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109:916-926, 2010. First published Apr 22, 2010; doi:10.1152/japplphysiol.00131.2010 J Appl Physiol
Matthew Shu-Ching Yan, Charles C. Matouk and Philip A. Marsden
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HIGHLIGHTED TOPIC Epigenetics in Health and Disease
Epigenetics of the vascular endothelium
Matthew Shu-Ching Yan,
1
Charles C. Matouk,
2
and Philip A. Marsden
1,2,3
1
Department of Medical Biophysics,
2
Institute of Medical Sciences, University of Toronto;
3
Renal Division and Department of
Medicine, St. Michael’s Hospital and University of Toronto, Toronto, Ontario, Canada
Submitted 9 February 2010; accepted in final form 16 April 2010
Yan MS, Matouk CC, Marsden PA. Epigenetics of the vascular endothe-
lium. J Appl Physiol 109: 916 –926, 2010. First published April 22, 2010;
doi:10.1152/japplphysiol.00131.2010.—Classical models of transcription in vas-
cular endothelial cells, specifically the cis/trans paradigm, have limitations. For
instance, how does the environment have chronic effects on gene expression in
endothelial cells after weeks or years? When an endothelial cell divides, how is this
information transmitted to daughter cells? Epigenetics refers to chromatin-based
pathways important in the regulation of gene expression and includes three distinct,
but highly interrelated, mechanisms: DNA methylation, histone density and post-
translational modifications, and RNA-based mechanisms. Together they offer a
newer perspective on transcriptional control paradigms in vascular endothelial cells
and provide a molecular basis for understanding how the environment impacts the
genome to modify disease susceptibility. This alternative viewpoint for transcrip-
tional regulation allows a reassessment of the cis/trans model and even helps
explain some of its limitations. This review provides an introduction to epigenetic
concepts for vascular biologists and uses topical examples in cell biology to provide
insight into how cell types or even whole organisms, such as monozygotic human
twins with the same DNA sequence, can exhibit heterogeneous patterns of gene
expression, phenotype, or diseases prevalence. Using endothelial nitric oxide
synthase (NOS3) as an example, we examine the growing body of evidence
implicating epigenetic pathways in the control of vascular endothelial gene expres-
sion in health and disease.
cell-specific expression; DNA methylation; histone code; endothelial nitric oxide
synthase
DOES THE CURE for cardiovascular disease, especially atheroscle-
rosis, lie in our genes? The promise of the postgenome period
argues that it is. In the contrary, we argue that the cure lies in
defining how the genome interacts with the environment in
which our cells exist. This view is significant because it
encompasses epigenetics.
The International Human Epigenome Consortium (IHEC)
was launched in January 2010 (1). Looking back, the proposal
for sequencing the human genome seemed a daunting task
when launched in 1990. The static genetic code is the same in
every diploid human cell, save for germline rearrangements in
the T-cell receptors and B-cell receptors in T- and B-cells,
respectively, and somatic DNA mutation or copy number
variations, among others. Although DNA sequence variation
can have important effects on epigenetic modifications, the
extent of this diversity is not fully appreciated as evidenced by
⬃250 distinct cell types in the human organism (1). Scientists
are also unsure whether important degrees of heterogeneity
exists in cells of the same lineage for epigenetic marks at
identical haplotypes, sets of alleles at multiple loci on the same
chromosome that are commonly transmitted together. Remem-
bering that the term “epigenetics” was initially used to refer to
the complex interactions between the genome and the environ-
ment that are involved in development and differentiation in
higher organisms, reminds us that the epigenetic code is
superimposed on the static genetic code. Today, the term
“epigenetics” is used to refer to heritable alterations in chro-
matin that are not due to changes in DNA sequence per se (6).
The potential exists, when taken together, therefore, for sub-
stantially higher levels of epigenetic diversity that is distributed
in time and space. This is significant as we have, for some time,
accepted that common diseases of the human cardiovascular
system are influenced by complex interactions between the
genome and environment. For example, atherosclerosis has
well-defined genetic determinants as well as environmental
risk factors. Although poorly understood, the epigenetic per-
spective is shedding new light on how the environment influ-
ences gene expression and disease susceptibility (27). Perhaps
most importantly, the dynamic nature of epigenetic modifica-
tion offers the possibility of therapeutic intervention. To date,
the roles of these pathways in vascular endothelial biology are
only beginning to be explored (61).
Address for reprint requests and other correspondence: P. A. Marsden, Rm
7358, Medical Sciences Bldg., Univ. of Toronto, 1 King’s College Circle,
Toronto, Ontario M5S 1A8 (e-mail: p.marsden@utoronto.ca).
J Appl Physiol 109: 916–926, 2010.
