Journal of Human Hypertension (2015) 29, 575582

2015 Macmillan Publishers Limited All rights reserved 0950-9240/15

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REVIEW
Hypertensive epigenetics: from DNA methylation to
microRNAs
J Wang, L Gong, Y Tan, R Hui and Y Wang

The major epigenetic features of mammalian cells include DNA methylation, posttranslational histone modications and RNA-based
mechanisms including those controlled by small non-coding RNAs (microRNAs (miRNAs)). An important aspect of epigenetic
mechanisms is that they are potentially reversible and may be inuenced by nutritionalenvironmental factors and through
geneenvironment interactions. Studies on epigenetic modulations could help us understand the mechanisms involved in essential
hypertension and further prevent its progress. This review is focused on new knowledge on the role of epigenetics, from DNA
methylation to miRNAs, in essential hypertension.

Journal of Human Hypertension (2015) 29, 575582; doi:10.1038/jhh.2014.132; published online 29 January 2015

INTRODUCTION hydroxymethylation), posttranslational modications of histone

Essential hypertension is a disease that develops due to complex
interactions of susceptibility genes and environmental factors.1 It
is estimated that the overall prevalence of hypertension appears
to be around 30%45% of the general population, with a steep
increase with aging.2 The number of adults with hypertension in
2025 is predicted to increase by ~ 60% to a total of 1.56 billion,
particularly projected in economically developing countries.3 As
we know, essential hypertension is a signicant risk factor for
coronary artery disease, stroke and kidney disease.4,5 Many
pathophysiologic factors have been implicated in the genesis of
essential hypertension such as increased sympathetic nervous
system activity, overproduction of sodium retention hormones
and increased or inappropriate renin secretion.6
Improved techniques of genetic analysis, especially genome-
wide linkage analysis, have enabled deep search for genes. Most
promising ndings suggest that there are many genetic loci, each
with small effects on blood pressure in the general population.7
Many genes of small effect, which display effect modication in
the presence of some environmental factors, are responsible for
the etiology of human hypertension.8 However, the genetic
contribution to blood pressure variation among individuals is
estimated to range from 30% to 50%.8 Epigenetic regulation,
which can alter gene expression without changing the nucleotide
base sequence of gene, may result from environmentgene
interactions.9 Epigenetics is emerging as one of important
regulators of transcription of specic genes involved in the
pathogenesis of essential hypertension.
DEFINITION OF EPIGENETICS
Epigenetic phenomena are dened as heritable mechanisms that
establish and maintain mitotically stable patterns of gene
expression without modifying the base sequence of DNA.10
The eld can be broadly categorized into three areas: DNA base
modications (including cytosine methylation and cytosine
proteins and RNA-based mechanisms that operate in the nucleus,
which collectively enable the cell to respond quickly to environ-
ment changes (Figure 1).11,12
GENE-SPECIFIC DNA METHYLATION AND ESSENTIAL
HYPERTENSION
DNA methylation is a covalent modication in which the
5-position of cytosine is methylated in a reaction catalyzed by
DNA methyltransferases with S-adenosyl-methionine as the
methyl donor.13 In mammals, this modication occurs exclusively
at the C5 position of cytosine residues (5-methylcytosine) and
predominantly in the context of CpG dinucleotides.14 However,
non-CpG methylation in adult mammalian tissues, such as CpA,
CpT and CpNpG sites, has been observed.11,15 The main function
of DNA methylation is to modulate the expression of the genetic
information, by modifying the accessibility of DNA to the
transcriptional machinery.10
HSD11B2 gene methylation
The HSD11B2 gene, encoding the kidney isoenzyme 11-hydro-
xysteroid dehydrogenase, is a candidate for essential
hypertension.16 The enzyme 11HSD type 2 (11HSD2) isoform
binds NAD with high afnity and catalyzes the dehydrogenation
of 11-hydroxyglucocorticoids.17 The absence of 11HSD2 pre-
vents conversion of cortisol into cortisone, resulting in a rise in
intracellular cortisol levels, overstimulation of the mineralocorti-
coid receptor and excessive sodium reabsorption (Figure 2).18 The
activity of the enzyme 11HSD2 can be indirectly evaluated by
measuring urinary tetrahydrocortisol/tetrahydrocortisone ratio; an
increase in urinary tetrahydrocortisol/tetrahydrocortisone ratio
indicates decreased 11HSD2 activity.19
Epigenetic modulation of the HSD11B2 gene has been
demonstrated in both rodent model and cultured human cell
lines.20 CpG islands covering the promoter and exon 1 of HSD11B2

State Key Laboratory of Cardiovascular Disease, Sino-German Laboratory for Molecular Medicine, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing, China. Correspondence: Dr Y Wang, State Key Laboratory for Cardiovascular Disease, Sino-German Laboratory for
Molecular Medicine, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Road,
Beijing 100037, China.
