High Blood Pressure & Cardiovascular Prevention

https://doi.org/10.1007/s40292-019-00356-y

REVIEW ARTICLE

From Endothelium to Lipids, Through microRNAs and PCSK9:

A Fascinating Travel Across Atherosclerosis
D. D’Ardes1,2 · F. Santilli1 · M. T. Guagnano1 · M. Bucci1,2 · F. Cipollone1,2 

Received: 16 September 2019 / Accepted: 17 December 2019

© Italian Society of Hypertension 2020

Abstract
Lipids and endothelium are pivotal players on the scene of atherosclerosis and their interaction is crucial for the establish-
ment of the pathological processes. The endothelium is not only the border of the arterial wall: it plays a key role in regu-
lating circulating fatty acids and lipoproteins and vice versa it is regulated by these lipidic molecules thereby promoting
atherosclerosis. Inflammation is another important element in the relationship between lipids and endothelium. Recently,
proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recognized as a fundamental regulator of LDL-C and anti-
PCSK9 monoclonal antibodies have been approved for therapeutic use in hypercholesterolemia, with the promise to subvert
the natural history of the disease. Moreover, growing experimental and clinical evidence is enlarging our understanding of
the mechanisms through which this protein may facilitate the genesis of atherosclerosis, independently of its impact on lipid
metabolism. In addition, environmental stimuli may affect the post-transcriptional regulation of genes through micro-RNAs,
which in turn play a key role in orchestrating the crosstalk between endothelium and cholesterol. Advances in experimental
research, with development of high throughput techniques, have led, over the last century, to a tremendous progress in the
understanding and fine tuning of the molecular mechanisms leading to atherosclerosis. Identification of pivotal keystone
molecules bridging lipid metabolism, endothelial dysfunction and atherogenesis will provide the mechanistic substrate to
test valuable targets for prediction, prevention and treatment of atherosclerosis-related disease.

Keywords  Atherosclerosis · Endothelium · Lipids · PCSK9 · miRNA

1 A Couple at the Beginning
of Atherosclerosis: Endothelium
and Lipids
Atherosclerosis is the result of complex pathogenic mecha-
nisms initiated by the lipid-endothelium intertwining, even-
tually leading to the development of atherosclerotic plaques.
The endothelium is characterized by intercellular tight junc-
tions and represents a selectively permeable barrier. It has
D. D’Ardes and F. Santilli equally contributed to this review
article.
* F. Cipollone
francesco.cipollone@unich.it
1
Department of Medicine and Aging, “G. d’Annunzio”
University, Chieti, Italy
2
Clinica Medica Division and European Center of Excellence
on Atherosclerosis, Hypertension and Dyslipidemia
“SS. Annunziata” Hospital, Chieti, Italy
a duplex role, having both sensory and executive functions,
and can also generate molecules thus influencing multiple
processes such as vascular tone and remodeling, inflamma-
tion and thrombosis. A lot of different forces act on endothe-
lial cells (EC), one being the fluid shear stress. In fact,
where blood flow is uniform and laminar, cells are ellipsoid
in shape and aligned in the direction of flow, showing ele-
vated nitric oxide (NO) production, reduced oxidant stress,
enhanced barrier function and limited proinflammatory
activity, through Erk5-dependent expression of Klf2, which
binds NF-κB and competes for binding of transcriptional
coactivators. Vice versa, where flow is altered, cells have
polygonal shapes without a particular orientation, showing
an increased permeability to macromolecules, for example
low-density lipoprotein cholesterol (LDL-C) [1], reduced
NO production and enhanced oxidant stress, endothelial
turnover and proinflammatory activation. Moreover, expo-
sure to laminar flow in culture actively suppresses endothe-
lial activation by cytokines and oxidized LDL-C by hinder-
ing NF-кB-dependent proinflammatory gene expression and

