miR-143 and miR-145: Molecular Keys to Switch the Phenotype of Vascular Smooth Muscle Cells

Advances in Genetics

miR-143 and miR-145Molecular Keys to Switch the Phenotype of Vascular Smooth Muscle Cells

Ashraf Yusuf Rangrez, PhD; Ziad A. Massy, MD, PhD;Valerie Metzinger-Le Meuth, PhD; Laurent Metzinger, PharmD, PhD

Vascular smooth muscle cells (VSMCs) are able toperform both contractile and synthetic functions, which

are associated with changes in morphology, proliferation, andmigration rates and are characterized by the specific expres-sion of different marker proteins. Under normal physiologicalconditions, VSMC rarely proliferate in adult tissues, butundergo major phenotypic changes from the contractile to thesynthetic in response to environmental cues, a phenomenonknown as switching, or phenotypic modulation.1,2 Phenotypicswitching is accompanied by production of abundant cyto-kines, extracellular matrix, and an increased rate of prolifer-ation and migration. Therefore, the transition of VSMCs froma differentiated phenotype to a dedifferentiated state plays acritical role in the pathogenesis of cardiovascular diseasessuch as hypertension, vascular injury, and arteriosclerosis.2,3

However, the molecular mechanisms involved in phenotypicswitching remain elusive.

The last decade has witnessed an exciting discovery thatled to a revolution in our understanding of the extensiveregulatory gene expression networks modulated by small,untranslated RNAs, microRNAs (miRNAs).4 miRNAs com-prise a novel class of endogenous, small RNAs of �20 to 25nucleotides. Although the mature miRNA is very small, it isderived from a transcriptional product of a few hundred to afew thousand nucleotides. This process of maturation isknown as miRNA biogenesis, extensively reviewed by Kim.5

Biogenesis of miR-143 and miR-145 is pictorially presentedin Figure 1. Functionally, miRNAs are noncoding RNAs thatnegatively regulate gene expression. In the current, generallyaccepted model, they act mostly by inducing an inhibition ofthe translation of their target mRNAs, and, in a minority ofcases, via their degradation.6,7 Very recently, however, Bar-tel’s team challenged this view by showing that, in a vastmajority of cases, mammalian microRNAs act by destabiliz-ing their target mRNAs and decreasing their levels.8 Theyfunction as posttranscriptional regulators of mRNA expres-sion by binding to the 3�UTR and repressing translation of thetarget gene.6,7 Note, however, that a few studies have de-

scribed that some miRNAs bind the coding region or 5�UTRof respective target mRNAs.9–14 One single miRNA is able toregulate the expression of multiple genes because it is able tobind to its mRNA targets as either a perfect or imperfectcomplement.6,7 Thus, 1 miRNA can regulate the expressionof multiple target genes. Similarly, 1 mRNA can be regulatedby several miRNAs. It is speculated that the human genomemay encode �1000 miRNAs15 that are abundant in manyhuman cell types. Thus, the process of regulation of mRNAexpression by miRNAs is complex, and explains that thesesmall RNAs may target about 30% to 60% of the mammaliangenes.16

Several miRNAs, including miR-21, miR-221, miR-222,miR-143, and miR-145, have a demonstrated role in VSMCdifferentiation. miR-21 negatively regulates programmed celldeath 4 (PDCD4),17 promoting VSMC differentiation,whereas upregulation of miR-221 and miR-222 promotesVSMC proliferation by targeting the negative regulators ofthe cell cycle, p27 and p57.18,19 Several recent studies havedemonstrated the critical role of miR-143 and miR-145 inVSMC phenotype switching.1,20–26 In this review, we specif-ically focus on the current, state-of-the-art information onmiR-143 and miR-145 (henceforth these 2 miRNAs togetherwill be designated as miR-143/145) and their involvement inphenotypic modulation of VSMCs and cardiovascular dis-eases. Further information about other miRNAs associatedwith VSMCs can be found elsewhere.27–33

miR-143/145 Are Transcribed as a Cluster That IsRegulated by Cardiac Transcriptional FactorsmiR-143 and miR-145 encoding genes are highly conserved(Figure 2), and lie in close proximity with each other onmurine chromosome 18 (�1.4 kilobases [kb]) and humanchromosome 5 (�1.7 kb).1,21,34 Concurrent genomic organi-zation suggests that miR-143/145 are cotranscribed from thesame gene (Figure 1). This fact was indeed validated byRT-PCR, when primers from stem loop sequences of the twomiRNAs were found to be transcribed as a bicistronic

