REVIEW
Cardiovascular Research (2014) 103, 452–460
doi:10.1093/cvr/cvu171
Emerging regulators of vascular smooth muscle
cell function in the development and progression
of atherosclerosis
Jason Lee Johnson*
Laboratory of Cardiovascular Pathology, School of Clinical Sciences, University of Bristol, Research Floor Level Seven, Bristol Royal Infirmary, Bristol BS2 8HW, UK
Received 13 January 2014; revised 1 July 2014; accepted 14 July 2014; online publish-ahead-of-print 22 July 2014
After a period of relative senescence in the field of vascular smooth muscle cell (VSMC) research with particular regards to atherosclerosis, the
last few years has witnessed a resurgence, with extensive research re-assessing potential molecular mechanisms and pathways that modulate
VSMC behaviour within the atherosclerotic-prone vessel wall and the atherosclerotic plaque itself. Attention has focussed on the pathological
contribution of VSMC in plaque calcification; systemic and local mediators such as inflammatory molecules and lipoproteins; autocrine and paracrine regulators which affect cell– cell and cell to matrix contacts alongside cytoskeletal changes. In this brief focused review, recent insights that
have been gained into how a myriad of recently identified factors can influence the pathological behaviour of VSMC and their subsequent
contribution to atherosclerotic plaque development and progression has been discussed. An overriding theme is the mechanisms involved in
the alterations of VSMC function during atherosclerosis.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Atherosclerosis † Vascular smooth muscle cells † Migration † Foam cell formation † Calcification † Matrix
metalloproteinases
1. The duality of vascular smooth
muscle cells in atherosclerosis
It is considered that the presence of VSMCs within advanced atherosclerotic plaques is beneficial and protective, due to their pivotal role in the formation and sustenance of the fibrous cap. It is the VSMC-rich fibrous cap
which guards advanced lesions from plaque rupture and subsequent
thrombosis.1 Conversely, the genesis of atherosclerotic lesions also
involves the preponderance of VSMCs.2,3 Atherosclerotic plaques
develop at a multitude of pre-defined sites within the arterial vasculature—these locations are termed ‘athero-susceptible’ and appear to be
shared between man and animal species used to model in vivo atherosclerosis.4 Such predilection is considered to be a response of endothelial cells
to alterations in blood flow including low shear stress, turbulence, and oscillating flow.4 When endothelial cells are exposed to these haemodynamic alterations they are rendered dysfunctional and are characterized
by impaired expression of endothelial nitric oxide synthase (eNOS).5
Decreased nitric oxide (NO) bioavailability facilitates a multitude of
pro-atherogenic effects including increased platelet adherence and aggregation, monocyte adhesion/infiltration, oxidative modification of accumulate lipoproteins, and vasoconstriction.6 Additionally, a reduction in
nitric oxide levels can result in the increased expression of matrix
metalloproteinases (MMPs)7,8 and exert pro-proliferative9 and promigratory10 effects on VSMCs. Saliently, while in mouse models, monocyte/macrophage accumulation is the first visible sign of atherogenesis,
in man, the fore-runner to nascent atherosclerotic plaques is adaptive
intimal thickening. This is a normal developmental process characterized
by the intimal accumulation of VSMCs embedded within a proteoglycanrich extracellular matrix, as alluded to, commonly at sites of altered
haemodynamic blood flow.11 The transition into the earliest recognized
form of an atherosclerotic lesion, termed a pathological intimal thickening,
is associated with morphological and biochemical alterations to the
VSMCs and their extracellular matrix.3 This evolution includes the
increased retention and modification of lipoproteins, VSMC uptake of
modified lipoproteins, VSMC apoptosis, infiltration of macrophages, presence of micro-calcifications, and the formation of a lipid core. Although
the precise sequence of mechanisms is yet to be resolved, a better understanding of these initial pathological processes may clarify how lipid/
necrotic cores form and expand into advanced unstable plaques. Interestingly, the pathological advancement of early atherogenesis in man cannot
be definitely modelled in animal models, as they regularly lack both adaptive and pathological intimal thickenings.3 Nonetheless, insights can be
gained from in vivo models of atherosclerosis to elucidate the contribution
of VSMC to plaque advancement but always require validation in human
cells and tissues.
