Comparative Analysis of miRNA Regulatory Pathways in Atherosclerosis: Unravelling

Similarities and Divergences across Different Stages and Phenotypes

Abstract

Atherosclerosis is a common cardiovascular disease that leads to multiple complications

including coronary artery disease, myocardial infarction, acute coronary syndrome, hemorrhagic
strokes, and ischemic. This occurs due to the presence of various predisposing factors involving
hypoxia, oxidative stress, hyperglycemia, hypertension, proinflammatory cytokines, and aging.
However, LDL accumulation is considered the primary cause of plaque formation which is
facilitated by the disruption of endothelial integrity and initiates the mechanisms to produce
prothrombic and pro-inflammatory effects. In actuality, the most important factors include the
diversion of expression of non-coding RNAs towards atherosclerosis to initiate various
prothrombic mechanisms. These couldn’t promote any particular gene or pathway upregulation
itself but it silences particular gene expression while the rest remain unaffected. So, the presence
of specific proteins transforms the whole micro-environment. Therefore, mi-RNAs are
considered important regulators in any disease due to their significant involvement in
transforming the micro-environment. In this article, the involvement of mi-RNAs in
atherosclerosis is discussed, which mi-RNAs are involved, what are their roles, how can we
identify them, their modulation in various phenotypes, their differential expression in animal
models and humans, and predisposing genetic and environmental factors that enhance the
chances of illness development in any individual. The mechanism of mi-RNA production in
response to the micro-condition of any individual that initiates atherosclerosis and promotes
progression is also discussed in detail.

Keywords: Atherosclerosis, Cardiovascular disease, Micro-environment, Inflammation,

Hyperglycemia

Graphical Abstract:
1. Introduction

Atherosclerosis being a highly widespread cardiovascular disease, is considered the primary

cause of death globally. It is classified into distinct phases: initiation, plaque progression, and
disruption. Endothelial ulcer, resulting from various predisposing factors such as hypertension,
smoking, or lipid metabolic problems, triggers a series of events including intra-plaque
angiogenesis, cellular infiltration, bleeding, and matrix disintegration [1], [2]. It is an intricate,
multifaceted disease marked by the development of ulcers linked to lipid accumulation under the
endothelium surface and the occurrence of mild inflammation in the vessels. Sub-endothelial
lipid build-up, specifically the modified low-density lipoprotein (LDL) accumulation is
considered a primary causative mechanism of plaque formation. The accumulation of lipids
beneath the endothelium layer is facilitated by the disruption of endothelial integrity, which is a
crucial regulator of the balance in the circulatory system. This disruption impairs vasodilation
and has both prothrombotic and pro-inflammatory effects, ultimately influencing the
development of early atheroma. Accumulated various lipoproteins trigger localized biochemical
reactions, characterized by persistent and dysfunctional inflammatory responses regulated by T-
cells and macrophages. These responses play an important role in the creation, progression, and
rupture of plaques [3]. Specifically, macrophages produced from monocytes take in modified
LDL and transform into foam cells, which then recruit T- and B- inflammatory cells to form a
growing layer in the artery intima. Furthermore, both arms of the immune system play a major
role in the formation of plaque. Atherosclerosis ulcers are typically absent in the linear sections
of the arterial network but are primarily located at branch sites where the flow of blood is
disrupted due to restricted forward movement.

The miRNAs are a particular transcriptomic group of single-stranded non-coding RNAs that are
highly conserved sequences among humans with generally 22 nucleotides long. They
downregulate the expression of genes at the post-transcriptional step, either by the arrest of
mRNA before protein synthesis or by facilitating the mRNA breakdown before translation [3],
[4]. The endothelium is frequently impacted by several causes, including hypoxia, oxidative
stress, shear stress, hyperglycemia, hypertension, proinflammatory cytokines, age, or necrosis.
These variables negatively affect the functioning of endothelial cells (EC), resulting in
heightened rates of cell division, movement, aging, cell death, blood vessel formation, and
inflammation. Accumulating data indicates that particular groups of non-coding RNAs
participate in various biochemical pathways regulation that impact endothelial cell function,
mainly involving the preservation of blood vessel integrity and the stimulation of cellular
proliferation and mobility [5].

