Atherosclerosis has been defined as an inflammatory disease developing primarily at
arterial branching points and curvatures, where disturbed flow constantly inflicts
low-grade injury to the endothelial monolayer lining the vessel wall. This step seems
to be crucial for atheroprogression, because the maladaptation of endothelial cells
(ECs) to this physiological disturbance increases the susceptibility of branching
points to a proinflammatory state. Consequently, maladapted ECs make arterial branching
points more prone to circulating and oxidized low-density lipoproteins (oxLDLs) influx
in the subendothelial space, and promote the recruitment of monocytes by releasing
proinflammatory molecules (i.e., chemokine and adhesion molecules). Subendothelial
monocytes differentiate into macrophages, which locally proliferate and take up oxLDL.
Exacerbation of lipid deposition into the plaque leads to insufficient lipoprotein
scavenging by macrophages, which turn into foam cells. Advanced lesion progression
is characterized by an accumulation of lipoproteins, macrophage-derived foam cell
apoptosis and necrosis due to defective efferocytosis, formation of cholesterol crystals,
and smooth muscle cell (SMC) cap formation. Hence, growing of advanced lesions leads
to a critical reduction of arterial lumen and blood flow, reduced oxygen supply, and
rupture or erosion of the plaques, which can cause thrombosis.
By analyzing aforementioned phases of plaque development, it has become evident that
inflammation plays a crucial role in atheroprogression. It is therefore not surprising
that many of the therapies currently used to treat atherosclerosis and thrombus formation
focus on counteracting the onset of an acute inflammatory state. Notably, statins
are capable not only of lowering cholesterol levels but also to exert anti-inflammatory
effects. Accordingly, studies conducted using colchicine, canakinumab, an antibody
to interleukin (IL)-1β, or other modalities targeting IL-6 or IL-1 receptors are aimed
at more specifically reducing inflammatory activity during atherosclerosis.[1] However, current anti-inflammatory and antithrombotic therapies are far from lacking
side effects, although the risk–benefit ratio still makes them an indispensable choice
improving the life expectancy of patients with cardiovascular diseases.
The exponential growth of genome-wide association studies (GWAS), single-cell sequencing
databases, and context-based text mining has brought to light the existence of complex
regulatory networks triggering cardiovascular disease. Among these, the heterogeneity
and interconnection of vascular cell populations, as well as their selective contribution
to distinct stages of atherosclerosis particularly stand out ([Fig. 1]). Moreover, GWAS have shown disease-linked genetic variation in the nonprotein-coding
sequence space, which are actively transcribed to noncoding ribonucleic acids (ncRNAs),
that is, micro- and long ncRNAs (miRNAs and lncRNAs). This novel class of ncRNAs is
differentially expressed in diseased tissues and act as epigenetic modulators of gene
expression. Indeed, the Part 2 of the Theme Issue on atherosclerosis and atherothrombosis
brings together the innovative and provocative view of expert scientists on novel
therapeutic perspectives. Among these, the identification of phenotype- and cell-to-cell
interaction-related molecules, as well as epigenetic modulatory-related molecules,
as novel and selective therapeutic targets to treat atherosclerosis.
Review articles by Busygina et al[2] and Pircher et al[3] underscore a fundamental problem with current immunotherapies. Indeed, antiplatelet
and collagen inhibitors, such as the first-in-class oral irreversible Bruton tyrosine
kinase (Btk) inhibitors (ibrutinib and acalabrutinib), show bleeding side effects.
These are partly ascribable to complex off-target mechanisms of drug concentrations
used to treat, for example, B cell malignancies and not required for the inhibition
of glycoprotein VI-mediated response of platelets to low collagen or plaque. Busygina
et al[2] summarized the last clinical trials on antithrombotic drugs and underlie the relevance
of a short-term application of novel reversible Btk inhibitors to avoid bleeding side
effects. At the same time, Pircher et al[3] introduce novel therapeutic targets with focus on cell-to-cell interaction. Following
plaque rupture, highly thrombogenic plaque content become exposed to blood stream
and mediate platelet and neutrophil recruitment. Neutrophils and platelets reciprocally
sustain their activation and promote inflammation by exposing adhesion molecules,
such as P-selectin, P-selectin glycoprotein ligand 1, and, as recently emerged, by
releasing citrullinated histone 3-rich extracellular traps (neutrophil extracellular
traps [NETs]). Notably, neutrophil-deriving NETs can serve as scaffold for platelet
aggregation, and sustain inflammation and thrombosis. Hence, Pircher et al[3] introduce novel short-term platelet inhibition treatments in acute thrombosis aimed
to inhibit neutrophil–platelet aggregation, with potentially less side effects than
those from established long antithrombotic therapies.
Monocyte-derived macrophages are a perfect example of stage-dependent cell phenotypic
change during atherosclerosis. Aside the concept that macrophage polarization can
be reprogrammed by inflammatory stimuli, Stremmel et al[4] here underline how the shift between inflammatory (M1-like) and proliferative (M2-like)
macrophages is the resultant of their metabolic changes. Notably, M1-like macrophages
utilize glycolytic metabolism while M2-like macrophages feature mitochondrial oxidative
phosphorylation and fatty acid metabolism. However, macrophages metabolism is altered
during atherosclerosis. Herein, Stremmel et al interestingly discuss how immunometabolism
might be used to target selective macrophage subpopulation in dependence of their
form of energy supply.
Heterogeneity of cells involved in atherosclerosis is therefore a critical point to
consider to generate more selective therapeutic drugs and to reduce side effects.