* Corresponding author. Tel: +44 117 342 3190. Email: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].
Emerging regulators of VSMCs in atherosclerosis
2. Modulators of VSMC migration
The proliferation and migration of VSMCs facilitate early lesion development but are equally important for maintaining plaque stability through
the maintenance of a protective fibrous cap overlying the thrombotic
lipid core of advanced lesions. Thus understanding key regulators of
VSMC growth within different stages of atherosclerosis will facilitate
and inform future strategies to modulate the disease process. MMPs
have been inexorably linked to VSMC growth and atherosclerosis.12 In
particular, MMP-9 has been demonstrated to play a fundamental role
in VSMC migration and although may promote early lesion development
appears to protect against advanced plaque progression.13 – 17 A recognized mechanism of action through which MMPs may modulate VSMC
behaviour is the processing of cell –cell and cell –matrix interactions.
N-cadherin represents a major cell–cell adhesion molecule in VSMCs
and has been demonstrated to be cleaved by several MMPs (MMP-7,
-9, and -12), affecting VSMC behaviour including migration, proliferation,
and apoptosis.18,19 Moreover, administration to atherosclerotic mice of
a soluble form of N-cadherin, acting as a mimetic, increased VSMC
number while concomitantly retarding plaque progression.20
More recently, oxidative stress has been proposed to regulate
MMP-directed shedding of N-cadherin from VSMCs and promote
their migration. Jagadeesha et al.21 revealed that thrombin utilizes
Nox1 to trans-activate the epidermal growth factor receptor (EGFR),
which in turn induces MMP-9 up-regulation/activity and subsequent
shedding of N-cadherin, resulting in augmented VSMC migratory
capacity. Similarly, a further study has demonstrated that Toll-like receptor (TLR)2 signalling also employs Nox1 to regulate MMP-induced
VSMC migration. Lee et al.22 revealed that the synthetic TLR2 ligand
Pam3CSK4, induced interaction of TLR2 with Nox1 and subsequent reactive oxygen species generation, which in turn increased MMP-2 activity
and subsequent VSMC migration. Furthermore, it was shown that VSMC
release of pro-inflammatory molecules involved in monocyte recruitment were also augmented. Together these findings imply that oxidative
stress within VSMCs, as a consequence of injurious insults such as
thrombosis, bacterial infection, or modified lipoproteins, may facilitate
the transition and development of early atherosclerotic lesions.
Further consequences of alterations in cell–cell and cell–matrix interactions are cytoskeletal changes, which have fundamental effects on cell
motility.23 Pro-migratory cytoskeletal re-arrangement requires the orchestration of numerous protein complexes to facilitate the generation
of new focal adhesions and associated lamellipodia protrusions from the
migratory leading edge of the cell, including small Rho GTPase members
such as Rac1.24 Scaffolding proteins are also necessary at the adhesionactin cytoskeleton interface to expedite migratory cues, such as
p130Cas.25 Indeed, PDGF-stimulated VSMC migration has been shown
to be mediated via p130Cas activation/phosphorylation.26 Moreover, in
parallel with the earlier study indicating that co-operation between oxidative stress and EGFR can modulate VSMC migration, via MMP-dependent
N-cadherin shedding,21 it has been shown that this signalling cascade also
augments p130Cas phosphorylation,27 further promoting the migratory
capacity of cells. Additionally, upon phosphorylation, p130Cas can
complex with focal adhesion kinase (Fak) at the leading edge of migratory
cells to activate and localize MMP-dependent motility.28,29 Taken together
these studies suggest that p130Cas activation plays a pivotal role in dictating the migratory potential of VSMC, and that modulating its activity or
expression may abrogate its pro-migratory effects.