First published April 22, 2010; doi:10.1152/japplphysiol.00131.2010.Review
8750-7587/10 Copyright ©2010 the American Physiological Society http://www.jap.org916
on September 10, 2010 jap.physiology.orgDownloaded from
This review takes some topical examples from general
biology to introduce how epigenetics is relevant in disparate
settings (Fig. 1). We also present a conceptual framework for
understanding the role of epigenetics in complex non-Mende-
lian diseases, including common cardiovascular diseases. We
will use studies performed on the endothelial nitric oxide
synthase (eNOS, NOS3) gene to illustrate key concepts that are
relevant to gaining an understanding of how epigenetic path-
ways regulate vascular endothelial gene expression in health
and disease.
EPIGENETIC BASIS FOR DIFFERENCES IN GENE EXPRESSION
WHEN THE DNA SEQUENCE IS IDENTICAL—APPLYING
CONCEPTS TO CARDIOVASCULAR DISEASE
Unlike familial monogenic disorders, non-Mendelian dis-
eases share some peculiarities that are difficult to explain using
current genetic paradigms. Similarly, cis/trans paradigms of
gene expression, the concept of particular transcription factors
(trans factors) binding to canonical promoter DNA elements to
mediate a distinct transcriptional program, also cannot fully
explain these pecularities. The pecularities that are commonly
demonstrated by non-Mendelian diseases include discordance
between monozygotic twins, sexual dimorphism, parent-of-
origin-dependent clinical differences, progression of disease
severity over time, and a relatively late age of onset (71). These
characteristics are true for a number of common cardiovascular
diseases, such as atherosclerosis and hypertension. The clas-
sical argument is to ascribe these disease characteristics to
poorly defined environmental influences (both internal and
external to the cell). We argue here that the molecular
mechanisms that translate environmental influences onto the
genome may well be epigenetic in basis by demonstrating
Fig. 1. Epigenetic pathways are broadly relevant to cardiovascular disease. A: somatic cell nuclear transfer is a cloning strategy that involves the transplantation
of a donor nucleus from an adult somatic cell (blue) into an enucleated oocyte (pink). In reproductive cloning, the nuclear transfer embryo is implanted into a
pseudopregnant female for the purpose of generating a genetically identical cloned animal. The prevailing model for the low efficiency of reproductive cloning
and the abnormal phenotypes of cloned animals is faulty nuclear reprogramming, a fundamental process governed by the epigenetic state of the nucleus,in
particular, DNA methylation (Œand , unmethylated and methylated CpG dinucleotides, respectively). B: discordance of monozygotic twins for disease
phenotype in common cardiovascular disease may reflect different epigenetic states at a given genetic locus. C: Hutchinson-Gilford progeria syndrome and
normal human aging evidences progressive epigenetic changes over time. The loss of heterochromatin is one example of these epigenetic changes.
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how epigenetics is relevant in three biological settings (Fig.
1) (27, 61, 71).
Reprogramming Differentiated Cells
Somatic cell nuclear transfer is a cloning strategy that injects
the nucleus from a donor somatic cell into a freshly fertilized
enucleated oocyte. In reproductive cloning, this embryo is
subsequently implanted into a pseudopregnant female to gen-
erate a genetically identical clone (46; Fig. 1A). The first
mammal cloned from a somatic cell is Dolly the sheep (93).
Accrued experimental evidence in a number of mammalian
species has demonstrated that reproductive cloning is an ex-
tremely inefficient process, with the vast majority of cloned
embryos dying in utero (102). The few nuclear transfer em-
bryos that do survive show abnormal phenotypes, such as
premature death, as exemplified by Dolly after accounting for
technical failures of nuclear transfer (46). The prevailing
model for the low efficiency of reproductive cloning is faulty
nuclear reprogramming. This is likely because much is asked
for successful reproductive cloning to occur. Namely, it re-
quires the somatic cell to dedifferentiate to a totipotent state
and redifferentiate to form a viable, adult organism (102).
Mechanistically, this is mediated by pronounced changes in
gene expression that reflect, in part, dynamic changes in the
cellular chromatin landscape. To date, the best understood
epigenetic mark in early embryonic development is DNA
methylation (102). In both male and female haploid gametes,
the amount of DNA methylation is high. Shortly after fertili-
zation, the male pronucleus is specifically actively demethyl-
ated, while the female pronucleus undergoes passive demeth-
ylation (63). In mice, genome-wide de novo methylation fol-
lows at the blastocyst stage. In cloned embryos, the male and
female contributions to the somatic cell nucleus might not be
adequately distinguished and widespread abnormalities, such
as defective genomic imprinting and X-chromosome inactiva-
tion, ensue from abnormal DNA methylation patterning (23,
46, 102). Interestingly, normal breeding of these mice yields
offspring with normal phenotype. If the abnormal phenotypes
were the result of genetic mutations, these would presumably
be faithfully inherited across generations. Thus these data
suggest an epigenetic basis for the abnormal phenotypes of
these “cloned” animals (46, 102).
