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Rupture of Vulnerable Atherosclerotic Plaques: MicroRNAs Conducting the

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 be performed at different levels, such
Rupture of Vulnerable Atherosclerotic as for instance proteomics, lipidomics,
Plaques: MicroRNAs Conducting or RNA analysis. With respect to RNA
analysis, the discovery of microRNAs
the Orchestra? (miRNAs) may yield new diagnostic
and 1even therapeutic options. Recent
Vincent G. Haver, Riemer H.J.A. Slart, Clark J. Zeebregts, studies suggest a role for miRNAs in
Maikel P. Peppelenbosch, and René A. Tio⁎ cardiovascular disease and atherosclero-
sis (Blake and Ridker 2001, Dong et al.
2009, Ji et al. 2007, Scalbert and Bril 2008,
Zhang 2009).
MicroRNAs (miRNAs) are tiny, endogenous nucleotides that bind to
Many genetic risk factors for athero-
mRNA and induce translation repression within metazoan cells. Since sclerosis and restenosis have been iden-
their discovery in 1993 in Caenorhabditis elegans and the demonstra- tified (e.g., Monraats et al. 2010). After
tion of miRNAs in Homo sapiens in 2000, research has been fruitful in transcription of genomic DNA, transla-
deciphering the role of these nucleotides in development, tissue tion of RNA (mRNA) will yield the gene
product—a protein. Interestingly, less
homeostasis, and pathologic processes. In humans, around 700 human than 20 years ago, it has been shown
miRNA nucleotides have been verified, which interfere with 30% of all that miRNAs are important players in
genes. Recently, the role of miRNA in cardiovascular research gained translational control of mRNA before
attention and the involvement of miRNAs in several cardiovascular translation takes place. They are short,
noncoding RNA molecules that inter-
diseases has been identified. In this review, we focus on the role of
fere with mRNA, thereby, inducing
miRNAs in atherosclerosis and in particular on the potential role of translational repression (Mishra et al.
miRNAs in the development of vulnerable atherosclerotic plaques. The 2009). They can act as important
role of miRNA in the main characteristics of these plaques, inflamma- regulators in inflammation as well as
tion, angiogenesis, and apoptosis will be discussed. Finally, the future angiogenesis, both important character-
istics of plaque vulnerability. After their
perspectives and miRNA-based diagnostic and therapeutic potentials first identification in the nematode
will be highlighted. (Trends Cardiovasc Med 2010;20:65–71) n 2010, Caenorhabditis elegans species, it has
Elsevier Inc. become evident that they are present in
a large variety of other species as well,
 such as worms, mice, and human
Introduction low-density lipoprotein-filled foam cells
(Pasquinelli et al. 2000).
(N40% of the plaque), a thin fibrous cap,
Hundreds of miRNAs have been
Atherosclerosis is one of the leading which is depleted of smooth muscle cells,
identified in vivo or predicted in silico
causes of mortality in the Western world. inflammatory cell infiltration (mainly
from the genomes of plants, worms,
Apart from its anatomical and physiologic macrophages), newly formed vessels (an-
flies, and mammals (MicroRNA.org).
characteristics, the vulnerability or biolo- giogenesis or neovascularization), intra-
Bioinformatic predictions indicate that
gy of a plaque is of the utmost importance plaque hemorrhage, and outward
the mammalian miRNAs can regulate
for its clinical sequelae. Vulnerable pla- remodeling (Hellings et al. 2007). Molec-
approximately 30% of all protein-coding
ques have a large lipid core composed of ular analysis of vulnerable plaques can
genes (Suarez and Sessa 2009). This
number of miRNAs is much higher than
previously expected and indicates the
Vincent G. Haver, Riemer H.J.A. Slart, Clark J. Groningen, Groningen, The Netherlands; importance of these regulatory noncod-
Zeebregts, and René A. Tio are at the and Maikel P. Peppelenbosch at the Depart-
ing RNAs. The number of a specific
Cardiovascular Imaging Group Groningen, ment of Gastro-enterology and Hepatology,
University Medical Center Groningen, Uni- Erasmus Medical Center Rotterdam, The miRNA can differ per cell type and per
versity of Groningen, Groningen, The Netherlands. tissue. In some cells, the number of a
Netherlands; Department of Cardiology, Uni- ⁎ Address correspondence to: René A. Tio, specific miRNA molecule can be as high
versity Medical Center Groningen, University Department of Cardiology, Thorax Center, as 10,000, whereas other miRNAs are
of Groningen, Groningen, The Netherlands; University Medical Center Groningen, Univer- only detectable by hybridization of total
Department of Nuclear Medicine and Molec- sity of Groningen, Hanzeplein 1, 9713GZ
RNA (Ambros 2004).
ular Imaging, University Medical Center Groningen, The Netherlands. Tel.: (+31)
Groningen, University of Groningen, Gronin- 503612355; fax: (+31) 503611347; e-mail: r.a.
The aim of this review was to sum-
gen, The Netherlands; Department of Surgery tio@thorax.umcg.nl. marize the potential role of miRNAs in
division of Vascular Surgery, University © 2010, Elsevier Inc. All rights reserved. atherosclerotic plaque formation and
Medical Center Groningen, University of 1050-1738/$-see front matter plaque rupture. After a short