Cysteine-rich proteins (CRP) are a family of LIM domain proteins
including CRP1, CRP2, and CRP4, which facilitate protein–protein
453
interaction along the actin-based cytoskeleton.30 Additionally, CRPs
can also localize to the nucleus where they interact with transcription
factors to mediate VSMC gene expression. Accordingly, CRPs have
been implicated in regulating VSMC differentiation, proliferation, migration, and survival.31 – 33 Interestingly, mice deficient for either CRP1 or
CRP2 or both are viable and fertile, while neointimal formation is
affected after arterial injury,32,34 implying differential regulatory mechanisms modulate VSMC CRP expression during embryonic development
and in response to vascular injury.35 Transforming growth factor beta
(TGF-b) signalling may represent one such mechanism, as two recent
independent studies have demonstrated that TGF-b stimulates CRP2
expression in VSMC.36,37 However, the responses to arterial injury in
mice lacking CRPs are divergent; neointimal formation was attenuated
in CRP1 global-deficient mice, increased in CRP2 whole-body deficient
animals, and unaltered in mice lacking both CRPs systemically.32,34 These
results intimate that the two CRPs are involved in different cellular
responses to arterial injury and may exert antagonistic effects on each
other. Thus understanding the molecular and cellular mechanisms
involved in CRP regulation of VSMC behaviour may elucidate their
pathophysiological role and reveal potential therapeutic approaches.
More recently, it has been proposed that CRPs may regulate VSMC
motility through interactions with p130Cas signalling complexes,
afforded to them through their LIM domains. Also, CRP2 cellular localization is regulated by alterations in the VSMC actin cytoskeleton,38
similar to p130Cas. Accordingly, in a recent issue of Cardiovascular Research, Chen et al.39 examined the role of CRP2 in p130Cas-induced
VSMC migration. Expectedly, PDGF-BB stimulated migration was
increased in CRP2 deficient VSMCs, independent of effects on adhesion
or spreading, but associated with increased lamellipodia formation.39
Using wild-type VSMC and through add-back experiments, they
observed that CRP2 localized with stress fibres of the cytoskeleton in
a LIM1-dependent manner, which is necessary for CRP2 to retard
lamellipodia formation and decrease motility. Importantly, CRP2 via its
LIM1 domain directly interacted with the substrate domain of
p130Cas at the site of focal adhesions, reducing lamellipodia and
VSMC migration. Moreover, the sequestering of p130Cas at focal adhesions by CRP2 abrogated its phosphorylation, intimating a critical role of
p130Cas phosphorylation in VSMC migration and neointimal formation.
Supportingly, high levels of phospho-p130Cas were observed in medial
and neointimal VSMC of arteries after carotid ligation.39 Also, pharmacological inhibition of p130Cas phosphorylation or siRNA knockdown
both reduced neointimal formation. However, it is also plausible that
other regulators of p130Cas phosphorylation may play a role, particularly as phospho-p130Cas levels were elevated within the ligated artery of
CRP2 deficient mice after 4 days compared with wild-type animals, possibly associated with alterations in VSMC phenotypic modulation.39
The above findings (summarized in Figure 1), demonstrate that CRP2 and
p130Cas play key regulatory roles in VSMC migration. However, direct
effects on atherogenesis and plaque progression have yet to be elucidated,
but would require VSMC-specific conditional knockout mice to enable an
informed extrapolation of the findings, particularly with regards to developing future strategies to modulate plaque progression. Nonetheless, alongside cadherins and MMPs, which CRP2 and p130Cas may effect, as well
as be affected by themselves, are worthy of further investigation. Indeed,
supporting proof-of-concept in vivo studies have already demonstrated
that targeting N-cadherin function20,40 or MMP levels and activity41 – 43
modulates VSMC behaviour, neointimal formation, and atherosclerosis.
Thus endorsing similar studies evaluating the therapeutic potential of
modulating CRP2 and p130Cas function in atherosclerosis.
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J.L. Johnson
Figure 1 Anti- and pro-migratory effects of CRP2 and p130Cas. (1) Cell-surface N-cadherin expression on adjacent VSMC maintains cell – cell contacts
and sustains an anti-migratory phenotype. CRP2 can also retard VSMC migration via its LIM1 domain, directly interacting with the substrate domain of
p130Cas, a pro-migratory molecule associated with lamellapodia formation. (2) Multiple MMPs are capable of shedding N-cadherin resulting in disruption
of cell – cell contacts while also triggering intracellular b-catenin signalling and favouring a pro-migratory phenotype. Moreover, MMPs can be directed and
co-localize to the lamellapodia of VSMC with phospho-p130Cas, permitting VSMC migration.