The endothelial progenitor cells (EPCs) can break down miR-221 to maintain optimum cellular
activity [6]. The increase in expression of miR-221 in EPCs was more significant in patients with
coronary artery disease (CAD) than in healthy individuals. Additionally, increased miR-221
expression reduces the cellular proliferation ability of EPCs [5]. as it facilitates the efficient ECs
functioning which induces apoptosis in older cells and replaces it with naïve and healthy cells
[7], [8]. Moreover, when miR-221and miR-222 bind with the c-kit receptor for stem cell factor,
they influence the process of angiogenesis in ECs directly [9] while indirectly control the
endothelial nitric oxide synthase (eNOS) synthesis [10] because the major nitric oxide synthesis
in vascular endothelium is performed by eNOS [11]. Nitric oxide is essential for regulating the
proliferation and migration of endothelial cells throughout the process of vascular remodeling
and angiogenesis. The reduced availability of nitric oxide is a characteristic feature of individuals
with atherosclerosis [12]. Endothelial cell-mediated angiogenesis in advanced plaques results in
more blood vessel development that invades an inner layer of the artery wall. This procedure is
mainly subjected to the formation, progression, and rupture of the plaque. Additional miRNAs
can control the process of angiogenesis formation. The study showed that miR-126, which is
only found in endothelial cells, promotes the growth of novel blood vessels throughout
development by decreasing the production of Spred-1 (sprout-related protein 1) mainly
indicating the important role of miR-126 in improving endothelial function [3]. However, the
excessive expression of miR-92a can inhibit naive blood vessel development and decrease the
movement of endothelial cells both in laboratory settings and in living organisms [13].
Additionally, miR-129-1 and miR-133 effectively inhibited important aspects of angiogenesis,
mainly the rate of cell division, the ability of cells to survive, and the movement of the HUVECs
(human umbilical vein endothelial cells) in laboratory conditions. This suppression was achieved
by specifically targeting FGFR1 (fibroblast growth factor receptor 1) and VEGFR2 (vascular
endothelial growth factor receptor 2) [14]. Moreover, the same study analyzed the functional
participation of specific miRNAs in remodeling of vascular response during plaque development
is a crucial aspect of atherosclerotic disease [15].

Various experiments have demonstrated that small RNAs could be utilized as diagnostic
biomarkers in different diagnostic settings, including cardiovascular illnesses, rheumatoid
arthritis, kidney diseases, diabetes, and cancer as explained in Fig. 1 enclosing the biogenesis
and involvement of intercellular communication in humans. Proteins-based biomarkers do not
meet the diagnostic requirements for heart disorders, so there is a need to enhance diagnostic
markers. Studies conducted on clinical samples have demonstrated that miRNAs are the primary
biomarkers for accurate diagnosis with the ability to be utilized as novel therapeutic agents for
multiple diseases including various cardiovascular diseases and cancer [16], [17]. miR-423-5p
exhibited increased expression in cardiac failure patients, regardless of age and gender, and has
the potential to be used as a sensitive tool for cardiac problems [18]. Contrastingly, coronary
artery disease patients exhibited reduced miR-126 and miR-145 expression [19]. Analysis of
tissue samples obtained from individuals with myocardial infarction (MI) reveals the reduction
of miR-133a/b and miR-1 expression, whereas miR-208 exhibited an increase [19]. Hence,
miRNAs could be considered diagnostic biomarkers for the detection, prediction, and
characterization of atherosclerotic lesions. The role of mic-RNAs in atherosclerosis is analyzed
and discussed by enclosing its various pathways, methodological approaches, and predisposing
factors in this review.

2. Methodological Approaches in Studying miRNA Pathways

Due to the significant role of miRNAs in therapeutics, there is ongoing development of various
methodologies and databases to detect miRNAs and conduct further analysis [20]. The cloning
and sequencing of mRNAs is an effective approach for identification at in vitro and in vivo
levels. The presence of short miRNAs in different genetic locations, and their methylation status
made the process time-consuming and laborious in identification [21]. The use of cloning and
computational approaches has greatly enhanced the process of identifying miRNA [22].
Currently, the combination of various interdisciplinary approaches encompassing biological and
bioinformatics methods is widely employed for the identification of miRNAs [23].

In modern transcriptomic analysis, precise miRNA sequences are obtained from the
transcriptome. The NGS strategy [24], [25] and Maxam-Gilbert's chemical cleavage approach
[26] have greatly transformed this process and serve as a fundamental component. However, the
commercialized Sanger's approach is refined and mechanized and it continues to be extensively
utilized for numerous applications but is still regarded as laborious and time-consuming. The
modifications include using in silico tools for high-throughput data analysis, fluorescent dyes for
detection, and optimized PCR sequences to reduce genetic material and improve efficiency and
precision [27]. There is a requirement for less expensive approaches to sequence huge genomes
including the automation of the Sanger sequencing method, which in turn prompted the
development of innovative techniques known as second-generation sequencing methods. This
sequencing method can be classified into two major categories, hybridization and sequencing
[28].
Fig. 1 miRNA biogenesis and intercellular communication through various biochemical
pathways. Primary miRNAs are expressed in nucleus, dicer cleaves the pre-miRNA to release
mature miRNAs in cytosol. These miRNA targets mRNA for degradation and translation
inhibition by loading into the RISC
The Illumina sequencing method utilizes a bridge amplification approach, in which a nucleic
acid template undergoes repetitive amplification on a solid substrate, leading to the production of
many sequence clusters. Currently, the Illumina platform provides a wide range of models,
including MiniSeq, MiSeq, NextSeq, NovaSeq, and HiSeq, each of which produces unique
results. Upon evaluating other sequencing technologies, it is apparent that the Illumina platform
has emerged as the most preferable option because of its exceptional resolution [27]. By
comparing the next-generation methodologies, it becomes evident that Illumina and HiSeq's
technologies exhibit superior accuracy and efficiency compared to other second-generation
sequencing procedures with cost-effectiveness [29], [30]. The exclusive sequencing of the short
transcriptome is becoming more popular because of its exceptional specificity for short RNA
transcripts, such as miRNAs. Therefore, the application of the short RNA-sequencing method in
combination with the comprehensive coverage offered by Illumina Sequencing emerged as the
preferable option for identifying novel miRNA [28].

