- Email: [email protected]
The salient role of microRNAs in atherogenesis
Accepted Manuscript The salient role of microRNAS in atherogenesis
Callum J. Donaldson, Ka Hou Lao, Lingfang Zeng PII: DOI: Reference:
S0022-2828(18)30766-1 doi:10.1016/j.yjmcc.2018.08.004 YJMCC 8782
To appear in:
Journal of Molecular and Cellular Cardiology
Received date: Revised date: Accepted date:
28 January 2018 5 August 2018 6 August 2018
Please cite this article as: Callum J. Donaldson, Ka Hou Lao, Lingfang Zeng , The salient role of microRNAS in atherogenesis. Yjmcc (2018), doi:10.1016/j.yjmcc.2018.08.004
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ACCEPTED MANUSCRIPT The Salient Role of MicroRNAs in Atherogenesis
Callum J. Donaldson 1*, Ka Hou Lao2*, Lingfang Zeng1,2
Department of Pharmacology and Therapeutics, 2Cardiovascular Division,
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1
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Faculty of Life Sciences and Medicine, King’s College London, London, SE5
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9NU UK
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* CJ Donaldson and KH Lao contributed equally to this work
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The correspondence should be addressed to: Dr Ka Hou Lao PhD,
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Cardiovascular Division, Faculty of Life Sciences and Medicine, King’s College London, 125 Coldharbour Lane, London SE5 9NU, United Kingdom. Tel: 44-2078485384, Fax: 44-2078485296. E-mail: [email protected]
ACCEPTED MANUSCRIPT
Abstract
Atherosclerosis, a chronic inflammatory condition that is characterized by the
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accumulation of lipid-loaded macrophages, occurs preferentially at the arterial
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branching points where disturbed flow is prominent. The pathogenesis of
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atherosclerotic lesion formation is a multistage process involving multiple cell types, inflammatory mediators and hemodynamic forces in the vessel wall in
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response to atherogenic stimuli. Researches from the past decade have
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uncovered the critical roles of microRNAs (miRNAs) in regulating multiple pathophysiological effects and signaling pathways in endothelial cells (ECs),
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vascular smooth muscle cells (VSMCs), macrophages and lipid homeostasis,
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which are key in atherosclerotic lesion formation. The expression of these
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miRNAs are either in response to biomechanical (flow-responsive) or
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biochemical (non-flow-responsive) stimuli. Recent evidences also indicate an important role for long non-coding RNAs (lncRNAs) in mediating several atherosclerotic processes. In this review, we provide a detailed summary on the current paradigms in miRNA-dependent regulation, the emerging role of lncRNAs in the initiation and progression of atherosclerosis, and clinical interventions targeting these in an attempt to develop novel diagnostics and treatments for atherosclerosis.
ACCEPTED MANUSCRIPT Keywords: MicroRNA; Long non-coding RNAs; Atherosclerosis; Shear stress;
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Inflammation; Lipoproteins
ACCEPTED MANUSCRIPT 1. Introduction Atherosclerosis is a serious condition that manifests a number of ischemic diseases including ischemic heart disease, stroke and peripheral arterial disease, and is the leading cause of mortality and morbidity in the developed world. The
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pathogenesis of atherosclerotic lesion formation is a multistage process
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involving multiple cell types in the vessel wall. Disturbed flow favours the
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subendothelial entry and modification of cholesterol-loaded low density lipoproteins (LDLs), which triggers a low-grade inflammatory response leading
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to the activation of vascular endothelial cells (ECs) and inflammatory cell
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infiltration. At the lesion site, the monocyte-derived macrophages internalize the retained and oxidised LDLs (ox-LDLs) to form macrophage-derived foam
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cells, while vascular smooth muscle cells (VSMCs) migrate, dedifferentiate and
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proliferate into the neointima to form VSMC-derived foam cells [1,2].
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microRNAs (miRNAs) are evolutionary conserved, small non-coding single-
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stranded RNAs that are produced by multistep processes of transcription, nuclear export and cytoplasmic cleavage [3], and work primarily as posttranscriptional repressors via targeting the 3’-untranslated region (3’UTR) of mRNA to provoke its degradation or its translational repression (Figure 1) [4,5]. Over 2000 miRNAs have hitherto been identified. With each miRNA targeting up to 100 to 200 different RNA strands, they cover an estimated 60% of human protein coding genes and are potentially involved in all physiological and
ACCEPTED MANUSCRIPT pathological processes [6]. Researches from the past decade implicate the importance of miRNAs in regulating multiple signaling pathways in ECs, VSMCs,
inflammatory cells
and
cholesterol homeostasis,
collectively
modulating various key processes of atherosclerotic development [7], while
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recent evidences also indicate an emerging role of long non-coding RNAs
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(lncRNAs) in atherosclerosis [8]. In this review, we provide a detailed summary
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on the recent advances in understanding the roles of miRNAs and lncRNAs, and possible clinical interventions targeting these, in the development of novel
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diagnostics and treatments for atherosclerosis.
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ACCEPTED MANUSCRIPT
Figure 1. The production and action of microRNAs. miRNA, microRNA; RNA, ribonucleic acid; Ran, Ras-related nuclear protein; GTP, guanosine triphosphate; TRBP, TAR RNA binding protein; PACT, Protein activator of
ACCEPTED MANUSCRIPT PKR; AGO, Argonaute; RISC, RNA-induced silencing complex; mRNA, messenger RNA.