3. Vascular smooth muscle foam
cell formation: contribution to
pathological intimal thickening
As mentioned previously, VSMC foam cell formation may represent a
pivotal step in the transformation of intimal thickenings into nascent atherosclerotic lesions. As recently reviewed,44 VSMCs harbour all the machinery necessary to accumulate modified lipoproteins and transform
into foam cells. Moreover, VSMC foam cells are prevalent in both
early and advanced human atherosclerotic plaques, however, due to
the differences in atherogenesis within animal models; their incidence
in plaques from hypercholesterolaemic animals is reduced and limited
to advanced lesions. Consequently, there are inherent difficulties in
assessing manipulations in animal models due to the different disease
aetiologies. Nonetheless, the mechanism underlying VSMC foam cell
formation is of obvious importance.
The VSMC-derived extracellular matrix within adaptive intimal thickenings at athero-susceptible sites facilitates the retention of lipoproteins,
increasing the susceptibly of their modification.3,45 Moreover, phenotypically altered VSMCs which populate adaptive intimal thickenings,
harbour a myriad of specific receptors which modulate the endocytosis
of modified lipoproteins, these include; SRA-I, SRA-II, CD36, lectin-type
oxidized LDL receptor 1 (LOX-1), and low-density lipoprotein
receptor-related protein 1 (LRP1).44,46 Concomitantly, ATP-binding
cassette transporter A1 (ABCA1) and apolipoprotein A1 (ApoA1),
which work in concert to mediate the efflux of cholesterol to form
nascent HDL particles, the primary canonical process of reverse
cholesterol transport, are down-regulated in intimal VSMCs.47 Interestingly, medial VSMCs show no such deficiencies,47 suggesting that
increased cholesterol influx alongside decreased efflux contributes to
VSMC foam cell formation in early atherosclerotic lesions. Diabetic
patients have an increased risk of atherosclerosis-related disorders
which can even be predicted in patients with pre-diabetes, as detected
by abnormal glucose tolerance tests.48 Concurring, high glucose levels
can induce VSMC foam cell formation due to an imbalance in lipid
uptake and efflux, as observed by increased CD36 expression and a
concomitant abrogation of the expression and function of ATP-binding
cassette transporter G1 (ABCG1), in cultured human VSMCs.49
The switching of VSMC from a contractile phenotype to a synthetic
one is considered a pre-requisite during the formation of the neointima,
as occurs during adaptive intimal thickening, and is associated with
increased migratory and proliferative capacity (as reviewed by Lacolley
et al.46). Recently VSMC lipid accumulation and foam cell formation
has been related with VSMC proliferation. Ma et al.50 demonstrated
that inflammatory stimuli (LPS) modulated low-density lipoprotein receptor (LDLR) expression through activation of the mammalian target
of rapamycin (mTOR) pathway and Rb phosphorylation, facilitating
Sterol Regulatory Element-Binding Protein (SREBP)-2 induction of
LDLR expression and ensuing cholesterol uptake. These data imply
that mTOR inhibition, especially mTORC1, may provide useful for
retarding early plaque progression.
Atherogenic lipoproteins have also been shown to induce VSMC proliferation through alterations in calcium (Ca2+) signalling/handling, primarily by modulating the sarco/endoplasmic reticulum Ca2+-ATPase
(SERCA).51 Moreover, impaired calcium handling is exhibited by
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Emerging regulators of VSMCs in atherosclerosis
VSMCs from athero-susceptible mice52 and also associated with altered
SERCA expression and concomitant changes in cell function.53 Furthermore, alterations in Ca2+ response can regulate the phenotypic switching
of in vitro human coronary artery VSMCs.54 A central role for SERCA in
controlling VSMC behaviour has been further demonstrated by in vivo
studies. Oxidative modification of SERCA through TGF-b1 up-regulation
of Nox4 (nicotinamide adenine dinucleotide phosphate-oxidase 4) promoted VSMC migration from obese Zucker rats. In these rats, knockdown
of Nox4 retarded SERCA oxidation and reduced neointima formation.55
Additionally, Lipskaia et al.56 demonstrated that SERCA2 inhibits VSMC
proliferation and subsequent balloon injury-induced neointima formation
in rats, via the transcription factor nuclear factor of activated T-cells
(NFAT). The same authors revealed SERCA2a (in synergy with protein
phosphatase inhibitor-1; I-1) regulated Ca2+ handling repressed VSMC
phenotypic switching to a synthetic state.57 Expectedly, VSMC proliferation and neointima formation were increased in I-1 knockout mice,
while overexpression of I-1 in the rat angioplasty model retarded VSMC
phenotypic switching and neointimal thickening.57 Therefore, SERCA2a
(and I-1) overexpression maybe considered therapeutic strategies
during specific stages of atherosclerosis.
