Download Nuclear Reprogramming and Its Role in Vascular Smooth Muscle

Curr Atheroscler Rep (2013) 15:352
DOI 10.1007/s11883-013-0352-6
CLINICAL TRIALS AND THEIR INTERPRETATIONS (J PLUTZKY, SECTION EDITOR)
Nuclear Reprogramming and Its Role in Vascular Smooth
Muscle Cells
Silvio Zaina & Maria del Pilar Valencia-Morales &
Fabiola E. Tristán-Flores & Gertrud Lund
Published online: 24 July 2013
# Springer Science+Business Media New York 2013
Abstract In general terms, “nuclear reprogramming” refers
to a change in gene expression profile that results in a
significant switch in cellular phenotype. Nuclear
reprogramming was first addressed by pioneering studies
of cell differentiation during embryonic development. In
recent years, nuclear reprogramming has been studied in
great detail in the context of experimentally controlled dedifferentiation and transdifferentiation of mammalian cells
for therapeutic purposes. In this review, we present a perspective on nuclear reprogramming in the context of spontaneous, pathophysiological phenotypic switch of vascular
cells occurring in the atherosclerotic lesion. In particular, we
focus on the current knowledge of epigenetic mechanisms
participating in the extraordinary flexibility of the gene expression profile of vascular smooth muscle cells and other
cell types participating in atherogenesis. Understanding how
epigenetic changes participate in vascular cell plasticity may
lead to effective therapies based on the remodelling of the
vascular architecture.
Keywords Vascular smooth muscle cell . Epigenetics .
Atherosclerosis . Nuclear reprogramming
This article is part of the Topical Collection on Clinical Trials and Their
Interpretations
S. Zaina (*)
Department of Medical Sciences, Division of Health Sciences,
León Campus, University of Guanajuato, 20 de Enero no. 929,
37320 León, Gto., Mexico
e-mail: [email protected]
M. del Pilar Valencia-Morales : F. E. Tristán-Flores : G. Lund
Department of Genetic Engineering, CINVESTAV Campus
Guanajuato, Irapuato, Gto., Mexico
Introduction
Nuclear reprogramming (NR) is defined as a change in gene
expression profile, which causes a cell’s phenotype to switch
to that of an unrelated cell type [1]. NR research was initiated
by experiments in amphibia showing that factors present in
oocyte cytoplasm can reset a somatic cell’s genome to reexpress oocyte-specific genes [2]. Because of obvious therapeutic implications, those early experiments have prompted
extensive efforts to devise techniques to reprogram somatic
cells for the production of pluripotent cells or to achieve
direct transdifferentiation. This vast field of research has
been reviewed recently with a special focus on the cardiovascular system by Ma et al. [3•]. In this review, after an
introductory overview of epigenetic mechanisms, we will
focus on their participation in the induction of the vascular
smooth muscle cell (VSMC) phenotypic switches as encountered in atherosclerosis. One well-established crucial step in
atherogenesis is the phenotypic switch of VSMCs from a
contractile, quiescent, fully differentiated cell type to a
“synthetic” cell capable of extracellular matrix synthesis,
migration and proliferation [4]. Another important
atherosclerosis-related phenotypic switch is that from
VSMCs to osteoblast-like cells during vascular calcification [5]. Furthermore, it has been shown that VSMCs
may transdifferentiate to a macrophage-like proinflammatory
phenotype. Remarkably, at least some of these phenotypic
switches are reversible. VSMCs can regress to a differentiated
phenotype, and even macrophages can undergo reverse
migration from the vessel to lymph nodes in advanced,
spontaneously regressing atherosclerotic lesions [6•].
The dominant view on vascular cell plasticity is that
full differentiation and quiescence is the default status
of adult vascular cells until a phenotypic switch is
triggered as a response to proinflammatory signals associated with atherogenic risk factors or to experimentally induced mechanical injuries [7]. Yet, this model
352, Page 2 of 9
has been challenged by the recent detection of atherosclerotic
lesions in preagricultural populations, suggesting that
such phenotypic plasticity may be an intrinsic vascular
feature regulated by an age-regulated cellular clock
[8].
One important and intensely debated issue is whether
resident differentiated VSMCs are the only source, or the
source at all, of phenotypically switched cells. The latter may
arise from recruited circulating progenitor cells or a resident
vascular multipotent population, in analogy with proposed
models of cancer initiation [9]. The topic of peripheral blood
VSMC progenitor cells is extremely promising and intellectually fascinating, but the experimental evidence is at best
controversial. Indeed, on the basis of results in animal
models, the existence of circulating VSMC progenitor cells
has been challenged [10]. The unresolved issues in the field
have been thoroughly reviewed elsewhere and will not be
touched on here [11•]. The topic of resident vascular
multipotent cells has been relatively little explored and is
on the rise. A recent report allegedly demolishes the view
that resident, pre-existing differentiated VSMCs are the origin of the dedifferentiated counterparts, as it claims that the
latter arise solely from resident multipotent cells [12].
Accordingly, previous findings suggest that resident vascular
stem-like cells are activated by turbulent blood flow, a
known local factor predisposing to atherosclerotic lesion
initiation (recently reviewed by Zhang et al. [13]). One
expected feature of an atherosclerotic lesion derived from a
tiny population of resident or recruited stem-like cells is
clonality. Experiments using X-chromosome inactivation as
a marker of clonality have been inconclusive, since normal
arteries are composed of patches of highly similar VSMCs
[14]. In summary, the jury is still out on the issue of whether
vascular progenitor cells are recruited from peripheral blood
or are resident. One can only expect that pluripotent cell
research will propose stimulating and paradigm-breaking
views of vascular and general cell biology. Owing to the
high degree of uncertainty in the field, our review takes the
simplified view that pre-existing vascular VSMCs undergo
phenotypic switches characteristic of the atherosclerotic
lesion.
