Citation: Jebari-Benslaiman, S.;
Galicia-García, U.; Larrea-Sebal, A.;
Olaetxea, J.R.; Alloza, I.;
Vandenbroeck, K.; Benito-Vicente, A.;
Martín, C. Pathophysiology of
Atherosclerosis. Int. J. Mol. Sci. 2022,
23, 3346. https://doi.org/10.3390/
ijms23063346
Academic Editor: Manfredi Rizzo
Received: 3 December 2021
Accepted: 18 March 2022
Published: 20 March 2022
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International Journal of
Molecular Sciences
Review
Pathophysiology of Atherosclerosis
Shifa Jebari-Benslaiman 1,2, Unai Galicia-García 2,3, Asier Larrea-Sebal 2,3, Javier Rekondo Olaetxea 4,
Iraide Alloza 1,5, Koen Vandenbroeck 1,5,6, Asier Benito-Vicente 1,2,* and César Martín 1,2,*
1 Department of Biochemistry and Molecular Biology, Universidad del País Vasco UPV/EHU,
48940 Leioa, Bizkaia, Spain; sjebari001@ikasle.ehu.eus (S.J.-B.); iraide.alloza@ehu.eus (I.A.);
koen.vandenbroeck@ehu.eus (K.V.)
2 Bioﬁsika Institute (UPV/EHU, CSIC), Barrio Sarriena s/n., 48940 Leioa, Bizkaia, Spain;
u.galiciag@gmail.com (U.G.-G.); alarrea040@ikasle.ehu.eus (A.L.-S.)
3 Fundación Bioﬁsika Bizkaia, Barrio Sarriena s/n., 48940 Leioa, Bizkaia, Spain
4 Cardiology Department, Basurto University Hospital, 48013 Bilbao, Bizkaia, Spain;
javiergregorio.rekondoolaetxea@osakidetza.eus
5 Inﬂammation & Biomarkers Group, Biocruces Bizkaia Health Research Institute,
48903 Barakaldo, Bizkaia, Spain
6 Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Bizkaia, Spain
* Correspondence: asier.benito@ehu.eus (A.B.-V.); cesar.martin@ehu.eus (C.M.); Tel.: +34-946-01-2741 (C.M.)
Abstract: Atherosclerosis is the main risk factor for cardiovascular disease (CVD), which is the
leading cause of mortality worldwide. Atherosclerosis is initiated by endothelium activation and,
followed by a cascade of events (accumulation of lipids, ﬁbrous elements, and calciﬁcation), triggers
the vessel narrowing and activation of inﬂammatory pathways. The resultant atheroma plaque, along
with these processes, results in cardiovascular complications. This review focuses on the different
stages of atherosclerosis development, ranging from endothelial dysfunction to plaque rupture. In
addition, the post-transcriptional regulation and modulation of atheroma plaque by microRNAs and
lncRNAs, the role of microbiota, and the importance of sex as a crucial risk factor in atherosclerosis
are covered here in order to provide a global view of the disease.
Keywords: atherosclerosis; endothelial dysfunction; atheroma plaque; risk factors; microbiota;
ocLDL; microRNA; lncRNA
1. Introduction
Atherosclerosis is a disease that is characterized by the accumulation of lipids, ﬁbrous
elements, and calciﬁcation within the large arteries. This process is initiated by endothelium
activation, followed by a cascade of events, which implies the vessel narrowing and
activation of inﬂammatory pathways leading to atheroma plaque formation. Altogether,
these processes result in cardiovascular complications that remain as the leading cause of
death worldwide.
In this study, we aimed to review the mechanism of development of atherosclerosis,
including endothelial dysfunction, fatty streak formation, ﬁbrous plaque formation, and
plaque rupture. This review approaches the pathophysiology of atherosclerosis from a
broad perspective, examining the pathological and biochemical processes of atherosclerotic
plaque formation and growth. In addition, this review summarizes the current
understanding of the involvement of microRNAs and lncRNAs in post-transcriptional
regulation and modulation of atheroma plaque and atherogenesis. Non-traditional drivers
of atherosclerosis—such as disturbed sleep, physical inactivity, the microbiome, the role of
microbiota, and the importance of sex as a crucial risk factor in atherosclerosis are covered
here in order to provide a global view of the disease.
Int. J. Mol. Sci. 2022, 23, 3346. https://doi.org/10.3390/ijms23063346 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2022, 23, 3346 2 of 38
2. Endothelium
The vascular endothelium is a heterogeneous monolayer formed by endothelial cells
(ECs), which face the luminal side of all blood vessels, representing the ﬁrst barrier for
molecules, cells, or pathogens circulating in the bloodstream [1]. In large vessels, the
vessel wall is lined with a single EC layer called the endothelium, which, together with
collagen and elastic ﬁbers, forms the luminal layer of the vessels or the intima. ECs are in
intimate contact with tunica media consisting of vascular smooth muscle cells (VSMC) and
elastic and collagenous tissue. Finally, surrounding this layer is tunica adventitia, which is
composed mainly of a dense matrix of connective tissue. On the other hand, the walls of
arterioles and venules are composed of the same three layers as the larger vessels, although
the media and adventitia are much thinner and less pronounced. Finally, post-capillary
venules completely lack media and adventitia and only consist of ECs and a basement
membrane [2].
Endothelium is strategically located between circulating blood and tissues, working as
a sensor and transducer of signals by the production of biologically active substances. All
changes in circulating blood are perceived by the endothelium, which then mediates signal
transduction to the other layers of the vascular wall. Such changes include mechanical
stress (elongation and wall shear stress (WSS)), as well as changes in the concentration of
metabolic factors.
Different mechanical forces acting on the arterial wall modulate several physiological
functions, such as the regulation of homeostasis, vascular tone, and vascular integrity.
Apart from their role in homeostasis, the involvement of hemodynamics in vascular disease
development has been shown to be crucial in the pathology of atherosclerosis, affecting
both disease onset and progression. The main forces acting on the arterial wall include
both tensile stress induced by blood pressure and WSS, a tangential force to the vessel wall
induced by blood ﬂow that plays an important role in atherogenic hemodynamics [3].
Vessel segments with low WSS or highly oscillatory WSS appear to be at the highest
risk for development of atherosclerosis [4,5]. WSS change can directly affect the morphology
and function of the vascular endothelium and stimulate the migration and proliferation of
VSMCs and mononuclear cells [6]. Whether low or unstable, changing WSS is an indicator
to evaluate hemodynamic changes that are closely related to atherosclerosis [7,8].
The magnitude and direction of WSS are recognized by mechanosensors on the endothelium
and are transduced as biochemical signaling. WSS-induced mechanotransduction
regulates the expression of numerous genes involved in cell morphology, adhesion,
and proliferation.
For example, shear stress induces characteristic EC alignment [9,10] (Figure 1A). In
tubular or straight regions of arteries, where the WSS presents a laminar ﬂow, ECs show a
ﬂattened shape and an elongated alignment in the direction of the ﬂow [11]. However, at
the bifurcation or high vessel curvature sites, ﬂow disturbance occurs and, as a consequence
of the turbulent and reversal ﬂow with lowered WSS at the outer vessel wall, ECs augment
their volume by adopting a cobblestone appearance [12] (Figure 1A). Moreover, hemodynamic
forces determine the early development of localized atherosclerotic plaques that are
not randomly distributed, neither in experimental animal models nor in humans [13,14].
Atherosclerotic lesions mainly occur in regions characterized by low WSS and ﬂow separation
(Figure 1B) and most frequently involve branch points and bifurcations. The extensive
correlative data indicating that low shear or disrupted ﬂow accounts for the localization
of atherosclerosis lesions emphasize the importance of arterial branches and bifurcations
when performing a diagnosis of atherosclerotic lesion development or progression.
Int. J. Mol. Sci. 2022, 23, 3346 3 of 38
Figure 1. Effect of ﬂow and WSS patterns at arterial bifurcations on atherosclerotic plaque development.
(A) In straight vessel segments, physiological WSS with laminar ﬂow leads to ECs and shows a
quiescent characteristic ﬂattened shape when ﬂow disturbance occurs. Lower WSS at the outer vessel
wall causes ECs to adopt a cobblestone appearance. (B) Turbulent ﬂow occurs at bifurcations and
branch points where the arterial curvature is higher due to ﬂow separation. Disturbed laminar ﬂow
or turbulent ﬂow reduces WSS and promotes endothelial dysfunction and LDL inﬁltration, which
constitutes the ﬁrst step of atheroma plaque formation. On the contrary, low curvature areas of the
vascular system subjected to higher shear stress are athero-protected.
Endothelium modulates the tone of underlying vascular smooth muscle; maintains a
non-adhesive luminal surface; and mediates hemostasis, cellular proliferation, and inﬂammatory
and immune response in the vascular wall [15]. In fact, endothelium releases both
agonists and antagonists in order to balance the effect in both directions. For instance, ECs
are able to produce both coagulants or anti-coagulants, vasodilators or vasoconstrictors
and pro-inﬂammatory or anti-inﬂammatory molecules [15,16].
3. Atherosclerosis Initiation and Fatty Streak Formation
Atherosclerosis initiates upon endothelial dysfunction accompanied by low-density
lipoprotein (LDL) retention and its modiﬁcation in the intima [17,18]. Modiﬁed LDLs,
Int. J. Mol. Sci. 2022, 23, 3346 4 of 38
together with additional atherogenic factors, promote the activation of ECs, leading to
monocyte recruitment within the intima. Modiﬁed LDLs are avidly captured by differentiated
monocytes and VSMC, which promote foam cell formation [19,20]. In addition,
several inﬂammatory signaling pathways are activated, allowing the fatty streak formation,
which represents the ﬁrst sign of atherosclerosis and is characterized by a substantial accumulation
of lipids both within the cells (macrophages and VSMC) and the extracellular
media [21].