E-mail: yibowang@hotmail.com
Received 25 August 2014; revised 18 November 2014; accepted 9 December 2014; published online 29 January 2015
 Hypertensive epigenetics
J Wang et al
576
Figure 1. The three areas of epigenetics: DNA base modications
(including DNA methylation and DNA hydroxymethylation), histone
modication and RNA-based mechanisms (including long non-
coding RNAs and short RNAs).
Figure 2. Physiological function of 11-hydroxysteroid dehydrogen-
ase type 2 (11HSD2).
are found to be densely methylated in tissues and cell lines with
low expression.20 Demethylation induced by 5-aza-2-deoxycyti-
dine and procainamide enhances the transcription and activity of
the 11HSD2 enzyme in human cells in vitro and in rats in vivo.20
Moreover, methylation of recognition sequences of transcription
factors, including those for Sp1/Sp3, Arnt and nuclear factor 1,
diminishes their DNA-binding activity.20 In a human study, the
HSD11B2 promoter region has an elevated methylation status in
glucocorticoid-treated patients who develop hypertension.21
Furthermore, the higher DNA methylation at HSD11B2 promoter
sites parallels a higher urinary tetrahydrocortisol/tetrahydrocorti-
sone ratio.21 These present results suggest DNA methylation is a
major mechanism for the regulation of HSD11B2 gene expression
by modulating the binding of transcription factors and has a role
in the pathogenesis of hypertension.
ACE gene methylation
Two isoforms, somatic and germinal angiotensin-converting
enzyme (ACE), are transcribed from two alternate promoters
within a single gene termed ace-1.22 Somatic ACE isoform is a key
regulator of blood pressure. It catalyzes the conversion of
angiotensin I into physiologically active angiotensin II, a substance
Journal of Human Hypertension (2015) 575 582
with potent vasopressive properties, and degrades the vasodilator
bradykinin.23,24 In mice, inhibition of ACE activity reduces
angiotensin II levels in blood and tissues.25 In clinical practice,
ACE inhibitors are of established benet for the treatment of
hypertension.
Two CpG islands are identied in the human ace-1 gene 3 kb
proximal promoter region and their methylation abolishes the
luciferase activity of ace-1 promoter/reporter constructs trans-
fected into human liver (HepG2), colon (HT29), microvascular
endothelial (HMEC-1) and lung (SUT) cell lines.26 Inhibition of DNA
methylation by 5-aza-2-deoxycytidine stimulates sACE mRNA
expression specically in in vitro cell type and in vivo tissue
type.26 5-Aza-2-deoxycytidine induces an increase (~25 mm Hg)
in blood pressure after a single injection in the rat, associated with
an elevated sACE expression in the lungs and the liver of treated
rats, and a substantial and unexpected decrease of plasma
angiotensin II levels.26 In low birth-weight children, DNA methyla-
tion status in three CpG sites (nucleotide positions +555, +561 and
+563) from the ACE gene promoter is decreased, simultaneously
with an increase in the ACE protein activity and high blood
pressure levels.27 Furthermore, systolic blood pressure levels and
ACE protein activity are inversely correlated with the degree
of DNA methylation.27 Epigenetic regulation of ACE gene
expression might be crucial in the control of blood pressure
through angiotensin II-dependent pathways.