Whatever the unresolved issues and detailed mechanisms,
cellular phenotype switching is a quintessential epigenetic
phenomenon, since it represents the reversible emergence of
phenotype diversity in the presence of genetic invariance, as
exemplified by twin studies [15]. This is possibly an
oversimplified view, as it cannot be ruled out that the frequency of somatic de novo mutations is perhaps higher than
previously appreciated and thus may play a significant role
in the phenomena described here (see later). Keeping the
latter caveat in mind, we will focus on epigenetic transcriptional regulation as an important mechanistic and potentially
therapeutic clue for vascular cell plasticity.
Curr Atheroscler Rep (2013) 15:352
Epigenetics in Cardiovascular Disease: A General View
Epigenetics is the study of nuclear information that is additional to the genetic information contained in a cell’s DNA
sequence. At the molecular level, the epigenetic information
consists of highly dynamic chemical modifications of cytosine residues of DNA and N-terminal tails of histone proteins. Besides these well-understood epigenetic “marks”,
non-coding RNAs (ncRNAs) are emerging as additional
epigenetic players. The transcriptional impact of epigenetic
marks is dependent both on the nature of the specific mark
and on the local sequence context. Epigenetic marks are
essential regulators of cellular differentiation and the organism’s development–for example the loss of DNA methylation is embryonically lethal in mice [16].
Epigenetics has become increasingly popular in the cardiovascular field in recent years, for at least two reasons. One
is the appreciation that cancer and a growing list of other
diseases are associated with aberrant epigenomes—defined
as the distribution of epigenetic marks across a cell’s genome
[17]. Secondly, a large body of evidence cemented the notion
that the epigenome can be shaped by a variety of exogenous
stimuli. Crucially, some of the latter fall within the same
categories as known cardiovascular risk factors: diet, environmental pollutants, smoking and even subtle factors such
as behaviour and stress [18–20]. As a result, a novel mechanistic model of cardiovascular disease emerged, according
to which risk factors act by imposing aberrant epigenetic
marks that result in pathological transcription profiles [21].
In the following sections, we provide a brief description of
the best-characterized epigenetic marks, followed by examples of the connection between specific epigenetic mechanisms and vascular cell plasticity in atherosclerosis.
DNA Methylation
DNA methylation involves the covalent addition of a methyl
group to carbon 5 of deoxycytosine, to yield 5methyldeoxycytosine (5mdC). In mammals, most of the
5mdC is in the 5′ position of CpG dinucleotides. DNA
methylation is performed by DNA methyltransferases
(DNMTs), of which the best characterized are DNMT1,
DNMT3A, and DNMT3B. A loose functional specialization
exists among these DNMTs, where DNMT1 is mainly involved in maintaining pre-existing DNA methylation profiles during mitosis, whereas the DNMT3A and DNMT3B
impose DNA methylation de novo [22]. DNA demethylation
is thought to be performed not by a single enzyme, but rather
via direct deamination of 5mdC to thymine, or by oxidation
of carbon 5 to produce 5-hydroxymethyldeoxycytosine. The
latter can be either further oxidized to formylcytosine and
carboxycytosine or directly deaminated to 5hydroxymethyluracil [23]. All these intermediates trigger
Curr Atheroscler Rep (2013) 15:352
the base excision repair pathway and replacement with an
unmethylated cytosine. Active DNA demethylation can occur in the absence of cell proliferation, for example during
monocytic differentiation [24•]. As for transcriptional impact, methylation of promoter sequences is generally a repressive mark. On the other hand, gene body methylation is
widespread and has been proposed to have pleiotropic functions such as regulation of RNA splicing, silencing of cryptic
promoters and elimination of transcriptional noise [25, 26].
Interestingly, gene bodies are targeted for methylation in
response to dietary factors in Apis mellifera [27]. Another
function of DNA methylation is silencing of transposable
elements [28]. A further important notion is that the probability of a given cytosine residue being methylated can be
affected by the surrounding DNA sequence, as shown by the
association between SNP genotype and local DNA methylation status [29]. Conversely, 5mdC deamination has been
proposed as the source for C–T transitions, the single most
abundant type of single-base mutations in eukaryotes (see
later). These observations demonstrate the epigenetic information and genetic information are not independent, but are
rather complementary and can modify each other.
Page 3 of 9, 352
results in trimethylation of histone H3 lysine 27 (H3K27me3)
[32].
Non-coding RNAs
High-throughout analysis of the transcriptome has revealed a
plethora of ncRNAs. These are generally divided into families relating to size and function to dynamically regulate
gene expression and silence transposable elements.
MicroRNAs (miRNAs) are approximately 22-nt small
ncRNAs that promote messenger RNA (mRNA) degeneration and/or inhibit their translation by complementarily binding on the 3′ untranslated region of mRNAs. Likewise,
ncRNAs of more than 200 nt, denominated long ncRNAs
(lncRNAs), have been implicated in both transcriptional and
post-transcriptional gene regulation (reviewed by Rin and
Chang [33] and Yoon et al. [34]). Transcriptional mechanisms include the association of ncRNAs with known silencing or activator protein complexes such as PcG and
Mediator, respectively [35]. Post-transcriptional mechanisms affected by lncRNAs include pre-mRNA splicing,
mRNA turnover, translation, miRNA function and, as recently demonstrated, transcriptional interference [34, 36].
Histone Post-translational Modifications
De Novo Somatic Mutations
Histones are small proteins tightly bound to DNA. Histones
are fundamental components of chromatin, a highly ordered
nucleoprotein polymer. The basic unit of chromatin is the
nucleosome, which consists of approximately 146 DNA base
pairs wrapped around an octamer of two copies of each of the
histones H2A, H2B, H3 and H4. Nucleosomes are characteristically absent from gene promoter transcription start sites
and generally co-localize with methylated DNA [30]. The Nterminal histone tail can undergo a plethora of posttranslational modifications: acetylation, methylation, phosphorylation, SUMOylation, ubiquitination and ADPribosylation [31]. In many cases, specific histone modifications are associated with transcriptionally permissive or repressive chromatin structure. One well-understood case is
acetylation, which is a mark of permissive chromatin.