Received October 26, 2010; accepted December 30, 2010.From INSERM-ERI 12 (EA4292) (A.Y.R., Z.A.M., V.M.-L., L.M.), Amiens, France; Faculty of Pharmacy and Medicine (A.Y.R., Z.A.M., L.M.),

University of Picardy Jules Verne, Amiens, France; the Divisions of Pharmacology and Nephrology (Z.A.M.), Amiens University Hospital, Amiens,France; and Universite Paris 13 (V.M.-L.), UFR SMBH, Bobigny, France.

The online-only Data Supplement is available at http://circgenetics.ahajournals.org/cgi/content/full/CIRCGENETICS.110.958702/DC1.Correspondence to Laurent Metzinger, PhD, INSERM-ERI 12, Faculty of Pharmacy and Medicine, University of Picardie Jules Verne, 1 Rue des

Louvels, F-80037, Amiens, France. E-mail [email protected](Circ Cardiovasc Genet. 2011;4:197-205.)© 2011 American Heart Association, Inc.

Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org DOI: 10.1161/CIRCGENETICS.110.958702

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transcript.34 Transcriptional regulatory studies of miR-143/145 revealed that an upstream region of �0.9 kb wassufficient for miR-143/145 cardiac and smooth muscle ex-pression.21 This region consists of highly conserved ciselements representing potential binding sites for transcrip-tional factors, such as serum response factor (SRF) andNkx2–5 (cardiac NK-2 transcription factor). Cordes et al21

showed that both SRF and Nkx2–5 could independentlyactivate the expression of miR-143/145. Myocardin is apotent transcriptional coactivator of SRF35 and is considereda component of a molecular switch for smooth muscledifferentiation.36 Therefore, SRF, in combination with myo-cardin, synergistically and robustly activates miR-143/145expression, whereas Nkx2–5 together with SRF and myocar-din have additive effects.21 In agreement with these results,Xin et al34 also demonstrated that both SRF and myocardinupregulate the expression of miR-143/145 in VSMCs.

miR-143/145 Are Highly Expressed in VSMCsMany recent studies have shown that miR-143/145 are highlyexpressed in VSMCs.1,20–22 In a screen for miRNA expres-

sion in different tissues, Elia et al22 found that miR-143 hashigher expression in heart than in other organs. The expres-sion studies of miR-143/145 in various mouse tissues bynorthern blotting establish that miR-143 is expressed in lung,skeletal muscle, heart, and skin and is most abundant in aortaand fat, where miR-145 is also at its highest expression level.In agreement with this, in situ hybridization of cross sectionsof the adult mouse heart revealed a very strong expression formiR-143/145 in the walls of the aorta and coronary vessels.22

Boettger et al2 performed a series of microarray hybridizationexperiments using various mouse tissues from different de-velopmental stages and found that the expression of miR-143/145 is proportional to the number of VSMCs in all organsstudied.1 One more study validated the abundance of miR-145 in rat carotid arteries and its selective expression inVSMCs and not in endothelial cells.20

Further expression studies at different developmentalstages in transgenic mice discovered that miR-143/145 isinitially expressed in the developing embryonic heart at E8.5to E9.5. During fetal stages (E16.5), the expression of themiR-143 gene gets confined to SMCs of various organs such

Figure 1. Biogenesis of miR-143/145.miR-143 and miR-145 are cotranscribedas a single primary-miRNA transcript,which is further processed by DGCR8(DiGeorge Syndrome Critical Region 8)and drosha to form the respective pre-miRNAs of miR-143 and miR-145. Pre-miRNAs are then exported to the cytosolof VSMCs by a nucleocytoplasmic shuttleprotein, exportin. In the cytoplasm, Dicercleaves the hair-pin of pre-miR-143 andpre-miR-145, yielding their respectiveimperfect miRNA:miRNA*duplexes. ThemiRNA duplex is finally unwinded to yielda mature miR-143/145, which gets incor-porated into the RNA-induced silencingcomplex (RISC) and acts on its mRNAtargets.