NKCC1 gene methylation
The solute carrier family 12 member 2 (SLC12A2), which is also
called Na+-K+-2Cl cotransporter 1 (NKCC1), mediates the symport
of sodium, potassium and chloride.28 NKCC1 is expressed in most
of the cells including vascular smooth muscle cells (VSMCs),
endothelial cells, cardiomyocytes, neurons, glial cells and blood
cells.28 Under baseline conditions ([Na+]o4 4[Na+]i and [Cl]2o44
2
[Cl ]i ), NKCC provides inwardly directed net ion ux and
maintenance of [Cl]i above values predicted by the Nernst
equilibrium potential.28 In VSMCs, the contribution of K perme-
ability (PK) to resting membrane potential (Em) is somewhat similar
with that of Cl permeability (PCl).29 NKCC-mediated modulation
of [Cl]i affects Em-dependent and Em-independent VSMC
contraction.30 High-ceiling diuretics, inhibitors of NKCC1, decrease
[Cl]i, hyperpolarize VSMCs, attenuate the activation of levo-type
Ca2+ channels and reduce smooth muscle contractions.31 NKCC1
has a role in the onset of high blood pressure, via an increased
cytosolic chloride accumulation in VSMCs.32
In cultured cells, WNK1 (with-no-lysine 1) is activated by either
hyperosmotic stress or hypotonic low Cl conditions.33,34 SPAK
(STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxida-
tive stress-responsive kinase 1) are physiological substrates for
WNK1, conditions that stimulate WNK1 activation in cells induces
the activation of endogenous SPAK and OSR1.35 CCT domain of
SPAK and OSR1 interacts with RFXV motifs on NKCC1 as well as
WNK1 and WNK4 with high afnity.36 Wnk1+/ mice exhibit a
signicant alteration in blood pressure and vasoconstricting
responses specic to 1-adrenergic vasoconstrictors in both
conductance and resistive arteries.37 WNK1 is a major protein in
the vasoconstriction pathway linking 1-adrenergic receptors and
its downstream effectors SPAK/OSR1 and NKCC1 (Figure 3).
Lee et al.38 analyzed promoter methylation of the NKCC1 gene
(ofcial gene symbol SLC12A2) in male Wistar Kyoto (WKY) and
spontaneously hypertensive rat (SHR). They revealed that NKCC1
promoter is hypomethylated in the aorta and hearts of SHR, that
is, coincided with upregulation of NKCC1 mRNA and protein
compared with WKY.38 Furthermore, the promoter region of
NKCC1 in WKY is methylated with age, while that in SHRs remains
hypomethylated during postnatal development of hypertension.39
The activity of DNA methyltransferases after development of
hypertension is about threefold higher in WKY than SHR, which is
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in accordance with the methylation status on the NKCC1 promoter
of WKY and SHR.39 Phenylephrine-induced vasoconstrictions are
signicantly attenuated by bumetanide (an inhibitor of NKCC1) in
both isolated aortae and second-order mesenteric artery of SHR
than those of WKY.38 This declined effect of phenylephrine by
bumetanide is partially caused by higher expression of NKCC1
mRNA and protein in SHR.38 These data provide new insights into
the epigenetic mechanism of the action of NKCC1 that may
contribute to the development of hypertension.
ADD1 gene methylation
ADD1 (-adducin) gene, a candidate gene for essential
hypertension,40 encodes one of adducin subunits and is expressed
in most tissues.41 Adducin selectively binds to the spectrinactin
network and recruits additional spectrins to actin laments.41 The
actin-based cytoskeleton interacts with the band 3-anion exchan-
ger, the epithelial Na channels, the Na-K-Cl cotransport and the
Na-K ATPase.42 Cells transfected with Milan hypertensive rats
adducin have shown a signicant increase in NaK pump activity
at Vmax and of NaK pump units compared with cells carrying the
Milan normotensive strain adducin.43 Thus, adducin modulates the
surface expression of multiple transporters and ion pumps, and
regulates cellular signal transduction and cytoplasmic ion
transport.40
An association study with 33 hypertensive patients and 28 age-
and gender-matched controls showed that DNA methylation
levels are closely correlated among CpG25, and ADD1 CpG25
methylation levels are signicantly associated with essential
hypertension.44 However, ADD1 CpG1 methylation level is
signicantly associated with essential hypertension in females
but not in males, and lower levels of ADD1 CpG25 methylation is
associated with increased risk of essential hypertension in males.44
ADRB1 gene methylation
The sympathetic nervous system has a crucial role in the
regulation of blood pressure, mainly through activating - and
-adrenergic receptors in the effector organs, including the heart,
kidney and the blood vessels.45 The 1-adrenoreceptor (ADRB1)
gene is one of the hypertension-susceptibility candidates as a
pivotal mediator of signal transduction in cardiac, vascular,
endocrine and sympathetic adrenal systems.46 The human ADRB1,
which mediates the actions of catecholamines in the sympathetic
nervous system, is a key cell surface signaling protein expressed in
multiple organs and tissues including the heart, kidney, brain and
the pineal gland.47
The ADRB1 promoter has many methylated sites.48 According
to the animal model experiment, SHRs that had better
Figure 3. The WNK-SPAK/OSR1 signaling pathway by which the Na+-
K+-2Cl cotransporter 1 (NKCC1) is regulated and affects vascular
contraction in essential hypertension.