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2. Roles of Endothelial miRNAs in Atherosclerosis
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Atherosclerotic plaques distribute discontinuously along the arterial tree,
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occurring preferentially at sites of arterial branch points, bifurcations and the lesser curvature of large and medium-sized arteries in mice and human subjects
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where flows are disturbed with low oscillatory shear stress (OSS) (Figure 2)
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[9,10]. ECs in these regions are prone to endothelial dysfunction that are hallmarked by increases in apoptosis, hyperpermeability (e.g. altered expression localization
of
intracellular
junctional
proteins
and
matrix
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and
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metalloproteinases (MMPs)) and inflammation (e.g. nuclear factor kappa b (NF-
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κB) activation and adhesion molecules upregulation), processes which are
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largely contributed by the lack of vasoprotective nitric oxide (NO) from impaired endothelial NO synthase (eNOS) expression and aberrant reactive oxygen species (ROS) production [11–13]. These collectively lead to disrupted monolayer integrity, inflammation and intimal extracellular matrix composition remodelling,
which
encourage oxLDL and
leukocytes
sub-endothelial
accumulation that characterizes the initial stage of atherosclerosis [1,2]. Chronic exposure to disturbed flow also promotes coordinated process of EC deaths and repair attempts involving proliferation and angiogenesis [9,14]. In contrary,
ACCEPTED MANUSCRIPT streamlined blood flow with high laminar shear stress (LSS) experienced at the straightened parts of arteries (Figure 2) confers anti-inflammatory, anti-adhesive and anti-thrombotic effects on the vessel wall via upregulation of flowresponsive master regulators Krüppel-like factor-2 and 4 (KLF2 and KLF4).
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Both factors are critical in maintaining vascular tone, EC permeability and
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metabolism, and inhibit NF-κB signaling through induction of eNOS and NO
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production [15–19].
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Over 200 miRNAs are expressed in human ECs, some of which have been found to be differentially regulated in response to different hemodynamic flow
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patterns (flow-responsive) and endothelial insults including inflammatory cytokines and hyperlipidaemia (non-flow-responsive) which target key
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signalling pathways critical in atherosclerotic development [7,20].
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ACCEPTED MANUSCRIPT
Figure 2. Flow patterns at atherosclerotic predilection sites. The flow is high shear and uniform in the obtuse angle of the aortic arch and in the descending aorta. The flow is disturbed in the acute angles of the aortic arch and at bifurcations, predisposing these areas to atherosclerotic plaque formation.
ACCEPTED MANUSCRIPT
2.1
Role of flow-responsive endothelial miRNAs
Shear stress plays a prominent role in regulating endothelial functions, where ECs sense mechanical forces through their surface integrins and transduce into
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signaling cascades via a network of cytoskeletal filaments and flow-responsive
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miRNAs to regulate gene expression [21,22]. These miRNAs have been largely
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identified and characterized in vitro to modulate gene expression mediating
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endothelial dysfunction and atherogenesis, using human ECs subjected to flows with uniform pattern or the more physiologically-relevant pulsatile flow (flow
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with periodic variation) in a high LSS or low OSS pattern. Many of these miRNAs were subsequently characterized and validated through either gain-of-
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function or loss-of-function approaches in vivo in genetic models of atherosclerosis and/or with partial carotid ligation to recreate disrupted flow at
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branched arterial sections (summarized in Table 1). Some of these flow-
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responsive endothelial miRNAs (e.g. miR-10, -19a, -21, -23b, 17-92a, -101, 126, -143/145, -155, -663, -712/205) have been previously discussed [20,23], therefore the following sections focus on the more recent findings on this miRNA class in atherogenesis.
2.1.1 Laminar flow-responsive anti-atherogenic endothelial miRNAs miR-10a
ACCEPTED MANUSCRIPT One of the first identified flow-responsive endothelial miRNAs, diminished expression of miR-10a was found in the atheroprone, lesser curvature region of the porcine aortic arch [24–26]. LSS-regulated KLF2 was recently identified as the master switch to regulate the binding of hormone receptor retinoic acid
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receptor-α (RARα) onto the miR-10a enhancer RA-responsive element (RARE)
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in ECs [26]. Therefore LSS increases, while OSS inhibits, endothelial miR-10a
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expression respectively through the effects of KLF2-mediated “binding enabler” retinoid X receptor-α or “binding inhibitor” HDAC3/5/7 on RARα-RARE
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interaction [26]. miR-10a exerts anti-inflammatory effects through inhibiting
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NF-κB signaling by either targeting both mitogen-activated protein (MAP) kinase kinase kinase 7 (MAP3K7) and β-transducin repeat-containing gene, or
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targeting GATA6/vascular cell adhesion protein-1 (VCAM-1) signalling
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leading to downregulation of adhesion molecules [24,26]. Its systemic anti-
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inflammatory role has been validated by ameliorating atherosclerotic lesions
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development in atheroprone ApoE−/− mice [26].
miR-30-5p family
All miR-30-5p family members (miR-30a, -30b, -30c, -30d and -30e) are upregulated in ECs by uniform flows, as examined in human umbilical vein ECs (HUVECs) exposed to prolonged LSS in vitro and in perfused mouse femoral arteries subjected to uniform or pulsatile laminar flows ex vivo [27]. Like miR-10a, KLF2 was also found to be the upstream regulator of LSS-
ACCEPTED MANUSCRIPT mediated miR-30-5p family. miR-30-5p exerts anti-inflammatory effects through targeting angiopoietin 2, thus downregulating adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), VCAM-1 and Eselectin, and thus impairing leukocyte adhesion to HUVECs [27]. Importantly,
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circulating miR30c-5p was recently identified to be inversely correlated with
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severity of human atherosclerotic plaques, and was found to restrain ox-LDL-
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induced proatherogenic events (IL-1β release, caspase-3 expression and apoptosis) in macrophages in vitro and is responsible for endothelial healing
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after carotid injury in vivo [28].
