PPARγ GENE AND ATHEROSCLEROSIS:
GENETIC POLYMORPHISMS, EPIGENETICS
AND THERAPEUTIC IMPLICATIONS
Grbić E, Peterlin A, Kunej T, Petrovič D
*Corresponding Author: Professor Daniel Petrovič, M.D., Ph.D., Institute of Histology and Embryology, Faculty of Medicine
University Ljubljana, Vrazov trg 2, Ljubljana 1000, Slovenia. Tel: +386-1-5437-360. Fax: +386-1-5437-361. E-mail:
Daniel.petrovic@mf.uni-lj.si
page: 39
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INTRODUCTION
Atherosclerosis is a long-term process characterized
by plaque formation in middle and large arterial blood
vessels [1]. Atherosclerosis is one of the leading causes
of stroke, heart attack and peripheral arterial disease [2,3].
The prevalence and degree of atherosclerosis increases
with increasing age, body mass index (BMI), increased
blood pressure (BP), and serum total cholesterol (TC)
and low-density lipoprotein cholesterol (LDL-C) [4]. An
elevated level of LDL is directly associated with development
of atherosclerotic cardiovascular disease (ASCVD)
[5]. According to the National Hearth, Lung and Blood
Institute (NHLBI) data, atherosclerosis begins when certain
factors such as smoking, high cholesterol, high BP and
high blood sugar levels due to insulin resistance or diabetes
damage the inner layers of the arteries. Other factors that
have a significant effect on the development of atherosclerosis
include family history, older age, unhealthy diet,
lack of physical activity [2]. Depending on the position
and size of the atherosclerotic plaque, microvascular and
macrovascular complications of atherosclerosis can seriously
damage the brain, heart, kidneys and other organs.
Atherosclerotic disease of the carotid and coronary arteries
appears to be highly prevalent in the ageing population [5].
Coronary atherosclerosis is the leading cause of
coronary artery disease (CAD) [6]. Three pathological
processes affect the formation of plaque: inflammatory
reaction, cell proliferation and differentiation of foam cells
[7]. The preliminary step in the formation of plaque is the
passage of monocytes into subendothelial space and their
differentiation into macrophages, which favor the oxidation
of LDL particles in the blood and their endocytosis
in the cells [8,9]. Endocytosis is mediated by scavenger
receptors, which are not inhibited by content of cell cholesterol,
that results in the accumulation of lipids in macrophages and foam cells. Activated macrophages secrete
inflammatory cytokines [macrophage-colony stimulating
factor (M-CSF), tumor necrosis factor α (TNFα) and interleukin
1 (IL-1)] that trigger inflammatory processes and
lead to proliferation of smooth muscle cells (SMCs). Then,
macrophages and foam cells necrotize that leads to the
release of the contents into the extracellular space, which
is the basis for the onset of atherosclerosis [8]. Peroxisome
proliferator-activated receptors (PPARs) modulate many
aspects of these processes [8,9].
The aim of this review was to discuss the relationship
between the peroxisome proliferator-activated receptor γ
(PPARγ) gene polymorphisms and macrovascular complications
of carotid and coronary arteries in patients with
type 2 diabetes mellitus (T2DM). Moreover, we discussed
epigenetic mechanisms affecting the onset of atherosclerosis
and therapeutic possibilities affecting epigenetic
mechanisms in order to prevent the onset and progression
of atherosclerosis.
The PPARγ and Its Role in the Development of
Atherosclerosis. Peroxisome proliferator-activated receptors
are members of the steroid/thyroid hormone receptor
superfamily of transcription factors that are encoded
by three PPAR genes: PPARα, PPARβ/δ and PPARγ. The
PPARγ gene has two isoforms of PPARγ1 and PPARγ2 that
are ligand-activating transcription factors [8]. The PPARγ
gene is most prevalent in fatty tissue and macrophages
and is very important in regulation of gene expression in
metabolism and inflammation [10]. The PPARγ transcriptional
activity was modulated by binding of numerous fatty
acid metabolites that activate PPARγ. Activated PPARγ
increases the expression of the scavenger receptor, which
transmits ox-LDL from blood to macrophages, which then
differentiates into foam cells [11,12]. By collecting foam
cells, necrotic tissue residues, migrating and proliferating
VSMCs (vascular smooth muscle cells), an atheromatous
plaque is formed [13]. Overweight patients, T2DM patients
and non diabetic patients, have increased PPARγ (γ1 and
γ2) values, associated with changes in BMI and fasting
insulin. Deviations from PPARγ values indicate a possible
role in the onset of insulin resistance of skeletal muscles
in obesity and diabetes [9]. Thus, PPARγ is involved in
the regulation of all the steps that precede the onset of
atheromatous plaque, therefore, the occurrence of mutations
in PPARγ might be an initial step for the onset of
atherosclerosis.