3.1. Endothelial Dysfunction in Atherosclerosis Development
Disruption of the mechanisms involved in vascular homeostasis regulation leads to
endothelial dysfunction [9,12,22]. Brieﬂy, when ECs lose their ability to maintain homeostasis,
vessel walls are predisposed to vasoconstriction, lipid inﬁltration, leukocyte adhesion,
platelet activation, and oxidative stress, among other things [23,24]. Together, these induce
an inﬂammatory response that is considered the ﬁrst step of atheromatous plaque formation:
the fatty streak [12,22]. In addition, endothelial dysfunction also plays a remarkable
role in subsequent steps of atherosclerosis by participating in plaque development and
in its rupture in the last steps of atherosclerosis [22]. Therefore, an increased endothelial
dysfunction is considered an early indicator of atherogenesis [25,26].
3.1.1. Hemodynamic Forces and Endothelial Dysfunction
Hemodynamic forces constitute a local risk factor of atherogenesis, as they promote
endothelial dysfunction [27]. As indicated above, lesion-prone regions are mainly located
in areas where the laminar ﬂow is disturbed due to ﬂow separation, recircularization, or
reattachment [28]. This turbulent ﬂow creates temporal and spatial gradients, which result
in a higher oscillatory index and a lower shear stress [11,29]. In addition, a disturbed ﬂow
also favors lipoprotein inﬁltration into the vessel intima, ﬁrstly because LDLs remain in
those areas for longer periods of time, and secondly because turbulent ﬂow induces a
physical disruption of endothelial integrity [30,31], thus facilitating lipoprotein inﬁltration
[30,32,33]. In addition, another fundamental link between hemodynamic forces and
atherogenesis relies on the expression of diverse endothelial genes regulated by blood
mechanical stimulus [34–36].
The effect of shear stress over endothelial gene expression has been studied during
the last 20 years; more than 40 genes implicated in the process have been discovered so
far [37–42]. Among them, several atherogenic genes, such as monocyte chemoattractant
protein 1 (MCP-1), which induces monocyte permeation into the arterial wall [43–45],
and platelet-derived growth factors (PDGFs), which enhance VSMC migration [46–48], are
upregulated in ECs. Interestingly, research evidence revealed shear stress response elements
(SSREs) in the promoters of these genes and others, such as eNOS or platelet adhesion
molecule-1 (PECAM-1), which contribute to plaque development [49–52]. Moreover, the
combination of two or more SSREs in the same promoter could have a synergistic effect
that enhances the expression of those genes [53]. On the other hand, in straight regions of
the vasculature where the laminar ﬂow drives high shear stress in the endothelium, some
pro-atherogenic genes are downregulated, while genes that induce cell-cycle growth arrest
or increase the antioxidant capacity are upregulated. Indeed, a long exposure of ECs to
undisturbed laminar ﬂow promotes the upregulation of endothelial nitric oxide synthases
(eNOSs), thus increasing their nitric oxide (NO) synthesis capacity [13,49].
These ﬁndings suggest a differential molecular response in endothelium depending
on the blood ﬂow pattern, highlighting the role of hemodynamic forces in endothelial
dysfunction.
3.1.2. The Role of Nitric Oxide in Endothelial Dysfunction
Endothelial dysfunction is also explained through a reduction in NO bioavailability
[54]. NO is synthesized from L-arginine in ECs in a reaction catalyzed by eNOS and
diffuses across cell membranes, reaching the smooth muscle tissue of the artery wall.
Int. J. Mol. Sci. 2022, 23, 3346 5 of 38
NO promotes smooth muscle ﬁber relaxation, known as endothelium-dependent vasodilatation
[55,56], and is considered an athero-protective molecule, because it counteracts
atherogenesis and its complications. Speciﬁcally, NO is involved in the reduction of platelet
aggregation; tissue oxidation and inﬂammation; the activation of thrombogenic factors; and
cell growth, proliferation, and migration, among other things [57–60] (Figure 2). Moreover,
it maintains metabolic homeostasis, as it reduces triglyceride content and steatosis and
increases insulin synthesis, glucose clearance, and mitochondrial efﬁciency [61]. However,
in the presence of cardiovascular risk factors, such as hyperlipidemia, hypertension,
smoking, or diabetes, NO production is reduced as a consequence of the increased oxidative
stress, which is commonly associated with those pathologies [28,62,63]. Oxidative
stress promotes the synthesis of pro-atherogenic cytokines (TNF-α and interleukins IL-1
and IL-6), adhesion molecules (VCAM-I and ICAM-I), and chemokines (MCP-1) through
NF-kB activation mediated by heat-shock proteins (HSP-60). These mediators inhibit the
activity of eNOS and, consequently, NO production [12] (Figure 2). In fact, studies carried
out in hypercholesterolemic patients demonstrated an impaired endothelium-dependent
vasodilatation due to a defect in the bioavailability of NO [64]. Hypertensive patients
also show a defect in the endothelium-derived NO system that may explain the abnormal
endothelium-dependent vasodilatation [65,66].
Figure 2. Nitric oxygen regulates cardiovascular metabolism and is compromised in the presence of
cardiovascular risk factors. eNOS catalyzes the production of NO from L-arginine. NO is an essential
metabolite that inhibits the progression of atherosclerosis improving vasorelaxation, angiogenesis,
endothelial function, insulin secretion, glucose clearance, and mitochondrial efﬁciency. On the other
hand, it reduces oxidative stress, inﬂammation, plasma lipid levels, and stenosis. Cardiovascular
risk factors, such as hyperlipidemia, hypertension, and diabetes, inhibit eNOS activity upon NF-kβ
induction, reducing NO and promoting atherosclerosis development.
3.2. LDL Inﬁltration
Accumulation of LDL in plasma favors transendothelial inﬁltration of circulating
LDLs to the intima. Although it has been traditionally accepted that LDLs cross the
endothelium by diffusion or paracellularly [67–70], it is now accepted that transcytosis
plays an important role in the transendothelial transport of LDLs [71,72]. More speciﬁcally,
it has been shown that LDL transcytosis is mediated by scavenger receptor B1 (SR-B1)
and activin A receptor-like type 1 (ALK1) receptor of the endothelium, which differs
from the classical LDL endocytosis pathway mediated by LDLR [73]. SR-B1 and ALK1
receptors co-localize with caveolae, indicating that LDL transcytosis by SRB1 and ALK1 is
mediated by a caveolae-dependent mechanism [74,75]. Moreover, an absence of caveolin-1,
Int. J. Mol. Sci. 2022, 23, 3346 6 of 38
the major structural protein of caveolae in ECs [76], signiﬁcantly impairs LDL transport
and retention within the arterial wall [77,78], and increased levels of caveolin-1 have been
found in atherosclerotic lesions [79]. Although additional experiments are needed to
elucidate the speciﬁc transport mechanism of both receptors, these ﬁndings suggest that
caveolae-dependent LDL uptake plays an important role in LDL transcytosis [80,81].
Although this review only summarizes the role of ECs in LDL inﬁltration, it is important
to note that other factors, such as the glycocalyx [82], pericytes [83], the subendothelial
extracellular matrix [84,85], and the role of shear stress [32], should be considered.
LDL Modiﬁcations in the Intima
Once in the subendothelial space, trapped LDL particles are oxidized, a process
facilitated by the absence of protective plasma antioxidants, such as tocopherol, ascorbate,
urate, apolipoproteins, or serum albumin [86,87]. Oxidized LDLs are key inﬂammatory
components that promote atherosclerotic plaque development, as they contain oxidized
lipids and products derived from their degradation that contribute to the physiopathology
of the disease [88,89].
LDLs can be oxidized by free radicals present in the extracellular media, such as
superoxide (O2
•–), hydroxyl radicals (•OH) [90], and others, such as HClO, produced by
the surrounding cells [91,92]. Additionally, LDLs can be directly oxidized by the enzymatic
activity of phospholipases and lipoxygenases [93,94]. In fact, the lipoxygenase pathway
has been highlighted to explain the initiation of LDL oxidation [95]. Interestingly, LDL
receptor-related protein (LRP) is involved in LDL recruitment, and 12/15 lipoxygenase is
translocated to the membrane where oxidation of LDL cholesterol esters takes place [94].
Independent of the mechanism involved in the initiation of the LDL oxidation, the
process is characterized by the loss of the antioxidants carried by LDLs, including alphatocopherol
and carotenoids [96,97]. This is followed by the small degradation of polyunsaturated
fatty acids (PUFAs), mostly arachidonic and linoleic acids, which are oxidized to
hydroperoxides. The latter leads to the formation of conjugated dienes and, upon further
oxidation, short-chain aldehydes [98].
On the other hand, apoB-100, the major protein of the LDL, also suffers different modiﬁcations
as a consequence of the oxidative environment. For instance, aldehydes generated
from lipid oxidation form adducts with lysine residues of apoB-100. Instead, apoB-100 can
be directly modiﬁed predominantly at tyrosine residues by oxidizing agents [99]. These
modiﬁcations inhibit LDL-LDLR recognition, thus increasing the uptake of LDL particles
through non-regulated receptors [87].