Histone acetylation is regulated by two enzyme families:
histone deacetylases (HDACs) and histone acetyltransferases
(HATs). An example of the complexity of histone regulation is
methylation of lysine 4 or 9 of histone H3 (H3K4 and H3K9),
which are marks of permissive and repressive chromatin,
respectively. Functionally coherent DNA methylation and
histone modifications are often co-imposed by complexes of
epigenetic regulators to reinforce repressed or activated states.
For example, promoters can be silenced by complexes of
DNMT3A/DNMT3B, HDACs and the H3K9
methyltransferase G9A [23]. Another important promoter silencing mechanism independent of DNA methylation is that
driven by Polycomb group (PcG) proteins. PcG occupancy
As mentioned already, the widely accepted definition of
epigenetics as the study of sequence-independent changes
in gene expression is based on the assumption that an organism’s genome content remains constant throughout its lifetime. However, enzymes belonging to members of the
activation-induced cytidine deaminase (AID/APOBEC) and
ADAR (adenosine deaminase) gene families are known to
induce specific point mutations in both DNA and RNA
(reviewed by Franchini et al. [37]). Indeed, apolipoprotein
B editing complex 1 (APOBEC1), which edits the apolipoprotein B RNA, was the first discovered example of an
AID/APOBEC deaminase [38]. In DNA, AID/APOBEC
enzymes specifically deaminate cytosine into uracil, which
if left unrepaired leads to a C:G transition mutation to T:A
following DNA replication [37]. Although AID plays a key
role in antibody diversification in B cells, increasing evidence implicates AID in active removal of 5-methylcytosine
from DNA (see earlier) [37, 39]. Following deamination of
5-methylcytosine (which results in a G:T mismatch) base
excision repair enzymes such as thymine DNA glycosylase
and methyl-CpG-binding domain protein 4 can remove the
thymine and reinsert cytosine. However, the fact that approximately 30 % of all human point mutations are transition
mutations in a CpG context, which cannot be explained by
spontaneous deamination [40, 41], implies that processes
leading to DNA demethylation are likely to contribute to
the development of de novo mutations in an individual’s
352, Page 4 of 9
lifetime. To our knowledge, the contribution of these phenomena has not yet been addressed in the context of NR or
cellular phenotype switching in atherosclerosis. However, it
can be anticipated that next-generation sequencing data will
provide interesting insights into the topic.
Dedifferentiation of VSMCs to a Synthetic Phenotype
Differentiation, proliferation and migration are pivotal cellular functions in the construction of the adult body’s complex structure during embryonic development. Those cellular functions are largely lost in the full-grown adult body.
Therefore, an obvious working hypothesis for VSMC dedifferentiation occurring in the atherosclerotic lesion is that at
least a partial reactivation of the embryo-specific transcription programme occurs in atherosclerosis-prone vessels. The
members of the homeobox (Hox) transcription factor family
are important regulators of animal embryonic development
and therefore good candidate regulators of VSMC differentiation [42]. Accordingly, class I vertebrate Hox genes have
been implicated in atherosclerotic lesion development, yet
only a limited number of studies have addressed their relevance in atherosclerosis. Following initial observations that
specific Hox members are associated with a VSMC synthetic
phenotype, a recent study detected differential expression of
a number of Hox genes, particularly Hoxa9, between
atherosclerosis-prone and atherosclerosis-resistant aortic
portions of 3-month-old, standard-chow-fed apolipoprotein
E (apoE)-null mice [43]. The rationale for choosing young
apoE-null mice was that they should reveal early or
predisposing transcriptional changes, although
macrophage-rich atherosclerotic lesions were already visible
in the mice used in the study. Importantly, the authors of the
study validated the mouse data in rat and pig models.
Intriguingly, all differentially expressed Hox gene members
were downregulated in the atherosclerosis-prone aortic portion, indicating an atherosclerosis-specific generalized transcriptional repression across the Hox gene cluster, as exemplified by HOXA9 and HOXA10. In the case of HOXA9, the
study confirms its role as an important player in vascular
biology, particularly the observed antagonism between an
endothelium-specific splice variant of HOXA9 (HOXA9EC)
and proinflammatory factors such as the transcription factor
NF-κB or tumour necrosis factor alpha [44]. The data are
consistent with the notion that HOXA9 is a transcriptional
activator of the cyclin-dependent kinase inhibitor and tumour suppressor CDKN2A (p16INK4a) [45]. The latter gene
maps to 9p21 in humans, a locus robustly linked to cardiovascular risk [46–48]. HOXA10 also displays a predictable
expression pattern considering its known role as a transcriptional activator of the proliferation inhibitor CDKN1A (also
known as p21; see later) [49]. Still, the data are at odds with
Curr Atheroscler Rep (2013) 15:352
previous evidence that two other Hox gene members,
HOXB7 and HOXC9, induce VSMC proliferation and are
overexpressed in atherosclerotic lesions [42]. In line with the
above data, the few genomics-based studies of DNA methylation in vascular tissues suggest that Hox genes are prominent targets for differential methylation in human arteries.
At least two studies, one comparing coronary atherosclerotic
lesion and lesion-free aortic root tissue, the other comparing
aortic and carotid atherosclerotic lesions, yielded several
Hox members among the most differentially methylated
genes [50, 51]. In particular, epigenetic marks in HOXA11
and HOXA5 were identified as markers of artery-typespecific atherosclerosis, as differential methylation between
aortic and carotid lesions was detected in the promoters of
those genes [51]. Overall, the involvement of Hox genes in
atherosclerotic lesion development is not yet fully understood. Establishing the causal implications of Hox gene
epigenetic regulation and differential expression for human
atherosclerosis is a challenging issue. First, a comparison
between portions of the same artery with differential susceptibility to atherosclerosis in humans is lacking. Second, the
results of the aforementioned study by Trigueros-Motos et al.