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Hypertensive epigenetics
J Wang et al
577
antihypertensive response to metoprolol showed a lower level
of DNA methylation modication and a higher expression of
ADRB1 in their myocardial tissues.48 In SHRs, hypomethlation of
the ADRB1 promoter is likely to improve the antihypertensive
efcacy of metoprolol.48 Thus, the epigenetic molecular mechan-
ism could lead to ADRB1 gene-directed therapy.
ENaC gene methylation
The epithelial sodium channel (ENaC) consists of three homo-
logous subunits , and , which are encoded by the sodium
channel, non-voltage-gated 1 (Scnn1a), (Scnn1b) and (Scnn1G)
genes, respectively.49 ENaC is expressed in the apical membrane
of salt-absorbing epithelia of kidney, distal colon and lung
airways.49 ENaC has a major role in Na+ reabsorption in the distal
tubule, and hence the regulation of Na+ balance, extracellular uid
volume and blood pressure.50 Of the three ENaC subunits, ENaC
appears to be critical to the overall salt balance. Mice with
targeted inactivation of ENaC in the connecting tubule/collecting
duct exhibit severe renal salt wasting characteristic of a
pseudohypoaldosteronism type I phenotype.51 Aldosterone is a
major regulator of epithelial Na+ absorption and acts in large part
through ENaC induction in the renal collecting duct.52,53 In this
region, aldosterone administration or hyperaldosteronism induced
by a low-Na+ diet increases ENaC gene transcription, without
increasing - or -subunit expression.54
A CpG island near the transcription start site of the ENaC
promoter is regulated by the control of promoter methylation status
(Figure 4). Under basal conditions, cytosine methylation in this
region of the ENaC promoter is evident in mIMCD3 cells.55 5-Aza-
2-deoxycytidine-mediated promoter demethylation enhances Sp1
binding to, and transactivation of the ENaC promoter, thus
increases ENaC mRNA expression.55 Aldosterone reprograms the
methylation status of the R3 subregion of the ENaC promoter from
a predominance of 5-methylcytosine under basal conditions to a
predominance of 5-hydroxymethylcytosine.55 This change is
accompanied by depletion of DNMT3b at this subregion and
enhances enrichment of Tet2.55 Aldosterone treatment disperses
DNMT3b from the ENaC R3 subregion and recruits Tet2 to convert
5-methylcytosine to 5-hydroxymethylcytosine at this subregion, to
contribute to the derepression of ENaC transcription.55
Aldosterone-dependent demethylation of the ENaC promoter
also facilitates Sp1 binding to the promoter, to allow further
transactivation of the ENaC gene.55
Figure 4. Two epigenetic mechanisms of ENaC for derepression:
disruption of Dot1a-mediated histone H3K79 methylation and the
targeted demethylation of the ENaC promoter.