The utilization of NGS platforms has facilitated the achievement of several objectives with a
high degree of precision. The aims encompass miRNA identification, miRNA expression
analysis, miRNA precursor prediction, variation identification, miRNA target identification, and
other associated characteristics [31], [32]. The effectiveness of miRNA research is improved by
the integrated use of NGS [33]. The cost-effectiveness and elevated sensitivity of NGS have led
to the production of vast amounts of microRNA (miRNA) data from human subjects. NGS and
various bioinformatic techniques can be employed to examine non-coding RNA transcripts that
are specific to a particular DNA strand with decreased quantities [34]–[36].

The methods employed for miRNA identification could be divided into two basic approaches:
the basic comparison and the advanced machine learning approach [37]. The primitive
comparative technique primarily utilizes flow-connected programming, whereas the machine
learning approach centers around predictive modeling derived from the given file. Both
approaches for miRNA identification involve two common processes: (a) performing secondary
structure folding, and (b) doing a similarity-based search against existing databases [38].

3. miRNA Regulatory Pathways in Early vs. Late-stage Atherosclerosis

3.1. Plaque Formation
The prolonged inflammatory response in vascular endothelium mainly based on dyslipidemia
leads to atherosclerosis. The liver is essential for controlling the body's cholesterol levels. It does
this by a mechanism called reverse cholesterol transfer (RCT), which involves absorbing LDL
and releasing HDL from cells and tissues [39], [40]. An abundance of LDL-C that is accumulated
in sub-endothelium initiates the inflammatory cascade which results in recruitment of monocyte
recruitment to the areas of inflammation with the activation of ECs. As scavenger cells, the
monocytes try to get rid of the deposited cholesterol.

Macrophages generated from monocytes ingest the aggregated or oxidized LDL (ox-LDL) and
mature into inflammatory foam cells. The underneath sheet of the blood vessel is lined by foam
cells that recruit the second wave of inflammation-producing agents. Furthermore, oxidized LDL
primes to the overproduction of adhesion molecules (vascular cell adhesion molecule-1 (VCAM-
1) intercellular adhesion molecule-1 (ICAM-1), and E-selectin) [41], [42], and enhances the
endothelial permeability [41], [43]. After the creation of foam cells, fatty streaks occur, which
then advance to susceptible plaques and result in the clinical presentation of myocardial
infarction or stroke. Various mechanisms including plaque rupture which involves the thinning of
the fibrous cap, the necrotic core development, the process of angiogenesis, and ultimately the
rupture of the plaque [44]. Recent findings have uncovered the crucial role of miRNA in the
cellular and molecular pathways that play an important role in the development of
atherosclerosis [41] as depicted in Table 1.

3.2. Initiation

The MiR-122 has a significant role in cholesterol balancing which contributes to 70% of total
miRNA expression in the liver [42], [45]. This experiment illustrates the significance of
antagomir-122 in reducing plasma cholesterol levels in mice [46]. Additional research employing
antisense oligonucleotides (ASO) has corroborated the reduction of overall cholesterol [42], as
well as the levels of HDL and LDL in the bloodstream, by inhibiting the activity of miR-122 in
cholesterol production [47], [48] as depicted in Fig. 2 with more detail. Hepatocytes are thought
to be affected indirectly by MiR-122 through genes that code for proteins in the lipid metabolism
pathway, such as sterol regulatory element-binding protein 2 (SREBP2) and fatty acid synthase
[45]. In addition, it has been found that miR-185 can reduce cholesterol synthesis by blocking
SREBP2 [45], [49].

Cardiometabolic disorders have been anticipated to be associated with miR-301b, miR-130b,

miR-148a, and miR-128-1, as shown by a genome-wide association study (GWAS) [50]. These
miRNAs have a crucial role in promoting the activity of the LDL receptor (LDL-R) and
increasing its abundance in the bloodstream [51]. ATP-binding cassette transporter A1 and G1
(ABCA1, G1) have a crucial function in eliminating cholesterol from the cellular compartment
through high-density lipoproteins (HDL) during the Reverse Cholesterol Transport (RCT)
process. This mechanism has an important role in down-stimulation of atherosclerosis [52].
Studies have demonstrated that these transporters are controlled by multiple miRNAs that have
important participation in various lipid pathways. These miRNAs include miR-223, miR-10b,
miR-19b, miR-26, miR-17, miR-33a/b, miR-27a/b, miR-128, miR-93, miR-148a, miR-144, miR-
758 and miR-302 [53], [54].
Fig. 2 Mechanism of orchestration of cholesterol homeostasis and macrophage activation in
atherosclerosis.

