SPOTLIGHT REVIEW
Cardiovascular Research (2012) 95, 233–240
doi:10.1093/cvr/cvs141
Wnt signalling in smooth muscle cells and its role
in cardiovascular disorders
Carina Mill and Sarah Jane George*
Bristol Heart Institute, School of Clinical Sciences, Research Floor Level 7, Bristol Royal Infirmary, Upper Maudlin St, Bristol BS2 8HW, UK
Received 1 November 2011; revised 28 March 2012; accepted 2 April 2012; online publish-ahead-of-print 4 April 2012
Abstract
Vascular smooth muscle cells (SMCs) are the major cell type within blood vessels. SMCs exhibit low rates of proliferation, migration, and apoptosis in normal blood vessels. However, increased SMC proliferation, migration, and
apoptosis rates radically alter the composition and structure of the blood vessel wall and contribute to cardiovascular
diseases, such as atherosclerosis, and restenosis that occur after coronary artery vein grafting and stent implantation.
Consequently, therapies that modulate SMC proliferation, migration, and apoptosis may be useful for treating cardiovascular diseases. The family of Wnt proteins, which were first identified in the wingless drosophila, has a wellestablished role in embryogenesis and development. It is now emerging that Wnt proteins also regulate SMC
proliferation, migration, and survival. In this review article, we discuss recently emerging research that has revealed
that Wnt proteins are important regulators of SMC behaviour via activation of b-catenin-dependent and b-cateninindependent Wnt signalling pathways.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Vascular smooth muscle † Proliferation † Migration † Apoptosis † Wnt
----------------------------------------------------------------------------------------------------------------------------------------------------------This article is part of the Spotlight Issue on: Smooth Muscle Cells and Vascular Diseases
1. Role of SMCs in cardiovascular
disease
Cardiovascular diseases (CVD) are the leading cause of mortality in
Western countries, accounting for over 16 million deaths per year.1
Atherosclerosis is the pathology behind the development of CVD including myocardial infarction, angina, and stroke, and it is caused by
the thickening of the arterial wall leading to reduced blood flow
and ischaemia.2
The coronary arteries are composed of three layers: tunica intima,
tunica media, and tunica adventitia. The smooth muscle cells (SMCs)
reside in the medial layer and exhibit a contractile phenotype with
low proliferative rates;3 however, during atherosclerosis endothelial
dysfunction and vessel wall injury results in activation and dedifferentiation of arterial SMCs (synthetic phenotype).4,5 This phenotypic switch permits arterial SMC migration into the intima where
they proliferate and deposit extracellular matrix (ECM).5 The resultant
thickened intima is the soil for the progression of the atherosclerotic
plaque.6 Intimal thickening, occurring in 30 –50% of patients, is also
an undesirable injury response after balloon angioplasty, bypass vein
grafting, and stent implantation.3
Paradoxically, fibrous cap SMC migration and proliferation is considered beneficial at the later stages of atherosclerosis, as it is
important for the formation of the fibrous cap (a structure composed
primarily of SMCs and ECM but also inflammatory cells such as
macrophages and lymphocytes, and foam cells) that is responsible
for plaque rupture resistance. Conversely, reduced ECM synthesis
leading to decreased tensile strength of the fibrous cap occurs as a
result of fibrous cap SMC apoptosis in unstable plaques.7 In normal
arteries, SMC apoptosis is rare; however, increased apoptosis
(1%) occurs in unstable atherosclerotic plaques.8 Arterial SMC
apoptosis is an important modulator of aneurysm formation,9 a
small but significant (2%) cause of sudden death in the UK.10
In summary, SMCs play a significant role in the formation and
stability of the atherosclerotic plaque and in intimal thickening,
which leads to restenosis after treatments of atherosclerosis. Consequently, a clear understanding of the mechanisms underlying SMC
migration, proliferation, and apoptosis may be useful for devising
new improved therapies for atherosclerosis and restenosis. Wnt
proteins regulate proliferation, migration, and apoptosis of numerous cells types and therefore, if expressed during atherosclerosis
and restenosis, are likely mediators of the cellular processes associated with these cardiovascular disorders. This review will focus
on the regulation of SMC behaviour by Wnt proteins with particular translation to their functional significance in atherosclerosis and
restenosis.