TCM Vol. 20, No. 2, 2010 65

 explanation of miRNA processing and
the role of miRNA in normal develop-
ment, the function of miRNAs in
pathologic processes related to athero-
sclerotic plaque development and vul-
nerability are discussed.

miRNA Processing
miRNA processing is a complex process
F1 (Figure 1) and has been investigated
intensely within the past 2 decades
(Suarez and Sessa 2009). miRNAs are
either intergenic, polycistronic, or intro-
nic. The primary miRNA (pri-miRNA)
capped, and polyadenylated transcript,
which can be thousands of nucleotides
long, is transcribed within the nucleus
by RNA polymerase III (or II) from any
of the aforementioned encoding regions
of miRNA. Drosha (an RNase III type
endonuclease) releases the stem loop
structure resulting in a 70 to 100
nucleotide hairpin-shaped precursor
(pre-miRNA) by cleaving the 5′cap and
polyadenylated tail. Drosha requires a
regulatory subunit called DGCR8 in
humans (Zeng 2006). The pre-miRNA
is transported from the nucleus to the
cytoplasm, where Dicer cleaves pre-
Figure 1. miRNA possessing. After transcription of intronic miRNA by RNA polymerase II, it
takes several steps before the mature miRNA nucleotide is loaded within the RISC complex
and induces either mRNA degradation or mRNA translational repression. See text for
further explanation. (Color version of figure is available online.)
66
miRNA into double-stranded transient
duplex miRNA (miRNA and a partially
complementary strand, referred to as
miRNA*) containing approximately two
nucleotides 3′ overhangs at both ends.
Proteins with double-stranded RNA-
binding domains (such as TRBP, TAR
RNA binding protein and PACT, protein
activator of PKR (protein kinase acti-
vated by dsRNA) bind to Dicer and
enhance the affinity of Dicer for RNA,
thereby, participating in the selection of
mature miRNA strands (Zeng 2006).
The double-stranded miRNA duplex is
incorporated into the RNA-induced si-
lencing complex (RISC), containing pro-
teins of the Argonaute family (e.g., Ago
2). Within this RISC complex, the
miRNA duplex is unwound into the
mature single-stranded form (guide
strand), and its complementary strand
(miRNA*) is degraded (Suarez and
Sessa 2009). The miRNA–RISC complex
regulates the concentration of a partic-
ular protein in a cell by either degrading
the complimentary mRNA with the use
of the RNAi mechanism or by inhibiting
the translational machinery of protein
dependent on the affinity for mRNA
molecules. In cases of perfect or near-
perfect complementary binding of
miRNA, target mRNA 3′untranslated
regions (UTRs) can be cleaved and
degraded, but in animals, generally,
binding is imperfect, and translation is
repressed without cleavage of mRNA
(Suarez and Sessa 2009). However, it
also has been stated that miRNAs in
animals lead to considerable cleavage of
target mRNAs (Jovanovic and Hengart-
ner 2006).
As mentioned previously, nonperfect
binding of miRNA leads to translational
suppression of target mRNAs. This impli-
cates that one particular miRNA can
regulate hundreds of genes, and one
gene can be regulated by a number of
miRNAs (Sen et al. 2009). This indicates
the major impact of miRNAs in transla-
tional control.
 miRNA Mechanisms
Altered miRNA concentrations within a
cell logically results in a modified level of
RNA interference. Because miRNA
destroys or blocks translation of mRNA,
up-regulation of miRNA leads to a down
regulation of target protein, whereas a
down regulation of miRNA causes an up-
regulation of the aimed protein.
The mechanisms behind the involve-
ment of miRNA in diseases are not
completely understood. However, some
hypotheses are raised and are shown
below. Probably, multiple mechanisms
are responsible for the changes seen in
diseases. Recently four ways of miRNA
involvement have been proposed (Son-
koly and Pivarcsi 2009).
- Loss or down regulation of
miRNA expression due to mu-
tation, epigenetic inactivation,
aberrant processing, or tran-
scriptional down regulation.
Down regulation is common
in cancer and inflammatory
diseases.
- Overexpression of miRNA due to
gene amplification or mutations
in the promoter region, for ex-
ample, oncomirs (miRNAs in-
volved in the development of
cancer).
- A mutation in the 3′UTR of an
mRNA may affect a miRNA-
binding site, leading to overex-
pression of the target gene.
TCM Vol. 20, No. 2, 2010
 - A mutation in the 3′UTR of a
gene generating a new miRNA-
binding site.