Polymorphisms of the PPARγ Gene, Genetic Biomarkers
for Atherosclerosis. Several studies have shown
an association between the PPARγ polymorphisms and
microvascular and macrovascular complications of coronary
and carotid arteries in patients with T2DM [13-15].
In a study with a relatively small number of subjects
(161) and a relatively young average lifespan (38.0 ± 15.3),
Al-Shali et al. [13] found a link between PPARγ genotypes
and carotid atherosclerosis. They measured the thickness
of carotid intima media (IMT) and total plaque volume
(TPV), and found that subjects with the PPARG A12 allele
had lower IMT (0.72 ± 0.03 mm; p = 0.0045), without
differences in TPV, and subjects with the PPARG c.1431T
allele have higher TPV (124.0 ± 18.4; p = 0.0079) without
differences in IMT [13]. According to Li et al. [16], the
Pro12Ala polymorphism modulates PPARγ activity and
leads to changes in the regulation of insulin sensitivity and
glucose tolerance that ultimately leads to ASCVD [allelic
model: odds ratio (OR) 0.80; 95% confidence interval
(95% CI) 0.66-0.98, p = 0.040; dominant model: OR 0.74,
95% CI; 0.58-0.95, p = 0.033). In their meta-analysis, 12
case-control studies (eight Caucasian, three Asian and one
African) were included with 10,189 cases with ASCVD
[myocardial infarction (MI), CAD and acute coronary
syndrome (ACS)] and 17,899 control subjects [17]. Yan
et al. [17] found that the CC genotype of the C16T polymorphism
(rs3856806) was associated with carotid lesions,
while the CT+TT genotype had a protective role, indicating
the important role of the C161T polymorphism in carotid
artery atherosclerosis. In the carotid artery of patients with
metabolic syndrome, CC genotype vs. CT+TT genotype
significantly increased IMT and plaque index (IMT: 0.84
± 0.3 mm; plaque index: 2.19 ± 1.21; p <0.05) [18]. In a
study on Thai subjects, Yongsakulchai et al. [12] found
that the combination of PPARγ polymorphisms rs3856806
(C1431T), rs8192678 (G482S) and liver X receptor-α
(LXRα) polymorphism rs12221497 (115G/A) predict the
development and progression of coronary atherosclerosis
in subjects at-risk for CAD, and that the central role in this
process belongs to rs8192678 polymorphism (OR 1.64,
95% CI: 1.01 ± 2.66, p: 0.048). In their study, 387 subjects
were included, aged between 35-85 years, of whom
225 had CAD and 162 non CAD subjects (CAD group =
stenosis ≥50.0% and non CAD group = stenosis ≤50.0%,
at least one of the major coronary arteries) [12].
Few studies have shown that there was no statistically
significant relationship between PPARγ polymorphisms
and atherosclerosis[18-20]. Wang et al. [18] in a metaanalysis
involving 29 studies (15 Caucasian, 13 Asian
and one African), with PPARγ polymorphisms rs1801282
(Pro12Ala)/ rs3856806 (C161T), did not find a statistically
significant relationship with the onset of atherosclerotic
diseases. In their meta-analysis, they analyzed different
genetic models and associations with atherosclerotic disorders, there were no statistically significant results for the
polymorphism rs1801282 [18].
However, for the polymorphism rs3856806, based
on ethnicity, they found a significantly increased risk for
ath-erosclerotic disease in Caucasians (2679 cases with
atherosclerotic disease and 5121 control subjects) for the
additive model (OR 1.72; 95% CI 1.12-2.66) and for the
recessive model (OR 1.71; 95% CI 1.11-2.62), whereas
the risk for the Asian population (1910 cases with atherosclerotic
disease and 1820 control subjects) was reduced,
for the dominant model (OR 0.70; 95% CI 0.62-0.81) and
for the recessive model (OR 0.63; 95% CI 0.47-0.84).