* Corresponding author. Tel: +44 117 3423154; fax: +44 117 3423581, Email: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: [email protected].
234
C. Mill and S.J. George
2. Introduction to Wnt signalling
The vertebrate Wnt family is a highly conserved group of 19 genes
that encode for cysteine-rich-secreted glycoproteins that act as important extracellular signalling ligands. Wnt genes were first identified
in mutant wingless Drosophila melanogaster in 1978 and were thus
termed Wingless genes.11 Subsequently, a similar sequence homology
was found in the Int-1 gene of vertebrates and the two terms were
eventually combined in 1991 to form the nomenclature ‘Wnt’.12
The Wnt signalling pathway is highly conserved in eukaryotes and is
essential for embryogenesis and development, where it regulates
diverse processes including cell proliferation, differentiation, cell polarity, and migration. Additionally, post-natal Wnt signalling regulates
numerous biological processes involved in cancer and neurological,
metabolic, and inflammatory disorders.13
Wnt ligands fine-tune cellular behaviour through intricate signalling
pathways involving their interaction with transmembrane-spanning
Frizzled receptors, of which 10 have been classified, as well as specific
co-receptors. The respective Wnt proteins, Frizzled receptors, and
co-receptors are differentially expressed among cells in a spatiotemporal manner to allow the highly specific and elaborate regulation
of both embryonic development and disease progression. The importance and complexity of Wnt signalling is revealed by the myriad of
both lethal and viable phenotypes resulting from Wnt-null mice, as
is described in detail by van Amerongen and Berns in their comprehensive review.14
Wnt signalling has been classically defined, for convenience, into
two intracellular pathways: canonical Wnt signalling, which is
b-catenin dependent and non-canonical Wnt signalling, which is
b-catenin independent. The activation of either pathway is largely dependent on the subsequent associations of Frizzled receptors with endogenous co-receptors including the low-density lipoprotein
(LDL)-related proteins 5 and 6 (LRP5/6) for canonical Wnt signalling
and receptor tyrosine kinase-like orphan receptor 2 (ROR2) for
non-canonical Wnt signalling. Thus, the specific pathway that is
activated upon Wnt ligand stimulation is highly dependent on cellular
context and the complement of cell surface Wnt receptor
subtypes.15 Activation of the canonical Wnt pathway leads to the
modulation of the transcription of .50 genes in mammals (a comprehensive list can be found at http://www.stanford.edu/group/nusselab/
cgi-bin/wnt). Binding of Wnt proteins to the Frizzled –LRP5/
6-receptor complex activates Dishevelled (Dvl) proteins, causing
the disassembly of the b-catenin destruction complex [composed of
axin, adenomatous polyposis coli, and glycogen synthase kinase-b
(GSK-3b), which usually phosphorylates b-catenin, targeting it to proteosome for degradation] (Figure 1). b-Catenin is an multi-faceted
protein, with signalling capacity, but also contributes to the adherens
junction complex, mediating homophilic cell-to-cell adhesion through
interactions with cadherin transmembrane proteins. Upon disassembly, the b-catenin degradation complex can no longer phosphorylate
b-catenin, therefore resulting in the rapid accumulation of dephosphorylated b-catenin and eventual translocation from the cytoplasm
to the nucleus. Once in the nucleus b-catenin displaces the transcriptional co-repressor proteins Groucho/transducin-like enhancer of
split from the T-cell factor (TCF) or lymphoid enhancer-binding
factor (LEF) transcriptional factors, leading to TCF regulation of
target genes such as those involved with cell-cycle activation
(cyclin D1,16 cMyc17), survival [survivin,18 Wnt-1-induced secreted
protein-1 (WISP-1),19 and insulin-like growth factor-1 (IGF-1)].20
Figure 1 A schematic diagram showing the Wnt canonical signalling pathway. In the absence of Wnt ligands (far left), GSK-3b, Axin
and APC and phosphorylate (p) b-catenin, targeting it to the proteosome for ubiquitination, thus regulating low cytosolic b-catenin
levels. Wnt binding to Frizzled receptors in complex with LRP5/6
recruits Dishevelled to Fzd and axin to LRP5/6 that promotes inhibition of the destruction complex, phosphorylating GSK-3b, and
allowing b-catenin translocation to the nucleus. In the nucleus
b-catenin assists in the activation of TCF/Lef transcription of target
genes.