miRNAs in Atherosclerosis
Many miRNAs have been found in heart
and vascular tissue. Of 180 miRNAs
arrayed, 140 miRNAs were found in
carotid arteries in rats (Ji et al. 2007),
and 157 miRNA of 233 miRNAs
arrayed were found in mouse hearts
(Cheng et al. 2007).
Endothelial cells are the first line of
defense against atherosclerosis. In a
recent paper, it was shown that sheer
stress is capable of inducing endothelial
miR-21. This miRNA causes up-regula-
tion of endothelial nitric oxide synthase
and reduces endothelial cell apoptosis
(Weber et al. 2010). The potential ather-
oprotective effects of this miRNA merit
further investigation in atherosclerotic
models. In addition, miRNAs are in-
volved in the prevention of leukocyte
adhesion. Vascular cell adhesion mole-
cule-1 (VCAM-1)-mediated adhesion
can be inhibited by miR-126 in human
umbilical vein endothelial cells. De-
creased expression of miR-126 up-reg-
ulates vascular cell adhesion molecule-
1-1 expression, which in turn enhances
leukocyte adherence to the endothelium
(Harris et al. 2008). Also, the expression
of E-selectin on vascular endothelial
cells can be manipulated by miRNAs
(miR-E1 and miR-E2). In human aorta
endothelial cells, suppression of E-selec-
tin expression and inhibition of leuko-
cyte adhesion has been found
(Yoshizaki et al. 2008). On the other
hand, endothelial cells can be stimulat-
ed by tumor necrosis factor to generate
miRNAs related to proatherogenic prop-
erties (Suarez et al. 2010).
In the development of atherosclerotic
plaques, the immune system plays a
crucial role. Toll-like receptors (espe-
cially TLR4) have extensively been im-
plicated in atherosclerotic plaque
development as well as destabilization
(Pasterkamp et al. 2004). Interestingly,
translational regulation of TLR4 by
miRNA has been described in a cholan-
gitis model (Chen et al. 2007). Whether
such regulatory control is possible in
other cell types remains to be deter-
mined. Furthermore, miR-147 is part of
a negative feedback loop in lipo-poly-
saccharide-stimulated monocytes (Liu
TCM Vol. 20, No. 2, 2010
et al. 2009). This further implies poten-
tial miRNA-related targets to modulate
plaque development and stability.
Recently, it has been shown that
mature miR-1 and -206 levels were
significantly up-regulated 1 week after
myocardial infarction induction (Shan
et al. 2009). Furthermore, both miRNAs
have been related to reduction of insulin-
like growth factor 1. As a result, the
extent of apoptosis increased (Shan et al.
2009, Yu et al. 2008). Interestingly cardi-
oprotective miRNA effects have been
shown (miR-21) resulting in less apopto-
sis (Dong et al. 2009, Yin et al. 2007). On
the other hand, differential miRNA ex-
pression in infracted, and border zone
areas were observed (Dong et al. 2009).
These findings imply an important role
for miRNA in myocardial infarction.
Therapeutic interference may add to a
further preservation of previously ische-
mic myocardium especially in the setting
of primary coronary interventions. Fur-
thermore, the possibility of systemic
detection may bring in new early diag-
nostic tools.
Angiogenesis is closely related to
plaque destabilization and rupture
(Figure 2). miRNAs have been reported F2
to influence angiogenesis (Bonauer et al.
2009). These miRNAs are called angio-
miRs. With the use of a hind limb
ischemia model, it has been shown that
miR-92a controls angiogenesis in mice.
Ischemic injury caused miR-92a up-
regulation and blockade of miR-92a