Vol.:(0123456789)

promoting NF-кB-dependent anti-apoptotic gene expression
[2].
The accumulation of LDL-C in the subendothelial matrix
is indeed one of the most important initiating events of ath-
erosclerosis. The higher the LDL-C levels, the greater the
accumulation: LDL diffuses passively through EC junctions
and its retention in the vessel wall involves interactions
between the LDL-C constituent apolipoprotein B (apoB)
and matrix proteoglycans [3].
However, atherosclerosis is not just a LDL-C-centered
process: other apoB-containing lipoproteins, namely
lipoprotein(a) and remnants, can also accumulate in
the intima and promote atherosclerosis. In particular,
lipoprotein(a) has been shown to be particularly atherogenic
for its effects on fibrinolysis and smooth muscle cells (SMC)
growth [4].
A strong relationship exists between LDL-C and arterial
wall: the LDL-C are somehow ‘modified’ in the vessel wall,
undergoing oxidation, lipolysis, proteolysis and aggregation,
thus contributing to inflammation as well as to foam-cell
formation. Lipid peroxidation is one of the most significant
modifications in terms of early lesion formation as a result
of the exposure of vascular cells to oxidative stress stimuli,
a combination of enzymatic, such as lipoxygenases, and
non-enzymatic, free-radical catalyzed reactions. The main
modification of LDL-C takes place inside the intima, where
proteoglycans can sequester LDL-C [5].
If endothelium influences lipids, their migration and mod-
ification, vice versa a large number of studies have shown
an impairment of vasodilatation mediated by endothelium-
derived NO in response to elevated total cholesterol and
particularly LDL-C. LDL-C effects have been demonstrated
at numerous sites of the NO signaling pathway including
G-protein-coupled receptors, NO synthase (NOS) and NO
bioactivity, with evidence for enhanced superoxide forma-
tion and the consequent production of the less potent dilator
peroxynitrite. The effects of lipids on endothelium-depend-
ent vasodilation can be reversed not only by reducing the
level of lipids but also by provision of the NOS substrate,
l-arginine and antioxidants, although the mechanisms under-
lying these effects are not fully elucidated [6].
Lipids and endothelium do not represent the only pro-
tagonists on the scene: a lot of other actors can influence this
relationship and modulate endothelial dysfunction. Platelets
are an example of potential “influencers”.
Interestingly, both platelet activation and endothelial dys-
function are early events in the natural history of atheroscle-
rosis. A continuous dialogue exists between platelets and
endothelial cells, dating back to the bone marrow, where
haematopoietic precursors, megakaryocytes, and sinusoidal
endothelium interact directly [7]. In addition, platelets are
a source of oxygen free radicals, and trigger a cascade of
events that ultimately leads to endothelial dysfunction: this
D. D’Ardes et al.
process is supported by observations showing a progressive
decrease in platelet formation of NO moving from patients
with hereditary deficiency in gp91phox (NOX-2) to obese
patients [8].
NOX2 represents the catalytic core of nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase, that is
believed to modulate arterial tone. In fact, vascular tone is
modulated by NADPH oxidase-generated oxidative stress,
affecting NO generation. Since platelets are a pivotal source
of systemic NOX-2, it is conceivable that they are the initia-
tors of this detrimental cascade in settings of persistently
enhanced platelet activation, such as obesity and metabolic
syndrome [9].
2 The Lipids–Endothelium Couple
and a Third Wheel: Inflammation
Inflammation is another important element in the relation-
ship between lipids and endothelium. If macrophages play
a fundamental role since the initial phases of the athero-
sclerotic process, thus participating to plaque progression, a
staggering array of immune cells migrate into the plaque and
release different kinds of cytokines contributing to a further
progression or to a regression of the plaque.
The cytokines associated with inflammatory macrophage
and Th1 T cell responses, such as interferon-γ (IFN-γ), inter-
leukin (IL)-1α, IL-1β, and tumor necrosis factor-α (TNF-α)
are responsible for encouraging atherosclerosis and these
pro-atherogenic cytokines enhance foam cell formation by
enhancing modified LDL-C uptake and inhibiting proper
reverse cholesterol transport in macrophages. Other kinds
of cytokines, such as IL-13, IL-10, IL-19 and transform-
ing growth factor-β (TGF-β), have been shown to reduce
atherosclerosis [10].
TNF-α and IFN-γ also contribute to macrophage and vas-
cular smooth muscle cells (VSMCs) apoptosis, promoting
the thinning of the fibrous cap and the growth of the necrotic
core, whose progression is linked to the formation of cho-
lesterol crystals in the cytosols, leading to inflammasome
activation and subsequent IL-1α/β production [11].
In addition, macrophages can contribute to oxidative
stress through production of hydrogen peroxide, superox-
ides, neopterin and myeloperoxidases [12].
These radicals can promote further oxidation of LDL-C
within the plaques, as well as the triggering of endoplasmic
reticulum (ER) stress that can initiate apoptosis. In contrast
to atherogenic inflammatory responses, the atheroprotec-
tive cytokines IL-10, IL-19 and TGF-β reduce leukocyte
recruitment and inflammation and slow down atherosclerosis
progression [13]. Thus, the plaque may be considered as a
dynamic environment in which plaque progression is the