Depending on the LDL oxidation level, oxidized LDLs are classiﬁed as minimally
modiﬁed LDL (mmLDL) or extensively oxidized LDL (oxLDL) [100]. Mm-LDLs differ
chemically from unmodiﬁed LDLs but are still recognizable by the LDLR and therefore are
internalized through regulated pathways. However, modiﬁed lipids within these particles
act as bioactive molecules conferring other biological activities not shown in unmodiﬁed
LDLs [100,101]. These bioactive lipids might induce a pro-inﬂammatory response in ECs
and macrophages [102–104]. On the other hand, when LDLs are extensively modiﬁed, they
become unrecognizable by the LDLR, while allowing recognition by a range of scavenger
receptors [105–108]. Oxidative modiﬁcations of apoB-100 underlie this lack of afﬁnity
toward LDLR and the increased afﬁnity for the scavenger receptors. Moreover, oxLDLs are
able to escape from proteoglycan retention, hence favoring their non-regulated uptake by
scavenger receptors [84,109]. Once internalized, products derived from oxLDL trigger the
expression of inﬂammatory molecules in macrophages, as outlined later in this review.
It is important to note that, even with LDL oxidation being the most common modiﬁcation,
a range of LDL modiﬁcations contributing to atherosclerosis development have
been well described in other works, including glycosylation, acetylation, and aggregation
[107,110–113].
Int. J. Mol. Sci. 2022, 23, 3346 7 of 38
3.3. Endothelial Activation
Endothelial stimulation, also known as endothelial type I activation, occurs when
inﬂammatory agents induce a response such as a change in microvascular tone, permeability,
or leukocyte diapedesis [114,115]. This phenomenon is an acute response with
short-term functional and morphological changes and does not require de novo protein
synthesis or gene upregulation [115]. However, in response to certain proinﬂammatory
agents, such as IL-1, TNF, endotoxins, modiﬁed lipoproteins, and advanced glycosylation
end products (AGE), as well as disturbed ﬂow derived biomechanical stimulation, the
endothelium can undergo a sustained phenotypic modulation, known as endothelium
type II activation [114,115]. This activation leads to a complex inﬂammatory response that
starts with an increased NF-kB production within the ECs, in response to the aforementioned
stimulus. NF-kB upregulates the expression of leucocyte adhesion molecules, such
as VCAM-1 and ICAM-1; secreted chemokines, such as MCP-1 and IL-8 [116,117]; and
prothrombotic mediators, such as plasminogen activator inhibitor or tissue factor.
3.4. Monocyte Recruitment and Foam Cell Formation
Activated ECs induce selective monocyte recruitment into the intima. This process has
been visualized in vitro [118], and it can be summarized in the rolling, adhesion, activation,
and transmigration of monocytes, as has been well documented [119–121].
Brieﬂy, monocyte recruitment starts with monocyte capture and rolling over the endothelium,
which is mainly mediated by P-selectin [122,123]. Monocyte-rolling is then
reduced, and monocytes remain ﬁrmly attached to the endothelium [124], a process mediated
by the binding of monocytes integrins to VCAM-I and ICAM-I of ECs [123,125,126].
In addition, while rolling over the endothelium, monocytes are activated by endothelial
surface-bound chemokines [127], such as CXCL1, CXCL2, CXCL4, and CCL5, and this
increases monocyte adhesiveness [124]. Afterward, monocytes transmigrate into the intima
space. This movement comprises the crossing throughout the EC barrier, its basement
membrane, and the pericyte layer [123,128]. The migration process is held by chemokines,
which have been previously secreted in response to proinﬂammatory signals.
Regarding monocyte recruitment, MCP-1 (also named as CCL2) is the most frequent
chemokine mediating monocyte transmigration; however, the effect of other chemokines,
such as CCL3, CCL4, and CCL5, has also been studied [129,130]. MCP-1 is produced
mainly by ECs, smooth muscle cells, and monocytes and macrophages of the intima, and
its expression is upregulated after proinﬂammatory stimulus or tissue injury, favoring the
transendothelial migration of circulating monocytes from the plasma to the intima [131].
This process is mediated by the paracellular and transcellular routes [120,123,132]. In
the paracellular route, monocyte migration is favored through EC junctions, due to the
redistribution of junctional molecules in the inﬂamed endothelium [133,134]. In addition,
some endothelial junction molecules actively mediate this type of migration [135,136]. On
the other hand, in the transcellular route, cells migrate through the body of ECs; however,
this type of transmigration has been observed in only 10–30% of the events in vitro [137,138].
New advances in live cell imaging will clarify this ﬁeld [118,139]. Finally, monocytes cross
the EC basement membrane, which is composed of a network of laminin and collagen, and
the pericyte sheath, which is found in most venules [132,140].
Once in the intima, monocytes are differentiated into macrophages that can be polarized
to the M1 (pro-inﬂammatory) or M2 (anti-inﬂammatory) phenotype [141,142].
Nonetheless, macrophages show sensitivity to the changes in inﬂammatory environment,
and, in response to new signals, they are able to switch their phenotype from
pro-inﬂammatory to anti-inﬂammatory [143–145]. Macrophage plasticity is fundamental
for a successful response with M1 predominating in disease progression and M2 in regression
[142,146]. M1 macrophages release inﬂammatory cytokines and chemokines and
produce NO and reactive oxygen species (ROS), which promote monocyte recruitment and
inﬂammatory response propagation [144]. In addition, macrophages express a battery of receptors
that mediate the internalization of modiﬁed and non-modiﬁed LDLs. As previously
Int. J. Mol. Sci. 2022, 23, 3346 8 of 38
mentioned, retained lipoproteins in the intima are prone to suffer modiﬁcations due to the
inﬂammatory environment, allowing their internalization through CD36, SRA-l, and LOX-I
scavenger receptors [105–107]. It is important to underline that the expression of those
receptors is not downregulated by cholesterol uptake. Thus, in an atherosclerotic context,
where oxLDL content is signiﬁcantly enhanced, cells internalize higher amounts of oxLDLs.
Within the cells, oxLDLs are degraded in the lysosomes, and the lipoprotein-contained
cholesterol is esteriﬁed by acyl CoA:cholesterol acyltransferase (ACAT) in the endoplasmic
reticulum (ER). Cholesterol esters are stored as lipid droplets located both in the cytoplasm
or linked to the ER [147,148]. Hydrolysis of these packed cholesterol esters mediated by
neutral cholesterol ester hydrolases, such as nCEH and NCEH1, generates free cholesterol
that is transferred from macrophages to apoA1 or HDLs (high-density lipoprotein), an
important step for the removal of cholesterol excess from peripheral tissues [149]. This
process is mediated by ABCA1 and ABCG1 ATP-binding cassettes and SR-B1, cholesterol
transporters that play an important role mediating cholesterol efﬂux from the cells and
preventing foam cell formation [105]. However, the pro-inﬂammatory microenvironment of
atherosclerotic lesions impairs the ABCA1 efﬂux system, both in M1 and M2 macrophages,
and promotes foam cell accumulation, as shown in experiments with murine macrophages
contributing to plaque development [150,151].
In addition, the excess of lipid uptake by macrophages perpetuates the inﬂammatory
response, and oxLDLs induce signaling cascades that activate NF-kB targets [152–154],
which maintain EC activation, monocyte recruitment, and foam cell formation [146]. The
uptake of oxLDLs by macrophages could be considered a protective mechanism, as they
remove cytotoxic elements from the intima. However, the increased migration of monocyte
to the intima and the subsequent differentiation into macrophages lead to a large number
of foam cells inducing the growth of the atherosclerotic lesion [147]. Therefore, cholesterol
accumulation is considered a hallmark of atherosclerotic lesions [155,156].
An accumulation of cholesterol in the subendothelial compartment also promotes the
formation of cholesterol crystals both inside and outside the cells and contributes to the
development of atherosclerotic plaques [157–159]. This process has been monitored both
outside and inside the cells, in macrophages incubated with oxLDLs [160]. Although cholesterol
crystals are a common feature of advanced atherosclerotic lesions, they are present also
in early plaques and can be used as a marker of early atherosclerosis development [161].
Cholesterol crystals within the plaque activate NLRP3 inﬂammasome in macrophages, leading
to activation of pro-inﬂammatory pathways. Inﬂammasomes are cytosolic multiprotein
complexes of the innate immune system responsible for the activation of inﬂammatory
pathways [162]. Although NLRP3 activation and assembly is not fully understood, it is
known that its activation leads to caspase-1 activation. Caspase-1 subsequently cleaves
the proinﬂammatory IL-1 family of cytokines into their bioactive forms, IL-1β and IL-18,
contributing to inﬂammation [88]. It has been suggested that uptake of oxLDLs mediated
by the CD36 receptor is responsible for NLRP3 activation [88]. Apparently, the CD36
scavenger receptor, along with TLR4-TLR6, takes up oxLDL, which results in intracellular
cholesterol crystals. These crystals cause lysosomal destabilization [163], inducing the
release of lysosomal contents, such as cathepsins or reactive oxygen species [164], leading
to NLRP3 inﬂammasome assembly and the subsequent activation of caspase-1.
3.5. Contribution of VSMCs to Foam Cell Population
VSMCs located in the intima are also able to internalize oxLDL in a non-regulated way
through different scavenger receptors, such as SR-A, CD36, and LOX-1 [165–169]. Indeed,
their contribution to the sum of foam cell population within the plaque is signiﬁcant [20].
In addition, VSMCs of the intima express fewer ABCA1 transporters than the ones of the
tunica media [170]. Therefore, the balance between cholesterol input and output is tilted in
favor of cholesterol accumulation and foam cell formation. At least 50% of the foam cells in
the human coronary intima are VSMC-derived rather than monocyte-derived, underlying
the importance of VSMCs in atherosclerosis development [170].