miR-181b
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miR-181b, upregulated by LSS and downregulated by hyperlipidaemia in ECs,
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is reduced in circulation in humans with coronary artery diseases (CAD)
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[25,29]. Mechanistically, it confers anti-inflammatory effects on arterial ECs at
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atheroprotective regions through targeting importin-α3, a protein that is critical in the nuclear translocation of NF-κB for activating downstream inflammatory cascades [29]. Systemic delivery of miR-181b mimics in ApoE-/- mice fed with high fat diet (HFD) reduces endothelial NF-κB activation in the atheroprone aortic arch, an effect that is sufficient to downregulate a range of proinflammatory genes and markedly suppress leukocyte recruitments in the vessel wall, thence ameliorating atherosclerosis development [29].
ACCEPTED MANUSCRIPT miR-143/145 The survival of ECs is paramount in maintaining endothelial integrity to protect against lesion formation. LSS-mediated miR-143/145 cluster maintains EC integrity and survival through targeting angiotensin-converting enzyme, thus angiotensin-2
that
is
largely
responsible
for
pathological
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reducing
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vasoconstriction and endothelial damages [30]. Notably, endothelial miR-145
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seems to inhibit monocyte recruitment to the arterial wall and lesion formation
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in HFD-fed ApoE-/- mice via targeting junctional adhesion molecule-A [31].
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2.1.2 Turbulent flow-responsive pro-atherogenic endothelial miRNAs miR-92a is upregulated by turbulent flows in human ECs in vitro [32,33], and
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found at sites prone to atherosclerosis in vivo [24,33,34] and in human
components
KLF2,
KLF4,
and
Phosphatidic
Acid
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anti-inflammatory
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atherosclerotic plaques [35]. Mechanistically, miR-92a targets flow-mediated,
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Phosphatase type 2B, aggravating ECs through reducing NO production and directing ECs towards a pro-inflammatory phenotype through upregulated NFκB-mediated gene profile, enhanced leukocyte adhesions and compromised monolayer integrity [32–34,36]. In vivo blockade of miR-92a expression in an experimental atherosclerosis model like HFD-fed Ldlr-/- mice (LDL receptor (LDLR)-deficiency) reduces endothelial inflammation, curtails atherosclerotic plaque development and promotes stable plaques through rescuing its target suppressor of cytokine signaling 5 [33,37]. On the other hand, miR-92a also
ACCEPTED MANUSCRIPT impairs endothelial repair through inhibiting EC proliferation and migration both in vitro and in vivo, an effect that has been associated with its targeting of the pro-proliferative integrin-α5 and sirtuin-1 (SIRT1) [38]. Therefore miR-92a might act in multiple fronts, inducing inflammatory activation together with
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impairing EC regeneration, collectively contribute to EC dysfunction and
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atherogenesis. Other OSS-induced miRNAs include miR-663 and miR-
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712/miR-205 worked by exacerbating EC hyperpermeability and inflammation [39,40], and treatment with anti-miR-712 derepresses metalloproteinase
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inhibitor 3 (TIMP3) expression and mitigates atherosclerosis progression in
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mice [40].
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2.1.3 Flow-responsive endothelial miRNAs with conflicting pleiotropic
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actions in atherosclerosis
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There are several miRNA candidates with discrepancies reported in response to
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different flow patterns and in their roles in atherosclerosis. This might due to the multivalent nature of miRNAs, where a single miRNA can be regulated similarly by different flow patterns, and can target a set of different genes and participate in different knots in the network depending on cell types, conditions or environment.
miR-21
ACCEPTED MANUSCRIPT miR-21 was reported to confer a pro-inflammatory EC phenotype through suppressing peroxisome-proliferator-activated receptor-α (PPARα), and its endothelial expression is elevated by disturbed flow produced by a parallel plate flow chamber [41], and in the inner curvature of porcine aortic arch [24], human
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atherosclerotic plaques [35,42] and porcine, murine and human vein graft
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neointimas where turbulent flows prevail [43]. However, its pro-atherogenic
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role is complicated by a study that links elevated endothelial miR-21 expression with LSS generated by a different shear system (cone-and-plate system),
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enhancing EC survival and NO production [44]. Importantly, miR-21 was found
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to exert anti-atherogenic effects through downregulating the dual specificity of MAP2K3 to enhance apoptosis and cholesterol efflux of macrophages during
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the early stage of atherosclerosis in HFD-fed LdlR-/- mice [45], while miR-21
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helps to stabilize plaque in advanced atherosclerosis through restraining
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macrophage activation and improving VSMC proliferation in the fibrous cap via
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targeting RE1-silencing transcription factor in plaque rupture model of HFD-fed ApoE-/- mice [46]. miR-21 was also shown to protect against arterial dysfunction and remodelling by inhibition of PTEN, with levels of miR-21 decreased in hypertension [47]. These indicate that miR-21 might play opposite roles in atherogenesis depending on cell-types and stages of atherosclerosis.