From Endothelium to Lipids, Through microRNAs and PCSK9: A Fascinating Travel Across…
result of the balance between pro-inflammatory and athero-
protective cytokines.
In this regard, a pivotal role has been reported for soluble
CD40 ligand (sCD40L), with particular reference to diabetic
patients. Upregulation of sCD40L as a consequence of per-
sistent hyperglycaemia in diabetic patients seems to result
in EC activation and monocyte recruitment to the arterial
wall, possibly contributing to accelerated atherosclerosis
development in diabetes. In fact, elevated sCD40L levels
have been associated with enhanced in vitro expression of
adhesion molecules, impaired migration in ECs, enhanced
­O2-generation in monocytes and with higher HbA1c; vice
versa, metabolic profile improvement has been associated
with a reduction of plasma sCD40L [14].
In addition to inflammation, endothelial cells at athero-
susceptible sites show signs of enhanced cellular injury as a
result of mechanical, oxidative and metabolic stress. Tran-
scriptome analysis of endothelial cells at athero-susceptible
sites suggests that ER stress may exert a pivotal role in ath-
erogenic endothelial activation [15].
ER stress is significant when protein rates overcome the
ER’s ability to promote proper protein folding. The accumu-
lation of misfolded peptides initiates the unfolded protein
response and this process results in an upregulation of ER
chaperone proteins. ER stress plays a role in inflammation,
oxidative stress and apoptosis [16].
It has also been noted that disturbed flow promotes ER
stress in endothelial cells and mimicking ER stress enhances
atherosclerotic plaque formation in laminar flow regions
[17].
In contrast, laminar flow is known to reduce endothe-
lial cell metabolism through Kruppel like factor 2 (Klf2)-
dependent repression of endothelial glycolytic enzymes
[18], eliciting in turn endothelial cell autophagy to protect
endothelial cells from oxidant-induced cell injury [19].
Moreover, oscillatory flow stimulates a c-Jun N-terminal
kinase (JNK)-dependent increase in mitochondrial super-
oxide production limiting autophagy, thus accumulating
oxidant injuries at sites of disturbed flow [20].
3 PCSK9: An Actor Not to be Missed
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a
fundamental regulator of LDL-C. Consistently, anti-PCSK9
monoclonal antibodies have been recently approved for the
treatment of heterozygous familial hyper-cholesterolemia
and clinical atherosclerotic cardiovascular disease as an
addition to diet and maximally-tolerated statin therapy in
adults who require additional LDL-C lowering [21].
PCSK9 interacts with the LDL-C receptor (LDL-R)
inducing its degradation by conveying it in lysosomes and
blocking its recycling on the cytoplasmic membrane [22].
Because PCSK9 induces the degradation of LDL-R, its
pharmacological inhibition would prolong the duration
of the receptor action, leading to a drastic reduction of
LDL-C [23].
Moreover, growing experimental and clinical evidence
is widening our understanding of the mechanisms through
which this protein may facilitate the genesis of athero-
sclerosis, independently of its impact on lipid metabolism
[24].
An increasingly emerging hypothesis suggests that the
impact of PCSK9 on lipids and on atherosclerosis involves
mechanisms going beyond the regulation of the clearance of
the LDL-C: PCSK9, in fact, does not bind exclusively the
LDL-R but promotes the degradation of other receptors such
as the receptor of very low density lipoproteins (VLDL),
CD36, ApoER2 and receptor of type 1 LDL (LRP1) [25,
26], whose contribution to the homeostasis of cholesterol
is still unclear, but seems to be linked to triglyceride-rich
lipoprotein (TRL) metabolism. TRLs represent important
elements in atherosclerosis development and the impact of
PCSK9 on these highly pro-atherogenic particles provides
an additional mechanism by which PCSK9 inhibitors can
reduce the risk of atherosclerotic cardiovascular diseases.
PCSK9 has also been shown to provide other proathero-
sclerotic effects not exclusively linked to plasma concentra-
tions of apoB-containing lipoproteins. Indeed, PCSK9 seems
to be expressed in the atheromatous plaque and in particular
at the level of VSMCs and in ECs, where it exerts parac-
rine effects on an array of foam cell receptors, including the
LDL-R and CD36. This represents a potential mechanism
to modulate atherogenesis independently of plasma LDL-C
concentrations, although its impact on each of these foam
cell receptors and its relevance to atherosclerosis remains
unclear [27].
PCSK9 released by VSMCs may influence the expression
of the LDL-R on the surface of macrophages [28]. PCSK9
has been also shown to promote the inflammatory response
in macrophages [29].
The expression of PCSK9 in VSMCs appears to be influ-
enced by shear stress. Moreover, in the absence of PCSK9,
a protective effect has been observed on the formation of the
neointima, after a vascular insult [30].
An interesting relationship may also exist between
PCSK9 and endothelial function. Indeed, treatment with a
PCSK9 inhibitor may improve endothelial function in sub-
jects with increased cardiovascular risk and this improve-
ment is proportional to LDL-C reduction [31].
One study reported an impressive improvement in
carotid-femoral pulse wave velocity after short-term treat-
ment with a PCSK9 inhibitor. This result was achieved
despite an increase in LDL-C, suggesting that the PCSK9
inhibitors may exert a beneficial impact on vasomotor func-
tion independently of their lipid-lowering effect [32].