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578
HISTONE MODIFICATION IN ESSENTIAL HYPERTENSION
Genomic DNA is compacted in the chromatin, whose basic unit is
the nucleosome that is formed by an octamer of histone proteins
H2A, H2B, H3 and H4. Posttranslational modications occurring
at residues in N-terminal tail of histone include phosphorylation,
sumoylation, biotinylation, acetylation and methylation,
and control the dynamics of chromatin to regulate gene
expression.56
ACETYLATION MODIFICATION
The acetylation/deacetylation balance among most of studies is
one of histone-related epigenetic mechanisms, and histone
histone or histoneDNA interplay regulates gene expression.57
Histone H3 is acetylated by histone acetyltransferase at different
lysine positions and the site is indicative of different events; for
example, acetylation at lysine-14 indicates active transcription of
DNA into RNA.58
Acetylation modication in the neurons of area postrema
Melatonin secretion from pineal gland may be disturbed by
almost all environmental factors, such as light and temperature.59
Melatonin as an appropriate mediator transfers the environmental
information to the area postrema (AP), where the high levels of
nuclear melatonin receptors are located.60 The rostral ventrolateral
medulla neurons receive excitatory input from AP, which responds
to blood and cerebrospinal uid signals, and provide excitatory
output to preganglionic neurons in the intermediolateral cell
column of the spinal cord, which provide sympathetic output to
target organs.60 Treatment with physiological (nanomolar) con-
centrations of melatonin for 24 h was reported to increase the
acetylation of histone H3 in progenitor cells, augment neurite-like
extensions and promote mRNA expression of nestin, a neural stem
cell marker.61 Melatonin was also reported to increase the mRNA
expression of various other histone acetyltransferase isoforms.61
Environmental stressors affect melatonin secretion and cause
epigenetic modications in the neurons of AP, shifting the blood
pressure set-point signal of the AP to a higher pressure.60 This
signal may then operate through efferent projections to key
medullary sympathetic nuclei in the rostral ventrolateral medulla,
thereby increasing brainstem sympathetic outow and explaining
the long-term alterations in sympathetic activity associated with
essential hypertension.60,62 The axis of pineal glandAProstral
ventrolateral medulla participates in adaptive responses to
environmental and internal stressors and the pathogenesis of
essential hypertension.
Acetylation modication at WNK4 promoter region
WNK4 is a member of the serinethreonine protein kinase family.
Human WNK4 (hWNK4) expresses mainly in the kidney and partly
in polarized epithelia.63 It acts as a multifunctional regulator of
diverse ion transporters, including NaCl cotransporter and renal
outer medullary K+ channel, and can vary the balance between
NaCl reabsorption and K+ secretion to maintain integrated
homeostasis.64 Therefore, it may be involved in pathophysio-
logical processes of uid and electrolyte perturbations and
hypertension.63
It has been demonstrated that glucocorticoid downregulates
hWNK4 expression through the negative glucocorticoid responsive
elements.65 The 2-adrenergic receptor stimulation leads to cyclic
adenosine monophosphate-dependent inhibition of histone
deacetylase-8 activity in the kidney and increases histone
acetylation in the WNK4 promoter region, resulting in transcrip-
tional modulation dependent on glucocorticoid receptors binding
to a negative glucocorticoid responsive element in this region.66
Trichostatin A, a histone deacetylase inhibitor, upregulates hWNK4
Journal of Human Hypertension (2015) 575 582
mRNA and protein expression within the hWNK4 promoter in
HEK293 cells.67 In rat models, salt loading suppresses renal WNK4
expression, activates Na+-Cl cotransporter and induces salt-
dependent hypertension.66 These ndings suggest epigenetic
modulation of WNK4 transcription in the development of salt-
sensitive hypertension.