[43] may imply that Hox gene upregulation is an initial event
resulting in remodelling of the vasculature to an
atherosclerosis-prone tissue, particularly for those Hox
members that favour cell proliferation. After this “priming”
phase Hox gene upregulation is reversed by
proinflammatory stimuli. Alternatively, it is possible that
only a small population of VSMCs undergo phenotypic
switching and overexpresses Hox genes in early atherosclerosis or prior to lesion formation. Subsequently, those cells
are diluted in the lesion mass. Ideally, younger, completely
atherosclerosis-free apoE-null mice should be used to verify
these hypotheses.
A clue to how global DNA methylation profiles may
change during VSMC differentiation comes from evidence
that during embryo and germ line development, DNA methylation tends to increase during the transition from totipotency to a differentiated state [23]. If ones takes this line of
reasoning simply, the dedifferentiation of VSMCs should be
associated with a relatively hypomethylated state. This
would imply that atherosclerotic lesions are globally
hypomethylated in comparison with control tissue. A tendency for hypomethylation in humans and the apoE-null
mouse model has been documented by at least two initial
studies, but firm evidence on an association between atherosclerosis and loss of DNA methylation is still lacking [52,
53]. To further complicate the issue, global hypermethylation in mice, when artificially imposed by the expression of a
bacterial DNMT transgene driven by a smooth muscle cell
(SMC)-specific promoter (SMC α-actin), displayed de novo
angiogenesis and tumours in SMC-rich organs, suggesting
that an increase in DNA methylation may favour VSMC
Curr Atheroscler Rep (2013) 15:352
dedifferentiation [54]. The latter hypothesis is indirectly
supported by evidence that triglyceride-rich lipoproteins induce global DNA hypermethylation in cultured human macrophages [55]. High-coverage epigenomics analysis of human atherosclerotic lesions is clearly needed to clarify these
issues.
The participation of histone post-translational modifications in differentiated VSMC marker genes (VSMC-MGs)
such as SMC α-actin and SM22-alpha has been studied in
detail and was thoroughly reviewed by Alexander and
Owens [56•]. This complex relationship can be summarized
as follows. The transcriptional co-activator serum response
factor binds to specific motifs in VSMC-MG promoters as a
complex with myocardin. Work in a cell culture model of
VSMC differentiation established that transcriptional activation by serum response factor–myocardin depends on local
chromatin remodelling to a permissive, acetylated histonerich state by retinoic acid [57]. These effects on chromatin
structure could be facilitated by a direct interaction between
myocardin and HATs. In vivo data show that the involvement of histone acetylation in the maintenance of VSMC
differentiation is complex and likely gene-specific, since the
HDAC inhibitors Scriptaid and trichostatin A inhibit proliferation and neointima formation in animal models by
inhibiting the positive regulator of cell proliferation cyclin
D1 (reviewed by Findeisen et al. [58•]). As for drivers of
VSMC-MG repression and VSMC dedifferentiation, the
transcription factor Krüppel-like factor 4 (KLF4) is a much
studied candidate as it is upregulated in atherosclerosis and
can repress myocardin and VSMC-MG expression
(reviewed in [56•]). Furthermore, KLF4 is a plausible main
trigger of VSMC dedifferentiation, since it is one of a small
group of prominent pluripotency inducers [59]. Yet, two
unexpected findings indicate that there is still much to be
learnt about KLF4 activity. First, conditional KLF4 inactivation in mice induces VSMC proliferation [60]. Second, the
aforementioned suppressive effects of HDAC inhibitors on
neointima formation were associated with KLF4 induction
[61]. Interestingly, KLF4 was shown to induce the cyclin D1
inhibitor CDKN1A [62]. Several explanations can be put
forward to explain these inconsistencies. As often pointed
out, findings in cell culture models and in vivo are often
different if not opposed [56•]. Furthermore, the phenotype of
KLF4 conditional knockout described by Yoshida et al. [60]
in mice is complex, as neointima formation is preceded by an
initial delay of SMC dedifferentiation. This phenotype may
be explained by attributing a temporally restricted “priming”
function to KLF4 in VSMC phenotype switching which
subsequently gives way to a different regulatory mechanism,
akin to the hypothesized scenario for Hox genes (see earlier).
A caveat is that KLF4 is downregulated during monocyte-tomacrophage differentiation; therefore, macrophage differentiation and atherogenesis may be accelerated in KLF4-
Page 5 of 9, 352
conditional mutant mice [24•]. Another important issue is
that manipulation of an individual transcription factor or
chromatin regulator expression is likely to exert global effects on several genes and thus produce complex downstream responses.
Evidence for the importance of lncRNAs in atherosclerosis has been provided by the analysis of the human 9p21
locus. This locus show a robust genetic association with
atherosclerosis that is independent of traditional risk factors
(see earlier). This region harbours the lncRNA antisense
ncRNA in the INK4 locus (ANRIL), also known as
CDKN2B antisense RNA (CDKN2BAS). Several ANRIL
transcripts, including circular transcripts, have been identified and are associated with atherosclerosis risk [63–65]
(reviewed by Holdt and Tuepser [66]). Interestingly, small
interfering RNA mediated targeted silencing of two different
exons differentially affects the expression of atherosclerosisrelated genes in VSMCs, suggesting independent functions
of ANRIL splice forms [67]. Furthermore, ANRIL is involved in epigenetic silencing of the cyclin-dependent kinase
inhibitor genes CDKN2B (encoding p15INK4b) and CDKN2A
(see earlier), both of which are well-known regulators of cell
proliferation and senescence, via its recruitment of PcG
proteins [68, 69]. Additional ncRNAs that mediate cellular
responses to angiotensin in VSMCs, including an lncRNA
that is responsible for the production of two miRNAs (miR221 and miR-222) implicated in cell proliferation, have
recently been described [70]. Likewise, several miRNAs
have been shown to participate in vascular remodelling
events (reviewed by Nazari-Jahantigh et al. [71]).