miR-126
ACCEPTED MANUSCRIPT miR-126 is among the most highly expressed endothelial miRNAs that has been shown to regulate both angiogenesis and inflammation in a flow-dependent manner [48–50]. Schober et al. have elegantly identified that the passenger strand miR-126-5p, and not the guide strand miR-126-3p, as the functional
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strand upregulated in LSS-exposed HUVECs in vitro [51]. Treatment with miR-
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126-5p mimics rescued EC proliferation in repairing damaged endothelium at
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the predilection sites by targeting Notch 1 inhibitor delta-like 1 homolog and limits atherosclerotic features in HFD-fed miR-126-/-ApoE-/- mice with partial
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carotid ligation [51]. Conversely, others have shown that OSS-induced
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endothelial secretion of miR-126-3p increased VSMCs turnover and reversed the atheroprotective effects of miR-126-deficiency in mice with complete
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carotid ligation-induced atherosclerosis [52,53]. This is in contrast with another
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study demonstrating the therapeutic effects of endothelial-derived apoptotic
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bodies of miR-126-3p in limiting atherosclerotic lesions in vivo [54]. In relation
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to human atherosclerosis, higher uptakes of both miR-126 strands by coronary plaques are observed in patients with more vulnerable plaques [55], while levels of miR-126-5p within human atherosclerotic plaque are correlated with increased luminal EC proliferation and decreased foam cell formation [51]. These demonstrate that both strands might play a protective role against plaque formation or being uptaken to stabilise vulnerable plaques. Overall, these conflicting findings indicate that further studies will be necessary to determine
ACCEPTED MANUSCRIPT the relative role of both miR-126 strands in response to differential biochemical and biomechanical stimuli and in different stages of atherosclerosis.
miR-155
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miR-155 is upregulated by LSS in HUVECs and in the intima of the
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atheroprotective thoracic aorta that is naturally exposed to atheroprotective
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unidirectional blood flow [56]. Mechanistically, miR-155 exerts differential functional effects on ECs, for instance, suppressing EC proliferation, migration
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and apoptosis through targeting myosin light chain kinase [56], and promoting
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angiogenesis by targeting E2F2 [57]. In contrary, miR-155 has been demonstrated to enhance monocyte adhesion to HAECs [58] and impairs
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endothelium relaxation in human arteries through targeting eNOS [59]. Studies
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for miR-155 in mouse models of atherosclerosis also suggest conflicting effects
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depending on the contexts. For instance, systemic delivery of miR-155 reduced
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atherosclerotic lesion formation in ApoE-/- mice [60], and hematapoietic deficiency of miR-155 increased atherosclerosis with reduced plaque stability in LdlR-/- mice [61]. Other studies, however, have demonstrated that both global and macrophage-specific miR-155-deficiency suppressed atherogenesis in ApoE-/- mice through reduced macrophage recruitment and inflammatory response, and increased cholesterol efflux [58,62,63].
ACCEPTED MANUSCRIPT 2.2
Role of non-flow-responsive endothelial miRNAs
Several EC-derived miRNAs are regulated by inflammatory insults such as cytokines and hyperlipidaemia rather than disturbed flow pattern to modulate atherosclerosis (Table 1) [64,65]. Ox-LDL particles modulate endothelial
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hyperlipidaemic stress through binding to scavenger lectin-like ox-LDL
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receptor 1 (LOX-1) [66], leading to exacerbation in endothelial permeability,
Ox-LDL-mediated
upregulation
of
LOX-1
is
obtained
through
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69].
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inflammation and apoptosis, while limiting EC regenerative proliferation [67–
downregulation of let-7a, let-7b and let-7g in ECs in vitro [70,71]. These three
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let-7 family members were found to be downregulated in hyperlipidaemic conditions and exert atheroprotective effects on ECs [70–72]. For instance,
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overexpression of let-7a and let-7b inhibited ox-LDL-induced EC apoptosis and repressed activation of NF-B and MAPK pathways in ox-LDL-treated
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HUVECs [70]. Moreover, let-7g inhibited endothelial ox-LDL uptake, and
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reduced inflammation and neointimal lesions in HFD-fed ApoE-/- mice through targeting three components in TGF-β signalling, and decreased senescence by indirectly upregulating SIRT1 [71,72]. Recently, endothelial miR-100 was also found to be repressed in response to pro-inflammatory cytokine tumor necrosis factor- (TNF-) in a NF-B-dependent manner, and inversely correlated with inflammatory cells and cholesterol contents in human atherosclerotic plaques [73]. miR-100 represses inflammation and atherosclerotic plaque formation
ACCEPTED MANUSCRIPT through directly targeting components of rapamycin complex 1-signalling (rapamycin and raptor), which stimulates EC autophagy, downregulated adhesion molecules (E-Selectin, ICAM-1 and VCAM-1) and attenuated NF- B signalling both in vitro in HUVECs and in vivo in HFD-fed LdlR-/- mice [73].
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miR-103, maintained by endothelial Dicer expression, is highly upregulated in
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ECs within atherosclerotic plaque, and has been found to supresses endothelial
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KLF4 signalling with an associated increase in NF-B mediated chemokines
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expression (CXCL-1, CX3CL1 and CCL2) [74]. In HFD-fed ApoE-/- mice, inhibiting the interaction between miR-103 and KLF4 led to a decrease in
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macrophage accumulation and atherosclerotic plaque formation, suggesting a
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pro-inflammatory and pro-atherogenic role of miR-103 by targeting KLF4 [74].