Figure 2. Sequence alignment of maturemiRNAs, miR-143, and miR-145, showingthe completely conserved sequences ofboth miRNAs across different speciesfrom mouse to human and other indi-cated species.

198 Circ Cardiovasc Genet April 2011

as the aorta, smaller blood vessels, esophagus, lung, smallintestine, colon, bladder, and umbilical cord. In adult animals,miR-143 expression is present in all VSMCs throughout thebody, including the aorta, heart, and coronary arteries.1,34

In Vitro Studies Postulated the Role ofmiR-143/145 in VSMC FateIndependent pioneering studies by Cheng et al20 and Cordeset al21 described the involvement of miR-143/145 in deter-mining the fate of VSMCs. The former group (Cheng et al)found that overexpression of miR-145 increased the expres-sion of VSMC differentiation marker genes, such as smoothmuscle �-actin (SM �-actin), calponin, and SM-myosinheavy chain (SM-MHC). Accordingly, levels of these markergenes were decreased in VSMCs treated with a miR-145inhibitor in cultured VSMCs. In addition to regulating VSMCdifferentiation markers, miR-145 alone was able to maintainthe differentiated spindle-like shape and inhibited VSMCproliferation. The regulatory effect of miR-145 on VSMCphenotype was further verified by the latter group, when theydemonstrated a strong regulatory effect of miR-145 overmiR-143 on VSMC differentiation.21 They also proved thatmiR-145 was sufficient for transforming multipotent neuralcrest stem cells into VSMCs. It is to be noted that multipotentneural crest stem cells normally populate the aortic smoothmuscle tissue, which is also the site of miR-145 expression.Additionally, both groups showed that myocardin is theregulator of miR-143/145 expression which is also a knownmaster regulator of smooth muscle phenotype. In an indepen-dent experiment, Cordes et al21 demonstrated that miR-145activity is required for myocardin-dependent conversion offibroblasts into VSMCs and that miR-145 strongly potenti-ates the effects of myocardin. These in vitro findings sug-gested a crucial role of miR-143/145 in VSMC phenotypedetermination and were further verified and validated by invivo studies described in later sections.

Knockout Studies in Mouse Provided FurtherInsights Into the Molecular and Mechanistic Rolesof miR-143/145

Phenotypic Observations in miR-143/145 Knockout MiceThree independent groups1,22,34 have generated mouse modelsof miR-143/145 knockout (KO) and shown that the expres-sion of miR-143/145 cluster is essential for VSMCs toacquire the contractile phenotype. The first group replacedthe miR-143/145-coding genomic region with a lacZ reporter,deleting the sequences coding for the mature miR-143 andmiR-145 and a 1.3 kb fragment located between the 2 genes.1

Elia et al22 generated a KO mouse model in which the exonspecifying miR-143 was replaced by lacZ reporter, and theynoticed that the expression of miR-145 was concomitantlydecreased by loss of miR-143. This observation furtherconcurred the cotranscriptional hypothesis for miR-143/145,discussed in earlier section of the review. Finally, the thirdgroup34 generated 3 separate deletion mutant mice lines formiR-143, miR-145, and miR-143/145 by introducing loxPsites for Cre-mediated recombination in the regions flankingthe premiR coding regions of each miRNA through homol-ogous recombination. All 3 groups found that the homozy-

gous miR-143/145 KO mice were viable, fertile, and did notdisplay gross macroscopic alterations, indicating that these 2miRs are dispensable for development.1,22,34 HomozygousKO mice for either miRNA were also viable, indicatingneither miR-143 nor miR-145 is essential for cardiovasculardevelopment in vivo.34 Developmental studies revealed thatmiR-143 locus was active during early stages of heartdevelopment from E8.5 onward but disappeared from cardio-myocytes at E16.5 and became exclusively confined to SMCsof the cardiovascular system, the gastrointestinal tract, blad-der, uterus, and lung.1 Interestingly, the expression of miR-143/145 was similar to that of other smooth muscle genessuch as SM �-actin and SM22�.1,22,37,38