Unexpectedly, given the body of evidence on endothe-
lial function, studies performed until now have shown no
correlation between PCSK9 and hypertension. Indeed,
PCSK9−/− mice display normal sodium balance and blood
pressure regulation [33]. Moreover, PCSK9 level is unre-
lated to blood pressure in Chinese hypertensive patients
[34]. Larger studies are needed to better clarify the link
between hypertension and PCSK9.
Another important element within the relationship between
inflammation, PCSK9 and atherosclerosis is represented by
the lectin-like oxidized low-density lipoprotein receptor-1
(LOX-1) whose expression is increased by inflammation and
that is involved in oxidized LDL-C uptake with a self-regula-
tion mechanism with PCSK9, that induces LOX-1 transcrip-
tion which in turn stimulates PCSK9 [35]. Furthermore, in an
inflammatory context, high levels of PCSK9 potently stimu-
late the expression of LOX-1 and oxidized LDL-C uptake
in macrophages and, moreover, PCSK9 has been shown to
promote atherosclerosis directly through the modulation of
proinflammatory monocytes in the arterial wall [36].
In conclusion, PCSK9 appears to have pleiotropic actions
and, consistently, the effect of anti-PCSK9 monoclonal anti-
bodies may determine not only a strong reduction of LDL-C,
but also other important effects in the atherosclerotic pro-
cess, that are still the object of ongoing investigations.
4 micro‑RNAs: The Directors Behind
the Scene of Atherosclerosis
MicroRNAs (miRNAs) play a key role in the context of the
crosstalk between endothelium and cholesterol [37] (Fig. 1).
Fig. 1  Summary of the influence of PCSK9 and hypercholesterolemia on the relationship between endothelium and lipids. In this context, also
microRNAs, shear stress and inflammation play an important role
D. D’Ardes et al.
MiRNAs are short non-coding RNA molecules (about
18–24 nucleotides), exerting their function via the seed
region, a sequence of 6–8 nucleotides that binds to messen-
ger ribonucleic acid (mRNA) [38] identified over 25 years
ago [39]. This interaction with mRNA often results in the
prevention of protein production by different mechanisms.
Both hyperlipidemia and shear stress are two important
pathogenetic elements in the vessel wall in terms of athero-
sclerotic lesion formation. When stimulated by biochemical
and biomechanical stimuli, endothelial cells undergo a series
of molecular and cellular conformational changes promot-
ing atherogenesis. First of all, early induction of adhesion
molecule expression such as vascular cell adhesion molecule
(VCAM)-1, intracellular adhesion molecule (ICAM)-1 and
E-selectin facilitates leukocyte recruitment to the vessel wall
and may be among the earliest hallmarks associated with
nascent plaques [40].
A lot of miRNAs have been implicated in atherogenesis
thanks to the possibility to target the 3′-UTRs of these mol-
ecules such as miR-17-3p, targeting ICAM-1, and miR-31,
targeting E-selectin [41].
4.1 MiRNAs, Endothelium and Inflammation
MiR-92a is expressed in ECs and is dynamically regulated
by shear stress both in vitro and in vivo [42]. When compar-
ing in vivo athero-prone areas of the aortic arch with athero-
resistant regions, miR-92a expression has been found highly
induced in the first ones [43].
In the context of the dynamic relation between endothe-
lium and physical forces that act within the vessels, a large
number of miRNAs, such as miR-126, miR-10a, miR-19a,
From Endothelium to Lipids, Through microRNAs and PCSK9: A Fascinating Travel Across…
miR-23b, miR-21, miR-663, miR-143/145, miR-101, miR-
712, miR-205, miR-155, 146a and miR-181b seem to have a
role as mechano-miRNAs. These mechano-miRNAs mainly
target key signaling pathways involved in endothelial cells
cycle, inflammation, apoptosis and NO signaling [44].
Moreover, miR-126 is one of the most widely expressed
miRNAs in the endothelium and it has been shown to play a
crucial role in regulating inflammation and angiogenesis [45].
In fact, miR-126 has been involved in endothelial dys-
function itself, by reducing VCAM-1 expression and thereby
leucocyte homing in the arteries, thus modulating, through
different pathways, vascular inflammation [46].
A similar role in the regulation of vascular inflamma-
tion is played by miR-155 and miR-221/222 through NOS
regulation [47].
Moreover, miR-155 increased untreated monocyte
adhesion to aortic endothelial cells by promoting ICAM-1