A summary of selected mechanisms that regulate VSMC
differentiation is presented in Fig. 1.
VSMCs Switch to a Macrophage-Like Phenotype
and to an Osteoblast-Like Phenotype
The participation of epigenetic mechanisms in the
transdifferentiation of VSMCs to macrophage-like or
osteoblast-like cells is a relatively scarcely studied topic.
The peroxisome-proliferator-activated receptor γ coactivator
1α, an activator of HAT p300, confers a proinflammatory
phenotype to VSMCs in response to high glucose concentrations [72]. As for transdifferentiation of VSMCs to
osteoblast-like cells, calcification is a common feature of
human atherosclerosis and is associated with
hyperphosphataemia. Cell-culture-based and aortic-tissueculture-based models of high-phosphate-concentration-induced conversion of VSMCs to osteoblast-like cells revealed
that promoter methylation and repression of the differentiated VSMC-specific SM22-alpha gene accompany the induction of osteoblast markers, although the detailed mechanisms
are not yet understood [73]. An unexplored domain of
352, Page 6 of 9
Curr Atheroscler Rep (2013) 15:352
Fig. 1 Overview of epigenetic mechanisms involved in vascular
smooth muscle cell (VSMC) phenotype switching. In differentiated
VSMCs (lower part, left to right) myocardin (MYOCD) in a complex
with serum response factor (SRF) homodimers keeps differentiated
VSMC marker genes (VSMC-MG) actively transcribed. Histone
acetyltransferase (HAT) activity induced by retinoic acid (RA) seeds
acetyl groups (Ac) in VSMC-MG promoters and facilitates transcriptional co-activation by MYOCD [56•]. Cell proliferation is restricted by
the activation of the cyclin-dependent kinase inhibitors CDKN1A and
CDKN2A by Krüppel-like factor 4 (KLF4) and the transcription factors
HOXA9 and HOXA10 [45, 49, 62]. In dedifferentiated VSMCs (upper
part, left to right) the long non-coding RNA ANRIL favours cell
proliferation by silencing the tumour suppressor CDKN2B through
recruitment of Polycomb group (PcG) proteins [68, 69]. Repressive
methylation marks are seeded at the promoter of the VSMC-MC SM22alpha gene (black lollypops) [73]. Furthermore, hypermethylation is
possibly a genome-wide mark of the atherosclerotic lesion [54]. KLF4
plays a complex role in these events, as it can promote cell proliferation
by silencing MYOCD and various VSMC-MGs. This effect is accomplished by releasing HATs from VSMC-MG promoters [56•]. HOXB7
acts as a driver of VSMC proliferation [42] by activating targets,
including possibly the epidermal growth factor receptor gene (EGFR),
which is upregulated in dedifferentiated VSMCs [79]. Arrows and
crosses in gene boxes indicate active and repressed transcriptional
states, respectively
epigenetic regulation is the generation of osteoclasts in atherosclerosis, which may be part of a regressive process with
potential therapeutic implications. It has recently been
shown that lesion osteoclasts originate from the recruitment
and NR of circulating monocyte/macrophages, thus
uncovering yet another layer of vascular cell plasticity in
which lesion macrophage functions are not limited to sustaining inflammation [74].
the same hurdles that are limiting transcription-factor-based
therapy. With the exception of peroxisome-proliferatoractivated receptors and oestrogen receptors, transcriptionfactor-based drugs have been difficult to design, as shown
by the fact that they account for only approximately 10 % of
prescribed medications [75]. One further problem is identifying critical epigenetic marks to be targeted. Quantitatively
robust epigenetic changes associated with pathological phenotypes in a population of cells or in a lesion are obvious
candidates. On the other hand, less abundant changes that are
specific for a critical subpopulation of cells—for example
selected VSMCs that secrete locally active proinflammatory
factors—may be disregarded for falling below a predefined
significance threshold value. This problem is particularly
relevant in the light of the cellular heterogeneity of the
atherosclerotic lesion and may not be completely eliminated
even by purifying homogeneous cell populations. Indeed,
Conclusions
Any detection of epigenetic changes in a gene promoter or
any other genomic sequence is an expected finding.
Epigenetic regulation is as ubiquitous as the regulation of
promoter activity by transcription factors. This means that
the design of effective epigenetic therapies is likely to face
Curr Atheroscler Rep (2013) 15:352
very recent single-cell RNA-seq data have revealed that cellto-cell variation in gene expression is remarkably high even
in supposedly homogeneous populations, implying that epigenetic changes are likely to show a comparable level of
variability [76]. In addition, only an extraordinary technological advance will allow the targeting of the desired epigenetic marks to or the erasing of unfavourable ones from
specific sequences in specific cells. One preliminary, successful example is the effectiveness of a methylated oligonucleotide in silencing the insulin-like growth factor II gene
in a murine model of hepatocarcinoma [77]. Another promising area that is very relevant to the topic of this review is
the attempt to target specific HDAC classes to modify the
histone signature of only a set of critical genes [61, 78].
Clearly, exciting research lies ahead to find creative solutions
to understand the pathobiological and therapeutic implications of vascular cell NR.
Conflict of Interest Silvio Zaina, Maria del Pilar Valencia-Morales,
Fabiola E. Tristán-Flores and Gertrud Lund declare that they have no
conflict of interest.
Human and Animal Rights and Informed Consent This article
does not contain any studies with human or animal subjects performed
by any of the authors.
References
Papers of particular interest, published recently, have been
highlighted as:
• Of importance
1. Gurdon JB, Melton DA. Nuclear reprogramming in cells. Science.
2008;322:1811–5.
2. De Robertis EM, Gurdon JB. Gene activation in somatic nuclei
after injection into amphibian oocytes. Proc Natl Acad Sci USA.