Structural Changes in miR-143/145 KO MiceHistological and electron microscopic studies revealed thatthe structure of the aorta of homozygous miR-143/145 KOmice is different from their wild-type counterpart,22 with asevere reduction in the number of contractile VSMCs andan increase in synthetic VSMCs in the aorta and femoralartery.1 However, the aorta showed less severe phenotypewhen compared with a femoral artery. Both groups havereported that there were no differences in the number ofproliferating or apoptotic VSMCs between control and KOgroups.1,22

A striking observation by Xin et al34 was that the smoothmuscle layers of the aorta and other arteries from miR-145and miR-143/145 KO mice were noticeably thinner thanthose of wild-type or miR-143 KO mice, indicating theprominent and distinct role of miR-145 compared withmiR-143. Further electron microscopic investigations re-vealed that the above observation was due to decreasedactin-based stress fibers in miR-145 and miR-143/145 KOmice, suggesting that these miRNAs modulate actin dy-namics and cytoskeletal assembly. Ultrastructural analysisof miR-143, miR-145, and miR-143/145 KO aorta alsodisclosed that the VSMCs had an increased and dilatedrough endoplasmic reticulum which is typical for synthet-ically active VSMCs.22,34

Functional Comparison Between miR-143/145 KO andControl MiceCell culture studies of VSMCs isolated from wild-type andmiR-143/145 KO mice aorta showed that the VSMCs fromwild-type mice were larger and migrated more than those ofKO mice.22,34 This fact was also confirmed by morphometricmeasurements, which indicated that the VSMCs of miR-143/145 KO mice were smaller when compared with controlanimals.1 Functional analysis of the vascular tone revealedthat miR-143/145 KO mice had statistically significant arte-rial hypotension in comparison with wild-type under steady-state conditions.34 Similar effects, for example, reducedsystolic and diastolic blood pressures, were seen in miR-143/145 KO compared with wild-type mice under anesthesia.1

Heart weight index and ventricular mass measurements sug-gested that the arterial hypotension was associated withreductions in cardiac and left ventricular mass.34 Similarly,miR-143/145 KO mice exhibited reduced systolic bloodpressure in response to angiotensin II (Ang II, a vasopressiveagent) stimulation. In an in vitro experiment using artery

Rangrez et al miR-143 and miR-145 in Vascular Biology 199

explants, there was a nearly complete loss and significantdecrease of the ability of the explants to contract when treatedwith Ang II and phenylephrine, respectively.1

Taken together, structural and functional observations,including reduced size and accumulation of syntheticVSMCs, increased proliferation, protein synthesis, and mi-gration and blunted hypertension response to receptor-mediated signals, suggest that the miR-143/145 cluster isrequired for maintaining VSMC phenotype, for normal con-tractility of arteries, and for controlling blood pressure.

miR-143/145 Act Through Regulating theRegulators Such as Myocardin and SRFWe discussed earlier that the master regulator of smoothmuscle contractile phenotype, SRF, in combination withmyocardin, regulates the miR-143/145 expression. However,one of the most striking observations revealed by Cordes etal21 was that miR-143/145 cooperatively target a network oftranscriptions factors such as myocardin, Kruppel-like factor4 (Klf4), Klf5, and so forth, to promote differentiation andrepress proliferation of VSMCs (Figure 3D). These results

Figure 3. Pictorial representation of the roles of miR-143/145 in VSMCs. Mechanisms of actions of miR-143/145 have been divided into4 sections in the figure, based on various vascular functions. Central oval-shaped structure represents the nucleus and divides the dia-gram in 2 halves. A, Upper left details about miR-143/145 and actin remodeling (adapted from Xin et al34); B, lower left section portraysthe association of miR-143/145 with contractility of VSMCs (adapted from Boettger et al2); C, upper right half describes the involve-ment of miR-143/145 in podosome formation and migration (adapted from Quintavalle et al24); D, lower right section shows the role ofmiR-143/145 in VSMC proliferation and differentiation (adapted from Cordes et al21).