A further example of applied epigenetics in reprogramming
is inducible pluripotent stem cell (iPS cells) generation. iPS
cells are somatic cells reprogrammed to an embryonic stem
(ES) cell-like state via the ectopic expression of ES cell-related
transcriptional factors, such as Oct3/4, Sox2, Klf4, c-Myc,
Nanog, Esrrb, and/or Lin2 (28, 41). iPS cells are ES-cell like
by virtue of their self-renewing and totipotent properties,
similar cellular phenotypes, gene expression patterns, and epi-
genetic profiles (41, 67). Clinicians are excited because it is
anticipated that patient-specific iPS cells may offer newer
approaches to treat cardiovascular diseases. Although the mo-
lecular mechanism behind iPS cell generation is unclear, epi-
genetic pathways appear to play a fundamental role. This was
demonstrated by studies in partially reprogrammed somatic
cells, which are characterized by reactivation of a distinct
subset of stem cell-related genes, incomplete repression of
lineage-specifying transcription factors, and incomplete epige-
netic remodeling (67). On treatment with 5-azacytidine, an
inhibitor of DNA methylation activity, these partially repro-
grammed cells undergo a rapid, stable transition to fully
reprogrammed iPS cells (67). Practically, we now know that
pharmacological agents that affect various chromatin modifi-
cations enhance the efficiency of iPS cell generation (41).
Monozygotic Twins
Human monozygotic (MZ) twins provide a natural experi-
mental system to explore the contributions of epigenetic mech-
anisms to phenotypic variance (Fig. 1B). Classically, twin
studies are used to determine the relative contributions of
genetic and environmental factors to a disease phenotype. MZ
twins are genetically identical, whereas dizygotic (DZ) twins
share approximately half of their genetic code with equal
contributions from each parent. A strong genetic component of
disease is inferred if disease prevalence is increased among MZ
versus DZ twins and this is quantified in the metric heritability
(61). Although heritability estimates a strong genetic contribu-
tion (30 –50%) for common cardiovascular diseases, MZ twin
pairs frequently show low concordance rates for disease phe-
notype (38, 62). The classical explanation for this apparent
paradox is the differential effect of the environment on genet-
ically identical individuals, which is arguably regulated by
epigenetic pathways.
In 2005, Fraga et al. (32) catalogued the global and locus-
specific differences in DNA methylation and histone H3 and
H4 acetylation in a large cohort of MZ twins of various ages.
Comparison of the epigenetic profiles within twin pairs re-
vealed several key observations. First, the most epigenetically
similar and dissimilar twins (at least for the three epigenetic
marks surveyed) were the youngest and oldest pairs, respec-
tively. Second, twin pairs with the most discordant epigenetic
profiles spent less of their lifetime together and/or reported the
greatest differences in natural health/medical history. Finally,
the degree of epigenetic discordance within twin pairs ap-
peared to correlate with the degree of intra-twin pair differen-
tial mRNA expression.
Although the study of this MZ twin study did not allow
correlation with disease discordance, studies by others have
demonstrated that discordance for some diseases, such as
Alzheimer’s disease, are associated with global or loci-specific
differences in DNA methylation status (53, 60, 100). It would
be of interest to directly assess epigenetic changes temporally
in individual MZ twins. Nonetheless, studies support the notion
that environment-dependent epigenetic modulations acquired
throughout an individual’s life span might affect human gene
expression and health (32, 61).
Hutchinson-Gilford Progeria Syndrome and Human Aging
Although common cardiovascular diseases demonstrate non-
Mendelian patterns of inheritance, much can be learned about
them by studying monogenic disorders. One example is the
Hutchinson-Gilford progeria syndrome (HGPS), a childhood
disease of premature aging that occurs in 1 of 4 million live
births. Affected children are diagnosed at a young age with
failure to thrive and prototypical skin abnormalities reminis-
cent of aging, such as prominent cutaneous vasculature. These
children develop severe atherosclerosis and die from myocar-
dial infarction and stroke at ⬃13 yr of age (66). HGPS results
from a specific mutation (a C-to-T substitution, 1824C¡T) in
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the LMNA gene encoding lamin A, the major structural com-
ponent of the nuclear lamina juxtaposed between the inner
membrane of the nuclear envelope and chromatin (25). This
genetic mutation activates a cryptic splice donor site resulting
in a new mRNA species that is translated to a novel protein,
progerin, with a 50 amino acid internal deletion. Progerin
induces dysmorphic nuclei with nuclear blebbing that progress
over time (25, 34). Although how progerin causes HGPS is
unclear, evidence suggests that changes in epigenetic pathways
are seminal. Similar to normally aged cells, the normal orga-
nization and structure of chromatin is disrupted in HGPS nuclei
(25, 34, 75, 76). In particular, these cells demonstrate dramat-
ically reduced heterochromatin, regions of limited transcription
that are preceded by the progressive loss of repressive epige-
netic marks including trimethylated H3K9 and H3K27 (79;
Fig. 1C). This loss of epigenetic control appears to be corre-
lated with widespread abnormalities in gene expression (76,
79). Since dramatic epigenetic changes occur in HGPS and
normal aging, and HGPS patients exhibit accelerated athero-
sclerosis, we argue that studies of epigenetic pathways in
human atherosclerosis are warranted and timely. This is further
supported by the fact that progerin activates the effectors of the
Notch signaling pathway, which plays a role in endothelial
dysfunction (35, 74). It is noteworthy that the abnormal phe-
notype of HGPS in cell culture and transgenic mice models can
be reversed by inhibiting progerin expression, thereby under-
scoring the dynamic nature of epigenetic pathways (73, 76).