The MiR-223 is an intracellular miRNA that relies on cholesterol and exerts a negative influence
on cholesterol production via post-transcriptionally regulating methylsterol mono-oxygenase 1
and HMG-CoA synthase that influences RCT. An experiment involving miR-223-/- in mice
reveals evidence supporting its involvement in the elevation of concentration of total cholesterol
levels and HDL-C [55].

3.3. Progression

The upregulation of miR-92a appears to contribute to the susceptibility to atherosclerosis by

regulating the transcription of Krüppel-like factor 2 (KLF2) and (KLF4) in the aorta of swine
[56], [57]. The abnormal regulation of genes that defend against atherosclerosis, such as eNOS
results in a decrease in KLF2 and KLF4 expression, ultimately leading to the atherosclerotic
lesions development [58]. In addition, an increase in oxidative conditions triggers the
simultaneous activation of miR-92a and SREBP2, which work together to stimulate innate
immune response and promote inflammation in ECs [59]. miR-92a has various athero-protective
effects involving the promotion of angiogenesis, speeding up the healing of ECs, preventing the
creation of new tissue inside blood vessels, and reducing plaque size and plaque rupture. These
effects have been documented in various lab animals including the mice and large animals in
both mice and large animal models that confirm its involvement in atherosclerosis [60].

Similarly, miR-126 has depicted its involvement in the angiogenesis processes and EC
proliferation. miR-126 and miR-126-3p exhibit functionality in the pathway as a negative
regulator by promoting VEGF signaling by SPRED1 (targeting sprout-related protein).
Furthermore, miR-126-3p functions in a protective manner by activating the antiapoptotic
CXCL12/CXCR4 signaling pathway. The increased concentration of miR-126-5p stimulates EC
cell proliferation after the activation of the Notch 1 signaling pathway [61]. Similarly, miR-126
has various roles in different kinds of cells such as CD34 +, macrophages, and adipocytes.
Likewise, miR-126 along with miR-92a miR-193b downregulates VCAM1 to elicit an anti-
inflammatory response.

Furthermore, miR-181b has a dual role in inflammation as it is downregulated in response to

inflammation generated by TNF or LPS. However, miR-181b is overexpressed via stimulation of
the NF-kB pathway in an anti-inflammatory response. Systemic administration of miR-181b
prevents atherosclerotic lesions in ApoE-/- mouse models [62].

Table 1: Different miRNAs are involved in various atherosclerotic pathways.

miRNAs Stimuli Targets Affected Effect on References

Signaling Atherosclerosis
Pathway
miRNA- Unknown ATP Binding Unknown Increase [63]
19b Cassette A1
miRNA- Macrophage Chitinase 3-Like- Extracellular Decrease [64]
24 Colony 1, Matrix signal-
 Stimulating Metallopeptidase regulated
factor 14 kinase
miRNA- Low ATP Binding Unknown Increase [65], [66]
33-5p cholesterol Cassette A1, ATP
levels, insulin Binding Cassette
G1
miRNA- Low shear Krüppel-like Nuclear Increase [67]
92a stress; High factor 2/4, Factor κB
Fat Diet Suppressor of
cytokine signaling
5
miRNA- Apoptosis Regulator of G- chemokine Decrease [68]
126-3p protein signaling (C–X–C
16 motif) ligand
12
miRNA- High shear Delta-like Notch Decrease [69]
126-5p stress homologue 1
miRNA- Unknown ATP Binding Unknown Increase [70]
144-3p Cassette A1
miRNA- Inflammation B cell lymphoma Nuclear Increase [71]
155 6 Factor κB
miRNA- High shear Importin α3 Nuclear Decrease [62]
181b stress Factor κB
miRNA- Differentiation Serine/threonine Nuclear Increase [72]
342-5p protein kinase 1, Factor κB
bone
morphogenetic
protein receptor
type II
miRNA- Unknown Lipoprotein lipase Unknown Decrease [73]
467b
 miRNA- Low shear Metalloproteinase Nuclear Increase [74]
205/712 stress inhibitor 3 Factor κB

The expression of miR-155 has shown enhanced proliferation in monocytes of patients with
coronary artery disease than in healthy individuals. The primary mediator of oxidized LDL leads
to increased transcription of miR-155 of macrophages of the M1 phenotype in atherosclerosis. It
appears to increase inflammation after stabilizing TNF-α mRNA. Moreover, the miR-155 with
monocyte/macrophage is largely involved in plaque development. Although miR-155 has been
demonstrated to have time-dependent effects, it is athero-protective in the initial stages and
atherogenic in the later stages [75]. Likely, miR-125a-5p has a role in the absorption of LDL and
the inflammatory responses linked with atherosclerosis [76].