Histone modication at ENaC promoter region
Dot1a (disruptor of telomeric silencing alternative splice variant
a) is a lysine methyltransferase that methylates the histone
H3K79 site of nucleosomes and disrupts the process of silencing
genes located in the telomeric regions of chromosomes during
DNA repair for maintaining telomere length.49 Af9, the fused
mixed-lineage leukemia and acute lymphoblastic leukemia gene
mapped to chromosome 9 (Af9), produces a sequence-specic
DNA-binding protein that binds to Dot1a.49
Af9, a putative transcription factor, physically and functionally
interacts with Dot1a to form a nuclear repressor complex, which,
via direct or indirect binding to specic sites in the ENaC
promoter, regulates histone H3K79 methylation at these sites and
represses basal transcription of ENaC.68 Aldosterone down-
regulates the Dot1aAF9 complex, at least in part by inhibiting
Dot1 and AF9 expression, leading to targeted histone H3K79
hypomethylation and transcriptional activation of ENaC.68
Furthermore, Af9 has been identied as a physiologic target for
Sgk1 (serum- and glucocorticoid-induced kinase-1) phosphoryla-
tion, and Sgk1 as a novel component and negative regulator of
the Dot1a-Af9 complex.53 Aldosterone not only downregulates the
abundance of the components of the Dot1a-Af9 complex but also
(via Sgk1-mediated phosphorylation of Af9) weakens their
interaction, leading to targeted histone H3K79 hypomethylation
at the ENaC promoter and derepression of ENaC transcription.53
The Dot1a-Af9 pathway is thus likely to inuence the expression of
this gene regulating sodium transport (permeability), contributing
to renal brosis and genetic predilections for salt-sensitive
hypertension.69 AF17 competes with AF9 for interaction with
Dot1a and antagonizes the epigenetic repressor effects of Dot1a-
AF9 on ENaC transcription, and augments ENaC-mediated Na+
transport under basal conditions.70 The positive regulatory effect
of AF17 on ENaC transcription appears to involve, at least in part,
enhances nuclear export of Dot1a to the cytoplasm, the resulting
reduction in nuclear Dot1a expression leads to H3K79 hypo-
methylation and de-repression of ENaC transcription.70
Aldosterone-induced ENaC transcription includes transactiva-
tion mediated by the binding of the liganded mineralocorticoid
receptor to glucocorticoid responsive elements and recruitment of
Sp1 to its cis-element, and at least two epigenetic mechanisms for
derepression: disruption of Dot1a-mediated histone H3K79
methylation and the targeted demethylation of the ENaC
promoter (Figure 4).55
MICRORNAS AND ESSENTIAL HYPERTENSION
microRNAs (miRNAs) are endogenously produced non-coding
RNA molecules, ~ 22 nucleotides long, that have a ubiquitous and
important role in regulating protein expression. At least 30% of
human genes are thought to be regulated by miRNAs, one of the
classes of small RNA.71 The human genome contains genes coding
at least 2588 mature human miRNAs. Some miRNA genes are
located in introns of protein-coding genes or form transcriptional
unit with adjacent protein-coding genes.72 Some miRNA genes are
located close to each other in the genome and form miRNA
cluster.72 In addition, some miRNAs, such as miR-149 and miR-29b,
are encoded by multiple copies of genes.72
Similar to mRNA, miRNAs are transcribed from endogenous
miRNA genes as primary transcript (pri-miRNA), containing 65- to
70-nucleotide stem-loop structure.7375 The hairpin structure is
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excised in the nucleus by the Drosha-DGCR8 complex to yield a
precursor miRNA.7375 Precursor miRNA is transported by exportin-
5 into the cytosol, where Dicer cleaves the Precursor miRNA,
producing a short double-stranded RNA fragment called miRNA:
miRNA duplex.7375 The duplex is successively incorporated into
the RNA-induced silencing complex.10 Within the RNA-induced
silencing complex the miRNA duplex is unwound and split into
two single strands; the mature miRNA single strand is retained in
the complex, determining the formation of miRNA-induced
silencing complex, while the other strand is lost (Figure 5).10 The
seed sequence of a miRNA in these protein complexes pairs with
complementary sites in the 3-untranslated region (3UTR) of
target mRNAs through RNARNA base pairing that involves not
only the WatsonCrick A:U and G:C pairs but also the G:U pair.76 In
the canonical pathway, base pairing is recognized between the 2
and 8 nucleotide seed sequence of the miRNA and the mRNA 3
UTR.77 In the non-canonical pathway, miRNA binding to its mRNA
targets relies less on seed sequence homology and more on
complementarity in coding regions or the 5UTR of the target
transcript.78
In general, miRNAs bind to the 3UTR of their target mRNA and
reduce the abundance of target proteins by repression of target
mRNA translation and removal of mRNA poly (A) tail (that is,
deadenylation), resulting in mRNA degradation.74 miRNAs emerge
recently on the scene of epigenetics as factors of important
mechanisms capable of modulating and controlling the expres-
sion of genes. Key miRNAs regulate the expression levels of
hundreds of genes simultaneously, and many types of miRNAs
regulate their targets cooperatively.79
AGT-regulated miR-584 and miR-31
Human angiotensinogen (AGT) gene, which is associated with
essential hypertension in Caucasians, Japanese and Asian Indian
subjects, has a C/A polymorphism at +11525 (rs7079) located in
the 3UTR.80 Both in HEK293 cells and Hep3B cells, transfection of
miR-31 or miR-584 downregulates the luciferase activity of
reporter construct containing only the 11525C allele and not the
11525A allele.80 Moreover, anti-miRNAs relieve the miR-584- and
miR-31-induced downregulation of the luciferase gene-containing
11525C allele of the 3UTR of hAGT gene.80 In addition,
transfection of either miR-31 or miR-584 also reduces the hAGT
mRNA/protein level in Hep3B cells.80 Owing to decreased binding
of miR-584 and miR-31, human subjects having the 11525A allele
may have increased hAGT expression compared with human
subjects with the 11525C allele (Table 1).80 This may lead to
increased plasma or tissue hAGT levels, ultimately resulting in
increased blood pressure in human subjects with the 11525A
allele compared with human subjects with the 11525C allele.80
REN-binding miR-181a and miR-663
The transcriptome-wide study of differential expression of mRNAs
and miRNAs in the kidney in human hypertension showed that
Figure 5. The biogenesis of miRNAs.