1977;74:2470–4.
3. • Ma T, Xie M, Laurent T, Ding S. Progress in the reprogramming
of somatic cells. Circ Res. 2013;112:562–74. This is a very recent
review on the complex topic of experimentally controlled NR, with a
focus on vascular tissue.
4. Nilsson J, Sjölund M, Palmberg L, Thyberg J, Heldin CH. Arterial
smooth muscle cells in primary culture produce a platelet-derived
growth factor-like protein. Proc Natl Acad Sci USA.
1985;82:4418–22.
5. Engelse MA, Neele JM, Bronckers AL, Pannekoek H, de Vries CJ.
Vascular calcification: expression patterns of the osteoblast-specific
gene core binding factor alpha-1 and the protective factor matrix gla
protein in human atherogenesis. Cardiovasc Res. 2001;52:281–9.
6. • Francis AA, Pierce GN. An integrated approach for the mechanisms responsible for atherosclerotic plaque regression. Exp Clin
Cardiol. 2011;16:77–86. This is obligatory reading to gain up-todate knowledge on atherosclerotic lesion regression and cellular
Page 7 of 9, 352
pathways that regulate a reversion in vascular cell phenotype
switching.
7. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med.
1999;340:115–26.
8. Thompson RC, Allam AH, Lombardi GP, Wann LS, Sutherland
ML, Sutherland JD, et al. Atherosclerosis across 4000 years of
human history: the Horus study of four ancient populations.
Lancet. 2013;381:1211–22.
9. Blanpain C. Tracing the cellular origin of cancer. Nat Cell Biol.
2013;15:126–34.
10. Bentzon JF, Sondergaard CS, Kassem M, Falk E. Smooth muscle
cells healing atherosclerotic plaque disruptions are of local, not
blood, origin in apolipoprotein E knockout mice. Circulation.
2007;116:2053–61.
11. • Bentzon JF, Falk E. Circulating smooth muscle progenitor cells in
atherosclerosis and plaque rupture: current perspective and methods
of analysis. Vascul Pharmacol. 2010;52:11–20. This is obligatory
reading to understand the conceptual and experimental complexity
faced by pluripotent cell research in the vascular system.
12. Tang Z, Wang A, Yuan F, Yan Z, Liu B, Chu JS, et al.
Differentiation of multipotent vascular stem cells contributes to
vascular diseases. Nat Commun. 2012;3:875.
13. Zhang C, Zeng L, Emanueli C, Xu Q. Blood flow and stem cells in
vascular disease. Cardiovasc Res. 2013;99(2):251–9.
14. Chung IM, Schwartz SM, Murry CE. Clonal architecture of
normal and atherosclerotic aorta: implications for atherogenesis and vascular development. Am J Pathol. 1998;152:913–
23.
15. Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, Ballestar ML,
et al. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci USA. 2005;102:10604–9.
16. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA
methyltransferase gene results in embryonic lethality. Cell.
1992;69:915–26.
17. Latham KE, Sapienza C, Engel N. The epigenetic lorax: geneenvironment interactions in human health. Epigenomics.
2012;4:383–402.
18. Haggarty P. Nutrition and the epigenome. Prog Mol Biol Transl Sci.
2012;108:427–46.
19. Cortessis VK, Thomas DC, Levine AJ, Breton CV, Mack TM,
Siegmund KD, et al. Environmental epigenetics: prospects for
studying epigenetic mediation of exposure-response relationships.
Hum Genet. 2012;131:1565–89.
20. McGowan PO, Szyf M. The epigenetics of social adversity in early
life: implications for mental health outcomes. Neurobiol Dis.
2010;39:66–72.
21. Ordovás JM, Smith CE. Epigenetics and cardiovascular disease.
Nat Rev Cardiol. 2010;7:510–9.
22. Smith ZD, Meissner A. DNA methylation: roles in mammalian
development. Nat Rev Genet. 2013;14:204–20.
23. Seisenberger S, Peat JR, Hore TA, Santos F, Dean W, Reik W.
Reprogramming DNA methylation in the mammalian life cycle:
building and breaking epigenetic barriers. Philos Trans R Soc Lond
B Biol Sci. 2013;368(1609):20110330.
24. • Klug M, Heinz S, Gebhard C, Schwarzfischer L, Krause SW,
Andreesen R, et al. Active DNA demethylation in human
postmitotic cells correlates with activating histone modifications,
but not transcription levels. Genome Biol. 2010;11:R63. This provides an example of biologically relevant epigenetic modifications
that occur in the absence of cell proliferation.
25. Flores K, Wolschin F, Corneveaux JJ, Allen AN, Huentelman MJ,
Amdam GV. Genome-wide association between DNA methylation
and alternative splicing in an invertebrate. BMC Genom.
2012;13:480.
26. Huh I, Zeng J, Park T, Yi SV. DNA methylation and transcriptional
noise. Epigenetics Chromatin. 2013;6:9.
352, Page 8 of 9
27. Lyko F, Foret S, Kucharski R, Wolf S, Falckenhayn C, Maleszka R.
The honey bee epigenomes: differential methylation of brain DNA
in queens and workers. PLoS Biol. 2010;8(11):e1000506.
28. Hsiao WL, Gattoni-Celli S, Weinstein IB. Effects of 5-azacytidine
on expression of endogenous retrovirus-related sequences in C3H
10T1/2 cells. J Virol. 1986;57:1119–26.
29. Kerkel K, Spadola A, Yuan E, Kosek J, Jiang L, Hod E, et al.
Genomic surveys by methylation-sensitive SNP analysis identify
sequence-dependent allele-specific DNA methylation. Nat Genet.
2008;40:904–8.
30. Nishida H, Suzuki T, Kondo S, Miura H, Fujimura Y, Hayashizaki
Y. Histone H3 acetylated at lysine 9 in promoter is associated with
low nucleosome density in the vicinity of transcription start site in
human cell. Chromosom Res. 2006;14:203–11.