4. Differential Expression in Vitro and in vivo

Earlier investigations have described the differential expression of miRNAs in clinical

cardiovascular disease in humans [77]. Notably, the angiogenesis investigation produced the
strongest evidence of miRNA involvement in ECs. For instance, miR-126 enhances the signaling
mechanism that encourages angiogenesis and preserves the structural integrity of existing blood
vessels, while miR-92a inhibits the vessel's development [78]. Furthermore, there are major
variations in expression profiles of miRNA expression across cardiac and vascular tissues that
may be relevant in clinical practices [79]. Likewise, variant miRNA profiles in monocytes of the
peripheral blood system activate the ox-LDL mediating cardiovascular disorders [79]. Different
miRNA expression patterns are associated with the development of carotid artery lesions, a
condition that differs from intimal hyperplasia [78]. miRNAs are also been utilized in the
treatment of arterial disorders such as athero-susceptible endothelium in pro-inflammatory
phenotypes are treated with miR-10a in vitro and in vivo [80].

5. Differential miRNA Regulation in Various Atherosclerosis Phenotypes

The distinct set of miRNAs with their specific living environment is expressed in the blood
vessels that are affected and susceptible to atherosclerosis. These studies have utilized different
comparison groups and employed different methodologies. The majority of researchers have
analyzed the real-time miRNA expression to evaluate the expression profile of an individual
[81]–[83]. However, the miRNA expression was analyzed through advanced microarrays and a
comparison was built among coronary, carotid arteries and blood vessels of healthy individuals
[84]–[86].

The groundbreaking microarray analysis identified 58 miRNAs that exhibited differential

expression among atherosclerotic plaques in different healthy and diseased atherosclerotic
arteries [87]. The increased expression of five microRNAs (miR-210a, -146a, -34a, -146b-5p,
and -21a) in atherosclerotic plaques was verified using real-time PCR on a broader set of arterial
samples. A total of 187 mRNA transcripts corresponding to these miRNA target genes exhibited
reduced expression levels in plaques. The proteins associated with these specific genes contribute
a distinct role in transcriptional control, signal transduction, and vesicular transport [88]. Only 31
miRNAs exhibit expression alterations in both carotid and coronary atherosclerotic plaques. The
miRNAs that were increased in plaques relative to controls (miR-22, -19b, -125a, -34a, -100, -
127, -133b, 133a, -155, -146a,) were commonly found in the carotid arteries. The levels of miR-
1, let-7f, and miR-92a were reduced in carotid atherosclerotic plaques. In several research,
contradictory findings have been documented regarding the effects of miR-21, -29b, -143, -145,
and -221 [88].

On the other hand, the coronary arteries displayed a significant decrease in the expression of
several miRNAs (let-7f, miR-19a, -1, -22, -34a -24, -106b, 133b -125a, -143, -145), and the
expression of other miRNAs (miR-9, -19b, -10a, -16, -10b, -21, -29b -25, -92a -29c, -100, -486, -
155, -497) showed changes in multiple directions across different studies. The miR-146a, -150, -
221, and -223 expression was increased in coronary atherosclerotic plaques. Altered expression
in the carotid and coronary arteries was observed for specific microRNAs such as, miR-146a
depicts up-regulation, whereas let-7f and miR-1 showed down-regulation. In contrast, miR-29b
and miR-21 findings are contradictory showing an increase in expression in a few samples while
a decrease in the rest of the samples [88].

The presence of miR-100, -21, -127, -125, -143/-145, -133, -221 in the carotid arteries is
generally linked with the instability of plaques [89], [90]. Unstable plaques exhibit increased
expression of several miRNAs (miR-127, -100, -133a/b, -145) and decreased expression of miR-
143 and miR-21. Inconsistent findings were found for miR-221. The decrease in miR-24
expression in the coronary arteries is linked to the instability of atherosclerotic plaques [91].

Similarly, [92] reported 155 miRNAs present in human blood that are associated with acute
coronary syndrome, coronary artery disease, myocardial infarction, and ischemic and
hemorrhagic strokes. Out of them, a total of 33 miRNAs have been identified in multiple studies.
Several miRNAs (let-7f, miR-16, -1, -24, -29b, -21, -133a, -106b, -155, -145, -146a, -486, -223)
were found to be present in both blood and tissues of coronary/carotid atherosclerotic plaques.
These miRNAs are of significant interest both as indicators for the complex progression of
atherosclerosis and as candidates for therapeutic intervention in this illness.