put forth a yet undescribed concept against the generallyaccepted view that miRNAs inhibit protein translation and/ordegrade target mRNA. This shows that miRNAs can self-regulate their expression by altering their transcriptionalregulators via feed-back, feed-forward, or double-negativefeedback mechanisms.21,39 Several targets (Table) and mech-anisms based on respective targets have been proposed andvalidated. We will discuss these mechanisms below in detail.Additionally, we also used dedicated software to detect theoverall predicted targets of miR-143/145 (online-only Sup-plement Table 1). This analysis shows that miR-143/145 havea wide range of targets, such as transcription and translationfactors, receptors, phosphatases, kinases, growth factors,RNA binding proteins, and so forth. These targets areinvolved in different cellular processes apart from VSMCplasticity and represent an example how 1 miRNA canregulate different related or unrelated cellular processes.

miR-143/145 Regulation of VSMC Proliferationand DifferentiationCordes et al21 proposed the model (adapted in Figure 3) inwhich they showed that miR-143/145 are positively regulatedby SRF and myocardin and function to repress multiplefactors that normally promote the less differentiated, moreproliferative smooth muscle phenotype (Figure 3, part D andcentral part). They suggested that miR-145 promotes VSMCdifferentiation in part by increasing myocardin expressionand functioning in feed-forward reinforcement of its own

expression by the SRF-myocardin complex. miR-145 re-presses Klf4, which otherwise interacts with SRF and alsorepresses myocardin,40 and calmodulin kinase II-� (CamkII-�),which was shown to be involved in multiple events includingneointimal proliferation.41,42 miR-143 represses Elk-1 (EtsLiKe gene 1), which competes with myocardin to bind SRFand exhibits an inhibitory effect on smooth muscle differen-tiation.43 Klf4 plays a key role in regulating VSMC pheno-type because it antagonizes proliferation, facilitates migra-tion, and downregulates VSMC differentiation markergenes.44

In another study, versican was identified as a new target formiR-143.45 Versican is a chondroitin sulfate proteoglycan ofthe extracellular matrix, produced by synthetic VSMCs, andpromotes VSMC migration and proliferation. Wang et al45

also demonstrated that myocardin coordinates VSMC differ-entiation by inducing transcription of miR-143, which in turnattenuates the expression of versican. Therefore, combinedand regulatory effects of miR-143/145 and SRF-myocardinare necessary to decide the proliferative or differentiatedphenotype of VSMCs.

miR-143/145 Regulation of Actin RemodelingAnother interesting mechanism of action of miR-143/145 wassuggested by Xin et al,34 based on the prediction andvalidation of multiple targets regulating actin dynamics (Fig-ure 3A). They identified a disproportionate number of thesame targets involved in actin dynamics, cytoskeletal func-tion, and phenotypic switching of VSMCs for both miR-143 andmiR-145. Among these are the actin-dependent SRF coactivatormyocardin-related transcription factor-B (MRTF-B)46,47 andAdducin-3 (ADD3), which caps the barbed ends of actinfilaments and acts as a bridge between the membrane andactin cytoskeleton.48,49 Rho kinase-dependent phosphoryla-tion of ADD3 results in enhanced F-actin binding and cellmotility.50 Sling-shot 2 (Ssh2) phosphatase, another target ofboth miR-143 and miR-145, promotes cell motility andenhances F-actin reorganization by dephosphorylating andactivating cofilin, an actin depolymerizing factor.51,52 miR-145 selectively targets the zinc finger proteins Klf4 and Klf5,which repress SRF activity and can either inhibit or promoteVSMC differentiation or proliferation, depending on thecontext.53–56 Slit-Robo GTPase-activating protein 1 (Srgap1)and Srgap2, also targeted by miR-145, modulate Slit-Robo-dependent repulsive cues and cell migration by inactivatingthe small GTPase, Cdc42 and inhibiting actin polymeriza-tion.57 The preferential targeting of these modulators ofcytoskeletal function by miR-145 may account, at least inpart, for the stronger phenotype (as discussed earlier), result-ing from miR-145 versus miR-143 deletion.34 The cycle ofactin remodeling starts with MRTFs sequestered in thecytoplasm by monomeric actin. On release from actin, MRTFtranslocates to the nucleus and interacts with SRF to activatethe transcription of genes encoding actin and other cytoskel-etal components, as well as miR-143 and miR-145. ThesemiRNAs repress the expression of a collection of regulatorsof actin dynamics and MRTF/SRF activity, thereby creating acomplex set of feedback loops to modulate cytoskeletalassembly and dynamics.