with systemic administration of antago-
mir-92a increased recovery of blood flow
(Bonauer et al. 2009). Some reviews have
been published addressing this topic in
the recent past (Kuehbacher et al. 2008,
Wang and Olson 2009, Wu et al. 2009).
Pro- as well as antiangiogenetic miRNAs
have been found (Table 1). Silencing of T1
Dicer in endothelial cells led to the up-
regulation of thrombospondin-1 that is a
known inhibitor of angiogenesis. It is
postulated that miRNAs Let-7 and the
miR-17-92 cluster are targeted at throm-
bospondin-1 and inhibitors of these two
miRNAs reduced endothelial cell sprout-
ing and matrigel tube formation in vitro
(Kuehbacher et al. 2008, Wang and Olson
2009). Down regulation of Drosha by
siRNA resulted in vitro in a significant
reduction of capillary sprouting and tube
formation but no reduced angiogenesis
in vivo(Kuehbacher et al. 2007). Finally,
miRNAs influence angiogenesis at the
level of vascular endothelial growth
factor (VEGF) and hypoxia. Blockade of
miR-210 inhibits hypoxia-induced vessel
formation and is a key player in VEGF-
driven endothelial cell migration (Fasa-
naro et al. 2009). Vascular endothelial
growth factor is also a target of miR15b,
-16, -20a, and -20b, and these miRNAs
are sharply down regulated after hypox-
ia. Inhibition of these posttranslational
regulators increased VEGF expression
significantly (Hua et al. 2006) indicating
that miRNAs are part of the angiogenic
switch in response to hypoxia. Interfer-
ence with angiogenesis in the plaque can
thus be envisioned at different levels. The
question whether systemic administra-
tion of specific inhibitors or antagonists
can be sufficiently targeted to (unstable)
plaques or whether local delivery should
be explored with the use of specific
devices remains.
 Diagnostic, Therapeutic, and
Future Perspectives
As described previously, miRNAs play
important roles in processes that also
contribute to plaque vulnerability. Anti-
angiogenic properties of the miR-17-92
cluster have recently been found (Doebele
et al. 2010). miRNA-based inhibition of
intraplaque angiogenesis may be a prom-
ising approach to prevent intraplaque
hemorrhage and subsequent rupture. In
addition, reducing inflammation by
interfering with cellular interactions or
with proinflammatory cell products (such
as metalloproteinases) may further stabi-
lize plaques.
The main difficulty with the use of
miRNAs for therapeutic purposes is the
delivery of the drug to target cells. There
have been a number of attempts to
overcome this problem, with the use of
techniques such as conjugation and
formulation. Conjugation includes direct
attachment of peptides or antibodies
to the noncoding RNAs that facilitate
cellular uptake. Formulation includes
complex lipid emulsions, synthetic lipo-
somes, and nanoparticles, among other
carrier complexes (Rooij van et al. 2008,
Silvestri et al. 2009), For example, the
conjugation of miRNAs to cholesterol
enhances cellular uptake of these sub-
stances. Cholesterol-bound nucleotides
(“antagomirs”) have been proven effec-
tive and specific silencers of endogenous
miRNAs in mice (Krutzfeldt et al. 2005).
67
Figure 2. miRNAs in atherosclerotic plaque formation. The potential positive or negative regulation by miRNAs on a number of atherosclerotic
plaque characteristics is shown. The main factors are neointimal formation, inflammation, apoptosis, and angiogenesis, including hypoxia. See
text for further explanation. SMC, smooth muscle cell; VCAM, vascular cell adhesion molecule-1.