Int. J. Mol. Sci. 2022, 23, 3346 9 of 38
4. Fibrous Plaque Development
During ﬁbrous plaque development, atheroma plaques undergo a transition from the
fatty streak to intimal growing, a step characterized by the presence of a cell-free and lipidrich
area known as the necrotic core (Figure 3). To stabilize the plaque, the necrotic core
is covered by ﬁbers, thus developing a ﬁbrous cap. The necrotic core and the ﬁbrous cap
constitute the hallmark of advanced atherosclerosis [171], and atheroma plaque regression
is unlikely to happen in this stage [172,173].
Figure 3. Schematic representation of atheroma plaque formation from a healthy artery to plaque
rupture underlying the most important events that contribute to its development in each stage.
4.1. Fibrous Cap
The ﬁbrous cap is a subendothelial barrier between the lumen of the vessel and the
atherosclerotic necrotic core consisting of VSMCs that have migrated to the luminal side
of the artery and extracellular matrix (ECM) derived from VSMCs [174,175]. The role of
the ﬁbrous cap is to serve as a structural support to avoid the exposure of prothrombotic
material of the core that otherwise would trigger thrombosis [174].
At the physiological situation, differentiated VSMCs of the tunica media show a
contractile phenotype that regulates the blood vessel diameter and blood ﬂow [176–178].
However, in response to injury, VSMCs switch their phenotype to the synthetic one in which
migratory and proliferation activities prevail [179,180]. For that purpose, neighboring cells
activate the healing process by producing several growth factors, which include epidermal
growth factor, ﬁbroblast growth factor, insulin-like growth factor, platelet-derived growth
factor (PDGF), transforming growth factor-β (TGF-β), and vascular endothelial growth
factor (VEGF) [179]. In atherosclerosis, in response to the growth factors produced by
foam cells (VSMC- or macrophage-derived) or ECs of the intima, VSMCs from the tunica
media migrate to the intima [175,179,181–183]. Moreover, IL-1 produced by macrophages
enhances the endogenous production of PDGF by VSMC, and, once in the intima, it autocrinically
leads to their proliferation [184,185]. In addition to migration and subsequent
Int. J. Mol. Sci. 2022, 23, 3346 10 of 38
proliferation, VSMCs with a synthetic phenotype also increase the production of ECM
components, such as interstitial collagen, elastin, and proteoglycans [183,186–188]. These
proliferating VSMCs, along with ECM production, generate a ﬁbrous cap that will cover
the developing atherosclerotic plaque, thus surrounding the lesion and preventing its
rupture [189]. Interestingly, if the mitogen production does not cease, VSMCs do not
switch back to the contractile phenotype [176,177,190,191], which facilitates lesion development.
Fibrous-cap features, such as thickness, cellularity, matrix composition, and collagen
content, are important determinants of plaque stability [192–194].
4.2. Necrotic Core
The necrotic core constitutes the nucleus of the atherosclerotic plaques. Covered
by the ﬁbrous cap, the necrotic core consists of a lipid-laden hipocellular region with
reduced supporting collagen [195–197]. While atherosclerotic plaque develops, the necrotic
core increases its size mainly as a consequence of two factors, macrophage death and
impaired efferocytosis, a process that removes apoptotic cells. Both events contribute to an
inﬂammatory microenvironment, oxidative stress, and thrombogenicity and promote the
death of neighboring cells, such as VSMCs, increasing plaque vulnerability [198].
In the early stages of atherosclerosis, macrophage apoptosis is programmed through
the coordinated caspase system, leading to cell death and efferocytosis [199]. However,
when atherosclerosis develops, apoptosis enhances in the macrophage and VSMCs due
to increased oxidative stress, the activation of receptors involved in death signaling, the
inhibition of survival pathways, and nutrient deprivation [200]. At this step, the phagocytic
activity of the macrophages is not able to handle the accumulation of apoptotic cells, which
undergo a secondary necrotic death with the concomitant release of intracellular oxidative
and inﬂammatory components. This situation aggravates inﬂammation and enhances
the death of neighboring cells [201]. Efferocytosis also becomes defective in advanced
atherosclerosis because the activity of several receptors that mediate the process, such as
MerTK, LRP1, CD36, and SR-B1, is impaired. Moreover, the oxidized phospholipids and
oxLDLs present in advanced atherosclerotic plaques inhibit the recognition of apoptotic
cells by efferocytotic receptors [201–205]. Efferocytosis impairment in advanced plaques
also favors cholesterol crystal accumulation coming from apoptotic cells. At physiological
conditions, small cholesterol crystals are rapidly sequestered from interstitial space by
phagocytic cells; however, while the lesion advances, phagocytic cells are unable to remove
the excess of crystals, which ﬁnally increase in size and remain in the subendothelial
space [206]. This process activates the complement and increases the proinﬂammatory
state of the plaque. Moreover, as phagocytic cells are unable to internalize large cholesterol
crystals by scavenger receptors, their lysosomal content is directly secreted to the interstitial
space, promoting a more intense immune response [207,208]. These events promote the
death cell accumulation and necrotic core growth. Furthermore, the metalloproteinases
released after cell death reduce the size of the ﬁbrous cap and increase plaque vulnerability
[209,210]. Finally, apoptotic and necrotic cells liberate tissue factor (TF), which, along
with oxidized lipids, increases the thrombogenicity of the necrotic core [211,212].
4.3. Plaque Calciﬁcation
Atheroma plaque calciﬁcation is another hallmark of advanced atherosclerosis. It exists
as a bone-like formation within the plaque and is initiated in inﬂammatory regions with a local
decrease in collagen ﬁbers [213,214]. The release of matrix vesicles upon the macrophage
and synthetic VSMC death initiates the calciﬁcation process of the plaque [215–218]. The
nucleation sites accumulate calcium orthophosphate, which is converted to amorphous
calcium phosphate and ﬁnally to crystalline structures [213]. In addition, other factors,
including reduced levels of mineralization inhibitors or increased osteogenic transdifferentiation,
contribute to the calciﬁcation process [213]. In particular, pericytes [219] and
VSMCs [220,221] transdifferentiate into osteoblast-like phenotypes during atherosclero-
Int. J. Mol. Sci. 2022, 23, 3346 11 of 38
sis development, acquiring the capacity to generate a mineralized matrix and leading to
calcium deposits, as it occurs in bone tissue [222–224].
Together, this contributes to microcalciﬁcations, the early stage of the vascular calciﬁcation
cascade in both intima and media [213,225]. Microcalciﬁcations then evolve into
larger calciﬁcations that extend from the bottom of the necrotic core to the surrounding
matrix [221]. Although calciﬁcation is a hallmark of advanced atherosclerosis (it correlates
very well with plaque size), the amount and size of calcium deposits are not correlated
with plaque vulnerability, which would rather be linked to other features, such as location,
calciﬁcation type, or the surrounding environment [213,214,226].
5. Plaque Stability and Rupture
5.1. Vulnerable Plaque
As mentioned above, atheroma plaques usually develop in branched areas where the
WSS is lower. In these areas, low shear stress contributes to local endothelial dysfunction
and eccentric plaque build-up. Initially, lumen narrowing is prevented by outward vessel
remodeling to maintain a normal lumen and to restore shear stress distribution. However,
this prolongs local, unfavorable, low-WSS conditions and aggravates eccentric plaque
growth. The eccentric plaques at preserved lumen locations experience increased tensile
stress on their shoulders, transforming the lesion into a rupture-prone [227]. A plaque
is considered vulnerable when the lesion shows a large necrotic core, a thin ﬁbrous cap,
and an increased inﬂammatory response [228,229] due to the continuous exposure to the
pro-atherogenic milieu.
The ﬁbrous cap separates the thrombogenic necrotic core from the circulating coagulation
factors and platelets, and its thickness correlates with the vulnerability of the
plaque [189]. As a result of VSMC death, ECM production is reduced, and the presence of
liberated matrix metalloproteinases (MMP) increases, making the ﬁbrous cap weaker [230]
(Figure 3).
As mentioned before, inﬂammation contributes to plaque development in all of the
steps from initiation to plaque rupture. Indeed, in this last stage, its relevance is remarkable,
as it promotes the instability of the ﬁbrous cap [231]. Certain pro-inﬂammatory cytokines,
such as IFN-γ, might inhibit collagen production in VSMCs. In addition, inﬂammatory
mediators usually found in atheroma, such as IL-1β, TNF-α, and the CD40 ligand (CD154),
may increase MMP expression in VSMCs, as observed in vitro [232,233]. This inﬂammatory
stage is commonly observed in the cap and shoulders of the plaque instead of a generalized
inﬂammation [189,234]. Together, the data show that, when inﬂammation prevails, the
maintenance of the strong and rigid ﬁbrous cap decreases, making the cap unstable and
susceptible to rupture when exposed to hemodynamic forces, the most common mechanism
of plaque rupture [189,227,231,235].
5.2. Plaque Rupture and Thrombus Formation
When the plaque ﬁssures or ruptures, the subendothelial space is exposed to blood,
triggering a coagulation process to cover the wound [186,236]. Initially, platelets adhere to
the subendothelial collagen and become activated, and more platelets are then recruited
and aggregated in the region in order to initiate wound healing [237]. Simultaneously, prothrombotic
elements of the lipid core are released and come into contact with coagulating
factors of plasma. More speciﬁcally, the tissue factor of the core reacts with factor VII
of the plasma, activating the coagulation cascade that leads to thrombin production, an
essential intermediate for ﬁbrin formation [189,238]. Fibrin is an insoluble protein that
forms networks of ﬁbrin threads and, along with platelets, covers the lesion, forming a
stable and well-arranged structure. This structure is known as the thrombus [231,239].