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Atherogenesis is exacerbated by disrupted EC integrity caused by dysregulated
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level of apoptosis, and several miRNAs participating in this process have been found elevated in atherosclerotic areas. One of these candidates, miR-181a,
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aggravates the rate of EC apoptosis by downregulating the pro-survival signalling B-Cell lymphoma 2 (BCL-2) [75]. miR-351, on the other hand, increased EC apoptosis by targeting the pro-survival signal transducer and activator of transcription 3 (STAT3) [76]. Not all endothelial atheroprotective miRNAs are downregulated in human atherosclerotic plaques however, and could be upregulated to prevent atherosclerosis in a negative feedback loop. For instance, miR-146a, found
ACCEPTED MANUSCRIPT elevated in human atherosclerotic plaques [42], are induced in ECs by proinflammatory cytokines TNF- and IL-1β, but restraining endothelial proinflammatory milieu by repressing NF-B and MAPK pathways both in vitro (in HUVECs) and in vivo (HFD-fed Ldlr-/- mice), while upregulating EC-
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protective eNOS through targeting RNA binding protein HuR [77,78].
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Similarly, both miR-31 and miR-17-3p can also be induced by TNF- in a
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feedback control of inflammation, and respectively targeting TNF--induced
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EC expression of E-selectin and ICAM-1 [79].
Table 1. Endothelial microRNAs involved in endothelial dysfunction and
Athe
RN
li &
ro-
l Systems
miRN genic
level
Effec
Effects on EC Re
Signaling
Dysfunctions
Pathways
and
f
Atherosclerosi
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A
Affected
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A
Experimenta Targets
D
Stimu
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mi-
MA
atherosclerosis
ts
s
chang es
Section 2.1.1: Laminar flow-responsive anti-atherogenic endothelial miRNAs mi
d-
Anti-
Porcine AA;
MAP3
↓ E-Sel,
Inhibits EC
[2
R-
flow↑
infla
In vitro
K7
VCAM-1 and
NF-κB
4]
ACCEPTED MANUSCRIPT m-
10a
βTRC
HAECs
mato
NF-κB
inflammatory
signaling
activation
ry LSS↑
Anti-
In vitro shear
R-
OSS↓
infla
by PP flow
Rα/RXRα &
m-
system on
↑miR10-a ->
mato
HAECs for 24
↓GATA6/VCA
ry
h; LSS (12
RI
SC
10a
GATA6 LSS↑KLF2/RA
M-1;
dyn/cm2) vs
prolif by PP flow
atherosclerosis in ApoE-/- mice
↓miR10-a -> ↑GATA6/VCA M-1
Cyclin
Cell cycle
Inhibits EC
[2
D1
arrest (G1/S)
proliferation
5]
E2F1
Cell cycle
Inhibits EC
[8
arrest (G1/S)
proliferation
0]
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R-
6]
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D TE
In vitro shear
EP
Anti-
inhibits
5/7/RARα &
dyn/cm2)
LSS↑
[2
OSS↑HDAC3/
OSS (0.5
mi
PreR-10a
PT
mi
19a
e-
system on
rativ
HUVECs for
e
12 h; LSS (12 dyn/cm2)
mi R-
LSS↑
Anti-
In vitro shear
prolif by PP flow
ACCEPTED MANUSCRIPT 23b
e-
system on
and ↑Rb hypo-
rativ
HUVECs for
phosphorylatio
e
24 h; LSS (12
n
dyn/cm2) Anti-
In vitro shear
R-
High
infla
by ibidi pump
30-
PSS↑
m-
system on
mato
HUVECs for
ry
72 h; LSS (20
↓E-sel, ICAM1 & VCAM-1
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leukocyte
[2 7]
adhesion to ECs
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dyn/cm2); ex
Inhibits
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5p
Ang2
PT
LSS↑
RI
mi
vivo shear by
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syringe pump
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on mouse
EP
femoral artery
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explant for 6 h; high PSS (76 dyn/cm2) vs low PSS (7.6 dyn/cm2)
mi R-
LSS↑
Anti-
In vitro shear
prolif by PP flow
mTOR
Cell cycle
Inhibits EC
[8
arrest (G1/S)
proliferation
1]
ACCEPTED MANUSCRIPT 101
e-
system on
rativ
HUVECs for
e
12 h; LSS (12 dyn/cm2) In vitro shear
ACE
R-
ather
by cone-plate
↑AMPKα2/
143/
o-
viscometer on
pp53/miR-
145
genic HUVECs for
D
In vitro shear
R-
infla
by ibidi pump
143/
mma tory
145
RI
miR-143/145 -
[3 0]
> impaired vasodilator
↓ACE in ECs
response in murine hindlimb
EP
JAM-A
system on
JAM-A-/-
[3
↓monocytes