Taken together, epigenetic pathways exert considerable in-
fluence on the genome’s structure and function from the
earliest time in development, throughout the normal and ab-
normal process of aging and nuclear reprogramming (summa-
rized in Fig. 1). Specifically, in the three examples presented
here, epigenetic mechanisms appear to be involved in regulat-
ing phenotypic characteristics that cannot be fully defined by
genetics or cis/trans regulation of gene expression. By trans-
lating the effects of environmental stimuli into coordinated
gene expression programs for cellular adaptation, epigenetic
pathways are potentially the mechanistic link between the
genome and environment that is important in understanding
common cardiovascular diseases.
OVERVIEW OF EPIGENETIC MECHANISMS
The molecular foundation of epigenetic theory is comprised
of three highly interconnected pathways: DNA methylation,
histone posttranslational modifications, and RNA-based mech-
anisms (Fig. 2). Together, they modulate the structure and
accessibility of DNA, thereby providing an important regula-
tory level of transcriptional control. Specifically, the three
mechanisms are involved in the formation of euchromatin and
heterochromatin. These older terms are still useful in convey-
ing concepts. In general, euchromatin represents decondensed
chromatin that is actively transcribed and affiliated with acti-
vating epigenetic marks. In contrast, heterochromatin is con-
densed chromatin that has limited transcription and is associ-
ated with repressive epigenetic marks (85). Over the last 20
years, significant progress has been made in understanding the
epigenetic marks, the processes that create and erase them, and
Fig. 2. Three fundamental mechanisms of epigenetic gene regulation. Epigenetic mechanisms of gene expression are subserved by three distinct, yet highly
interrelated, mechanisms. 1) DNA methylation refers to the addition of a methyl group to the 5-position of cytosine in the context of CpG dinucleotides to define
the “fifth base of DNA.” 2) The fundamental repeating unit of chromatin is the nucleosome comprised of an octamer of core histone proteins. Posttranslational
modifications of the amino-terminal tails of histone proteins (light and dark blue balls) and the density of these proteins per unit length of DNA, can importantly
affect chromatin structure and constitute a putative “histone code.” 3) RNA-based mechanisms have also recently been shown to impact on the higher-order
structure of chromatin.
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the readers that interpret them. This section provides a brief
review of epigenetic pathways in mammals.
DNA Methylation
DNA methylation refers to the addition of a methyl group to
the 5-position of cytosine to create 5-methyl-cytosine (68).
There is an inverse correlation between DNA methylation at
promoter regulatory regions and gene transcription (64). As
such, this review will focus on its repressive function.
DNA methylation has functional roles in X chromosome
inactivation, genomic imprinting, embryonic development, and
lineage specification (7, 68). Its dysregulation, in part, defines
the tumor cell phenotype. DNA methylation at cytosine resi-
dues occurs almost exclusively in the context of the sequence
CpG. However, non-CpG methylation has been observed in
early development (58), endogenous LINE-1 retroelements
(95), and integrated plasmid DNA (17). DNA methylation is
catalyzed by three distinct enzymes that are collectively known
as DNA methyltransferases (DNMTs). DNMT1, the “mainte-
nance” methyltransferase, is believed to transmit DNA meth-
ylation patterns during mitotic cell division. In contrast,
DNMT3a and DNMT3b function act as de novo methyltrans-
ferases that establish DNA methylation patterns during embry-
onic development (68). The mechanisms responsible for DNA
demethylation remain poorly defined, but include both passive
(replication dependent) and active (replication independent)
processes (68). Interestingly, 5-methyl-cytosine can be hy-
droxylated into 5-hydroxymethylcytosine in murine ES cells
and the purkinje and granule cells of the brain. 5-Hydroxym-
ethylcytosine might be an intermediate of either DNA demeth-
ylation processes (52, 81).
Three general mechanisms have been proposed for 5-methyl-
cytosine-mediated gene repression. First, 5-methyl-cytosine can
sterically interfere with transcription factors to their cis-DNA
binding elements. This mechanism has been described for
several transcription factors, including hypoxia-inducible fac-
tor-1␣(HIF1␣), but not others (5, 20, 37, 92). Second, methyl-
CpG binding proteins, such as MeCP2, can interfere with the
recruitment of transcriptional machinery, such as DNA-bind-
ing trans factors. Third, methyl CpG binding proteins can
recruit large chromatin modifying complexes that reduce DNA
accessibility (68). For example, MeCP2 can recruit HDACs,
histone methyltransferases, and the ATP-dependent Swi/Snf
chromatin remodeling complex (68).