Typically, experiments on expression profiling of human carotid and coronary plaques analyze
individual miRNA individually. Considering the intricate and prolonged process of
atherosclerotic plaque formation, which is marked by significant cellular diversity, it is
reasonable to hypothesize that various miRNAs may collectively alter their expression and create
co-expression components during the progression of atherosclerosis or alterations in arterial cell
composition [93], [94].

6. Role of Genetic and Environmental factors in miRNA regulation in Atherosclerosis

The activation or inhibition of atherogenesis under flow circumstances is mediated by the gene
regulation of EC. The promotion or inhibition of atherogenesis under various flow circumstances
is mediated through the regulation of endothelial cell (EC) gene expression. This regulation
governs various biochemical processes including, size, cell shape, migration, survival,
proliferation, and inflammatory response. Several functional investigations indicate that miRNAs
have regulatory functions to control EC phenotype, function, and vascular inflammation [95].
Crucially, these miRNAs that are sensitive to shear forces can function as key controllers in the
field of endothelium biology and illness as described in Fig. 3. Various extracellular miRNAs
have been demonstrated to be controlled by distinct flow conditions including genetic and
environmental factors. Different genetic variants of particular miRNAs in various populations
are depicted in Table 2.
MiR-21 can regulate tissue fibrosis and regulate the apoptosis induction in vascular smooth
muscle cells (VSMCs) [96] after the increase in gene expression which further triggers the
activation of an inflammatory signaling cascade downstream [97]. miR-21 levels were
significantly enhanced (5.2-fold) by Unidirectional shear stress (USS) rather than oscillatory
shear stress (OSS), as observed in experiments employing human umbilical vein endothelial cells
(HUVECs) [98]. Another research revealed that miR-21 suppressed the expression of
phosphatase and tensin homolog (PTEN), which is a protein that inhibits the PI3K/Akt pathway
which results in a decrease in endothelial cell (EC) apoptosis and an increase in eNOS activation
and the availability of nitric oxide [99]. Although not specifically related to shear forces,
previous studies have demonstrated that miR-21 can suppress the production of RhoB protein,
which in turn restricts endothelial cell motility [91]. Additionally, miR-21 has been found to
inhibit CXCR2-mediated chemotaxis and intracellular protein trafficking [100].

Table 2: Gene polymorphism of miRNAs in different cardiovascular diseases in various

populations.

MiRNA rs Number Disorder Population References

MiR-149 rs2292832 Coronary artery Korean [101]
disease
Pre-MiR- rs895819 Myocardial infarction Chinese [102]
27a
Han population
Pre-MiR- rs2910164 Acute coronary Chinese [101], [103], [104]
146a syndrome
Coronary artery Korean
disease
Coronary artery Iranian
disease
MiR-196a rs1161491 Cardiovascular Polish [105]
3 disease
MiR-499 rs3746444 Coronary artery Chinese [106]–[109]
disease
Ischemic stroke Chinese
 Coronary artery Chinese
disease
Myocardial infarction Chinese
MiR-4513 rs2168518 Coronary artery Chinese [109]
disease
Fig 3. Endothelial miRNAs regulate vascular inflammation. miRNAs modulate specific targets in
endothelial cells (ECs) in response to biochemical and biomechanical stimuli, influencing the
equilibrium of pro- or anti-inflammatory signaling pathways.

The miR-145 and miR-143 genes are co-expressed as a contiguous intergenic cluster. The
expression of these genes was significantly upregulated in HUVECs subjected to 72 hours of
unidirectional steady shear stress (USS) utilizing a pump system [110]. KLF-2 is a shear-
responsive transcription factor, that facilitates the increase of gene expression [110]. It is
noteworthy that miR143 and miR-145, which are found in endothelial cells (ECs), have been
seen to be released and taken up by VSMC in co-culture. These microRNAs inhibit cell
proliferation by directly inhibiting the function of many transcription factors, including
myocardin, KLF-4, and KLF-5 [111]. Injecting miR-145 and miR-143 derived from ECs, into
the circulation of ApoE-/- mice led to a decrease in atherosclerosis [110].

miR-17∼92a host gene cluster contain precursor sequences of various miRNAs including 19a
and 92a and it is depicted in the experiment that miR-19a expression has significantly increased
on exposure to 12 hours of USS in a parallel plate flow chamber (PPFC) [111]. These miRNAs
arrest the cell cycle at G1 phase by promoting antiproliferative activity of USS that
downregulates the cyclin-D1 expression [111]. Similarly, the miR-92a expression using PPFC
technique leads to enhanced miR-92a expression under OSS [112] and it is confirmed by the in
vivo analysis in swines which describes the increased miR-92a expression in atherosclerosis-
prone regions of the aorta [113] which leads to the inhibition of KLF-2 and KLF-4 [114]. These
non-coding factors have principal role in balancing and stability of ECs by provoking
antithrombic and anti-inflammatory activity [115].