Although the aim of this process is wound healing, triggering of the biochemical
cascade promotes the expansion of the intima to the luminal side. For instance, activated
platelets release TGF-β, which, as indicated earlier, promotes the production
of interstitial collagen and, therefore, ﬁbrous-cap thickening [240]. Consequently, the
Int. J. Mol. Sci. 2022, 23, 3346 12 of 38
atherosclerotic lesion expands, leading to lumen constriction. This all supposes an absence
of clinical complication.
5.3. Clinical Complications
Thrombus development triggers a range of reactions that makes the lesion more
ﬁbrous and stable and, therefore, less prone to rupture. However, due to plaque growth,
the risk of vessel obstruction increases. Consequently, blood ﬂow in coronary arteries
is reduced, generating ischemic cardiopathies, such as cardiac insufﬁciency or angina
pectoris [231,241,242]. Moreover, if the obstruction is complete or almost complete, it leads
to myocardial infarction or stroke [243]. Detachment of the thrombus from the arterial wall
would produce a clot, known as the embolus, that circulates within the cardiovascular
system. Eventually, the embolus lodges in distal arteries, where it obstructs blood ﬂow,
leading to local ischemia, organ dysfunction, or potential infarction [244].
However, if the inﬂammatory response ceases in time, for example, due to a lipid
lowering treatment, a stable plaque with enough lumen for correct blood circulation would
be acquired [231].
6. Inﬂammation in Atherosclerosis
As indicated through the previous sections of this review, several inﬂammation processes
participate in all phases of atherosclerosis [245,246] At early stages of atherosclerosis,
LDLs accumulate in the subendothelial region, where they become modiﬁed. VSMCs
exposed to modiﬁed LDL release chemoattractants, including chemokine 2 (CCL2) and
CCL5 [247,248], which promote the recruitment of monocytes [249]. Moreover, ox-LDL
and mm-LDL are able to induce a pro-inﬂammatory response in ECs and macrophages,
increase endothelial damage, and recruit leukocytes. Furthermore, mmLDLs can bind to
the TLR2 and Class 4 of PRRs and induce the secretion of pro-inﬂammatory cytokines
IL-1β, IL-6, and TNF-α in an NF-kB-dependent fashion [250,251]. CD36-mediated oxLDL
uptake activates the NLRP3 inﬂammasome, resulting in the robust secretion of the inﬂammatory
cytokine IL-1β36 [252]. In addition, oxLDL can bind to speciﬁc antibodies
forming immune complexes that induce inﬂammatory responses in macrophages and
dendritic cells. These immune complexes induce cellular activation, inﬂammatory cytokine
production, and foam cell formation, mainly by signaling through the immune complex
receptor Fc gamma receptor I (FcγRI) [253]. In addition, they can activate inﬂammasome
via an FcγR/TLR4/CD36-dependent mechanism [254]. At early stages of atherosclerosis,
the release of CCL2 and T-cell chemoattractants recruits monocytes and lymphocytes into
the inner arterial wall, where monocytes are differentiated into macrophage foam cells
under the inﬂuence of M-CSF. Upon fatty-streak lesion formation, T-cells secrete TNF-β,
IFN-γ, ﬁbrogenic mediators, and growth factors that induce the migration and proliferation
of VSMCs. Activated T-cells also stimulate MMP production by macrophages in the lesion.
Cholesterol crystals activate NLRP3 inﬂammasome through lysosomal damage [255].
Several studies have shown that activated ECs overexpress cell adhesion molecules, such as
VCAM-1, ICAM-1, PECAM-1, E-selectin, and P-selectin, on their surfaces [256]. Thus, these
activated ECs are the local source of leukocyte recruitment into atherosclerotic lesions [257].
ECs, macrophages, and SMCs express LOX-1, which binds to oxLDL, leading to increased
ROS production and VSMC death. This contributes to oxLDL inﬁltration into
endothelium that disrupts the normal endothelial function, monocyte adhesion, and inﬁltration
[258].
Prostaglandin (PG) E2 is an important lipid mediator that in a pathophysiological
context mediates functions such as pyrexia, pain sensation, and inﬂammation. PGE2 is
synthesized from arachidonic and by the participation of the cyclooxygenase (COX) enzyme
and PGE2 synthase (PGES) afterward. It has been shown that a depletion of myeloid cell
microsomal PGES, not in ECs or VSMCs, fosters atherogenesis in mice [259]. In addition,
prostaglandin receptors (EP receptors) have been related to early atherosclerosis and the
inﬂammatory process, which induce plaque erosion and rupture [260].
Int. J. Mol. Sci. 2022, 23, 3346 13 of 38
7. Post-Transcriptional Regulation of Atherosclerotic Plaques
Several studies have suggested that distorted non-coding RNA (ncRNA) expression
and function are involved in the atherosclerotic process. Particularly, microRNAs (miRNAs)
and long non-coding RNAs (lncRNAs) have been determined as important regulators in the
progress of atherosclerotic plaque. While miRNAs are known to regulate gene expression
post-transcriptionally, mainly through mRNA degradation, lncRNA-mediated regulation
is less characterized, mainly due to its low sequence conservation and low expression.
However, lncRNA can activate and repress genes by a variety of mechanisms at both
transcriptional and translational levels, and its relevance in atherosclerosis development
has been updated. It is now accepted that they play a crucial role in the atherosclerotic
context [261,262].
7.1. miRNAs
miRNAs are crucial molecules for the maintenance of cardiovascular homeostasis [263].
Indeed, many studies in recent years have indicated that miRNA expression deregulation
affects cellular and molecular processes that contribute to atherosclerosis. The pathological
basis of atherosclerotic plaque formation and development is related to the expression
levels of miRNAs (Table 1). Atherosclerotic coronary arteries revealed upregulated levels
of miR-29, miR-100, miR-155, miR-199, miR-221, miR-363, miR-497, miR-508, and miR-181;
on the contrary, miR-1273, miR-490, miR-24, and miR-1284 were found to be downregulated
[264–266]. In the case of aorta, femoral, and carotid arteries, atherosclerotic plaques
exhibited increased levels of miR-21, miR-34, miR-146, and miR-210 [267]. In addition,
studies performed in carotid plaques showed an upregulation of miR-15, miR-26, miR-30,
miR-98, miR-125, miR-152, miR-181, miR-100, miR-127, miR-133, miR-145, and miR-422
and a downregulation of miR-520 and miR-105 [268,269].
Table 1. Differential microRNA expression levels in atherosclerotic arteries.
Location Upregulated Downregulated
Coronary arteries
miR-29, miR-100, miR-155,
miR-199, miR-221, miR-363,
miR-497, miR-508 and
miR-181 [264–266].
miR-1273, miR-490, miR-24
and miR-1284 [264–266].
Aorta, femoral, and
carotid arteries
miR-21, miR-34, miR-146 and
miR-210 [267].
Only in carotid plaques:
miR-15, miR-26, miR-30,
miR-98, miR-125, miR-152,
miR-181, miR-100, miR-127,
miR-133, miR-145 and
miR-422 [268,269].
Only in carotid plaques:
miR-520, miR-105 [268,269].
7.1.1. miRNAs in Atherosclerotic Plaque Initiation and Progression
The ﬁrst steps in atherosclerosis development include endothelial dysfunction, followed
by inﬂammatory response and foam cell formation. There is an extensive list of
miRNAs that regulate endothelial function, such as miR-221, miR-503, miR-217, miR-34a,
miR-181b, miR-155, miR-126, miR-1, miR-223, miR-145, miR-146a, miR_92a, or miR10a
(Table 2). Expression of miR-155 induces the downregulation of endothelin-1 and angiotensin
II type I receptor, suggesting a role in endothelium protection [270]. Moreover,
miR-10a showed an athero-protective role by repression of GATA6/VCAM1 signaling
within ECs [271]. MiR-10a, miR-31, and miR-17-3p regulate inﬂammation modulating the
expression of adhesion molecules in ECs [272]. MiR-126, one of the most studied miRNAs,
plays a role in the prevention of atherosclerosis by regulating the vascular endothelial
growth factor (VEGF) pathway and inhibiting endothelial permeability [273]. Another
regulator of endothelial inﬂammatory response is miR-181b, which regulates the NF-kB
Int. J. Mol. Sci. 2022, 23, 3346 14 of 38
pathway [274]. MiR-146a does not only decrease the lipid uptake in macrophages, suggesting
an athero-protective role, but also inhibits endothelial activation by promoting
eNOS expression [275]. MiR-125a reduces the lipid uptake, as well, through stimulated
macrophages [276]. The expression of miR-223 negatively regulates cholesterol synthesis
and exhibits an anti-inﬂammatory effect due to the attenuation of pro-inﬂammatory
cytokine production [277]. On the contrary, miR-92a, a miRNA that reduces endothelial
inﬂammation and atheroma plaque size, is upregulated in atheroma plaques via the regulation
of Kruppel-like factor 2 (KLF2) [278,279]. Some miRNAs also modulate EC senescence.
MiR-let-7g inhibits cellular senescence by regulating anti-aging gene sirtuin 1 (SIRT1) and
the insulin growth factor (IGF) 1 pathway [280], while miR-126a was found to induce EC
senescence via the activation of NF-kB signaling [281].