Histone Protein
In the nucleus, DNA is packaged into chromatin as repeating
units of nucleosomes, which form a “beads-on-a-string” struc-
ture that can compact into higher order structures to affect gene
expression. Nucleosomes are composed of 146-bp DNA
wrapped in histone octamers (composed of two H2A, H2B,
H3, and H4) and are connected by a linker DNA, which can
associate with histone H1 to form heterochromatin (86). His-
tone proteins contain a globular domain and an amino-terminal
tail, with the latter being posttranslationally modified. Currently,
more than 60 modifications have been described, including the
posttranslational modification of lysine (acetylation, methylation,
ubiquitnation, sumolyation), arginine (methylation), and serine
and threonine (phosphorylation) (7, 89). Many of these modifica-
tions are known to play functional roles in transcription (Table 1).
The histone code hypothesis proposes that the combination of
histone posttranslational modifications encode regulatory infor-
mation interpretable by the cell (80). An increasing number of
proteins that specifically recognize unique posttranslational mod-
ifications are being uncovered (89).
The functional roles of lysine acetylation and methylation on
gene expression are best understood. The most important nucleo-
somes here are commonly at the promoter regions. Histone
acetylation is associated with transcription activation and is dy-
namically regulated by the competing enzymatic activities of
histone acetlytransferases (HATs) and histone deacetylases
(HDACs), which mediate its addition and removal, respectively.
Although HATs and HDACs can non-specifically regulate the
acetylated states of proteins, their specificity in histone modifica-
tion is achieved, in part, by their recruitment to chromatin in
multiprotein complexes (89). Histone acetylation is believed to
enhance transcription by neutralizing the basic charges of lysine
residues and recruiting bromodomain-containing proteins, includ-
ing other HATs and chromatin remodeling enzymes, that prevent
chromatin compaction (78).
In contrast to histone acetylation, the impact of histone
lysine methylation on gene expression is dependent on the
specific lysine residue. For example, genome-wide profiles of
histone methylation show that H3K4 and H3K36 methylation
are associated with transcriptionally permissive chromatin,
whereas H3K9 and H3K27 methylation are markers of tran-
scriptionally silent chromatin (4). In addition, single lysine
residues are variably methylated to mono-, di-, and trimethyl-
ated states. This can be contrasted with addition of a single
acetyl group. Some lysine residues can be modified by either
methylation or acetylation, but never both together. The dif-
ferent histone methylation states are functionally relevant.
Active promoters are enriched in trimethylated H3K4, while
enhancer elements are enriched in monomethylated H3K4
(39). Similar to histone acetylation, histone methylation status
at a particular lysine is dynamically regulated by histone meth-
yltransferases and histone demethylases (18). The molecular
Table 1. Histone posttranslational modifications
and gene transcription
Histone Posttranslational Modification Transcriptional Effect
Histone H3
Acetylation (K9, K14) 1
Methylation
K4 (Trimethyl) 1
K9 (Trimethyl) 2
K27 (Trimethyl) 2
K36 (Trimethyl) 1
K64 (Trimethyl) 2
R2 (Dimethyl) 2
Phosphorylation
S10 1/2
T6 1
T11 1
Histone H4
Acetylation (K5, K8, K12, K16) 1
Methylation
K20 (Trimethyl) 2
Histone H2A
Ubiquitination (K119) 2
Histone H2B
Ubiquitination (K120) 1
K denotes lysine; R, arginine; S, serine; T, threonine.
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mechanisms behind the functional effects of histone methylation
marks are being uncovered. For instance, trimethylated H3K4 is
implicated in recruiting PHD finger-containing proteins to recruit
chromatin remodeling complexes and transcription machinery to
promote transcription (87, 97). In contrast, trimethylated H3K9
recruits heterochromatin protein 1 to form transcriptionally silent,
constitutive heterochromatin (7, 89).
Another facet of histone biology that is involved in tran-
scriptional regulation is histone density. In general, the histone
density at the transcriptional start site of expressed genes is
depleted relative to non-expressed genes, suggesting that low
histone density is associated with transcription (77). However,
histone density can be altered to activate or repress specific
genes in response to cellular activation. For instance, T-cell
activation results in the loss of histones at the IL-2 promoter.
This acute change in histone density is functionally relevant to
enhanced IL-2 gene transcription (14). In contrast, the repres-
sion of cyclin A, a cell cycle regulator, in quiescent cells is, in
part, due to the maintenance of histone density at its promoter
by Brahma containing chromatin remodeling complexes (19).
RNA-Based Mechanisms
RNA-based mechanisms of epigenetic gene regulation involve
the coordinated activities of noncoding RNAs (ncRNA) with
other epigenetic activities, such as DNA methylation and histone
posttranslational modifications. Studies suggest that long and
short ncRNAs, which are distinguished by an arbitrary size cutoff
of 200 nucleotides, can regulate the chromatin state of genomic
loci (65). A large number of large intervening non-coding RNAs
(lincRNAs) have just been described in the genomes of humans
and mice. A total of ⬃3,300 lincRNAs have been identified with
computationally predicted roles in various cellular processes,
including cell-cycle regulation (36, 50). lincRNAs can recruit
chromatin modifying activity and regulate gene expression at
target loci (50). An excellent example is HOTAIR, which aids in
HoxD silencing by recruiting polycomb repressive complex 2 and
its H3K27 trimethylation activity (65). Long ncRNAs that overlap
protein-coding genes can also recruit chromatin modifying com-
plexes to regulate gene expression at target loci. Examples of this
include Xist, which is involved in X chromosome inactivation.