It has been demonstrated that VSMC exhibit plasticity within early
intimal thickenings2 which may give rise to a considerable diversity of
VSMC responses to prolonged exposure of modified lipoproteins. Aargmann et al. 58 demonstrated divergent sub-populations of VSMCs in
primary culture from the same human artery which differently
responded to LDL exposure implying that a number of VSMCs from
the same arterial site can be reprogrammed in response to modified
lipoproteins. It has also been suggested that VSMC harbour the ability
to trans-differentiate to a macrophage-like state on cholesterol
loading, including the expression of genes considered macrophagespecific.59 These intriguing mechanisms, devised largely from in vitro
studies and in mouse cells, provide a major challenge for the field to
further define and validate the mechanisms of VSMC plasticity in response to modified lipoproteins. Notably, recent evidence has shown
that cholesterol-loaded lysosomes can be transported from foam cell
macrophages to VSMCs in vitro, inducing VSMC foam cell formation.60
This novel pathway may in part account for the prevalence of VSMC
foam cells in early atherosclerotic lesions, which are admixed with
macrophage foam cells in pathological intimal thickenings.
The down-stream effects of VSMC foam cell formation on plaque progression are numerous. For instance, perpetual cholesterol accumulation can induce cell death of the lipid-laden VSMC, promoting the
migration and proliferation of adjacent VSMCs within the intima.61
VSMC apoptosis and cell death can also instigate inflammation
through the monocyte chemo-attractant protein-1 (MCP-1)-mediated
recruitment of circulating monocytes and the release of other
pro-inflammatory mediators.62 Moreover, both MCP-1 and chemokine
(C-C motif) ligand 19 (CCL19) can directly modulate VSMC phenotype,
growth, and their production of MMPs.63,64 Furthermore, continual
apoptosis of VSMCs promotes calcification.65 All of the above would
be expected to precipitate the early stages of atherosclerosis formation,
recently coined pathological intimal thickening.3 The above findings are
summarized in Figure 2.
A further pro-atherosclerotic consequence of VSMC foam cell
formation and subsequent apoptosis, arises due to their impaired
clearance.66 Particularly within a hyperlipidemic setting, the reduced
phagocytosis/efferocytosis of apoptotic VSMCs can result in secondary
necrosis, which triggers the release of pro-inflammatory cytokines
including MCP-1, IL-1a, and IL-1b from the dying cells and surrounding
viable VSMCs.67 Interestingly, IL-1b can induce VSMC growth, phenotypic switching, MMP production, and facilitate intimal thickening.68 – 71
Accordingly, atherogenesis is reduced in mice with a global deficiency
in IL-1b72,73 or when treated with a IL-1b neutralizing antibody.69
These studies would support targeting IL-1b to inhibit the development
and progression of atherosclerosis. Indeed, a phase IIb randomized,
placebo-controlled trial entitled CANTOS is currently underway to
assess the anti-inflammatory and anti-atherosclerotic effects of a monoclonal anti-human IL-1b antibody (canakinumab) in stable but high-risk
cardiovascular patients.74 However, it must be noted that despite its
proven anti-inflammatory effects in man,74 it is possible that blocking
VSMC IL-1b signalling in advanced atherosclerotic plaques may exert
deleterious effects on fibrous cap stability, such as reduced VSMC
growth and phenotype-specific matrix remodelling.
As mentioned earlier, the transition of adaptive intimal thickenings
into the earliest stage of atherosclerosis involves the accumulation of
modified lipoproteins by intimal VSMCs and their subsequent transition
into foam cells, upon which they take on macrophage-like properties and
aid disease progression. Accordingly, targeting lipid-laden VSMCs at this
early stage represents a therapeutic opportunity to halt disease development and the later formation of advanced clinically relevant plaques.