miR-10a expression was decreased in arteries which are susceptible to the development plaque
because it functions to reduce inflammation at a particular site [113]. Which results into
enhanced NF-Kβ recruitment into the HAECs where mi-R10a was suppressed. This is associated
with the significant rise in the production of VCAM-1, E-selectin and inflammatory cytokines
such as MCP-1 and IL-6, IL-8, etc. [113].
The microarray analysis of HUVECs upon exposure to USS and OSS for one day using cone-
and-plate viscometer system reveals that the miR-663 shows steep rise in expression in response
to OSS [116]. A particular inhibitor prevent upregulation of miR-663 in endothelial monocyte
but do not induce apoptosis. miR-663 target several transcription factors including KLF-4,
C/EBPB, and ATF3 that have role in expression of genes involved in anti-inflammation. Similar
results are also found in mouse using cone-and-plate viscometer system [74].

Similar to miR-663, miR-155 gene expression is increased in HUVECs upon exposure to USS in
a cone-and-plate viscometer system for 24 hours than to static or oscillatory shear stress
conditions [117], [118]. The increased miR-155 expression and rhoA and MYLK downregulation
leads to decreased movement and growth and promotes apoptosis in ECs. Moreover, it elicits the
rearrangement of actin cytoskeleton in arteries subjected to USS indicating a protective role in
atherosclerosis.

The expression of miR-23b∼27b∼24-1 cluster is increased in tissues associated with blood

vessels and ECs [119]. Multiple experiments in shear models reveal higher miR-23b expression
in response to 24 hours of USS [119], [120]. The shear-sensitive transcription factor, KLF-2, is
identified to increase the expression of miR-23b [121]. In addition, Wang and colleagues have
shown that 24 hours of pulsatile shear in HUVECs utilizing a parallel plate flow chamber leads
to an increase in miR-23b levels. MiR-23b inhibited the phosphorylation of the retinoblastoma
(Rb) protein, resulting in the stoppage of the cell cycle and growth [122]. Additional research is
required to clarify the mechanism by which shear forces regulate the members of the miR-
23b∼27b∼24-1 cluster and to determine their subsequent impacts.

7. Therapeutic Potential of Targeting miRNA Pathways

One potential advantage of miRNAs as a therapy is their capacity to counter numerous genes that
are part of the same disease pathway. Conventional medicines, in contrast, primarily focus on
one specific protein, while discounting the others. As a result, the remaining proteins may
continue to contribute to the diseased process [123]. Presently, the techniques employed for this
novel treatment strategy involve the utilization of microRNA mimics overexpression, anti-
miRNAs (antimiRs), or inhibitors. Regarding this matter, the anti-miR method is more suited for
promoting gene suppression, whereas miR mimics are used to increase gene expression [124].

Table 3. Presents a summary of the many cell kinds and cellular processes that are complicated
in the advancement of atherosclerosis. These are the specific targets of the miRNAs discussed in
this study. Multiple cell types have been demonstrated to be regulated by several miRNAs
concurrently, affecting various functions. This emphasizes the therapeutic possibility of focusing
on miRNAs to offer a comprehensive strategy for preventing atherosclerosis. It is well known
that miR-21 influences leukocyte polarisation and survival, thereby promoting the proliferation
and differentiation of smooth muscle cells into synthetic phenotypes, facilitating embryonic stem
cell differentiation, and enhancing endothelial progenitor cell migration and function during the
early stages of atherosclerosis. In addition, studies have shown that miR-34 protects the
endothelium by preventing oxidative stress damage [125].

Table 3: List of miRNAs highlighted in this review and their role in different cellular processes in
different cell types involved in atherosclerosis advancement.

References Stage of the Disease Process Cell type miRNA

[45] [47], [48] [52]
, , [63] Homeostasis and miR-24
transport of miR-122
cholesterol Biosynthesis of miR-185
Hepatocytes
Cholesterol miR-223
miR-486

[52] [53], [54]

, Cholesterol Macrophage miR-10b
Efflux s miR-20a/b
miR-23a/b
miR-30e
miR-33a/b
miR-92a
miR-101
miR-144
 miR-148
miR-302a

[62] [88]
[7], , , [101], Atherosclerotic let-7g
[117], [118] [128]
, plaque (initiation miR-17-3p
and miR-31
progression) miR-146a
miR-155
Endothelial
Inflamamation miR-181a-
Cells
3p/-5p
miR-181b
miR-221
miR-222

[5] [9] [10] [13] [53],

, , , , let-7a/b
[54] [125]
, miR-19b
miR-20a
miR-34
miR-98
miR-142-3p
Endothelial
Oxidative stress miR-199a-
Cells
3p/-5p
miR-200c
miR-221
miR-222
miR-328

[75]
Endothelial miR-155
barrier integrity Endothelial
modification Cells
[88]
, [117] Senescence Endothelial let-7g
 miR-216a
Cells
[88]
Recrument of miR-21
Monocytes
monocytes
[53], [54] [75]
, miR-22
miR-27a/b
Differentiation
miR-33
of monocytes to Monocytes
miR-34a
macrophages
miR-155