These modified RNA molecules have
sequences complementary to target miR-
NAs. Intravenous injection of an antago-
nist of the hepatocyte-specific miR-122
(antagomir-122) led to decreased plasma
cholesterol levels and significantly im-
proved liver steatosis in an obese mouse
model (Esau et al. 2006). The first human
phase I studies are currently being per-
formed with the use of locked nucleic
acid–modified oligonucleotide (SPC3649)
complementary to miR-122, which suc-
cessfully suppressed hepatitis C virus
concentration in the bloodstream in
primates (Lanford et al. 2010). Other
promising miRNA-based therapeutics
are under investigation for treatment of
oncologic pathologic conditions such as
prostate cancer (Bonci et al. 2008),
leukemia (McLaughlin et al. 2007), and
breast cancer(Liang et al. 2007, Valastyan
et al. 2009).
In the cardiovascular field, miRNA-145
has been studied intensively for its
expected therapeutic power in atheroscle-
rotic disease. It is the most abundant
miRNA in arteries (Ji et al. 2007) and in
differentiated vascular smooth muscle
cells (Cheng et al. 2009). Adenovirus-
mediated gene transfer of miRNA-145 in
rat balloon-injured carotid arteries inhib-
ited neointimal lesion formation (Cheng et
al. 2009). Knockout of miRNA-145
resulted in formation of neointima in
mouse arteries (Boettger et al. 2009). In
addition, inhibition of miR-21 expression
via antisense oligonucleotide-mediated
miRNA depletion significantly decreased
neointima formation after angioplasty in
rats (Ji et al. 2007). Suppression of miR-21
via antisense-mediated depletion has also
been reported to act antihypertrophic in
animal models (Cheng et al. 2007), al-
though contrary results have also been
published (Tatsuguchi et al. 2007). The
results presented previously are promis-
ing, but still a lot of experiments are
necessary before clinical therapy can be
initiated; for example, the cellular and
molecular mechanisms and potential side
effects of miRNA-based therapy need to be
determined (Zhang 2009). Among the
difficulties of therapeutic interventions
are the invasive administration by injec-
tion and the potential off-target effects due
to the broad spectrum of cellular pathways
regulated by single miRNAs (Yang and
Mattes 2008).
The discovery of myocardial-specific
miRNAs and the fact that they can be
detected in peripheral blood samples
have paved the way for diagnostic appli-
cation. On the one hand, a large group of
patients where this may be of great
benefit are those presenting with acute
chest pain. The triage of these patients is