1]
recruitment and
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LSS↑
TE
Anti-
mi
Low level of
143/145 ->
MA
dyn/cm2)
SC
72 h; LSS (12
LSS->
NU
LSS↑
PT
Anti-
mi
HAECs for 48
↓
h; LSS (5 or
atherosclerosis
20 dyn/cm2);
in
in vivo
ApoE−/− mice
ApoE−/− mice mi
LSS+
Anti-
In vitro shear
IRAK
↓NF-kB
miR-146a
[8
ACCEPTED MANUSCRIPT EC/sS
infla
by PP flow
(miR-
pathway
↓neointima
146
MC
m-
system on
146);
activation
formation in
a,
co-
mato
HUVECs for
IKK-γ
mouse CA
708, culture ry
6-24 h; LSS
(miR-
ligation model
451, ↑
(12 dyn/cm2);
708);
in vivo mouse IL-6R
flow↓
partial CA
RI
d-
(miR-
ligation model 451);
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CHUK
SC
98
2]
PT
R-
MA
(miR98)
LSS↑
Anti-
ApoE-/- mice
R-
HLD↓
infla
with HFD
mato
TE
b
EP
m-
AC C
181
D
mi
ry
IPOA3
↓E-sel, ICAM-
i.v. injection of
[2
1 & VCAM-1
miR-181b
5,2
& NF-kB
mimics
9]
signaling in
↓inflammation,
AA of ApoE-/-
leukocyte
mice; ↓IPOA3
recruitment &
& NF-kB-p65
atherosclerosis
translocation in in ApoE-/- mice lesional ECs in ApoE-/- mice +
+ HFD
ACCEPTED MANUSCRIPT HFD Section 2.1.2: Turbulent flow-responsive pro-atherogenic endothelial miRNAs ↓eNOS, NO
s.c. treatment
[4
by PP flow
production,
with pre-92a
1]
m-
system on
TM
↓eNOS-
mato
HUVECs for
ry
8 h; LSS (12
In vitro shear
R-
PSS↓
infla
92a
OSS↑
KLF2
PT
Pro-
dependent
RI
LSS↓
dyn/cm2),
NU
PSS (12±4
D
dyn/cm2) d-
Pro-
Ex vivo
R-
flow↑
infla
Porcine AA,
KLF2
↑E-sel,
miR-92a KD
[3
KLF4
VCAM-1 &
↓leukocyte
4]
MCP-1 ↓eNOS
adhesion to
EP
m-
TE
mi
in vitro
AC C
92a
MA
dyn/cm2) OSS (4±4
mato
vasodilatation
SC
mi
HAECs
HAECs
ry
Pro-
In vitro
ITGA5
pre-miR-92a
[3
R-
infla
HCAECs; in
SIRT1
↓EC
8]
92a
m-
vivo Tie2-
proliferation &
mato
Cre;miR-
migration;
mi
NA
ACCEPTED MANUSCRIPT ry &
92a(fl/fl)-
anti-miR-92a
anti-
mice
& ECconditional KO
-
of miR-92a
genic
↑re-
PT
angio
D
MA
NU
SC
RI
endothelization
Low
Pro-
In vitro shear
R-
LSS+
infla
by PP flow
92a
HLD↑
m-
flow↑
EP
system on
AC C
d-
TE
mi
SOCS5
& ↓leucocyte recruitment & neointima formation in mice wireinjured artery
↓NOS3, KLF2,
AntagomiR-
[3
KLF4;
92a ↓EC
3]
↑NF-κB
inflammation
signaling
& monocyte
mato
HUVECs for
ry
24 h; high
adhesion in
LSS (15
vitro;
dyn/cm2), low
↓atherosclerosi
LSS+HLD (4
s & ↑stable
dyn/cm2+ox-
plaque in LdlR-
ACCEPTED MANUSCRIPT /-
LDL);
mice+HFD
AA of LdlR-/mice+HFD mi
OSS↑
Pro-
In vitro shear
PPAP2
miR-92a ↓LSS- PPAP2B KD -
R-
d-
infla
by cone-plate
B
induced
92a
flow↑
m-
viscometer on
KLF2/PPAP2B
mato
HAECs for 24
-> ↑LPA
ry
or 72 h; LSS
PT
RI
SC
↓EC alignment & monolayer
&↑MCP-1,
integrity &
VCAM-1 &
↑leukocyte
SELE
adhesion
D
dyn/cm2); in
MA
dyn/cm2),
6]
inflammation:
signalling
NU
(12-40
OSS (5
> ↑EC
[3
TE
vivo mouse
EP
partial CA
mi R663
OSS↑
AC C
ligation model
Pro-
In vitro shear
infla
NA
EC
[3
by cone-plate
inflammation:
9]
m-
viscometer on
↑monocyte
mato
HUVECs for
adhesion &
ry
24 h; LSS (15
inflammatory
dyn/cm2),
markers in ECs
ACCEPTED MANUSCRIPT OSS (5 dyn/cm2) mi
d-
Pro-
In vivo mouse TIMP3
↑MMPs
EC
[4
R-
flow↑
infla
partial CA
activity
inflammation:
0]
712/ OSS↑
m-
ligation
mi
mato
model;
R-
ry
In vitro shear
PT
↑monocyte
SC
RI
adhesion,
viscometer on
MA
iMAECs &
NU
by cone-plate
205
↑TNFα release. anti-miR712 ↓EC inflammation &
h; LSS (15
atherosclerosis
D
HAECs for 24
dyn/cm2),
TE
in vivo
EP
OSS (5
AC C
dyn/cm2)
Section 2.1.3: Flow-responsive endothelial miRNAs with conflicting pleiotropic
↑Akt & eNOS
↑EC survival
[4
by cone-plate
phosphorylatio
&NO
4]
viscometer on
n& NO
bioavailability
HUVECs for
production
Anti-
In vitro shear
R-
apo-
21
totic
mi
LSS↑
actions in atherosclerosis PTEN
ACCEPTED MANUSCRIPT 24 h; LSS (15 dyn/cm2) LdlR-/-
MAP2
↓p38-CHOP &
miR-21-/- BM
[4
R-
infla
mice+HFD
K3
JNK signaling
↑macrophage
5]
21
m-
with BM
-> ↑ABCG1
arterial
mato
transplanted
degradation
ry
from WT or
RI SC
NA
PT
Anti-
mi
Inducible
R-
infla
plaque rupture
21
m-
EP
TE
Anti-
model of miR-
AC C
mi
NA
D
MA
NU
miR-21-/- mice
REST
infiltration, cytokine releases, & apoptosis-> ↑foam cell formation & atherosclerosis miR-21-/-ApoE-
[4
/-
6]
mice +HFD:
↓VSMC
mato
21-/-ApoE-/-
proliferation
ry
mice+HFD
and ↑plaque rupture, lesion size, foam cell formation and arterial
ACCEPTED MANUSCRIPT macrophage infiltration in an inducible plaque rupture
PT
model.
plaque rupture phenotype.