[54] [75]
, miR-155
Polarization of Macrophage
miR-33
macrophages s

[53], [125] miR-23a-5p

miR-27
miR-34
Foam cell Macrophage
miR-212
formation s
miR-590
miR-758-5p

[19] [88] [91]

, , Vascular
miR-1
smooth muscle
miR-21
cell Smooth
miR-143/145
proliferation Muscle Cells
and
differentiation
[88] [117], [118] [121]
, , Atherosclerotic Fibrous cap Smooth miR-21
plaque rupture thinning Muscle Cells miR-29b
miR-124-3p
miR-133b
miR-181b
 miR-362

Necrotic core Macrophage miR-155

formation s miR-378a

Additionally, miR-34 plays an important role in the differentiation of macrophages and the
generation of foam cells [125]. miR-155 possesses the capacity to simultaneously impact several
characteristics that influence endothelial dysfunction and macrophage differentiation. These
effects vary depending on the specific stage of macrophages within the atherosclerotic lesion
[126]. The miR-143/miR-145 cluster has a crucial role in maintaining and promoting the growth
of vascular smooth muscle cells (VSMCs). It not only promotes the transformation of
multipotent stem cells into VSMCs but also plays a significant role in regulating the adaptability
of VSMCs, which can have a significant impact on the blood vessel wall [127]. Moreover,
miRNAs can exert contrasting effects on various cell types, as demonstrated by the two closely
related miRNAs, miR-221 and miR-222, which can stimulate the proliferation of vascular
smooth muscle cells (VSMCs) while simultaneously inducing programmed cell death (apoptosis)
in endothelial cells (ECs) [128]. Additional investigation into the extensive function of these
miRNAs, along with the study of other developing miRNAs, will offer a valuable understanding
of how to optimally utilize their characteristics for therapeutic purposes.

8. Conclusion and Future Prospective

The current review highlights the crucial role of miRNAs in controlling the various stages and
phenotypes of atherosclerosis. miRNAs played an important role as modulators in atherosclerosis
in different stages from early endothelial dysfunction to late plaque stability. The comparative
analysis showed the similarities and divergence in the regulation of miRNAs in different stages
and phenotypes. Moreover, it also reveals the miRNA regulation in response to genetic and
environmental factors.

The field of microRNAs (miRNAs) in atherosclerosis is expanding rapidly, and there are a lot of
promising new directions that could lead to better knowledge and applications of miRNA-
mediated regulatory mechanisms. Single-cell analysis and CRISPR-based methods should be
used for further miRNA detection and functional studies. These methods help researchers to
miRNA function, cell-specific role, and links with cellular proteins. For a better understating of
the crucial rule of miRNA in the regulatory network of atherosclerosis, integrated omics
approaches (genomics, proteomics, and metabolomics) must be utilized.

The individual variation in miRNA expression offers intriguing prospects for treating
atherosclerosis. The tailor-made approaches for the diagnosis and treatment of a specific patient’s
miRNA profile possess the potential to be more specific and efficient. To know the time-
dependent effect of miRNA treatments, as well as the biosafety of the treatment, comprehensive
longitudinal studies at the clinical level are necessary to bring miRNA research towards clinical
practices. However, more studies should be conducted to know the role of novel miRNA in
Atherosclerosis regulatory networks which may help find new therapeutic targets. To solve the
problems with the delivery of miRNA, extracellular vesicle-mediated miRNA transfer should
prove to be an effective method. Furthermore, Bioinformatics tools should be employed for a
better understanding of miRNA interaction with mRNA. Through this, it is possible to identify
new regulatory pathways that lead to atherosclerosis. It also uncovers novel therapeutic targets.

To be safe from atherosclerosis and improve lifestyle, environmental and lifestyle factors should
be investigated for their impact on miRNA expression. There is one way to open regulatory
components and disease-specific markers to compare miRNA profiles among cardiovascular
diseases. Most of the cardiovascular complications might have similar aetiologies and
therapeutic options by comparing them. This approach proved to be very effective in improving
the accuracy of diagnosis and monitoring of possible miRNA biomarkers, which opens ways to
use less intrusive diagnostic techniques utilized in clinical setups on a routine basis.

miRNA research is required to be accelerated via collaboration and data sharing across the globe.
To improve the accuracy and reliability of investigations, Trials of diverse patient groups should
be conducted at large scales. Ethical questions have been raised over the clinical trials of
miRNA-based therapies. To make sure the miRNA research follows the standards and values, the
ethical guidelines for the utilization of genetic information must be followed. This will improve
the public faith in groundbreaking therapies to tackle cardiovascular complications. Overall, the
integration of multiple approaches in miRNA research for atherosclerosis improves the quality of
diagnosis and treatment options.