More knowledge is obviously required on the underlying mechanisms
of intimal VSMC plasticity and the need for lineage-specific markers
when assessing the contribution of foam cells within all stages of atherosclerosis. Indeed from numerous in vitro and in vivo animal studies, the
current dogma postulates that resident VSMC that undergo phenotypic
switching participate in early atherosclerotic formation and the development of advanced lesions. However, recent cell lineage studies have
challenged this tenet, highlighting that in mouse models of atherosclerosis, blood-born cells foster the ability to express markers considered
smooth muscle cell-specific, while VSMCs themselves can display proteins associated with macrophages at the expense of their smooth
muscle differentiation markers.75 As such, uncertainty remains as to
which cells within atherosclerotic plaques are actually of VSMC origin.
Moreover, Tang et al.76 have suggested that a population of ‘multipotential vascular stem cells’ propagate the media of the blood vessel
wall and harbour the ability to reconstitute both medial cells and
foster neointima formation after vascular injury, although this paradigm
has been robustly challenged.77 Taken together, there is a need for refinement and accepted definitive characterization markers of VSMC
lineage alongside compelling tracing studies in an array of in vivo cardiovascular models. In the absence of definitive evidence that allows the delineation of VSMCs from other VSMC-like cells within the blood vessel
wall and the atherosclerotic plaque, caution should be heeded when
interpreting both in vitro and in vivo studies.
4. VSMC and calcification: role
of Wnt/b-catenin pathway
and galectin-3
Calcification of atherosclerotic plaques is common and further increases
with age, and is associated with plaque burden rather than directly with
vulnerability.1 In general, plaque calcification does not correlate with
symptomatic plaques, but contrastingly confers stability to plaques.78
Nonetheless, plaque calcification can represent as two major forms:
spotty (also referred to as micro-calcification) and dense (also termed
macro- or lamellar calcification), and it has been recently identified
that spotty calcification typifies culprit plaques as opposed to stable
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J.L. Johnson
Figure 2 Formation and consequences of vascular smooth muscle foam cell formation in atherogenesis. Within an adaptive intimal thickening, VSMC
acquire a synthetic phenotype (1) where they secrete and maintain a proteoglycan and extracellular matrix-rich environment (2). This VSMC-derived
extracellular matrix facilitates the retention of lipoproteins at so called athero-susceptible sites (3). Ensuing lipoprotein modification, alongside the
up-regulation of VSMC receptors specific for lipoprotein uptake, and concomitant down-regulation of mediators of cholesterol efflux, precipitate
VSMC foam cell formation (4). Perpetual cholesterol accumulation can induce cell death of the lipid-laden VSMC (5). Moreover, VSMC foam cell formation,
apoptosis, and cell death can also initiate inflammation through MCP-1-mediated recruitment of circulating monocytes and the release of other
pro-inflammatory mediators (6). All of the above would be expected to precipitate the early stages of atherosclerosis formation, recently termed pathological intimal thickening.
lesions.79,80 Accordingly, the pattern rather than the total amount of
plaque calcification is more informative for plaque risk prediction. Interestingly, spotty calcification is also a hallmark of early atherosclerosis in
man, associated with the formation of pathological intimal thickenings.3
Moreover, a recent fate-mapping study in a mouse atherosclerotic
model demonstrated that VSMCs represent the largest number of
calcific cells in atherosclerotic lesions.81 Furthermore and as mentioned
earlier, chronic VSMC apoptosis promotes atherosclerotic plaque
calcification and progression.65 Supportingly, insulin-like growth
factor-1 (IGF1) enhances VSMC survival and blocks calcification, in
part through moderate calcium induction of osteoprotegerin.82
Smooth muscle foam cell formation can induce apoptosis as alluded
to earlier,83 and has also been associated with VSMC calcification.