Additional examples include Air and Kcnq1ot1, which are in-
volved in genomic imprinting, a process that mediates the expres-
sion of only one allele of a gene in a parent-of-origin-dependent
manner (65). In addition, long ncRNAs can potentially mediate
transcriptional activation via recruitment of the H3K4 methyl-
transferase MLL1 (65). Transcriptional silencing by small ncRNA
in mammals may also occur, but is poorly understood. DNA
methylation and repressive histone modifications can be elicited at
target gene promoters following treatment of cells with exogenous
administration of small interfering RNAs (siRNAs)(69). Interest-
ingly, a similar phenomenon may be mediated by endogenous
miRNAs (69). Taken together, these observations suggest a fun-
damental role of RNA-based mechanisms in gene regulation.
eNOS: FIRST CLUE TO THE IMPORTANCE OF
EPIGENETIC REGULATION OF VASCULAR ENDOTHELIAL
GENE EXPRESSION
The constitutively expressed endothelial NOS (NOS3) is the
main source of NO in the vascular endothelium and is pivotal
for its function (61). eNOS-null mice are characterized by
systemic and pulmonary hypertension, impaired wound heal-
ing and angiogenesis, impaired mobilization of stem and pro-
genitor cells for neovascularization, and reduced vascular leak-
age during acute inflammation (2, 9, 42, 55, 72). Due to the
pivotal physiological role of eNOS in the vascular endothe-
lium, its regulation has been extensively studied.
eNOS is a member of an unique set of endothelial-restricted
genes that define endothelial cell identity. In contrast to other
cell types such as skeletal muscle or adipocytes, there are no
known “master regulators” of gene expression, such as MyoD
or PPAR-␥, respectively, that are specifically expressed only in
ECs (56, 82). A number of transcription factors have been
shown to be preferentially expressed in differentiating endo-
thelial progenitor cells and mature ECs and have been argued
to orchestrate the expression of a wide number of endothelial
genes. Indeed, the promoters and enhancers of endothelial-
restricted genes are commonly enriched with cis-binding ele-
ments recognized by such factors, including Sp-1, forkhead,
and Ets proteins, among others (21, 29). Additionally, a 44-bp
enhancer containing the composite cis-binding element of
Forkhead and Ets proteins has been found to be present at
many endothelial-restricted gene enhancers and is sufficient for
directing endothelial-specific expression (22). However, the
paradox is that these transcription factors are not restricted in
expression to the vascular endothelium. Thus the concept of a
master transcription factor (trans factor) binding to a canonical
promoter DNA element (cis element), the cis/trans paradigm,
that is uniquely evident in EC-enriched genes has, to date, not
been substantiated by published work. Nonetheless, is there
additional regulatory information that allows ubiquitous tran-
scription factors to distinguish and induce the appropriate
expression of endothelial-restricted genes? One possible source
of information is their chromatin accessibility.
eNOS is the most well-characterized example of an endo-
thelial-restricted gene that is regulated by its chromatin acces-
sibility. eNOS evidences a TATA-less promoter with two 5=
cis regulatory element, known as positive regulatory domain I
and II, that are situated ⫺104/⫺95 and ⫺144/⫺115, respec-
tively, from its single major transcriptional start site (TSS) (49,
59), In addition, eNOS has a 269-bp enhancer that is ⫺4.9 kb
from the TSS (54). Similar to other endothelial-restricted
genes, the regulatory DNA elements of eNOS can bind ubiq-
uitous transcription factors, including Sp1 and the Ets (54, 61).
Although transient transfection of eNOS promoter-reporter
constructs into various expressing and non-expressing cells
show robust promoter activity (12), eNOS promoter-reporter
transgenic mice show endothelial-restricted expression (83).
These observations suggest that the chromatin context of eNOS
is involved in regulating its endothelial-restricted expression.
Indeed, the chromatin structure at the eNOS promoter is tran-
scriptionally permissive in endothelial cells and repressive in
nonendothelial cells. Specifically, the eNOS promoter in endothe-
lial cells was found to be DNA hypomethylated and enriched with
activating histone posttranslational modifications, including acety-
lated H3K9, acetylated H4K12, di- and trimethylated H3K4, by
sodium bisulfite genomic DNA sequencing analysis and ChIP
analysis, respectively (12, 30) (Fig. 3). In contrast, similar analysis
of the eNOS promoter in non-expressing cell types, such as
vascular smooth muscle cells (VSMCs), showed DNA hyper-
methylation and a lack of activating histone posttranslational
modifications. Consistent with the differences in the chromatin
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structure of the eNOS promoter, ChIP analysis showed selected
recruitment of Sp1, Sp3, Ets transcription factors and RNA
polymerase II to the eNOS proximal promoter in endothelial cells,
while MeCP2 and HDAC1 were specifically localized to the
promoter in VSMCs (12, 30, 33) (Fig. 3). The functional impor-
tance of DNA methylation and histone posttranslational modifi-
cations at the eNOS promoter was demonstrated by pharmaco-
logical inhibition studies. Namely, treatments of VSMC with
5-azacytidine, a DNMT inhibitor, and trichostatin A, a HDAC
inhibitor, upregulated eNOS mRNA levels. In contrast, eNOS
expression was downregulated in endothelial cells when treated
with methylthioadenosine, a H3K4 methylation inhibitor.