Table. Function-Based Known Targets of miR-143 andmiR-145 Involved in VSMC Fate Determination

Function Target Reference

miR-143 Proliferation anddifferentiation

Ets LiKe gene 1 (Elk1) 21

Versican 45

Podosome formationand migration

Protein kinase C-� 24

Platelet derived growthfactor receptor-�

24

miR-145 Proliferation anddifferentiation

Myocardin 20, 21

Krüppel-like factor 4 (Klf4) 21

Calmodulin kinase II-� 21

Actin remodeling Klf4 34

Krüppel-like factor 5 (Klf5) 34

Slit-Robo GTPase-activatingprotein 1

34

Slit-Robo GTPase-activatingprotein 2

34

Myocardin 34

Contractility Klf5 63

Podosome formationand migration

Fascin 24

Adducin-3 34

miR-143/145

Actin remodeling Myocardin relatedtranscription factor-B

34

Sling-shot 2 34

Contractility Tropomyosin 4 1

Angiotensin-convertingenzyme

1


The mechanism proposed by Xin et al34 has further beenstrengthened by a recent report in which authors havegenerated VSMC-specific deletion of Dicer in mice.27 Theyfound that inactivation of Dicer in VSMC results in impairedactin cytoskeleton and defects in VSM-specific gene expres-sion. Cytoskeletal defects caused by Dicer deletion could bepartially rescued by miR-145 confirming its involvement inthe control of actin dynamics.27

miR-143/145 Regulation of ContractilityBoettger et al1 have identified tropomyosin 4 (TPM-4) andangiotensin-converting enzyme (ACE) as major miR-143/145targets (Figure 3B). TPM-4 is a structural protein that isspecifically upregulated in synthetic VSMCs,58 whereas ACEconverts circulating Ang I into its active form, Ang II.59 AngII is a potent agonist for the contraction of VSMCs and alsoa major regulator of the contractile phenotype ofVSMCs.3,60–62

An interesting observation was the identification of addi-tional changes in the transcript and/or protein expressionlevels of molecules known to influence the VSMC pheno-type. These changes would be the consequences of secondaryevents.1 In principle, many elements of signaling cascadesthat govern contraction and migration of VSMCs werechanged, including a downregulation of angiotensin receptor1 (AT-1) and of molecules that control the trafficking ofplasma membrane receptors such as Caveolin-2 (Cav-2) andCav-3. On the other hand, regulator of G-protein signaling(RGS)-interacting molecule GNB5, as well as components ofthe Rho-signaling cascade (Rock1, Rnd2/3, Cdc42ep3,Arggef17) and molecules that direct calcium handling andsignaling (SERCA, caldesmon-1, Camk2g) of VSMCs, wereupregulated.1 The effect on the Rho signaling cascade was ofparticular significance because it affects nuclear translocationand/or activation of SRF.1,47 Collectively, modulation of ACEand consequently Ang II by miR-143/145 explains, at least inpart, the shift from the contractile to the synthetic phenotypein miR-143/145 KO mice.

Recently, Liu et al63 have documented that Klf5 is involvedin Ang II–induced VSMC proliferation through transactivat-ing cyclin D1 expression. Ang II induced expression andactivation of Klf5 via the ERK and p38 MAPK pathwaystriggered by AT-1.63 Klf5 is one of the targets of miR-145, aknown repressor of myocardin expression and a negativeregulator of VSMC differentiation,20,64 which further comple-ments and illustrates the partial mechanism by which Ang IInegatively regulates the contractile phenotype of VSMCs.

miR-143/145 Regulation of Podosome Formationand MigrationA very recent study has proposed a novel mechanism dem-onstrating the role of miR-143/145 in VSMC migration andpodosome formation.24 Podosomes are important morpholog-ical actin-rich membrane protrusions involved in the migra-tion of several cell types including VSMCs.65,66 Src tyrosinekinase activity promotes podosome formation67,68 and thePKC induction of podosomes is a result of Src activation.69