Moreover, Wnt signalling regulates the expression of several ECM
components including fibronectin21 and versican,22 and matrixdegrading metalloproteinases (MMPs) such as MMP-723,24, MMP-225,
MMP-326, MMP-925, MMP-1327, MMP-1428, and MMP-26.29 Both
ECM composition and MMP activity regulate SMC migration, proliferation, and apoptosis (see review)30 and as a consequence canonical
Wnt/TCF signalling may alter SMC behaviour during intimal thickening
and atherosclerosis via modulation of the expression of matrix proteins and MMPs. However, there is little direct evidence for this in
the literature except that highlighted in the section 5 for MMP-7.31
Although less well understood, Wnts also activate divergent
(b-catenin independent) pathways collectively named under the umbrella term, non-canonical signalling (Figure 2). These pathways
involve the alternative binding of Wnt ligands to Frizzled receptors
alone or in complex with discrete co-receptors (other than LRP5
and 6). Non-canonical Wnt pathway activation can involve the
release of Ca2+ from intracellular stores to activate co-effector
kinases such as protein kinase C (PKC), calcium- and calmodulindependent kinase II (CAMKII), and Jun N-terminal kinase (JNK) or
can activate JNK directly. Individually these kinases regulate the
235
Wnt signalling and SMCs
Figure 2 A schematic of Wnt signalling pathways. Wnt also activates non-canonical signalling pathways. The interaction of Wnts with the Frizzled
receptors and with the participating co-receptor ROR2 leads to the activation of the Ca2+ pathway. Ca2+ is released from the endoplastic reticulum
via the enzyme phospholipase C (PLC). Calcineurin (CaCN) and CAMKII are activated, which in turn trigger NFAT and NFkB, respectively. PKC is also
activated and in turn triggers NFkB and CREB. NFAT, NFkB, and CREB translocate to the nucleus and transcribe downstream regulatory genes. Wnt
can bind to ROR2 in the absence of Frizzled receptors to release Ca2+ and activate JNK. The activation of this pathway may also inhibit canonical Wnt
signalling. The PCP pathway is controlled by Frizzled and Dishevelled (Dvl). Upon Wnt– binding, small-G-proteins Rho-kinase and Rac-kinase in addition to JNK are activated. These proteins directly regulate the cytoskeletal reorganization in addition to cell polarity and migration. Wnt activates
protein kinase A (PKA) through an unknown pathway, resulting in CREB activation and additional inhibition of GSK-3b.
transcription factors nuclear factor of activated T cell (NFAT), activating protein-1, and nuclear factor k-B (NFkB) leading to the coordination of gene transcription to regulate a multitude of cellular
behaviours including cytoskeletal organization, cell polarity, and cell
motility. Rho and Rac-kinases can also be activated by Wnt via a noncanonical pathway known as the planar cell pathway (PCP), which can
in turn activate JNK or rho-associated protein kinase (ROCK). There
is increasing evidence of cross-talk between these pathways, for
example, the activation of the non-canonical pathways can lead to
the inhibition of canonical Wnt signalling outcomes (see review).32
This effect is largely dependent on the cell type and the corresponding receptor expression.15
Both canonical and non-canonical Wnt signalling pathways are controlled by several endogenous inhibitory proteins such as secreted
Frizzled-related proteins (SFRPs), which act as decoy receptors,33
and Wnt-inhibitory factor-1, which binds directly to the Frizzled
receptor.34 Additionally, the canonical Wnt pathway is specifically
inhibited by members of the Dickkopf (DKK) family, whose inhibitory
mechanism involves binding to the canonical specific LRP5/6
co-receptor.35
3. Wnt signalling in SMC
proliferation
The important role that Wnt-b-catenin signalling plays in SMC proliferation has been demonstrated in a number of important studies over the
last decade. This has been reviewed comprehensively in the following
review.36 Briefly, b-catenin activation and TCF signalling up-regulates
pro-proliferative genes including cyclin D137,38 and down-regulates
p2137 in arterial and venous SMCs. Moreover, enhanced proliferative
rates are associated with the activation of b-catenin signalling in atherosclerotic plaques in vivo; however, in this study, it was not determined in
which cell type this occurred.38 b-Catenin activation and proliferation
have, however, been linked in arterial and venous SMCs in vitro and
in vivo after balloon injury of the rat carotid artery.39 – 42 Retarding Wnt
236
signalling using a dominant negative LRP significantly reduced arterial
SMC proliferation42 and a sFRP (FrzA), which acts as a decoy receptor
for Wnt ligands, delayed arterial SMC entry into S-phase.43 Since
b-catenin is regulated downstream of Wnt activation, this suggests
that canonical Wnt signalling may play a role in arterial and venous
SMC proliferation.