-response
inflammation:
1]
system on
element->
↑monocyte
HUVECs for
↑AP1
adhesion &
activation,
inflammatory
(12 dyn/cm2),
MCP-1,
markers in ECs
OSS (0.5
VCAM-1
infla
by PP flow
21
mmato
PPARα
TE
D
R-
6-24 h; LSS
AC C
ry
vulnerable
[4
In vitro shear
EP
OSS↑
rescues this
↓PPARα/PPAR ↑EC
Pro-
mi
MA
NU
SC
RI
miR-21 mimic
dyn/cm2) ↓STAT3,
miR-21-/-
[4
prolif murine and
PTEN &
↓mouse vein
3]
e-
BMPR2
graft
mi
Vein
Pro-
R-
graft↑
21
porcine,
human vein
NA
ACCEPTED MANUSCRIPT rativ
graft models
neointimal
e Pro-
In vitro shear
FOXJ2
R-
differ by PP flow
34a
e-
system on
ntiati
eEPCs for 6-
on
24 h; LSS (15
↑EC markers,
↑EC
[8
↓SMC markers
differentiation
3]
PT
of eEPCs
RI
LSS↑
dyn/cm2)
↑NF-kB
EC
[8
signaling,
inflammation:
4]
↑ICAM-1 &
↑monocyte
VCAM-1
adhesion and
LSS↓
Pro-
In vitro shear
R-
OSS↑
infla
by PP flow
m-
system or
mato
ibidi pump
ry
system on
inflammatory
HUVECs for
markers
EP
TE
D
MA
NU
mi
34a
SIRT1
SC
mi
formation
AC C
24 h; LSS (15 dyn/cm2), OSS (5 dyn/cm2) ↑EC repair &
i.v. injection of
[5
angio partial CA
cell cycle
miR-126-5p
1]
-
progression
mimics rescues
mi
d-
Pro-
R-
flow↓
126
LSS↑
In vivo mouse Dlk1
ligation
ACCEPTED MANUSCRIPT genic model; In
(G2/M)
EC
;
vitro shear by
proliferation &
Anti-
ibidi pump
↓atherosclerosi
ather
system on
s in miR126-/-
o-
HUVECs for
ApoE-/-
PT
-5p
mice+HFD
RI
genic 72 h; LSS (10
Anti-
R-
OSS
prolif by ibidi pump
155
↔
e-
system on
rativ
HUVECs for
e;
0.5-4 h; LSS
anti-
(15 dyn/cm2),
migr
OSS (5
MYLK
↑RhoA activity
↓EC
[5
cytoskeleton
6]
rearrangement -> ↓migration,
D
proliferation &
EP
TE
apoptosis
dyn/cm2); ex
AC C
a-
In vitro shear
NU
LSS↑
MA
mi
SC
dyn/cm2)
tory
vivo mouse AA
Pro-
In vivo mouse BCL6
↑NF-kB
↑atherosclerosi
[6
R-
infla
partial CA
signaling
s
2]
155
m-
ligation model
mi
NA
mato
in vivo
ACCEPTED MANUSCRIPT ry HLD↑
In vitro
R-
infla
155
NA
miR-151
[5
HAECs
mimics
8]
m-
exposed to
↑monocyte
mato
HLD
adhesion to
PT
Pro-
mi
HAECs;
MA
NU
SC
RI
ry
miR-151-/↓atherosclerosi s in ApoE-/mice
Section 2.2: Non-flow-responsive endothelial miRNAs in atherosclerosis In vitro
infla
HUVECs
Let-
m-
exposed to
7b
mato
TE
LDL↓
EP
7a,
ry
D
Anti-
ox-LDL
AC C
Let- ox-
LOX-1
↓NF-κB
Overexpression
[7
signaling and
of Let-7a and -
0]
p38MAPK
7b inhibits oxLDL-induced EC apoptosis, NO deficiency, ROS overproduction and inflammation
ACCEPTED MANUSCRIPT in HUVECs ↓TGF-β
Let-7g mimics
[7
infla
HUVECs
THBS1
signalling
↓HUVEC
1,7
m-
exposed to
TGFBR (pSmad2/PAI-
TGF-β-
2]
mato
ox-LDL; In
1
1)
dependent
ry
vivo ApoE-/-
SMAD
↑SIRT1
mice+HFD
2
RI SC NU
MA D
PT
LOX-1
TE
LDL↓
In vitro
EP
7g
Anti-
AC C
Let- ox-
cytokines secretions (VCAM-1, MCP-1, IL-6) & SIRT1dependent senescence in vitro. Let-7g lentiviral overexpression ↓macrophage infiltration, TGF-β signalling & lesion size in
ACCEPTED MANUSCRIPT ApoE-/mice+HFD TNF
Anti-
In vitro
Rapam
↓NF-κB
miR-100
[7
R-
↓
infla
HUVECs
ycin
signaling &
mimics ↓
3]
m-
treated with
Raptor
↓E-Sel, ICAM-
macrophages
mato
TNF; In
ry
vivo LdlR-/-
RI
1, VCAM-1
SC
100
PT
mi
↑EC autophagy in HUVECs; miR-100 mimcs ↓plaque size in LdlR-/mice+HFD
mi
TNF
Anti-
In vitro
E-SEL
miR-31or miR-
[7
R-
↑
infla
EP
TE
D
MA
NU
mice+HFD
infiltration &
(miR-
17-3p mimics
9]
AC C
HUVECs
31, mi
m-
treated with
31);
↓neutrophil
mato
TNF
ICAM-
adhesion to
1 (miR-
TNF-treated
17-3p)
HUVECs
HuR on ↓NF-κB
miR-146a
ry
R173p mi