31. Badeaux AI, Shi Y. Emerging roles for chromatin as a signal integration and storage platform. Nat Rev Mol Cell Biol. 2013;14:211–24.
32. Simon JA, Kingston RE. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell. 2013;49:808–24.
33. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs.
Annu Rev Biochem. 2012;81:145–66.
34. Yoon JH, Abdelmohsen K, Gorospe M. Posttranscriptional gene
regulation by long noncoding RNA. J Mol Biol. 2012. doi:10.1016/
j.jmb.2012.11.024.
35. Lai F, Orom UA, Cesaroni M, Beringer M, Taatjes DJ, Blobel GA,
et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature. 2013;494:497–501.
36. Latos PA, Pauler FM, Koerner MV, Şenergin HB, Hudson QJ,
Stocsits RR, et al. Airn transcriptional overlap, but not its
lncRNA products, induces imprinted Igf2r silencing. Science.
2012;338:1469–72.
37. Franchini D, Schmitz KM, Petersen-Mahrt SK. 5-Methylcytosine
DNA demethylation: more than losing a methyl group. Annu Rev
Genet. 2012;46:419–41.
38. Navaratnam N, Morrison JR, Bhattacharya S, Patel D, Funahashi T,
Giannoni F, et al. The p27 catalytic subunit of the apolipoprotein B
mRNA editing enzyme is a cytidine deaminase. J Biol Chem.
1993;268:20709–12.
39. Morgan HD, Dean W, Coker HA, Reik W, Petersen-Mahrt SK.
Activation-induced cytidine deaminase deaminates 5methylcytosine in DNA and is expressed in pluripotent tissues:
Implications for epigenetic reprogramming. J Biol Chem.
2004;279:52353–60.
40. Duncan BK, Miller JH. Mutagenic deamination of cytosine residues in DNA. Nature. 1980;287:560–1.
41. Cooper DN, Mort M, Stenson PD, Ball EV, Chuzhanova NA.
Methylation-mediated deamination of 5-methylcytosine appears
to give rise to mutations causing human in-herited disease in
CpNpG trinucleotides, as well as in CpG dinucleotides. Hum
Genom. 2010;4:406–10.
42. Gorski DH, Walsh K. Control of vascular cell differentiation by
homeobox transcription factors. Trends Cardiovasc Med.
2003;13:213–20.
43. Trigueros-Motos L, González-Granado JM, Cheung C, Fernández
P, Sánchez-Cabo F, Dopazo A, et al. Embryological-origindependent differences in homeobox expression in adult aorta: role
in regional phenotypic variability and regulation of NF-κB activity.
Arterioscler Thromb Vasc Biol. 2013;33:1248–56.
44. Patel CV, Sharangpani R, Bandyopadhyay S, DiCorleto PE.
Endothelial cells express a novel, tumor necrosis factor-alpharegulated variant of HOXA9. J Biol Chem. 1999;274:1415–22.
45. Martin N, Popov N, Aguilo F, O’Loghlen A, Raguz S, Snijders AP,
et al. Interplay between homeobox proteins and Polycomb repressive complexes in p16INK4a regulation. EMBO J. 2013;32:982–95.
46. Helgadottir A, Thorleifsson G, Manolescu A, Gretarsdottir S,
Blondal T, Jonasdottir A, et al. A common variant on chromosome
Curr Atheroscler Rep (2013) 15:352
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
9p21 affects the risk of myocardial infarction. Science.
2007;316:1491–3.
McPherson R, Pertsemlidis A, Kavaslar N, Stewart A, Roberts R,
Cox DR, et al. A common allele on chromosome 9 associated with
coronary heart disease. Science. 2007;316:1488–91.
Liu Y, Sanoff HK, Cho H, Burd CE, Torrice C, Mohlke KL, et al.
INK4/ARF transcript expression is associated with chromosome
9p21 variants linked to atherosclerosis. PLoS One. 2009;4:e5027.
Bromleigh VC, Freedman LP. p21 is a transcriptional target of
HOXA10 in differentiating myelomonocytic cells. Genes Dev.
2000;14:2581–6.
Castillo-Díaz SA, Garay-Sevilla ME, Hernández-González MA,
Solís-Martínez MO, Zaina S. Extensive demethylation of normally
hypermethylated CpG islands occurs in human atherosclerotic arteries. Int J Mol Med. 2010;26:691–700.
Nazarenko MS, Puzyrev VP, Lebedev IN, Frolov AV, Barbarash
OL, Barbarash LS. Methylation profiling of human atherosclerotic
plaques. Mol Biol (Mosk). 2011;45:610–6.
Laukkanen MO, Mannermaa S, Hiltunen MO, Aittomäki S,
Airenne K, Jänne J, et al. Local hypomethylation in atherosclerosis
found in rabbit ec-sod gene. Arterioscler Thromb Vasc Biol.
1999;19:2171–8.
Lund G, Andersson L, Lauria M, Lindholm M, Fraga MF, VillarGarea A, et al. DNA methylation polymorphisms precede any
histological sign of atherosclerosis in mice lacking apolipoprotein
E. J Biol Chem. 2004;279:29147–54.
Carpinteyro-Espín P, Jacinto-Ruíz S, Caballero-Vazquez P,
Alvarado-Caudillo Y, Lund G, Rodríguez-Rios D, et al.
Organomegaly and tumors in transgenic mice with targeted expression of HpaII methyltransferase in smooth muscle cells.
Epigenetics. 2011;6:333–43.
Rangel-Salazar R, Wickström-Lindholm M, Aguilar-Salinas CA,
Alvarado-Caudillo Y, Døssing KB, Esteller M, et al. Human native
lipoprotein-induced de novo DNA methylation is associated with
repression of inflammatory genes in THP-1 macrophages. BMC
Genom. 2011;12:582.