eNOS is also regulated by RNA-based mechanisms. A 27-nt
RNA duplex produced at the variable number tandem repeat
region (VNTR) of intron 4 in eNOS was found to be expressed
and localized to the nucleus of endothelial cells exclusively
(103, 105). Interestingly, exogenous administration of the
small RNA to endothelial cells induced H3K9 and H4K12
hypoacetylation at the eNOS promoter, DNA methylation at
exon 3 of eNOS, and reduced eNOS transcription (103, 104).
The repressive function of the small RNA was supported by the
ability to salvage eNOS expression in the small RNA trans-
fected endothelial cells by HDACIII depletion and treatments
with trichostatin A and 5-azacytidine (104). Although the
biological relevance of micromanaging eNOS transcription by
the 27-nt RNA duplex is unknown, it is clinically relevant that
copy number polymorphism of the eNOS VNTR is associated
with risk for ischemic heart disease (10).
Taken together, chromatin-based mechanisms of gene regula-
tion ensure that eNOS expression is restricted to endothelial
cells at, perhaps, an appropriate level. It is important to note
that chromatin-based gene regulation is observed in other
endothelial-restricted genes, including vWF, Notch4, and
E-selectin (24, 70, 96).
THE EPIGENETIC PERSPECTIVE ON HUMAN
CARDIOVASCULAR DISEASE
Recent years have witnessed an increased appreciation for
the potential of modulating epigenetic pathways to treat dis-
ease. For example, pharmacological HDAC inhibitors are un-
der investigation in treating cancers (40) and have shown
promise in treating chronic inflammatory diseases, including
rheumatoid arthritis among others (3, 57, 88). However, the
Fig. 3. eNOS—a model system for studying epigenetic pathways in the vascular endothelium. A: proximal eNOS promoter in expressing endothelial cells is
defined by an open chromatin configuration, lack of DNA methylation, and the preferential enrichment of activating posttranslational histone modifications. This
enables the efficient recruitment of the transcriptional machinery, including RNA polymerase II, to the eNOS proximal promoter and productive transcription.
B: in contrast, the proximal eNOS promoter in nonexpressing cell types demonstrates a closed chromatin configuration. DNA methylation is a prominent feature
with the recruitment of repressive proteins, for example, methyl-CpG-binding protein, MeCP2. Activating histone posttranslational modifications are absent, and
the gene is not actively transcribed.
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demonstration that TSA treatment of atherosclerosis-prone
Ldlr
⫺/⫺
mice exacerbates neointimal lesions underscores the
need for improving our understanding of epigenetic pathways
in cardiovascular disease (16). From this perspective, the
contribution of epigenetic pathways in the endothelial response
to external stimuli, including the physical forces of circulation
(e.g., shear stress), hypoxia , cytokines (11, 45, 90), and entry
into the cell cycle (61), are being explored (Fig. 4).
Interestingly, laminar shear stress can elicit both global and
gene-specific histone modification changes in cultured human
endothelial cells (43). Shear stress can affect changes in global
histone modification in mouse ES cells and promote their
differentiation to an endothelial cell lineage (44, 99). Laminar
shear stress can also induce histone modifications at specific
sites in the genome as demonstrated by the dependency on
p300/HAT-mediated H3 and H4 acetylation in laminar flow-
induced eNOS expression (13). Since laminar flow can affect
gene regulation via epigenetic pathways, disturbed flow may
impinge on them to regulate gene expression. Whether epige-
netic pathways contribute to the susceptibility of different
regions in the vasculature to atherosclerosis is worth consid-
ering, especially since the expression of eNOS, an atheropro-
tective gene, is lower at regions of the mouse aorta with high
probability (HP) of developing atherosclerosis compared with
regions with low probability (LP) of developing the disease
(84, 94).
Hypoxia has major effects on endothelial phenotype. In
general, hypoxia decreases global transcriptional activity (48).
The hypoxia-inducible factor (HIF) transcription paradigm is
an ancient eukaryotic response that allows cells to adapt to
changes in oxygen supply or availability. Evidence suggests
that epigenetic pathways are also relevant. In contrast to the
HIF cis/trans transcriptional paradigm, which is well studied,
the effects of hypoxia on chromatin-based pathways is a ripe
area for detailed study. Concomitant with this, hypoxia induces
a global decrease in H3K9 acetylation in various cells as a
possible consequence of HDAC upregulation (47, 48). How-
ever, acetylated H3K9 is enriched at the promoters of hypoxia-
activated genes, such as VEGF (31, 47, 48).