Moreover, it could also explain why current therapies do not show the same beneficial
effects in all patients. The epigenetic aspect could be the missing link to develop
more selective and effective therapies. The review articles by Eckardt et al,[5] Paloschi et al,[6] and Holdt et al[7] exactly describe glycan-binding proteins, miRNAs, and lncRNAs selective epigenetic
biomarkers as novel and potential therapeutic targets to treat atherosclerosis. Vascular
glycans are dynamically influenced by the physiological state of the cells, which
is read and translated into function by glycan-binding proteins. Therefore, glycans
represent the “footprint” of the cells at a selective pathophysiological stage. Eckardt
at al[5] discuss how glycans could be used as intermediate between a drug and its target
to increase drug specificity. Notably, mass spectrometry and nuclear magnetic resonance
studies identified lectins and galectins as therapeutic biomarkers and thrombotic
targets. However, the complexity of glycan structures makes their identification and
structural function analysis difficult, and at the moment only heparins have been
customized for anti-inflammatory therapies.
ncRNAs are a new class of — “mechanistically easy-to-study” — epigenetically modulated
molecular markers that offer a selective therapeutic advantage. Paloschi et al[6] and Holdt et al[7] describe selective inhibition/overexpression of miRNAs and lncRNAs as more off-target,
easy delivery, and less side effect therapeutic alternative to conventional immunotherapies.
In particular, Paloschi et al overview the role of ncRNAs in myeloid cells, or released
from arterial cells for myeloid recruitment, to modulate inflammatory cascade during
vascular disease. Notably, administration of miR-126–5p mimics restore impaired endothelial
proliferation, whereas miR-181b and miR-92a inhibition reduces inflammation. Similarly,
exosome-released lncRNCR3 protect against hypercholesterolemia-induced EC and SMC
dysfunction, whereas lincRNA-p21, MeXis, and MALAT1 promote macrophage anti-inflammatory
role and cholesterol efflux. As underlined in the review by Holdt et al,[7] current RNA therapeutic trials exclusively involve small interfering RNAs and mRNAs,
but not yet lncRNAs. However, to consider lncRNA-centered therapies we should first
take some issues into consideration. First, some lncRNAs are poorly conserved between
mouse and human. Second, some of the ncRNAs reported as potential therapeutic candidates
show different effects depending on the disease considered. For example, MALAT1 overexpression reduces atherosclerosis, but it is also cancer-promoting. Third, miRNAs
and lncRNAs can affect their activation, playing opposite roles during atherosclerosis.
Indeed, sponge activity of lncRNA MEG3 and MALAT1 on miR-21–3p and miR-22–3p, respectively,
affect EC proliferation and apoptosis during atherosclerosis. Moreover, one miRNA
can target bot mRNAs and lncRNAs, such as athero-miR-103, which promote EC inflammation
by targeting KLF4,[8] and impairs EC regeneration by targeting lncWDR59.[9] To avoid side effects and off-targeting, Holdt et al[7] describe the use of synthetic circular lncRNAs, which can play even a different
role compared with their endogenous counterparts, that can be packaged in lipid vesicles,
conjugated to antibodies, or (current modern approach) eluted from coated stents and
perivascular hydrogels. Accordingly, while linear ANRIL promote atherosclerosis, circular
ANRIL mediates opposite, protective effects.
Reading these reviews reveals how innovative but at the same time how complex it will
be to consider epigenetically relevant molecules as more selective alternative therapies.
The context-based atheMir database introduced by Joppich et al[10] is a database of miRNA–gene interaction networks that offer a good starting point
to investigate epigenetic molecule interconnections in a cell-to-cell and phase–context
dependent manner during atherosclerosis. Based on scientifically supported evidences
and reported interactions, atheMir explores new selective miRNA–gene interaction hypothesis
in cardiovascular diseases, that is, endothelial and monocytes miR-126 and miR-155/222
role in early atherosclerotic stages, and SMC miR-98 and miR-504 role at later stages.
Although considered the “dark side of the genome,” this ncRNA force in humans probably
derives from the ability (or attempt) of nature being to develop a new evolutionary
stage to adapt more easily to the cumulatively higher risk of cardiovascular diseases
related to a longer life time.
Fig. 1 Micro (miRNAs)- and long noncoding ribonucleic acids (lncRNAs') role during atherosclerosis.
Graphical overview of the major miRNAs and lncRNAs reviewed in the Theme Issue. MiRNAs
and lncRNAs contribute to atherosclerosis alone or by affecting their reciprocal activation.
Hyperlipidemia impairs endothelial regeneration inhibiting miR-126–5p expression and
promoting proinflammatory miR-92a and miR-103. The majority of lncRNAs are downregulated
during atherosclerosis, suggesting that their overexpression might be promising to
prevent plaque progression. Indeed, MALAT1, HOTAIR, lncWDR59, and circANRIL sustain
endothelial regeneration and prevent their apoptosis and inflammation. However, linear
ANRIL, lncMRC3, and PACER promote endothelial inflammation and smooth muscle cell
(SMC) cup formation. Monocyte recruitment is enhanced by endothelial release of miR-10a.
At early stages of atherosclerosis, miR-155 and miR-195 inhibit M2-like macrophage
proliferation. However, miR-342–5p-mediated increase of miR-155 expression, miR-21
overexpression, and miR-195 inhibition, all together sustain M1-like inflammatory
macrophage polarization and defective efferocytosis. CircANRIL, MeXis, and lncRNA
HOCX-AS1 are downregulated during these stages, and therefore cannot inhibit chronic
inflammation. SMC miRNAs and lncRNAs, such as miR-146a/145/21/26a, NEAT1, SENCR, and
ANRIL, seem to synergistically promote their proliferation and to contribute at cup
formation. CircANRIL is instead acting as SMC proliferative agonist.