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Hypertensive epigenetics
J Wang et al
579
several of the mRNAs identied have miRNA targets in their 3
UTR.81 Functional experiments in cultured kidney cells showed
that two selected miRNA, miR-181a and miR-663, may exert
posttranscriptional control of renin (REN), apolipoprotein E (APOE)
and apoptosis-inducing factor mitochondrion-associated 1 (AIFM1)
mRNAs via their 3UTR.81 In human hypertension, endogenous
REN gene is overexpressed and the expression of miR-181a and
miR-663 is reduced.81 REN mRNA may be regulated via binding of
miR-181a and miR-663 to its 3UTR (Table 1).81
MIRNA AS BIOMARKERS
miR-483-3p
miR-483-3p is one of the 22 VSMC-specic miRNAs, whose
expression is decreased on chronic angiontensin I receptor
activation.82 This miRNA is encoded within intron 2 of the
insulin-like grow factor 2 (IGF2) gene in humans and rodents.82
IGF2 gene is known to be regulated by angiontensin II signaling
and in turn IGF2 signaling affects reninangiotensin system (RAS)
functions.82
The miR-483-p targets of the components of RAS were
predicted using multiple commonly used miRNA target prediction
algorithms (Target-Scan, PITA, DIANA and MicroCosm). Prediction
results showed that 3UTRs of AGT, angiotensin-converting
enzyme-1 (ACE-1), angiotensin-converting enzyme-2 (ACE-2) and
angiontensin II receptor (AT2R) each contain a single site in the
genes 3UTR for miR-483-3p.82 Cotransfection experiments clearly
demonstrated that miR-483-3p can effectively initiate the RNA
interference process on the target 3UTRs of AGT, ACE-1, ACE-2 and
AT2R, suggesting that this miRNA could be a global regulator of
tissue RAS.82 In the miR-483-3p-expressing RASMC (human aortic
smooth muscle cells)angiontensin I receptor cells, protein levels
of these miR-483-3p targets consistently decrease.82 Decreased
levels of AGT and ACE-1 in these cells can be rescued by
transfection with an antagomir (one of miRNA inhibitors) to
miR-483-3p.82 In the presence of miR-483-3p, there is no change in
AGT, ACE-1, ACE-2 and AT2R transcripts.82 Thereby, miR-483-3p
does not induce degradation of transcripts of these target genes,
but acts on posttranscriptional levels of these genes.82 Four
different RAS components could be as targets of the angiontensin
I receptor -regulated miR-483-3p (Table 1).82 Thus, miRNA
regulation by angiontensin II to affect cellular signaling is a novel
aspect of RAS biology, which may lead to discovery of potential
candidate prognostic markers and therapeutic targets.82
miR-296
hWNK4 3UTR is highly conserved and functions as an enhancer in
HEK293 and BGC823 cells, no matter whether under a hetero-
logous or a homologous promoter.63 The enhancer and promoter
of the hWNK4 gene interact physically, with the intervening DNA
Table 1. RAS components and target miRNAs
RAS genes Target miRNAs
AGT miR-584
miR-31
REN miR-181a
miR-663
AGT miR-483-3p
ACE-1
ACE-2
AT2R
Abbreviations: ACE-1, angiotensin-converting enzyme-1; ACE-2,
angiotensin-converting enzyme-2; AGT, angiotensinogen; AT2R, angion-
tensin II receptor; RAS, renin-angiotensin system; REN, renin.