Direct evidence for the role of Wnt signalling in SMC proliferation
has been provided in a small number of studies. Wnt1 and Wnt3a are
capable of inducing b-catenin signalling and cyclin D1 expression in arterial SMCs in vitro 42,44 but there is no evidence for expression of
these Wnt proteins by proliferating SMCs in vitro or in vivo.
However, our group has recently identified that Wnt4 is a critical
modulator of arterial SMC proliferation in vitro and contributes to
pathological intimal thickening in vivo.45 In addition, Wnt4 is expressed
in human internal mammary artery SMCs.44 Wnt4 was the only Wnt
protein that is significantly induced in arterial SMCs when stimulated
to proliferate with growth factors in vitro.45 The important role that
Wnt4 plays in proliferation and intimal thickening in vivo was demonstrated using a Wnt4 heterozygous mouse, which experienced significantly reduced intimal thickening and proliferation following ligation of
the right carotid compared with wild-type controls.45 Interestingly,
this proliferative effect of Wnts may be regulated by age since the
pro-proliferative effects of Wnt1 and Wnt3a were lost with increasing
age despite sustained Wnt signaling.44 Since Wnt-b-catenin signalling
can regulate the expression of many genes, it is likely that other genes
in addition to cyclin D1 and p21 are regulated by this pathway and
may therefore affect SMC proliferation. In fact recently, Reddy
et al.46 demonstrated that interleukin-8-induced venous SMC proliferation is dependent on the Wnt-b-catenin responsive gene WISP-1, a
member of the family of connective tissue growth factors.
Together these findings clearly demonstrate that canonical Wnt signalling is pro-proliferative in arterial and venous SMCs, both in vitro
and in vivo. However, despite the lack of direct evidence there is indirect evidence for the involvement of b-catenin-independent noncanonical Wnt signalling in the promotion of SMC proliferation. For
example, NFATc1 is essential for human aortic SMC proliferation
in vitro, via cyclin D1 gene expression, and after a balloon-induced vascular wall injury in vivo.47 In addition, CAMKII promotes rat aortic
SMC proliferation48 and JNK is activated in proliferating SMCs after
a balloon injury; however, these findings are not directly related to
pathway activation by Wnt proteins.49 Moreover, it has been revealed
that SMC proliferation is retarded by bone morphogenetic protein 2
(BMP2) via a novel tandem inter-dependent activation of both
Wnt-b-catenin and Wnt-JNK pathways, which converge to ultimately
activate RhoA-Rac1-dependent arterial SMC motility, but simultaneously repress further b-catenin accumulation and proliferation.50
These studies highlight the complexity of Wnt signalling pathways
and their potential to cross-talk in order to achieve fine regulation
of SMC cell behaviour. Nevertheless, it remains to be determined
whether and which Wnt proteins cause the activation of the noncanonical pathways via NFAT, CAMKII, and JNK during SMC proliferation in vivo.
Characterization of the subtypes of Wnt and Frizzled receptors
involved in SMC proliferation is incomplete. We observed expression
of both Frizzled-1 and Frizzled-6 in quiescent and proliferating mouse
arterial SMCs and in rat intimal SMCs in the carotid artery after a
balloon injury.45 Interestingly, however, only the silencing of
Frzzled-1 retarded Wnt-4-induced arterial SMC proliferation.45 Puzzlingly though, the down-regulation of Frizzled-1 and Frizzled-2 was
C. Mill and S.J. George
observed at 1 and 4 h after an arterial injury of the carotid artery in
rats and in proliferative arterial SMCs in vitro, suggesting that they
may have a negative relationship to proliferation.51 On the other
hand, increased expression of Fzb-1, an endogenous antagonist of
the Wnt cascade, was observed in the rat arterial wall at 4 days
and 3 weeks after an in vivo injury and in quiescent isolated SMCs.51
Since Fzb-1 is a sFRP that inhibits Wnt signalling by binding and
thereby preventing their interaction with Fzds, it was suggested that
this increased Fzb-1 expression, when an arrest of arterial SMC proliferation is observed in the media and neointima, indicates that Fzb-1
may suppress proliferation. Consequently this observation also suggests that Wnt signalling has a positive role in proliferation prior to
the up-regulation of Fzb-1.