Moreover, based on ethnicity, in the subgroup with
MI: they reported an association for rs3856806 for the
additive model (OR 2.68; 95% CI 1.10-6.54) and for the
recessive model (OR 2.58; 95% CI 1.09-6.10), whereas for
CAD they found a decreased risk for the additive model
(OR 0.67; 95% CI 0.51-0.88) and for the dominant model
(OR 0.69; 95% CI 0.61-0.79) [18]. In the Korean Population
Study, they did not find any statistically significant
association between Pro12Ala polymorphism and CVD
development (p = 0.824). In their prospective study, 267
subjects were included, divided into four groups, in the
number of stenotic coronary arteries, the values of stenosis
≥50.% were considered significant. The percentage
of patients with normal arterial lumen was 43.8%;
33.0% of patients had a stenosis of one coronary artery,
14.6% had a stenosis in two coronary arteries, and 8.6%
had a stenosis of three coronary arteries [19]. Similarly,
Wan et al. [20] reported that there was a significant link
between the C161T genotype and the vessels disease in
a group of Chinese patients with CAD and T2DM (OR
1.22; 95% CI: 1.03-1.45, p = 0.019), but that there was
no significant association with CAD risk (p = 0.695). In
their study, a group of patients (CAD+T2DM) with CC
genotype (70.3%) had severe stenosis >75.0% of one of
the major coronary arteries. Moreover, they discovered
that the C161T polymorphism was associated with adipose
metabolism, which suggests that by modulating it, the risk
of atherogenesis could be reduced in the group of patients
with CAD and T2DM [20].
Epigenetics. So far, no study has reported about epigenetics
of PPARγ in atherosclerosis in humans. However,
epigenetic mechanisms have been implicated in the onset
of atherosclerosis [21-23]. Previous research has shown a
significant effect of epigenetic factors on gene expression,
affecting adhesion, migration, differentiation of leukocytes,
proliferation and migration of VSMCs, or all key
processes in the onset and development of atherosclerosis.
In addition to direct changes to human DNA, there are
three other important epigenetic pathways that are important
for the regulation of gene expression, which are
DNA methylation, histone posttranslational modification
and RNA-based mechanisms [21].
DNA methylation represents the covalent binding of
the methyl group to the 5 position of the cytosine, and
plays a very important role in the organization of chromatin
and in that way, leads to the silencing of certain genes, the
complex formed is called 5 methyl-cytosine [24]. Key role
in DNA methylation, which is mainly related to the CpG
region, is played by three methyl transferases (DNMT1,
DNMT3a and DNMT3b) with the S-adenosyl methionine
donor of the methyl group [24]. The exact mechanism of
the development of atherosclerosis by changes in DNA
methylation is not fully known, but many studies of highfat
diet-fed apoE null mice, human SMCs, and ballooninjured
rabbit have shown a link between hypomethylation
with the onset of atherosclerosis [24-26]. In these studies,
hypomethylation was associated with increased expression
of DNA methyltransferase (DNMT) in atherosclerotic
lesions, removal of the methyl group increases the transcription
activity. Migration and proliferation of VSMC
are the central axis in the development of atherosclerosis
[25]. Lund et al. [27], showed in a study with ApoE null
mice that changes in DNA methylation profiles might be
markers of atherosclerosis in diabetics.
The four classes of histones (H2A, H2B, H3 and
H4) form an octameric complex, with two copies of each
of these four histones, and 147 bp chromosomal DNA
wrapped on octameric complex, forming the onset and
functional unit of chromatin nucleosomes. Cell DNA is
packaged in chromatin [28]. The unstructured N-terminal
tail histone is subject to numerous modifications such
as acetylation, methylation and phosphorylation [28].
The most common histone modification is acetylation.
With the enzyme histone acetyltransferase (HATs) and
histone deacetylase (HDACs), gene transcription activity
is monitored, HATs add the acetyl group to the histone
tail, thereby activating the gene, and HDACs inhibiting,
removing the acetyl group [29]. Many studies show the
relationship between acetylation and deacetylation status
and atherosclerosis [25,30].
Peroxisome proliferator-activated receptor induces
the expression of the nuclear receptor in macrophages
(LXRα), which increases the expression of ATP Binding
Cassette Subfamily A Member 1 (ABCA1) (a member of
the ABC transporter protein family) leading to the elimination
of cholesterol from the macrophage. Deacetylation
of PPARγ inhibits the pathway: PPARγ, LXRα, ABCA1,
which leads to the blocking of cholesterol efflux, increased production of proinflammatory macrophages and the development
of an inflammatory reaction, leading to the
onset and development of atherosclerosis [31,32].
According to Cao et al. [32], the HDAC9 expression
is associated with the onset of atherosclerotic plaques in the
arteries, the onset of stroke, and the increased expression
of macrophages acts atherogenetic. In the study with LDLr
[/] mice, the atherogenetic effect is reduced by deletion
of HDCA9, which leads to an increase in macrophage
cholesterol efflux and the prevention of the formation of
foam cells, and reduces the production of inflammatory
cells by translating macrophages from inflammatory M1
phenotype into an antiinflammatory M2 phenotype [33].