Goettsch et al.84 identified an osteogenic role of the nuclear factor of
activated T cells (NFAT) signalling pathway in VSMCs exposed to
oxLDL, in part through up-regulation of Runt-related transcription
factor 2 (RUNX2), which is known to promote VSMC-specific vascular
calcification.85 Intriguingly, NFAT signalling is in part inactivated by
Glycogen synthase kinase 3 beta (GSK3b) which has been recently
shown to retard VSMC osteoblast differentiation and mineralization.86
Furthermore, members of the Wnt signalling pathway can modulate
NFAT and GSK3b activity, and accordingly Wnt5a promotes VSMC calcification.87 Additionally, MMPs can modulate Wnt/b-catenin signalling
through the cleavage of VSMC cadherins88 and numerous MMPs have
been linked to atherosclerotic plaque calcification.42,89 It is therefore
conceivable that MMPs, through cadherin-mediated Wnt/b-catenin signalling, induce VSMC calcification and plaque progression, highlighting
culpable MMPs and pivotal members of the Wnt/b-catenin signalling
pathway as viable therapeutic targets.
Galectin-3, a carbohydrate-binding protein belonging to the galectin
gene family [also called by synonyms such as Mac-2, and carbohydratebinding protein 35 (CBP-35)], mediates cell –cell and cell –matrix interactions and modulates a myriad of biological functions.90 Galectin-3
cleavage has been proposed as a surrogate marker of MMP activity91
and its role in atherosclerosis has received abundant attention of late,
providing conflicting results. Human and mouse atherosclerotic
plaques studies have intimated that galectin-3 promotes plaque progression, primarily through amplification of inflammation and increased
foam cell formation.92 – 94 Conversely, it was shown that galectin-3 can
also retard plaque progression through down-regulation of proinflammatory pathways and removal of modified lipoproteins, partly
through decreased expression of the receptor for advanced glycation
end-products (RAGE).95 Interestingly, galectin-3 can also directly act
as a receptor and therefore mediate uptake of advanced glycation endproducts (AGE); however, in the presence of modified lipoproteins,
galectin-3 mediated endocytosis of AGE is reduced due to ligand
Emerging regulators of VSMCs in atherosclerosis
457
Figure 3 VSMC and calcification—role of galectin-3 and b-catenin. Similar to RAGE, galectin-3 can mediate uptake of advanced glycation end-products
(1); however, in the presence of modified lipoproteins, galectin-3-mediated endocytosis of AGE is reduced due to ligand competition with modified lipoprotein (2). Furthermore, galectin-3 endocytosis down-regulates RAGE expression (3). Moreover, VSMC galectin-3 ligation (i.e. modified lipoproteins)
augments Wnt/b-catenin regulated osteoblastogenesis and macro-calcification, which is associated with stable atherosclerotic plaques (4). Conversely,
RAGE ligation (i.e. advanced glycation end-products) favours vascular osteoclastogenesis and micro-calcification, promoting an unstable plaque phenotype
(5). MMPs such as MMP-12 may indirectly induce micro-calcification through the cleavage of galectin-3 and removing its inhibitory action on RAGE.
competition with modified lipoproteins.96 Furthermore, Galectin-3mediated uptake of AGE enhances VSMC proliferation.97 With regard
to vascular calcification, Menini et al.98 have recently demonstrated divergent effects of AGE on the type of calcification formed in atherosclerotic
plaques, and therefore plaque phenotype, dependent on ligation of
galectin-3 or RAGE. VSMC content and galectin-3 expression was associated with stable lesions exhibiting sheet-like/lamelatted macrocalcification, whereas RAGE expression prevailed in unstable plaque
with abundant spotty/granular micro-calcification.98 The divergent
effects on plaque calcification are in part explained by a regulatory role
of galectin-3 on RAGE expression (as alluded to above 99) and further
strengthened by the demonstration that RAGE expression is increased
in galectin-3 deficient VSMCs and modulates osteogenic differentiation.98
In fact, this novel galectin-3/RAGE dyad in VSMC-mediated atherosclerosis calcification may be mediated by the Wnt/b-catenin signalling
pathway, which we have discussed can control the calcification process,
but is also regulated by galectin-3,100,101 in part through modulation
of GSK-3b activity.102 Accordingly, it has been proposed that VSMC
galectin-3 ligation (i.e. modified lipoproteins) augments Wnt/b-catenin
regulated osteoblastogenesis and macro-calcification. In opposition,
RAGE ligation (i.e. advanced glycation end-products) favours vascular
osteoclastogenesis and micro-calcification, promoting an unstable
plaque phenotype.98 The above findings are summarized in Figure 3.