expression during homeostasis and lipopolysaccharides
(LPS)-induced inflammation [48].
On the contrary, a number of miRNAs with anti-inflam-
matory properties has been identified. For example, miR-10a
is expressed in endothelial cells where it seems to contrast
pro-inflammatory gene expression in athero-susceptible
regions in vivo, thus exerting a possible atheroprotective
role [49].
4.2 MiRNAs, Liver and Cholesterol Metabolism
In addition to modulate inflammation, miRNAs are
also involved in cholesterol homeostasis by controlling
LDL-C and high density lipoprotein cholesterol (HDL-C)
abundance.
Since the liver has a key role in the production and clear-
ance of lipoproteins, a great number of hepatic-enriched
miRNAs have been identified that functionally regulate lipo-
protein metabolism. First of all, miR-122 has been implicated
in lipoprotein metabolism. Studies based on loss-of-function
experiments in animals, identified miR-122 as a crucial regu-
lator of cholesterol and lipoprotein homeostasis [50].
In addition, miR-223 and miR-27b have been identified as
post-transcriptional regulatory hubs involved in the control of
cholesterol and lipoprotein metabolism genes [51, 52]. Con-
cerning miR-223, it represses genes involved in cholesterol
biosynthesis (HMGS1, SC4MOL) and HDL-C uptake (SRB1)
[51], whereas miR-27b represses a number of targets (PPARG​,
GPAM, ANGPTL3 and NDST1) involved in lipoprotein metab-
olism and remodeling [52].
MiRNA regulation of plasma levels of LDL-C through tar-
geting of the LDL-R has also been reported. LDL-R expres-
sion in the liver promotes the clearance of circulating LDL-C
particles and is a major determinant of plasma cholesterol
levels. MiRNAs are also involved in the regulation of LDL-C
plasma levels: miR-148a is a negative regulator of expression
and activity of LDL-R, that usually promotes the clearance of
circulating LDL-C particles [53].
MiRNAs have also been reported to act as critical regula-
tors of HDL-C biogenesis and cholesterol efflux. In fact, a
great number of miRNAs (miR-33 [54], miR-758 [55], miR-26
[56], miR-106 [57], miR-144 [58]) have been shown to target
ABCA1 to reduce cholesterol efflux to apoA1 in vitro. MiR-33
inhibition in mouse models of atherosclerosis plays a pivotal
role in the modulation of circulating HDL-C and regression of
atherosclerosis. Targeted deletion of miR-33 increased plasma
levels of HDL-C and reduced atherosclerotic plaque size in
Apoe−/− mice and furthermore treatment of Ldlr−/− mice
increased plasma levels of HDL-C associated with a regres-
sion of atherosclerosis [59].
MiRNAs have also been implicated in macrophage choles-
terol efflux in stable plaque. In fact, adenosine triphosphate
(ATP)-binding cassette (ABC) transporters A1 and G1 are the
main transporters involved in this process and we showed that
miR-758 and miR-33b are potential modulators of ABCA1
expression in atherosclerotic plaques [60].
4.3 MiRNAs and Atherosclerotic Plaques
MiRNAs have been also implicated in plaque instability and
related cardiovascular events, such as stroke and myocardial
infarction. We demonstrated that profound differences exist
in the expression of 5 miRNAs (miR-100, mi-127, miR-145,
miR-133a, and miR-133b) in symptomatic versus asympto-
matic plaques. These data were confirmed by in vitro experi-
ments on endothelial cells transfected with miR-145 and miR-
133a that further remarked the importance of these miRNAs
in the modulation of stroke-related proteins [61].
Moreover, hypertension may upregulate miR-145 expres-
sion in human atherosclerotic plaques [62].
In light of all these data, it seems clear that miRNAs rep-
resent crucial messengers of the fascinating dialogue between
endothelium and lipids. This relation has attracted the attention
of researchers and it will continue to deepen our knowledge,
thanks to further studies, with important implications in terms
of diagnosis and therapies for our patients.
5 Conclusions and Perspectives
Advances in experimental research, with development of
high throughput techniques, have led, over the last century,
to a tremendous progress in the understanding and fine tun-
ing of the molecular mechanisms leading to atherosclerosis.
Identification of pivotal keystone molecules bridging lipid
metabolism, endothelial dysfunction and atherogenesis, such
as LOX-1, PCSK9 and others, are emerging as valuable tar-
get for prediction, prevention and treatment of atheroscle-
rosis-related disease.