In summary, the data presented above provides strong evidence for
the role of Wnt proteins in the stimulation of arterial and venous
SMC proliferation; however, considerably more remains to be
learnt concerning the precise mechanisms underlying the induction
of SMC proliferation. The manipulation of Wnt proteins and Frizzled
receptors in animal models of proliferative cardiovascular disorders in
future studies may help to elucidate the precise role of the Wnt family
in the pathogenesis of atherosclerosis and intimal thickening. Although
the majority of the data so far are gained from arterial and venous
SMCs, it is possible that Wnts may regulate venule SMC proliferation.
Interestingly, DKK-1 promoted SMC proliferation in rat mesenteric
microvasculature,52 which may indicate differential regulation of proliferation in SMCs from different blood vessel types.
4. Wnt proteins in SMC migration
In comparison with proliferation, less is known about the role that
Wnt proteins play in the promotion of SMC migration. However,
the involvement of the Wnt-b-catenin pathway appears likely, since
animal models of intimal thickening (in which both SMC proliferation
and migration occur) have shown increased b-catenin levels.39,41
Moreover, it is likely that Wnt proteins will play a role in SMC migration since they promote the migration of other cell types during development53 and migration of other cell types such as monocytes and
endothelial cells.54,55 We currently have studies underway to directly
examine the role of Wnt-b-catenin signalling in arterial SMC migration and have demonstrated that arterial SMC migration is regulated
by Wnt2.56 To date the role of the non-canonical Wnt pathways
(Wnt-Ca2+ and Wnt-JNK) in SMC migration has not been examined
directly; however, indirect evidence suggests that these pathways may
be involved. For example, NFATc1 promotes the arterial SMC response to injury, and the inhibition of GSK3b is required for the
activation of NFAT during arterial SMC migration in wound
repair.57 Additionally, CAMKII is required for arterial SMC migration,58 and JNK is activated after a rat carotid artery balloon injury
when SMC migration is induced.49 Furthermore, BMP-2 consecutively
and interdependently activates the Wnt-b-catenin and Wnt-noncanonical planar cell polarity (PCP) signalling pathways to facilitate
arterial SMC motility.50
5. Wnt proteins in vascular smooth
muscle apoptosis
The apoptosis of SMCs in CVD can be induced by a number of extracellular agents and inflammatory mediators, including oxidative stress
237
Wnt signalling and SMCs
induced by reactive oxygen species,59 – 61 reactive nitrogen species,62
inflammatory mediators/cytokines such as IL-1b and IFN-g,63 and oxidized low-density lipoprotein (oxLDL).59,64,65 As discussed previously
fibrous cap SMC apoptosis leads to thinning and weakening of the
fibrous cap and contributes to aneurysm formation. Consequently,
the identification of factors that provide an anti-apoptotic signal for
fibrous cap SMCs may be useful for reducing plaque rupture and
aneurysm formation. In addition, a pro-apoptotic strategy may be
useful for the reduction in intimal thickening by inducing the apoptosis
of arterial or venous SMCs that are responsible for intimal thickening.
Wnt-dependent survival signalling predominantly concerns the
activation of the b-catenin-dependent canonical Wnt signalling
pathway. Over 54 b-catenin target genes have been identified, of
which a handful of genes play a direct defined role in cell survival
including survivin, IGF-1, and members of the connective
tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed
(CCN) family of growth factors. However, numerous transcription
factors, including c-jun, fra-1, Sox9, and ID2, are b-catenin responsive
genes, which could activate the indirect expression of other genes
involved in cell survival.