Platelet-derived growth factor (PDGF) is a known regulatorof VSMC differentiation and migration3 by stimulating the

PDGF-receptor (PDGF-R), which in turn activates Src,70

Protein kinase C (PKC)-�,71 and downstream signal transduc-tion pathways.72 Quintavalle et al24 have shown that miR-143/145 inhibits podosome formation, whereas Src andPDGF reduce expression of miR-143/145. Moreover, theydeduced the underlying pathway demonstrating that in re-sponse to PDGF, Src downregulates miR-143/145 expressionthrough p53 inhibition. These findings also support previousobservations indicating that PDGF can reduce miR-145expression,20 and p53 increases miR-143/145 levels in cancercells.73 PKC-� and PDGF-R� were new targets for miR-143,whereas fascin was identified as a new target for miR-145.Each of these targets were required for podosome formationin VSMCs.24 On the basis of all these results, Quintavalle etal24 have described a new pathway summarized in Figure 3C.They hypothesized the presence of an auto-regulatory loop,initiated by PDGF production in response to vessel injury.This leads to activation of Src, which in turn inhibits p53 andthus represses miR-143/145 expression, leading to upregula-tion and Src phosphorylation of key podosome formingproteins (PKC-�, PDGF-R�, and fascin) and increased ex-pression of PDGF-R, which further boosts signaling. Insummary, this cascade of events promotes migration ofVSMCs and podosome formation.

miR-143/145 Expression Directly Correlates WithImportant Cardiovascular Diseases Such asAtherosclerosis and Coronary Artery DiseaseSeveral in vitro and in vivo studies have suggested a criticalrole for miR-143/145 in maintaining VSMC contractile phe-notype by both inducing VSMC differentiation markers andsuppressing dedifferentiation markers. However, what wasmore interesting was the demonstration of a direct associationof miR-143/145 with cardiovascular disease in human andexperimental animal models.20–23 Initial strong evidencecame from the studies involving mouse ligation-inducedcarotid artery, rat carotid balloon-injured carotid artery andatherosclerotic aortas of ApoE KO mice.20,21 Both studiesdocumented a significant decrease in the expression ofmiR-143/145 in study models described above, comparedwith respective controls. Interestingly, miR-145 expressionwas downregulated to nearly undetectable levels in athero-sclerotic lesions containing neointimal hyperplasia.21 miR-143 and miR-145 were also shown to be downregulatedduring neointimal formation in the rat carotid artery.74 An-other study elucidated that miR-143/145 KO mice developneointimal lesions even in the absence of hyperlipidemia,lipid depositions, and foam cells, which highlights the poten-tial role of miR-143/145 and VSMCs in the pathophysiolog-ical process leading to atherosclerosis.1 In an independentexperiment, Elia et al22 reported similar effects by anotherexperiment where they generated a traverse aortic constric-tion (TAC) in wild-type mice and found that the expression ofmiR-143/145 was dramatically reduced in aortas upstreamand downstream of the TAC site.

Elia et al22 have also evaluated the expression levels ofmiR-143/145 in aorta obtained from patients having an aorticaneurism. As expected, they found a significant decrease inexpression of miR-143/145 in patients when compared with a


control group.22 This result strongly suggests that the expres-sion of miR-143/145 can be downregulated in human vascu-lar diseases. Further strengthening this observation, a recentstudy reported a significant reduction of miR-145 in the bloodof patients with coronary artery disease (CAD) when com-pared with healthy individuals.23 They concluded that thecirculating levels of several vascular miRNAs are signifi-cantly downregulated in patients with CAD. However,whether downregulation of miR-143/145 is the primary causeof aortic aneurism and/or CAD or it is a secondary event dueto the disease remains to be established. Notwithstanding,quantifying the expression of miRNAs such as miR-143/145could be a useful tool for the diagnosis and evaluation ofCAD and other vascular diseases, as will be discussed infurther details below.23,75