This study demonstrates the important role of HDCA9 in
the development of atherosclerosis, and the possibility of
developing epigenetic therapy aimed at inhibiting HDCA9
isoforms in macrophages.
The third epigenetic model, the RNA-based mechanism,
is relatively new. Currently the greatest attention of
scientists attracts non coding RNA (ncRNA), including
small RNAs [34]. Considering the length of the fragment,
we distinguished two main types of ncRNA: long ncRNA
(>200 nucleotides) and short ncRNA (<200 nucleotides)
and several subtypes that modulate gene expression [34].
Short RNA [i.e., microRNA (miRNA)] performs genome
repression by complementary binding to the 3 or 5 UTR
(untranslated region) of targeted mRNA, activates the
miRNA-induced silencing complex (miRISC) through
which it silences gene expression [34]. Long non coding
RNA (LncRNA) has a wide range of effects in various
processes from increasing to reducing gene expression in
combination with other epigenetic enzymes, plays an important
role in chromatin modulation, transcriptional and
post-transcriptional regulation, cell apoptosis, etc. [34].
MicroRNA has a leading role in the regulation of
ath-erosclerotic process [35]. Previous studies of miRNA
indicate a role in the prediction of certain diseases such
as atherosclerosis, due to its role in protein production
and the impact of one miRNA on several hundred target
genes, so far about 1100 miRNA are known in humans
[35]. In the study, Zhao et al. [36] point out the important
role of miR-613 in blocking the signaling pathway of
PPARγ, LXRα and ABCA1, which leads to the stopping
of cholesterol efflux and the development of atherosclerosis.
Also, indicating that activated PPARγ increases the
expression of LXRα and ABCA1, through the negative
control of miR-613, acting anti-atherogenetic [36]. In the
Tampere Vascular Study, a significantly expression of miR-
21, miR-210, miR-34a, and miR-146a/b was reported in
aortic, carotid, and femoral atherosclerotic arteries in relation
to non ath-erosclerotic left internal thoracic arteries
[37]. MicroRNA-21 and miRNA-34a show a significant
relationship with the proliferation of VSMCs [38,39]. MicroRNA-
146a is associated with CADs and increased LDL
release [39]. The levels of ox-LDL play an important role
in the onset of atherosclerosis, increasing the expression
of miRNA-29b. These effects are achieved by repression
of DNA methyl-transferase 3 Beta (DNMT3b), which
increases cellular migration of VSMC through increased
regulation of matrix metalloproteinase 2 (MMP-2) and
matrix metalloprotease 9 (MMP-9) [39]. In the study Cipollone
et al. [40], a significant difference in the expression
of miRNA-100, miRNA-127, miRNA-145, miRNA-133a
and miRNA-133b was found in the tissue of patients with
endarterectomy of the carotid and control group. In short,
this study, as well as the above studies, points to the role of
miRNA in the onset of atherosclerosis, and highlights the
possibility of using miRNAs as biomarkers for the onset
and development of atherosclerosis.
Therapy. In vitro and in vivo studies have shown a
positive effect of TZDs (tiazolidinediones) on the function
and pharmacology of β-cells through the mechanism of
mediated PPARγ, increasing the expression of PDX-1 (pancreatic
duodenal homeobox) on β-cells in pre diabetics and
T2DM patients [41]. Clinical studies have shown that TZDs
are PPARγ agonists that reduce inflammatory reactions,
modulate two ATP-binding cassette transporter (ABCA1
and ABCG1) expression and inhibit key VSMC processes
associated with atherosclerosis and protect blood vessels
of T2DM patients [42]. Several studies have shown positive
effects of TZDs (Troglitazone, Pioglitazone) in T2DM
patients on intima media thickness reduction and restenosis
processes in T2DM patients with stent [42-44]. On the other
hand, several studies describe the opposite effect of TZDs
on PPARγ, which modulates adipocyte activity and leads
to metabolic disorders and heart disease such as T2DM and
CAD [16,45]. So far, delivery of miR-150 may represent a
potential approach to prevent macrophage foam cell formation
in atherosclerosis by inhibition of the formation of macrophage
foam cells through targeting adiponectin receptor 2
[46]. Also, PPARγ agonists, by activating PPARγ, increase
the concentration of adiponectin in plasma and expression
of AdypoR2 in macrophages, and act anti-arteriogenic [47].
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