There are numerous other regulatory processes which can propagate
VSMC-driven calcification within the atherosclerotic plaque, including
developmental factors such as bone morphogenetic proteins (BMPs),
matrix Gla protein, and the osteoprotegerin/RANKL axis; inflammatory
factors including TNF-a, Gas6, Fetuin A, and TGF-b; and metabolic
factors incorporating oxidant stress and oxidized lipids, hyperphosphataemia, and vitamin D.103 However, there remains a paucity of therapeutic approaches to reverse atherosclerotic plaque calcification and,
accordingly, this area of cardiovascular disease has received heightened
attention. HMG-CoA reductase inhibitors (statins) are currently under
investigation, primarily due to their pleiotropic effects, including many of
the aforementioned regulatory factors of vascular calcification. Supportingly, simvastatin was demonstrated to reduce intra-plaque calcification
within advanced lesions of Apoe-deficient mice.104 However, a recent
coronary computed tomographic angiography (CCTA) study revealed
an association of statin use with an increased prevalence and burden
of calcification in patients with coronary artery atherosclerosis.105
There is also a body of evidence suggesting an association between
MMPs and vascular calcification.106 Two independent studies have
demonstrated that Apoe-knockout mice which are also deficient for
MMP-289 or MMP-1242 exhibit reduced atherosclerotic plaque calcification compared with their relevant wild-type controls. Disappointingly, a
prospective, randomized, double-blind trial determining the effect of the
non-specific MMP inhibitor doxycycline on MMP expression within atherosclerotic carotid plaques, failed to decrease the presence of calcification despite reducing MMP-1 expression.107 These findings may imply
that a more targeted approach is necessary. Consistent with this, administration of an MMP-12 specific inhibitor to Apoe-deficient mice suppressed atherosclerotic plaque calcification and was associated with
promoting a stable plaque phenotype.42 As stated previously, MMPs
can modulate cadherin mediated cell– cell interactions and also cleave
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J.L. Johnson
effects (as summarized in Figure 4) may inform future therapeutic strategies targeting VSMC with regards to preventing atherosclerotic plaque
progression and its clinical sequelae.
Conflict of interest: none declared.
References
Figure 4 Recent factors implicated in altered VSMC behaviour. Multiple factors have been implicated in the modulation of VSMC behaviour (migration, phenotypic switching, calcification, and foam cell
formation) during atherosclerosis. Interestingly, some factors are associated with multiple behavioural changes such as MMPs, N-cadherin,
TGF-b1, ECM proteins, RAGE, and galectin-3. LOX-1, lectin-type oxidized LDL receptor 1; LRP-1, low-density lipoprotein receptor-related
protein 1; LDLR, low-density lipoprotein receptor; AGE, advanced glycation end-products; RAGE, receptor for advanced glycation endproducts; NFAT, nuclear factor of activated T-cells; TGF-b1, transforming growth factor-b; mTORC1, mammalian target of rapamycin
complex 1; MMPs, matrix metalloproteinases; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; p130Cas, Crk-associated substrate;
TLR2, toll-like receptor-2; EGFR, epidermal growth factor receptor;
ECM, extracellular matrix; CRP2, cysteine-rich protein 2; PDGF,
platelet-derived growth factor; IL-1b, interleukin-1b; MCP-1, monocyte chemo-attractant protein-1; CCL19, chemokine (C-C motif)
ligand 19; VSMC, vascular smooth muscle cell.
galectin-3, processes which would be proposed to drive calcification.
Therefore selective MMP inhibition may also serve as salient therapeutic
approach to reduce atherosclerotic plaque calcification.
Collectively, novel approaches to reduce plaque calcification may
provide an esoteric approach to stabilize atherosclerotic lesions,
particularly mechanisms driven by VSMCs. The galectin-3/AGE axis
may represent one such avenue, either directly or through modulators
of the axis such as MMPs.
5. Conclusion
In conclusion, VSMCs appear to play a dual role in atherosclerosis. With
regards to maintaining plaque stability they are fundamental through
retaining fibrous cap stability. Conversely their phenotypic modulation,
growth, and transformation into foam cells, may drive atherogenesis and
also contribute to plaque development by orchestrating calcification.
Furthermore, dependent on the micro-environment they can modulate
the calcific response and exert disparate effects on plaque phenotype.
Consequently, elucidating the underlying regulators of these divergent
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