We and others have demonstrated that in arterial SMCs,
Wnt-b-catenin signalling promotes SMC survival.39,42,66,67 Recent
findings suggest that a reduction in b-catenin protein levels through
the inhibition of the cell cycle protein peptidyl-prolyl cis/trans isomerase (Pin1) in arterial SMC increases the rate of apoptosis.67 The perturbation of Wnt-b-catenin signalling, using a dominant negative form
of TCF-4 or LRP, increased arterial SMC apoptosis,39,42 although the
precise mechanisms of action were not investigated. We showed that
Wnt5a treatment of mouse aortic SMCs results in b-catenin activation and enhanced TCF signalling.66 Interestingly, when challenged
with H2O2, a pro-apoptotic mimic of oxidative stress associated
with CVD, we demonstrated that Wnt5a reversed H2O2-induced
apoptosis of mouse aortic SMCs.66 This effect was reversed by canonical Wnt pathway inhibition using the gene silencing of LRP5/6 canonical co-receptors and treatment with the canonical-specific pathway
inhibitor DKK-1.66 Intriguingly, in these experiments, the survival of
arterial SMCs in the presence of Wnt5a and H2O2 was paralleled
with a reduction in TCF signalling, suggesting that there is downstream
inhibition of the canonical Wnt pathway by H2O2.66 In these conditions, arterial SMC survival appeared to be dependent on the
up-regulation of the b-catenin-dependent gene, WISP-1, which was
regulated by an alternative Wnt5a-dependent pathway involving
CREB transcription factor activation.66 Further studies are, however,
essential to completely dissect the precise role of b-catenin signalling
in fibrous cap SMC apoptosis.
6. Wnt and SMC differentiation
Differentiation of venous and arterial SMC may also be regulated by
Wnts; however, this research area has received little attention. The
canonical Wnt/TCF pathway has been identified as a strong SMC
lineage inducer in the chick embryo.68 Moreover, the Notch
pathway acts together with the BMP and Wnt pathways in coordinating mesoderm ventralization, ventral mesoderm lineage induction, and
lineage segregation events.68 Specifically, Wnt3a (but not Wnt1 or 5a)
promotes the expression of the early myofibroblast marker
SM-22-a.69 Studies in the lung have also highlighted Wnts as potentially important regulators of SMC differentiation. Specifically,
Wnt7b is thought to play a role during the early events of pulmonary
SMC differentiation, through canonical signalling via Fzd1 and 10 and
LRP5.70 Interestingly, Wnt7b2/2 embryos and newborn mice exhibit
severe defects in the smooth muscle component of the major pulmonary vessels, due to increased apoptosis of SMCs.71
6.1 Relation of Wnt proteins to
cardiovascular disorders
The evidence presented above outlines the current data that clearly
demonstrate a novel role for Wnts in SMC proliferation, migration,
and survival. Consequently, one can suggest that Wnt proteins are
likely to play an important role in atherosclerosis, aneurysm formation, and intimal thickening. Although a direct assessment of the involvement of Wnt signalling in atherosclerosis has not been
presented, a few descriptive studies have quantified the expression
of Wnt proteins and b-catenin as well as Wnt receptors and endogenous soluble inhibitors in atherosclerotic lesions and provide a
powerful argument to this notion. Elevated levels of active
b-catenin were detected in disrupted atherosclerotic plaques compared with stable plaques and associated with the evidence of elevated cell proliferation, suggesting that b-catenin signalling
contributes to plaque instability.38 However, in this study it was not
examined in which cell type this occurred. Moreover, the expression
of Wnt5a in human and murine atherosclerotic lesions has indicated a
potential role for Wnt5a signalling in atherosclerosis.72 Furthermore,
in our group we observed a significant up-regulation of Wnt5a in unstable plaques compared with stable atherosclerotic plaques and
normal and intimal thickened coronary arteries.73
The most direct evidence to suggest the importance of canonical
Wnt signalling in CVD is supported by studies in which the expression
and significance of the canonical Wnt pathway co-receptor LRP5/6 in
CVD has been examined.74,75 Reduced LRP6 receptor expression
levels were identified in human carotid atherosclerotic lesions, indicating that diminished canonical Wnt signalling may contribute to atherosclerosis perhaps by reducing the SMC survival, proliferation, and
migration that is required in healing plaque ruptures.76 The identification of a rare familial missense mutation in the LRP6 gene encoding a
defective form of the canonical co-receptor in vivo was associated
with the increased incidence of early coronary artery disease,
hypertension, type 2 diabetes, and high-circulating LDL cholesterol
levels.74,76 Magoori et al.75 also suggested an augmentation of atherosclerotic lesion formation in LRP5 null mice. Additionally, elevated
levels of the canonical Wnt inhibitor DKK-1 are present in advanced
carotid plaques, since DKK-1 promotes platelet-mediated endothelial
cell activation, it was proposed that this could lead to enhanced
inflammation in the atherosclerotic lesion; however, it may also
lead to reduced SMC survival, proliferation, and migration, thereby
reinforcing the potential complexity of Wnt participation in
atherosclerosis.77
7. Regulation of Wnt and Frizzled
expression
Although the key regulators of Wnt and Frizzled expression have not
been determined in CVD, previous studies in other cell types have
indicated possible mechanisms of regulation. The tumour suppressors
p63/p73.78 and Wilms tumour 179 positively regulate Wnt4 expression, whereas p21 has the opposite effect,80 suggesting that the maintenance of appropriate Wnt4 expression is potentially modulated by
238
C. Mill and S.J. George
Figure 3 Endogenous and synthetic inhibitors of the Wnt/b-catenin signalling pathway. The Wnt/b-catenin signalling pathway is regulated by several
endogenous inhibitors shown in the diagram and in the upper table. Recently described small-molecule inhibitors are also shown in the diagram
(A– H ), with their sites of action detailed in the lower table.