Furthermore, miRNAs also regulate the inﬂammatory response of macrophages during
atherosclerosis development. These include, among others, miR-155, miR-222, miR-424,
miR-503, miR-146a/b, and miR-147. MiR-125b overexpression induces the activation
of macrophages by suppressing interferon regulatory factor 4 (IRF4) [282]. In addition,
miR-342-p was found to enhance proinﬂammatory macrophage factors, such as IL-6. In
animal models, it has been shown that the inhibition of miR-342-p reduces atheroma formation
[283], while the inhibition of miR-33, a regulator of ABCA1 and ABCG1, reduces the
cholesterol efﬂux to HDL in macrophages and promotes the development of atherosclerotic
plaques in mice [284].
Table 2. Role of microRNAs in atherosclerosis initiation and progression.
Cell Line Athero-Protective Pro-Atherogenic
Endothelial cells
miR-155 [270]
miR-10a [271], miR-31 and
miR-17-3p [272]
miR-146a [275]
miR-181b [274]
miR-92a [278,279]
miR-let-7g [280]
miR-216a [281]
Macrophages
miR-146a [275]
miR-125a [276]
miR-223 [277]
miR-125b [282]
miR-342-p [283]
miR-33 [292]
VSMCs *
miR-21 [285]
miR-221 and miR-222 [286]
miR-26a [287]
miR-663 [290]
miR-143, miR-145 and
miR-1 [288]
miR-29a and miR-24 [289]
miR-133a [288]
miR-15b and miR-16 [291]
* Anti-atherogenic, considered as VSMC activation and phenotype switching.
The regulation of VMSC proliferation is also regulated by miRNAs. MiR-21 overexpression
induces the switch of VSMCs toward the synthetic phenotype, the one related to
proliferation and ECM production [285]. Similarly, the inhibition of miR-221 and miR-222
diminishes the proliferative rate of VSMCs via the inhibition of c-Kit, p27 (Kip1), and p57
(Kip2) [286]. The synthetic VSMC phenotype is also induced by miR-26a through modulation
of the TGF-β signaling pathway [287]. On the contrary, miR-143, miR-145, and miR-1
also play a role in preventing a VSMC phenotypic switch via the Kruppel-like factor 4
(KLF4), KLF5, and/or myocardin [288]. Myocardin also promotes a contractile phenotype
via the inhibition of PDGF-β by miR-29a and miR-24 [289]. Similarly, miR-133a promotes
the contractile phenotype in VSMCs, and its levels are decreased in atherosclerosis [288].
Moreover, the differentiation of VSMCs was also induced by miR-663, which promotes the
expression of transcription factor JunB and myosin light chain 9 [290]. In addition, targeting
the oncogene yes-associated protein (YAP) by miR-15b and miR-16 induced a more
contractile phenotype in VSMCs [291]. Finally, several other miRNAs have been associated
with a contractile and synthetic phenotype in VSMCs. Contractile-phenotype-associated
Int. J. Mol. Sci. 2022, 23, 3346 15 of 38
miRNAs include miR195, miR-638, miR-7d, miR-100, miR-10a, miR-204, miR-199a, and
miR-424; synthetic-phenotype-associated miRNAs include miR146a, miR-208, and miR-31.
Here, we classiﬁed miRNAs related to a synthetic phenotype as anti-atherogenic (Table 2).
7.1.2. miRNAs and Atherosclerotic Plaque Rupture
The mechanisms involved in plaque rupture are not completely understood, but
plaque vulnerability is associated with ﬁbrous cap thickness, necrotic core development,
and the inﬂammatory response [293,294]. However, there is growing evidence that miRNAs
play a role in processes leading to plaque rupture. MiR-322 is upregulated in vulnerable
plaques compared with stable plaques, and its inhibition produces a downregulation of the
proinﬂammatory cytokine IL-6 [295]. MiR-712 also leads to plaque rupture, as it activates a
disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMST4) [296]. As
mentioned before, the thickness of the ﬁbrous cap depends on ECM synthesis and degradation,
and it is associated with plaque vulnerability. The ECM is composed mainly of
collagens, which are degraded by MMPs, and some miRNAs have been shown to target
MMPs (i.e., miR-24 [265] and miR-133a [297]). MiR-494, which is expressed in atheroma
plaques, downregulates TIMP3 and reduces the collagen content in the plaque [298]. Moreover,
miR-29 helps to maintain the ﬁbrous cap integrity targeting IFN-γ, which activates
procollagen genes in VSMCs [299]. The inhibition of genes that produce ECM proteins by
miR-29 may lead to plaque rupture [300]. Regarding inﬂammation in the necrotic core,
miR-21 has been demonstrated to promote efferocytosis and suppress innate immune
response [301]. In addition, miR-223, as a negative regulator of the NLRP3 inﬂammasome,
prevents associated inﬂammatory response [302]. MiR-155 also contributes to the necrotic
core formation in atherosclerosis, leading to vulnerable plaque through the induction of
apoptosis [303]. In addition, miR-365 induces endothelial apoptosis, promoting plaque
rupture [304]. Table 3 summarizes the role of these miRNAs in plaque rupture.
Table 3. Role of microRNA in plaque rupture.
Anti-Atherogenic Pro-Atherogenic
miR-24 [265] miR-322 [295]
miR-133a [297] miR-712 [296]
miR-29 [299,300] miR-494 [298]
miR-21 [301] miR-155 [303]
miR-223 [302] miR-365 [304]
Although some miRNAs are considered signature molecules in atherosclerosis, more
studies on miRNAs are needed to unravel the precise potential roles of miRNAs as targets
for therapy.
7.1.3. Distinct miRNA Regulation of VSMC and EC Function
Deciphering the role of different miRNAs in vascular biology as direct or indirect
post-transcriptional regulators of VSMC and EC function constitutes a major challenge
in the smooth muscle biology ﬁeld. The discovery that the expression of miR-143/145
is critical for maintaining the contractile phenotype of VSMCs [305,306] highlighted the
role of the bicistrionic unit encoding for miR-143 and miR-145 in the regulation of smooth
muscle physiology [306,307]. The role of miR-143/145 was demonstrated in mice with
a genetic deﬁciency of miR-143/145, which displays a reduced vascular tone and blood
pressure control [308]. Additionally, the functional relevance of miR-143/145 in human
vessel pathology has been suggested by the observed downregulation of the miR-143/145
cluster in human aortic aneurysms compared with normal aortic tissue [309].
Several studies have shown that cells can release miRNAs, which can then exert their
speciﬁc effects by modulating processes in recipient cells. It has been shown that VSMCs
communicate with ECs via miR-143/145 [310]. Cell-to-cell VSMC/EC contact induces the
transcription of miR-143/145 in VSMCs, promoting the transfer of these miRNAs to the
Int. J. Mol. Sci. 2022, 23, 3346 16 of 38
endothelium [310]. In particular, VSMCs can deliver miR-143/145 to ECs via membrane
nanotubes or tunneling nanotubes [310]. The transfer of miR-143/145 from VSMCs to
ECs is stimulated by TGF-β secreted by ECs. VSMC-derived miR-143/145 diminishes the
angiogenic potential of the ECs by repressing hexokinase II and integrin β8 [310].
On the other hand, miR-126 acts as an intercellular messenger mainly released by ECs
and internalized primarily by monocytes and VSMCs [311]. MiR-126 plays a critical role in
modulating vascular development and homeostasis, targeting speciﬁc mRNAs, including
the Sprouty-related protein 1 (SPRED-1), CXCL12, SDF-1, and phosphoinositol-3 kinase
regulatory subunit 2 (PIK3R2) [312–317]. MiR-126 has also been related to the endothelial
dysfunction associated with the development of diabetes and its complications [318].
7.2. lncRNAs
The progress achieved by next-generation sequencing technologies has revealed an
increased number of lncRNAs associated with the pathogenesis of atherosclerosis [319].
Several lncRNAs identiﬁed in plaque tissues play a protective role in vascular disease.
lncRNA MeXis (Macrophage-expressed LXR-induced sequence) is involved in cholesterol
transport. Animal models lacking MeXis showed an increased atherosclerotic load and a
decreased expression of the atheroprotective protein ABCA1 in atheroma plaques [320].
Reduced expression of lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript
1) was also found to be associated with the development of atheroma plaques [321].
The knockdown of MALAT1 promotes lipid uptake in foam cells by inducing the transcription
of scavenger receptor CD36 [322]. Moreover, lncRNA CHROME (cholesterol
homeostasis regulator of miRNA expression) protects against atherosclerosis promoting
cholesterol efﬂux by inhibiting miRNAs, such as miR-33 [323,324]. However, few lncRNA
have shown athero-protective effects by inhibiting apoptosis and senescence. In particular,
lncRNA CERNA1 promotes plaque stabilization by inhibiting cellular apoptosis via
inducing API5 expression [325], and lncRNA SNHG12 in pig and human atheroma plaques
inhibits DNA damage and senescence [326]. Human carotid atherosclerotic plaques show
decreased levels of lncRNA NEXN-AS1 and lncRNA MANTIS [327,328]. NEXN-AS1 upregulates
the expression of NEXN, a gene that exerts atheroprotective effects in VSMCs
and ECs [327]. Furthermore, one lncRNA, called SENCR (smooth muscle and EC-enriched
migration/differentiation-associated long noncoding RNA), plays a role in atherosclerosis
development by protecting the endothelial layer [329]. Hence, Boulberda et al. [330] found
altered levels of lncRNA SENCR in vascular tissue. Moreover, the newly identiﬁed lncRNA
RP11-714G18.1 is also downregulated in atheroma plaques. This lncRNA inhibits VSMC
and EC migration by downregulating the expression of MMP1, inhibiting atherosclerosis
progression. In addition, RP11-714G18.1 inhibits the adhesion of monocytes to ECs [331].