Intensive LDL-C lowering, achieved through the com-
bined therapy with PCSK9 antibody and statin, has been
associated with coronary plaque regression in a patient with
double-heterozygous familial hypercholesterolemia with a
LDL-R mutation and a PCSK9 V4I mutation. This finding
provides the mechanistic framework for studies assessing the
efficacy of combined therapy including PCSK9 antibodies in
reducing cardiovascular events in patients with established
cardiovascular disease [63].
The existence of a functional miRNAs machinery in
human cells have deepen our knowledge about the molecular
phenotype in health and disease, and opens up the possibility
of modulating gene expression using therapeutic RNAs to
prevent atherogenesis [64].
An additional layer of complexity has been provided, over
the last 10 years, by the publication of large-scale genome-
wide association studies (GWAS), identifying dozens of can-
didate genetic loci associated with risk for coronary artery
disease (CAD). Indeed, genetics accounts for approximately
50% heritability of CAD. Since the role and function of
identified loci or genes remains often elusive, this kind of
studies has promoted a novel wave of functional studies to
translate GWAS findings into novel discoveries involving
the pathogenesis of atherosclerosis and atherothrombosis. In
this regard, the novel GWAS-identified CAD risk gene CAD/
KIAA1462 has been shown to promote endothelial dysfunc-
tion and atherosclerosis. Indeed, junctional protein JCAD
triggers the expression of inflammatory genes in endothelial
cells, driving an inflammatory process via the recruitment of
monocytes and resulting in the formation of atherosclerotic
plaques in ApoE−/− mice [65]. This approach, starting from
the genetic loci associated with the risk of disease and mov-
ing back to the pathophysiology, has also therapeutic impli-
cations. Indeed, targeting GWAS-identified genes relevant
to cardiovascular disease, such as JCAD, may represent a
novel strategy for designing innovative drugs to prevent or
delay atherosclerosis and thrombosis.
Compliance With Ethical Standards  Ponte A, Santucci A, Cuccurullo F, Mezzetti A. Enhanced
Conflict of Interest  On behalf of all authors, the corresponding author
states that there is no conflict of interest.
Research Involving Human Participants and/or Animals  Not applicable.
Informed Consent  Not applicable.
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