several factors. Also there was evidence that the ELL-associated
factor and Wnt4 form an auto-regulatory negative feedback loop
in vivo.81 WNT5A is also transcribed due to multiple mechanisms,
such as NFkB, Hedgehog, JAK-STAT3, TGF-b, and Notch signalling activation.82 Demethylation of the promoter region of Wnts may also
be an important regulator of Wnt gene expression in SMC.83,84
Although the data regarding Frizzled promoter regulation is not extensive, it is apparent that differential regulation of the Frizzled may
occur. ERG promotes Frizzled4 gene,85 SRY-related HMG-box 4
(Sox4); POU5F1 and POU2F subfamily members regulate Frizzled5
expression,86,87 and ELK1- and PAX4-binding sites were conserved
in Frizzled8 promoters,88 whereas the binding sites for PU.1, SP1/
Krüppel-like, CCAAT-box, and TCF/LEF/SOX transcription factors
were conserved among the 5′ -promoter in Frizzled7 orthologs.89
The triggers for the activation of Wnts in arterial and venous SMCs
in CVD are currently unknown. Evidence in the literature from other
cell types suggest various regulatory factors, for example, retinoic
acid,90 cytokines including IL-6,82 low oxygen,91 toll-like receptor
activators, for example, lipopolysaccharide,92 and LDL may be regulators of Wnt activity.93 Further analyses are essential to determine
which of these regulators are important for the modulation of
Wnt gene expression in SMC during intimal thickening and
atherosclerosis.
In this review, we have described Wnt –Frizzled signalling in SMCs
in a variety of processes associated with cardiovascular disorders. Our
understanding of the underlying mechanisms of Wnt proteins in cardiovascular disorders, however, remains incomplete. The exploitation
of small-molecule Wnt inhibitors may advance our understanding of
the Wnt pathways in CVD, but also facilitate the development of
therapeutic reagents for the treatment of CVD, associated with deregulation of normal Wnt signalling such as atherosclerosis and
intimal thickening. Recent studies have proposed that many of the components of the Wnt signalling pathway are ‘druggable’ (Figure 3). A
number of synthetic inhibitors that act at different steps in the Wnt/
b-catenin signal transduction pathway have been identified.94 – 106
However, because binding of Wnts to receptors can activate both canonical and non-canonical intracellular pathways, inhibitors that retard
membrane-adjacent events could possibly have undesirable farreaching effects. It is apparent that inhibiting b-catenin signalling activity
may be advantageous but since b-catenin is an important component of
the adherens junction (composed of cadherins and catenins), the inhibition of b-catenin function may have undesirable effects in addition to
the retardation of Wnt/TCF signalling. Recently, an RNAi-based
modifier screen identified b-catenin responsive transcription (CRT)
inhibitors and highlighted their specificity in antagonizing the transcriptional activity of nuclear b-catenin.94 It will be of great value to evaluate
Wnt signalling and SMCs
the potential of various inhibitors such as CRT on intimal thickening
and atherosclerosis in future studies.
Conflict of interest: none declared.
Funding
C.M.’s research was funded by the British Heart Foundation FS/08/042/
25378.
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