Medicine.

Abbreviations


VSMCs

vascular smooth muscle cells

SM

smooth muscle

MLCK

myosin light chain kinase

SM MHC

smooth muscle myosin heavy chain

microRNA

miR

3′-UTR

3′ untranslated region

mRNA

messenger RNA

YAP

yes-associated protein

Lats

large tumor suppressor kinase

qRT-PCR

quantitative reverse transcription PCR

PDGF-BB

platelet-derived growth factor BB

TEAD1

TEA domain family member 1

GFP

green fluorescent protein

LCA

left carotid artery

RCA

right carotid artery

H&E staining

hematoxylin and eosin staining

HCASMCs

human coronary artery smooth muscle cells

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Significance

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Phenotypically switched VSMCs are the major contributor to the neointima formation after arterial injury. Previously we found that YAP is induced and plays a crucial role in the phenotypic switch of VSMCs in response to arterial injury. However, the mechanism by which YAP was up-regulated during VSMC phenotypic switching is unknown. In this study, we demonstrate that, YAP is a direct target of miR-15b/16. We find that expression of miR-15b/16 is significantly reduced in concomitant with the up-regulation of YAP following arterial injury. Over-expression of miR-15b/16, mimics the effects of silencing YAP and promotes the smooth muscle contractile phenotype thereby attenuating neointima formation after arterial injury in vivo. Our study not only provides a novel mechanistic insight into the regulation of YAP expression at the posttranscriptional level, but also suggests restoring miR-15b/16 expression in VSMCs would be a potential therapeutic strategy for treatment of occlusive vascular diseases.

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Figure 1. Expression of miR-15b/16 is reduced during smooth muscle phenotypic switching from contractile to synthetic phenotype

A. qRT-PCR was performed to evaluate expression of miR15b/16 or smooth muscle contractile marker gene Hic-5 using the samples either from the freshly isolated rat thoracic aorta or rat thoracic aorta cultured in 20% FBS medium for 3 days. N=4. Culture vs Tissue, *p

Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation.

Mindin regulates vascular smooth muscle cell phenotype and prevents neointima formation.

Caveolin-3 Promotes a Vascular Smooth Muscle Contractile Phenotype.

Vascular smooth muscle contractile elements. Cellular regulation.

Mixed-lineage kinase 3 deficiency promotes neointima formation through increased activation of the RhoA pathway in vascular smooth muscle cells.

Genistein suppresses leptin-induced proliferation and migration of vascular smooth muscle cells and neointima formation.

PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Defective autophagy in vascular smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis.

Interferon regulatory factor 7 protects against vascular smooth muscle cell proliferation and neointima formation.

Nicotine exposure alters human vascular smooth muscle cell phenotype from a contractile to a synthetic type.

Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

Endoglin Mediates Vascular Maturation by Promoting Vascular Smooth Muscle Cell Migration and Spreading.

LincRNA-p21 regulates neointima formation, vascular smooth muscle cell proliferation, apoptosis, and atherosclerosis by enhancing p53 activity.

Puerarin attenuates calcification of vascular smooth muscle cells.

Telomerase Inhibition by Everolimus Suppresses Smooth Muscle Cell Proliferation and Neointima Formation Through Epigenetic Gene Silencing.

Dysfunction of vascular smooth muscle and vascular remodeling by simvastatin.

Wnt16 attenuates TGFβ-induced chondrogenic transformation in vascular smooth muscle.

The novel mineralocorticoid receptor antagonist finerenone attenuates neointima formation after vascular injury.

Syndecan-1 regulates vascular smooth muscle cell phenotype.

Chronic kidney disease alters vascular smooth muscle cell phenotype.

Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ.

New endoplasmic reticulum stress regulator, Gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury.

Vascular smooth muscle hyperplasia underlies the formation of glomeruloid vascular structures of glioblastoma multiforme.

Vascular Smooth Muscle Cells.