In contrast to histone acetylation, hypoxia-mediated changes
in histone methylation are more complicated and also a newer
area for study. Consistent with decreased global transcriptional
activity under hypoxic conditions, increased global H3K9
dimethylation, a repressive histone mark, has been observed
across different cells and is attributed, in part, to increased G9a
histone methyltransferase expression (47, 48). Although other
repressive histone methylation marks increase globally, global
di- and tri-methylated H3K4 levels, which are activating his-
tone marks, are paradoxically elevated (48). It is tempting to
attribute this to the decreased catalytic activities of oxygen-
dependent JmjC-demethylase domain-containing histone dem-
ethylases. This is because structural studies on the JmjC
domain of JmjD1a show a similarity to Fe(II)- and 2-oxoglu-
tarate-dependent dioxygenases, whose catalytic activities are
responsive to cellular oxygen levels (15). However, the mRNA
levels of 17 of 22 JmjC-domain family members are upregu-
lated by hypoxia (98). In fact, JmjD1A, JmjD2B, JmjD2C,
JARID1B are directly regulated by the HIF transcription fac-
tor, the heterodimeric master regulator of the hypoxia-induced
gene transcription program (8, 98, 101). Histone demethylase
upregulation might be a compensatory mechanism for mini-
mizing increases in global histone methylation levels as dem-
onstrated by an increase in global trimethylated H3K4 levels in
hypoxic cells with a disrupted HIF pathway (98). This is
significant, as it suggests that HIF is involved in maintaining
global transcriptional silencing, as well as directing gene re-
pression at specific genes (26, 91). However, in contrary to the
compensatory role of histone demethylase in global histone
methylation levels, depletion of methyl H3K9 demethylases,
JmjD2B and JmjD1A, does not affect global di- and tri-
methylated H3K9 levels (8).
How is a distinct epigenetic signature established in hy-
poxia-regulated gene promoters? One possibility is that the
original epigenetic signature of a hypoxia-regulated gene is
reset and established anew. In support of this, Fish et al. (31)
demonstrated that rapid eNOS transcriptional repression in
hypoxic endothelial cells is associated with a decrease in
histone H3 and H4 acetylation levels at the eNOS proximal
promoter. This is mediated by histone eviction and subsequent
reincorporation of histones that lack substantial modifications.
Although not observed at eNOS, it is possible that the reincor-
Fig. 4. The epigenetic perspective on human cardiovascular disease. Epigenetic pathways, which are important in the transcriptional control of gene expression,
are responsive to various physiological and pathophysiological cues relevant to the health of the vascular endothelium. Some of these cues are shear stress (blood
flow), hypoxia, cytokines, and entry into the cell cycle. Their ability to act as molecular integrators of environmental signals internal and external to the cell forms
the basis for this fundamentally new, epigenetic perspective on human cardiovascular disease.
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porated histones are modified to establish a distinct hypoxic
epigenetic signature at other hypoxia-regulated genes.
The effects of hypoxia on global levels of DNA methylation
are just beginning to be studied. Fish et al. (31) recently
reported that acute (4 h) or chronic (24 h) hypoxia does not
have a major effect on global levels of endothelial cell DNA
methylation. Little is known about whether DNA methylation
levels are altered at specific genes under hypoxic conditions to
regulate transcription.
Our current understanding of hypoxia-regulated epigenetic
pathways, as discussed above, is relatively sparse. Future
genome-wide mapping of specific acetyl and methyl histone
modifications, histone demethylases, histone density, and DNA
methylation in hypoxic cells will be necessary to fully under-
stand their importance in transcriptional regulation and forma-
tion of distinct hypoxia-mediated epigenetic signatures at hy-
poxia-regulated genes. This may be therapeutically useful as
shown by the finding that TSA can blunt hypoxia-inducible
angiogenesis of mature endothelial cells (51). This finding
suggests that manipulation of the epigenetic pathways may be
clinically relevant in inhibiting tumor angiogenesis.
SUMMARY
So pervasive is the role of epigenetic pathways in the
response of endothelial cells to physiological and pathophysi-
ological stimuli that it represents a fundamentally new perspec-
tive on human cardiovascular disease. This perspective is
exciting given the possibility of therapeutic intervention by
environmental and pharmacological modulation of epigenetic
pathways. Additional studies that expand our understanding of
chromatin-based regulation of endothelial restricted gene ex-
pression are important because of their translational implica-
tions for regenerative medicine and blood vessel diseases.
ACKNOWLEDGMENTS
We gratefully thank members of the Marsden lab for critical review of this
manuscript.
GRANTS
P. A. Marsden is a recipient of a Career Investigator Award from the Heart
and Stroke Foundation of Canada and is supported by a grant from the
Canadian Institute of Health Research (CIHR MOP 79475). M. S. Yan is a
recipient of a CIHR Frederick Banting and Charles Best Canada graduate
scholarship.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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Review
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