A recent study highlighted the role of lncRNA SMILR in atherosclerosis and showed
that its expression is signiﬁcantly higher in unstable plaques than in stable plaques and
increases VSMC proliferation by regulating the expression of the proximal gene HAS2 [332].
Moreover, lncRNA SMILR binds directly to the mRNA of the mitotic protein CENPF (centromere
protein F) driving the proliferation of smooth muscle cells [332]. The aberrant
proliferation and migration of VSMCs are critical factors in atherosclerotic plaque formation,
and several molecular mechanisms that involve lncRNAs control these processes.
Among the lncRNAs that suppress VSMC migration, lncRNA RP11-714G18.1 acts by directly
targeting LDL-related receptor 2 binding protein (LRP2BP) in atherosclerosis [331].
Very recently, it has been found that lncRNA ZNF800, which is highly expressed in human
atherosclerotic plaque tissues and predominantly in VSMCs, suppresses VSMC proliferation
and migration through the AKT/mTOR/HIF-1α signaling pathway by activating
PTEN [333]. Another study demonstrated that lncRNA RNCR3 expression is signiﬁcantly
higher in atherosclerotic lesions, leading to reduced VSMC proliferation and migration
via the RNCR3/Kruppel-like factor 2/miR-185-5p regulatory network [334]. Furthermore,
lncRNA RP11-714G18.1 impairs VSMC migration in atherosclerosis through the LRP2BPmediated
downregulation of MMP1 [331]. The expression of lncRNA p21 is also reduced
Int. J. Mol. Sci. 2022, 23, 3346 17 of 38
in atherosclerotic plaques, and it represses VSMC proliferation and atherosclerosis by
enhancing TP53 activity, thus playing an atheroprotective role in atherosclerosis [335].
Similarly, lncRNA CCL2 is upregulated in unstable atherosclerotic plaques compared
to stable atherosclerotic plaques. Moreover, lncRNA CCL2 modiﬁes the mRNA levels
of the pro-inﬂammatory chemokine CCL2 (or MCP-1) by interacting with RNA binding
proteins (HNRNPU and IGF2BP2) [336]. Recently, lncRNA GAS5 (growth arrest-speciﬁc 5)
has received attention as a potential biomarker for atherosclerosis [337]. Levels of GAS5
were upregulated in atherosclerotic plaques, and GAS5 binds and suppresses the microRNA
miR-221, increasing the production of MMPs and pro-inﬂammatory molecules
within the atherosclerotic plaque [338]. Arslan et al. [321] have identiﬁed the upregulation
of lncRNA MIAT (myocardial infarction-associated transcript) in atheroma plaques.
MIAT increases VSMC proliferation by binding and suppressing miR-181b [339]. Another
lncRNA that displayed effects on VSMC proliferation and migration is BANCR
(BRAF-regulated lncRNA 1) [340]. Finally, lncRNA ANRIL (antisense non-coding RNA in
the INK4 locus) is also a crucial molecule in atherogenesis, as it affects several cell types in
the atherosclerotic plaque, where it is upregulated and is directly correlated with atherosclerosis
severity [341,342]. Recently, circular forms of ANRIL (circANRIL), comprising different
exons and opposed to the linear form, have been identiﬁed. Conversely, circANRIL is
inversely correlated with atherosclerosis risk [343]. CircANRIL has been found in vascular
tissue, smooth muscle cells, and macrophages, where it exhibits an athero-protection
function [342].
In summary, recent ﬁndings suggest that lncRNA plays a crucial role in the development
of atherosclerotic plaque (Table 4). However, for most of them, the speciﬁc mode
of action is not completely understood, and further research is needed to understand the
complex role of lncRNA in atherosclerosis.
Table 4. Role of lncRNA in atherosclerosis.
Athero-Protective Pro-Atherogenic
MeXis [304]
MALAT1 [321,322]
CERNA1 [325]
SNHG12 [326]
NEXN-AS1 [327]
MANTIS [328]
SENCR [329]
RP11-714G18.1 [331]
CHROME [323,324]
circANRIL [342]
SMILR [332]
CCL2 [336]
GAS5 [337,338]
MIAT [321,339]
BANCR [340]
ANRIL [341,342]
8. Microbiota
The human body hosts many bacteria species (microbiota) that have co-evolved with
humans, and they have generated a symbiotic relationship that mutually beneﬁt each other.
Microbiota is able to positively affect human physiology through the synthesis of different
metabolites or the training of the immune system; however, it has also been associated with
the development of different diseases, such as atherosclerosis or cardiovascular disease
(CVD) [344,345].
The presence of microorganisms in the atheroma plaque is well established, and some
studies have shown that the abundance of certain phyla could correlate with plaque stability
and overall inﬂammation [346,347]. For instance, bacterial DNA from pathogenic families,
such as Helicobacteraceae or Neisseriaceae, has been shown to be more abundant in plaques
of symptomatic patients [348,349]. Nevertheless, these results need to be corroborated in
larger studies.
Gut microbiota also contributes to the development of atherosclerosis and CVD. The
gut hosts the largest microbial population in humans and is able to regulate many biologi-
Int. J. Mol. Sci. 2022, 23, 3346 18 of 38
cal functions, such as energy storage, the absorption and processing of nutrients, or the
maturation of the immune system [350,351]. However, when gut microbial homeostasis
is disrupted (referred to as dysbiosis) toward underrepresented colonies, it can induce
the development of different diseases, such as cancer, type 2 diabetes, obesity, CVD, and
atherosclerosis [346,352,353]. In addition, gut dysbiosis increases intestinal permeability,
reducing the expression of tight junction proteins and allowing the translocation of
lipopolysaccharides that induce low-grade inﬂammation via Toll-like receptors [354]. On
the other hand, several metabolites produced by the gut microbiota can modulate systemic
inﬂammation and contribute or prevent atherosclerosis development. The most studied
one is trimethylamine-N-oxide (TMAO) [355], the oxidized form of trimethylamine (TMA).
TMA is synthesized by gut microbiota after metabolizing diet choline and carnitine. It
is then transferred to the bloodstream and oxidized to TMAO in the liver through ﬂavin
mono-oxigenase [356]. TMAO increases vascular wall inﬂammatory responses and suppresses
reverse cholesterol transport favoring cholesterol accumulation in the intima [357].
Moreover, APOE−/− mice fed with a choline-rich diet showed increased TMAO levels and
atheroma plaque development, a process that was reversed by antibiotic intervention [358].
Indirect infections in distant areas from atheroma plaques also contribute to its development
and destabilization. Indeed, in recent years, many studies have correlated
the incidence of atherosclerotic cardiovascular disease (ACV) with bacterial infections.
Epidemiological studies support the idea of a causative association between periodontal
disease or chlamydia infections and CVD [359]. A large study that included almost
12,000 participants demonstrated that poor oral hygiene is associated with an increased
rate of CVD and increased lower-grade inﬂammation [360]. Moreover, a small study with
92 participants showed that some bacteria phyla were more abundant in the oral cavity of
patients with symptomatic atherosclerosis than in control patients, indicating a possible
association between oral bacteria and atheroma plaque development [361]. Nevertheless,
the direct association of bacteria in the oral cavity and ACV is still controversial [362].
9. Sex as an Important Risk Factor in Atherosclerosis
Several modiﬁable and non-modiﬁable atherosclerotic risk factors have been well
described. Among them, unhealthy diet, physical inactivity, dyslipidemia, hyperglycemia,
high blood pressure, obesity, sex, and age [363–366]. Here, the impact of sex on atherosclerosis
will be covered in detail.
The pathophysiology of atherosclerosis shows different patterns between women
and men due to inherent biological and social differences (e.g., Younis, N.). In 1948, the
US Public Health Service initiated the Framingham study, which indicated that sexual
dimorphism in major modiﬁable risk factors, including cigarette smoking, dyslipidemia,
hypertension, and diabetes mellitus, could be responsible for the observed differences in
atherosclerosis development and/or complications [367]. Since then, large datasets have
shown that the impact of these risk factors on the associated risk of myocardial infarction
(MI) is higher in women compared to men, with odds ratios of 1.3, 1.5, and 1.6, for
smoking, hypertension, and diabetes mellitus, respectively [368,369]. In addition, modern
epidemiological data show that younger women have a decreased risk to develop CVD
and lower rates of MI relative to men [370,371]. However, the observed cardioprotective
effect in women is lost between the age of 60 and 79 years, and CVD risk in women
surpasses that in men by the age of 80 years [371]. Nonetheless, this pattern of disease
onset is not recapitulated in stroke, which shows a greater prevalence in women until the
seventh decade of life and a greater probability of recurrent stroke within the ﬁrst 5 years
after stroke compared to men [372]. Additionally, coronary artery disease (CAD) is often
underdiagnosed and viewed as a leading cause of female mortality. In Europe, CVDs
account for 43% of deaths in men and 55% in women [373]. When analyzing the different
components of CVDs, coronary heart disease (CHD) represents 21% of deaths in men and
23% in women, whereas, as indicated above, stroke is a more frequent cause of death in
women than in men (18 and 11%, respectively), as well as the other CVDs (15% in women
Int. J. Mol. Sci. 2022, 23, 3346 19 of 38
and 11% in men). These demographic statistics evidence sex differences in CVD risk and
highlight the need to take into consideration sex as an important variable to be included
from animal models through clinical trials [372].