68 TCM Vol. 20, No. 2, 2010

Table 1. Atherosclerotic plaque characteristics affected by miRNAs
Characteristic Facilitating Inhibiting Effect Model

Angiogenesis miR-126, -17-92 miR-221/miR-222 Decreases plaque Hind limb ischemia

cluster, Let7b,f (Scalbert and Bril 2008), stability model, Dicer
(Scalbert and Bril 2008); miR-15b/miR-16, knockout, HUVECs,
miR-130a, -210, -378, miR-20a/b, -328 and the like
Hypoxia -296 (Wu et al. 2009); (Kuehbacher et al. 2008, Initiates HUVECs
and miR-27b Wu et al. 2009) angiogenesis
(Kuehbacher et al. 2008)
miR-210 (Panutsopulos
et al. 2005)
miR-210
(Fasanaro et al. 2008)
Apoptosis miR-214 miR-1 (Tang et al. 2009) Apoptosis of Ischemia reperfusion
(Cheng et al. 2005) miR-1d, 7, 148, 204, 210, VSMCs rat model
miR-15, 16 216, and 296 Decreases plaque (Tang et al. 2009)
(Guo et al. 2009) (Cheng et al. 2005) stability Antisense inhibitor
Reduces Bcl-2, in HeLa cells
increases (Cheng et al. 2005)
caspases 3, 8, 9 Culture-activated
HSCs
Inflammation miR-155 Down regulation Human monocyte-
(Ceppi et al. 2009) of VCAM-1 derived dendritic cells
miR-126 (Ceppi et al. 2009)
(Harris et al. 2008) HUVECs
(Harris et al. 2008)
MMPs/TLRs miR21 miR188 Decreases plaque
(Gabriely et al. 2008) (Mishra et al. 2009) stability
Cell adhesion miR-126 Inhibits VCAM-1 Knockdown
(Harris et al. 2008) expression on transfections
endothelial cells in HUVECs
Neointimal lesion miR-21 Initiation of VSMC Balloon injury and
formation (Ji et al. 2007) proliferation, dedifferentiated
decreased VSMCs in vitro
apoptosis (Ji et al. 2007)
The effects of different miRNAs on plaque (in)stability are summarized. Facilitating and inhibiting miRNAs and their specific modes of action are
mentioned. HUVEC, human umbilical vein endothelial cell; HSC, hepatic stellate cell; MMP, matrix metalloproteinases; TLR, Toll-like receptor;
VSMC, vascular smooth muscle cell; VCAM-1, vascular cell adhesion molecule 1.

now based on history, electrocardio-
gram, and biomarkers (especially tropo-
nin). Levels of miR-208, a cardiac-
specific nucleotide, correlate with the
classic biomarker cardiac troponin I (Ji
et al. 2009). Furthermore, miR-1 con-
centration in plasma of myocardial
infarction patients was lower than
healthy controls (Ai et al. 2010). Another
approach aiming at early diagnostics
may be to look for miRNAs related to
plaque rupture instead of myocardial
damage. Interestingly, the diagnostic
potential of miRNAs in yet another
large group of cardiac patients, those
with (suspected) heart failure, has re-
cently been explored (Tijsen et al. 2010).
Before implementation of such diagnos-
tics in the triage of (suspected) acute
coronary syndrome patients, the analyt-
ic procedure should be thoroughly vali-
dated and even more important fast to be
performed. Furthermore, more knowl-
edge about miRNA regulation in the
context of plaque biology and whether
certain miRNAs are plaque specific
could give research in this field a boost.
Finally, measurable circulating miR-
NAs within the body raise the hypothesis
that the miRNA signature could discrim-
inate between patients with stable and
unstable atherosclerotic plaques. Dis-
crimination between these forms of
plaques may lead to a patient-tailored
approach and help to choose between
intervention and watchful waiting and
thus decrease the number of unnecessary
and costly interventions.
 Ambros V: 2004. The functions of animal
Conclusions
This review summarizes the experimen-
tal data published thus far on the role of
miRNA in vulnerable plaque develop-
ment. The evidence, although indirect,
clearly indicates a role for miRNAs in
processes related to the risk of athero-
sclerotic plaque rupture. More plaque-
specific miRNA research is needed. Fu-
ture developments with the use of
miRNA-based strategies may give us the
possibility for ultrarapid diagnosis in
suspected acute coronary syndrome and
heart failure patients, to discriminate
between high- and low-risk patients and
to stabilize unstable plaques.
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