Therapeutic Potential and Future PerspectivesAll the discussion thus far leads to the conclusion thatmiR-143/145 is an important molecular key required toswitch the VSMC phenotype. Initial studies have demon-strated the importance of these small RNAs in determiningthe fate of VSMCs, whereas recent studies substantiated theirassociation with human vascular diseases such as aorticaneurism, atherosclerosis, restenosis, and CAD. VSMCs havethe ability to undergo profound phenotypic changes in re-sponse to changes in their extracellular environment, asoccurs in vascular diseases such as atherosclerosis, restenosis,aortic aneurism, and CAD. These changes often includededifferentiation, enhanced migration, proliferation, and apo-ptosis of VSMCs. Several recent studies have established anegative correlation between the expression of miR-143/145and the dedifferentiation of VSMCs. Therefore, downregula-tion of miR-143/145 induced by pathological stimuli in thevascular diseases cited above facilitates VSMCs dedifferen-tiation. This fact indicates that modulating the expression ofmiR-143/145 can potentially be used for the treatment ofvascular diseases. One should, however, keep in mind thatmodulating the phenotype of VSMCs by using miRNA couldprove to be beneficial in some vascular disease states, inwhich phenotype switching may be helpful, such as aneurysmof the abdominal aorta, and harmful in others, such aspostangioplasty restenosis.76

In these lines, Cheng et al20 have shown that the restorationof the downregulated miR-145 is sufficient to inhibit theneointimal lesion formation in rat carotid arteries after angio-plasty. Similar results were obtained where balloon-injury–induced neointimal formation was significantly reduced whenthe carotid artery was locally perfused with adenovirusexpressing either miR-143 or miR-145.22 Several other ther-apeutic approaches and modes of delivery are available formiRNA-based treatment. They include chemically modifiedmimics of miRNAs, vector-based delivery of miRNAs, anti-sense oligonucleotides, miR-masks, miR-sponges, and soforth, and are summarized in recent reviews.77–79 Importantly,one has to be cautious when considering miRNA basedtherapy due to the involvement of a single miRNA in morethan 1 physiological process, because 1 miRNA can controlmultiple related or unrelated targets. However, as stated, thisaspect may also be advantageous because targeting multiple

genes with a single mimetic could modulate complex pro-cesses such as neovascularization, atherosclerosis, and simi-lar vascular or other physiological processes.28

SummaryUntil recently, miRNAs, which are cytoplasmic components,have been quantified in healthy/pathological tissues, a proce-dure that usually involves a surgical act. A recent clinicalbreakthrough occurred when miRNAs were detected in cir-culating body fluids together with other types of noncodingRNAs, DNAs, and mRNAs.80,81 They are detected in serumand EDTA-treated plasma, are very stable, can stand repeti-tive freeze-thaw cycles and are protected from RNases.23

Moreover, a growing number of studies have shown that thelevels of these circulating miRNAs vary significantly inpathological conditions such as various cancers,82 myocardialinfarction,83,84 diabetes,85 and so forth. An interesting articleby Gupta et al86 provides the current knowledge aboutcirculating miRNAs during CAD, myocardial infarction, andheart failure. Recently, Fichtlscherer et al23 performedmiRNA profiles using RNA isolated from the blood ofhealthy volunteers and patients with stable CAD. They foundthat the expression of several miRNAs was significantlyaltered in patients. In the same study, they validated theirresults by quantitative PCR in 2 independent cohorts ofpatients and showed that the expression of miR-145, amongother miRNAs, was significantly reduced in patients withCAD compared with healthy control patients. Thus, quanti-fying total blood levels of miR 143/145 and other experimen-tally validated miRNA candidates may provide a specificsignature that could be an independent predictor of diagnosis,prognosis and/or etiology of CADs, keeping in mind that thisconcept will need comprehensive investigations to validatethe theory.

As a consequence, apart from being considered as potentialtherapeutic targets and the feasibility of these strategiesbecome routinely available, we believe that the relationshipbetween circulating miR-143/145 levels and CAD may atpresent serve as novel markers for diagnosis and prognosis ofCAD and other vascular diseases.

AcknowledgmentsWe thank Kumari Manju and Bengrine Abderrahmane for criticalreading and useful suggestions.

Sources of FundingThis work was supported by funds from Region Picardie.

DisclosuresNone.

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KEY WORDS: biomarker � gene regulation � microRNA � vascularcalcification


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miR-143 and miR-145: Molecular Keys to Switch the Phenotype of Vascular Smooth Muscle Cells