Although great efforts have been made to understand molecular mechanisms and
discover novel drug targets for atherosclerosis, data examining sex differences are still
relatively limited. A robust study, in which 771 preclinical articles on atherosclerosis and
other vascular diseases were analyzed, showed that the sex of animals is not reported
in the 18.8% of them. When the sex was speciﬁed, 55.4% of the studies were performed
on males, 20.4% were performed on females, and less than 25% studied both males and
females [374]. The proportion of studies including both sexes was very similar (21–28%) in
recently published articles [375,376]. Moreover, fewer than half of the studies including
both sexes directly compare males and females with the appropriate statistical analysis
to consider sex as an independent variable or the interaction of sex with a treatment or
genotype [374–376]. Different patterns in CVD incidence between women and men are
shown in Figure 4.
Figure 4. Different patterns in CVD incidence between women and men.
9.1. Impact of Plaque Size and Morphology between Sexes
Frequently, exertional ischemia (i.e., angina and claudication) occurs when an individual
plaque enlarges sufﬁciently to impair blood ﬂow to meet tissue demand (usually >70%
stenosis). On the other hand, plaque rupture leads most of the morbidity and mortality
events from atherosclerosis, such as MI, stroke, disabling peripheral artery disease, and
eventually death. Non-invasive imaging has shown that men develop plaques earlier
and have a greater plaque burden than women, even after accounting for differences in
risk factors. In addition, post-mortem pathological studies have shown that plaques with
30–40% stenosis are more likely to rupture, leading to vascular occlusion and death [377].
These studies suggest that an overall plaque burden, a plaque inﬂammatory state, and an
unstable plaque morphology contribute to acute MI and stroke risk, in agreement with the
greater incidence of ischemic events in males, although this relationship changes later in
Int. J. Mol. Sci. 2022, 23, 3346 20 of 38
time [378–380]. Additionally, it has been shown that women do not present as much of an
atherosclerotic plaque burden as men and have fewer high-risk plaque features [381–383].
Signiﬁcantly more atherosclerotic and calciﬁed plaques, higher rates of CAD, and major
adverse cardiac events over 5.6 years of follow-up have been observed in men compared
with women [380]. Moreover, the cross-sectional REFINE-Reykjavik study, which included
21,132 patients, showed that 50% of women had normal computed tomography scans
compared with 31% of men [378].
New imaging techniques allow one to assess individual atherosclerotic plaque features
that are associated with adverse events. Intravascular ultrasound, which provides
information about both the degree of stenosis and the extent of necrotic core in the plaque,
has shown that women suffering from acute coronary syndrome present a similar number
of culprits compared to men [379]. Nevertheless, despite a higher mean age and multiple
comorbidities, women present fewer non-culprit lesions, fewer involved coronary arteries
with lesions, a lower frequency of plaque rupture, and a smaller total necrotic core
volume [381,384].
9.2. Clinical Implications
The importance of understanding age and gender differences in culprit plaque composition
is crucial to choose more appropriate pharmacological and interventional therapies
[385]. The signiﬁcantly higher prevalence of thin-cap ﬁbroatheroma in elderly women
patients compared to men shows the importance of intensive lipid-lowering therapy for
women [386]. However, recent studies found that women were less likely to be eligible
for statin administration than men, due to women being offered statins at a lower rate
by their caregivers [387–389]. Sufﬁcient intensity and duration of lipid-lowering therapy,
including statins, should be provided for women as that provided for men [386]. With the
imbalance of cholesterol metabolism being a key factor in the progression of atherosclerosis
by increasing age in women, ezetimibe in combination with statins might also be effective
to stabilize coronary plaque in women [390,391].
In contrast to the treatment and prevention of vulnerable plaque, the therapeutic strategy
for coronary calciﬁcation may be more complex and challenging. For the interventional
treatment of culprit lesions with large calciﬁcation, intracoronary imaging techniques may
provide safer procedures and better clinical outcomes [392,393]. Nonetheless, lesions with
large plaques also require the use of a debulking device, and a subsequent wider stent
area is also required for calciﬁcation [394]. In addition, lifestyle modiﬁcation may be more
important in women than in men regarding the risk reduction of atherosclerotic change
in plaque components and subsequent disease onset [395,396], as they are considered
modiﬁable factors.
10. Cigarette-Smoking-Induced Atherosclerosis
Cigarette smoking is a powerful independent risk factor for atherosclerosis and
ACV events, since chemical constituents of smoke have high oxidant and inﬂammatory
capacities that can directly induce endothelial damage and potentiate inﬂammatory
response [397–401]. Clinical evidence has shown a direct proportional dose-dependent
association of smoking exposure with the presence of extensive and calciﬁed atherosclerotic
plaques, and smoking cessation at any age is one of the most important health interventions
for reducing risks of ACV, cancer, and mortality [402]. Smoking cessation has been associated
with less progression of carotid plaque, but not with intima media-thickness [403].
The effects of smoking cessation on carotid atherosclerosis were related to the degree of
abstinence and persisted after adjusting for baseline smoking heaviness and ACV risk
factors [403].
Int. J. Mol. Sci. 2022, 23, 3346 21 of 38
Molecular Mechanisms Underlying Clinical Smoking-Induced Atherosclerosis
It has been shown that single smoking compounds (e.g., nicotine, carbonyl compounds,
acrolein, and methyl vinyl ketone) and/or their combined action can affect each stage of
the atherosclerotic process [401].
Exposition to smoke can promote oxidative stress, which constitutes one of the main
mechanisms underlying endothelial injury. As mentioned above, oxidative stress inﬂuences
the activity of numerous enzymes (e.g., eNOS and NADPH–oxidases) [404] and leads to
irreversible modiﬁcation of different proteins, thereby deeply altering intracellular signaling
pathways. Smoke is able to increase LDL levels through metabolic alterations and the
induction of LDL oxidation due to the direct oxidant capacity of smoke components [401].
Smoke exposition also leads to NF-κB activation in ECs, increasing the expression of
adhesion molecules on the plasma membrane [405] and the upregulation of inﬂammatory
genes, including IL-1 and COX-2 [406]. Furthermore, nicotine triggers the secretion of
pro-inﬂammatory adipokines from the perivascular adipose tissue [407].
Moreover, smoking induces the proliferation and migration of VSMCs and the switch
from contractile to secretory phenotype by increasing the expression of IFN-β and
PDGF [305,408]. Consequently, VSMCs are able to release pro-inﬂammatory factors and
extracellular matrix components.
Atherosclerotic plaques of smokers are characterized by a predominance of the lipid
core, and the ﬁbrotic cap is thinner than in non-smokers [409]. This conﬁguration is partly
due to augmented MMP activity in the plaque of smokers [410,411]. Smoking also triggers
the inﬁltration and activation of macrophages inside the lesion, as well as their conversion
in foam cells, contributing to the growth of the lipid core. Smoking is also essential in
the process of platelet activation and adhesion to endothelium [412]. In addition to these
effects, smoking can cause an increase in blood pressure, a determinant for plaque damage
and instability.
Several microRNA pathways that are involved in the development of atherosclerosis
are also regulated by cigarette-smoke exposure [413,414]. It has been shown that exposure to
high-dose cigarette smoke leads to the upregulation of miRNA-155 and miRNA-21, which
target PPAR-α. The downregulation of PPAR-α leads to the upregulation of VCAM-1,
ICAM-1, and MCP1 through the activation of transcription factor AP-1 [415,416]. VCAM-1
and ICAM-1 mediate the ﬁrm adhesion of leukocytes to the ECs and play a critical role in
subsequent leukocyte transmigration, leading to atherosclerosis [417,418], whereas MCP-1
regulates the migration and inﬁltration of monocytes/macrophages [419].
In addition, the upregulation of miR-155 and miR-21 modulates the EGFR/ERK/p38
MAPK and the PI3K/Akt/eNOS pathways in ECs, respectively [420,421].
11. Conclusions
During the last few years, we are witnessing an increased burden of atherosclerotic
disease that contributes to CVD risk, which is becoming a global epidemic. The study of
cellular and molecular biology mechanisms of atherosclerosis has provided remarkable
insights into the processes that lead to atheroma development and the clinical manifestations
of this disease. Knowledge and continued research about the functions of non-coding
RNAs in plaque development have improved, showing that miRNAs and lncRNAs alter
the transcription of genes implicated in atherosclerosis. In addition, microbiota have been
linked to the development of atherosclerosis by identifying microbial ecosystems residing
in different habitats of the human body that contribute to metabolic and cardiovascular
disorders. The role of microbiota in atherosclerosis development is supported by increasing
mechanistic evidence; however, further studies are needed to understand the contribution
of microbiota to atherosclerosis. Finally, the importance of analyzing sex-speciﬁc
differences as risk factors associated with atherosclerosis is important for individualized
risk-management strategies to prevent the development and progression of atherosclerosis.
Int. J. Mol. Sci. 2022, 23, 3346 22 of 38
The progress in understanding the mechanisms that lead to atherosclerosis development
will surely provide therapies to address the unacceptable burden of persistent risk
and will ultimately improve the diagnosis and prognosis of the disease.
Author Contributions: Conceptualization, S.J.-B., A.B.-V. and C.M.; writing—original draft preparation,
S.J.-B., A.L.-S., U.G.-G., A.B.-V., J.R.O., I.A., K.V. and C.M.; writing—review and editing, S.J.-B.,
A.B.-V. and C.M. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Basque Government (Grupos Consolidados IT-1264-
19). A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la
UPV/EHU (2019) 2019/2020; U.G-G. was supported by Margarita Salas Grant; and S.J. and A.L-S
were supported by a grant PIF (2017–2018) and PIF (2019–2020) Gobierno Vasco, respectively.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conﬂicts of Interest: The authors declare that there is no conﬂict of interest.
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