Journal of Human Hypertension (2015) 575 582
 Hypertensive epigenetics
J Wang et al
580
looped out in hWNK4-expressing HEK293 cells, but not in non-
hWNK4-expressing Hela cells.63 The transcriptional factor GATA-1
and its motif could be a part of the bridge between the promoter
and 3UTR of hWNK4.63 After overexpressing miR-296, the level of
hWNK4 protein decreases but not the mRNA. Thus, miR-296
targets on the hWNK4 3UTR to downregulate gene expression at
the posttranscriptional level.63 A coordinated modulation by
miR-296 and cofactors may be attributed to hWNK4 physiopatho-
logical function and provide a novel therapeutic clue for
hypertension.63
miR-9 and miR-126
A recent study in mice has revealed that the nuclear factor of
activated T cells c3 and myocardia, which participate in the
aldosterone-induced hypertrophic pathway, can both be targeted
by miR-9.83 miR-126, which is one of the most abundant miRNAs
in endothelial cells, has been found to regulate angiogenic
signaling and vascular integrity.84 Based mainly on experimental
animal studies, it has been suggested that miR-9 and miR-126
could have a role in human hypertension.85
To conrm their involvement in the pathophysiology of
hypertension in humans, 60 patients with hypertension and 29
healthy volunteers were enrolled in this study.86 The expression
levels of miR-9 and miR-126 are lower in hypertensive patients
than those in healthy controls.86 miR-9 expression levels in
peripheral blood mononuclear cells are positively correlated with
left ventricular mass index in patients with essential
hypertension.86 Administration of miR-9 mimic (double-stranded
RNA oligonucleotide) could reverse the hypertrophic response
induced by isoproterenol and aldosterone in cellular model.83
Examination of miR-9 and miR-126 expression levels in essential
hypertensive patients in relation to 24-h ambulatory blood
pressure monitoring parameters revealed signicant positive
correlations with the 24-h mean pulse pressure.86 These studies
indicate that miR-9 and miR-126 may have the potential to be
used as prognostic biomarkers and possibly as therapeutic targets
in essential hypertension.86
miR-145, miR-132 and miR-212
Recent studies have also found that miR-145 is signicantly more
expressed in atherosclerotic plaques of hypertensive patients than
in plaques from the control patients.87 A post-hoc analysis showed
that treatment with angiotensin II receptor blocker is associated
with higher levels of miR-145, whereas treatment with ACE
inhibitors is associated with lower levels of miR-145, although
these data do not show any statistical signicance (P = 0.06)
probably due to the limited sample size (n = 22).87 Plasma levels of
circulating miR-132 and miR-212 are highly elevated in the heart,
aortic wall and the kidney in hypertensive rats, following chronic
infusion of angiotensin II,88 whereas these two miRNAs levels are
signicantly decreased in human arteries from bypass-operated
patients treated with angiotensin II receptor blockers, suggesting
that circulating miR-132 and miR-212 are involved in angiotensin
II-induced hypertension and might be used for therapeutic targets
during hypertension management.89
CONCLUSION
Epigenetic studies on hypertension should be focused, because
not only there is an increasing prevalence with hypertension but
also hypertension is a major modiable risk factor for heart
disease, kidney disease and stroke. Epigenetic events, differently
from genetic sequence, are potentially reversible through their
interface with environmental and nutritional factors. A proven
effective intervention is lifestyle changes, including weight loss,
reduced intake of dietary sodium, low alcohol of consumption,
potassium supplementation, modication of eating habits and
Journal of Human Hypertension (2015) 575 582
increased physical activity.4 Epigenetic pathways offer a new
perspective in gene regulation. With further research, epigenetics
might make a signicant contribution for preventive and
therapeutic approaches for essential hypertension.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGEMENTS
The study was supported by the Ministry of Science and Technology of China with
973 grant 2012CB517804 and the National Natural Science Foundation of China with
grants 81322002 and 81270333 to Dr Wang.
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