• Alexander MR, Owens GK. Epigenetic control of smooth muscle
cell differentiation and phenotypic switching in vascular development and disease. Annu Rev Physiol. 2012;74:13–40. This is a
thorough and very informative review of VSMC phenotype
switching, with a focus on histone post-translational modifications.
Manabe I, Owens GK. Recruitment of serum response factor and
hyperacetylation of histones at smooth muscle-specific regulatory
regions during differentiation of a novel P19-derived in vitro
smooth muscle differentiation system. Circ Res. 2001;88:1127–34.
• Findeisen HM, Kahles FK, Bruemmer D. Epigenetic regulation of
vascular smooth muscle cell function in atherosclerosis. Curr
Atheroscler Rep. 2013;15:319. This is an exhaustive review on
chromatin remodelling and vascular cell phenotype modulation.
Jauch R, Kolatkar PR. What makes a pluripotency reprogramming
factor? Curr Mol Med. 2013;13:806–14.
Yoshida T, Kaestner KH, Owens GK. Conditional deletion of
Krüppel-like factor 4 delays downregulation of smooth muscle cell
differentiation markers but accelerates neointimal formation following vascular injury. Circ Res. 2008;102:1548–57.
Findeisen HM, Gizard F, Zhao Y, Qing H, Heywood EB, Jones KL,
et al. Epigenetic regulation of vascular smooth muscle cell proliferation and neointima formation by histone deacetylase inhibition.
Arterioscler Thromb Vasc Biol. 2011;31:851–60.
Kee HJ, Kwon JS, Shin S, Ahn Y, Jeong MH, Kook H. Trichostatin
A prevents neointimal hyperplasia via activation of Krüppel like
factor 4. Vascul Pharmacol. 2011;55:127–34.
Folkersen L, Kyriakou T, Goel A, Peden J, Mälarstig A, PaulssonBerne G, et al. Relationship between CAD risk genotype in the
chromosome 9p21 locus and gene expression. Identification of
eight new ANRIL splice variants. PLoS One. 2009;4(11):e7677.
Curr Atheroscler Rep (2013) 15:352
64. Burd CE, Jeck WR, Liu Y, Sanoff HK, Wang Z, Sharpless NE.
Expression of linear and novel circular forms of an INK4/ARFassociated non-coding RNA correlates with atherosclerosis risk.
PLoS Genet. 2010;6:e1001233.
65. Holdt L, Beutner F, Scholz M, Gielen S, Gäbel G, Bergert H, et al.
ANRIL expression is associated with atherosclerosis risk at chromosome 9p21. Arterioscler Thromb Vasc Biol. 2010;30(3):620–7.
66. Holdt LM, Teupser D. Recent studies of the human chromosome
9p21 locus, which is associated with atherosclerosis in human
populations. Arterioscler Thromb Vasc Biol. 2012;32:196–206.
67. Congrains A, Kamide K, Katsuya T, et al. CVD-associated noncoding RNA, ANRIL, modulates expression of atherogenic pathways in VSMC. Biochem Biophys Res Commun. 2012;419:612–6.
68. Yap KL, Li S, Munoz-Cabello AM, Raguz S, Zeng L, Mujtaba S,
et al. Molecular interplay of the noncoding RNA ANRIL and
methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell. 2010;38:662–74.
69. Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M,
et al. Long non-coding RNA ANRIL is required for the PRC2
recruitment to and silencing of p15(INK4B) tumor suppressor gene.
Oncogene. 2011;30:1956–62.
70. Leung A, Trac C, Jin W, Lanting L, Akbany A, Sætrom P, et al.
Novel long non-coding RNAs are regulated by angiotensin II in
vascular smooth muscle cells. Circ Res. 2013. doi:10.1161/
CIRCRESAHA.112.30084.
71. Nazari-Jahantigh M, Wei Y, Schober A. The role of microRNAs in
arterial remodelling. Thromb Haemost. 2012;107:611–8.
72. Wallberg AE, Yamamura S, Malik S, Spiegelman BM, Roeder RG.
Coordination of p300-mediated chromatin remodeling and TRAP/
Page 9 of 9, 352
73.
74.
75.
76.
77.
78.
79.
mediator function through coactivator PGC-1alpha. Mol Cell.
2003;12:1137–49.
Montes de Oca A, Madueño JA, Martinez-Moreno JM, Guerrero F,
Muñoz-Castañeda J, Rodriguez-Ortiz ME, et al. High-phosphateinduced calcification is related to SM22α promoter methylation in
vascular smooth muscle cells. J Bone Miner Res. 2010;25:1996–2005.
Byon CH, Sun Y, Chen J, Yuan K, Mao X, Heath JM, et al. Runx2upregulated receptor activator of nuclear factor κB ligand in calcifying smooth muscle cells promotes migration and osteoclastic
differentiation of macrophages. Arterioscler Thromb Vasc Biol.
2011;31:1387–96.
Konstantinopoulos PA, Papavassiliou AG. Seeing the future of
cancer-associated transcription factor drug targets. JAMA.
2011;305:2349–50.
Shalek AK, Satija R, Adiconis X, Gertner RS, Gaublomme JT,
Raychowdhury R, et al. Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature. 2013.
doi:10.1038/nature12172.
Yao X, Hu JF, Daniels M, Shiran H, Zhou X, Yan H, et al. A
methylated oligonucleotide inhibits IGF2 expression and enhances
survival in a model of hepatocellular carcinoma. J Clin Invest.
2003;111:265–73.
Di Micco S, Chini MG, Terracciano S, Bruno I, Riccio R, Bifulco G.
Structural basis for the design and synthesis of selective HDAC
inhibitors. Bioorg Med Chem. 2013. doi:10.1016/j.bmc.2013.04.036.
Saltis J, Thomas AC, Agrotis A, Campbell JH, Campbell GR,
Bobik A. Expression of growth factor receptors in arterial smooth
muscle cells. Dependency on cell phenotype and serum factors.
Atherosclerosis. 1995;118:77–87.