IL-1β
Anti-
In vitro
[7
ACCEPTED MANUSCRIPT HUVECs
ECs;
signaling and
mimics ↓NF-
7,7
146
m-
treated with
SORT1
MAPK
κB and
8]
a
mato
TNF or IL-
in liver
↓ IL-1β -
monocyte
ry
1β; In vivo
induced E-Sel,
adhesion to IL-
LdlR-/-
ICAM-1,
1β -treated
mice+HFD &
VCAM-1,
MiR-146a-/-
MCP-1
RI
SC
LdlR-/-
EP
TE
D
MA
NU
mice+HFD
AC C
↑
PT
infla
R-
HUVECs in vitro; MiR146a-/-LdlR-/mice + WT BM:↑EC activation & plaque burden MiR-146a-/LdlR-/- mice + MiR-146a-/LdlR-/- mice BM: impaired hematopoietic cells & ↓circulating LDL
ACCEPTED MANUSCRIPT ↑NF-κB
miR-103
[7
HAECs;
signalling;
mimics
4]
m-
In vivo ApoE-
↑CXCL1, CX3
↑monocyte
mato
/-
CL1 & CCL2
adhesion to
mi
Dicer↓ Pro-
In vitro
R-
Ox-
infla
103
LDL↑
KLF4
mice+HFD
HAECs; i.v.
PT
ry
mi
H2O2
Pro-
In vitro
R-
↑
apop
EP
TE
D
MA
NU
SC
RI
injection of
BCL-2
HUVECs
AC C
a
blockers in ApoE-/mice+HFD ↓lesion size & lesional macrophages
-totic
181
miR-103/KLF4
Anti-miR-181a
[7
↓H2O2-
5]
induced apoptosis in HUVECs
Pro-
In vitro
R-
apop
mouse
351
-totic primary
mi
HLD↑
STAT3
miR-351
[7
mimics ↑
6]
H2O2-induced
ACCEPTED MANUSCRIPT CAECs from
apoptosis in
ApoE-/- mice
mouse CAECs
Table 1. Endothelial microRNAs involved in endothelial dysfunction and
PT
atherosclerosis. Abbreviations: ->, leads to; ↑, upregulation; ↓, downregulation;
RI
↔, no change; AA, aortic arch; ACE, angiotensin-converting enzyme;
SC
AMPK2, 5'-AMP-activated protein kinase catalytic subunit 2; Ang2, angiopoieting-2; ApoE, apolipoprotein E; TRC, -transducin repeat-containing
NU
gene; BCL, b-cell lymphoma; BMPR2, bone morphogenetic protein receptor 2;
MA
CA, carotid artery; CAECs, coronary artery endothelial cells; CCL2, chemokine (C–C motif) ligand 2; CHUK, conserved helix-loop-helix ubiquitous kinase; d-
D
flow, disturbed flow; CXCL1, chemokine C–X–C motif chemokine 1;
TE
CX3CX1, chemokine (C–X3–C) ligand 1; Dlk1, delta-like 1 homolog; E-sel, E-
EP
selectin; EC, endothelial cell; eEPCs, early endothelial progenitor cells; FOXJ2,
AC C
forkhead box J2; GATA6, GATA-binding factor 6; H2O2, hydrogen peroxide; HAECs, human aortic ECs; HBVPs, human brain vascular pericytes; HCAEC, human coronary artery ECs; HDAC, histone deacetylase; HFD, high fat diet; HLD, hyperlipidemia; HuR, ELAV-like RNA binding protein 1; HUVECs, human umbilical cord ECs; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1 beta; IL-6R, IL-6 receptor; IKK-, inhibitor of nuclear factor kappa-B kinase subunit ; iMAECs, immortalized mouse aortic ECs; i.p.,
ACCEPTED MANUSCRIPT intraperitoneal; IPOA3, importin-a3; IRAK, interleukin-1 receptor-associated kinase 1; ITGA5, integrin subunit alpha 5; JAM-A, junctional adhesion molecule A; JNK, c-jun N-terminal kinase; KD, knockdown; KLF, Kruppel like factor; KO, knockout; LdlR, low-density lipoprotein receptor; LOX-1, lectin-
PT
like oxidized LDL receptor-1; LSS, laminar shear stress; MAP2K3, mitogen-
RI
activated protein kinase kinase 3; MAP3K7, MAP kinase kinase kinase 7;
SC
MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; miR, microRNA; mTOR, rapamycin; MYLK, myosin light chain
NU
kinase; NA, not assessed; NF-B, nuclear factor B; NO, nitric oxide; NOS3,
MA
nitric oxide synthase 3; OSS, oscillatory shear stress; ox-LDL, oxidized lowdensity lipoprotein; p38-CHOP, p38 MAP kinase-C/EBP homologous protein;
D
PP, parallel plate; PPAP2B, phosphatidic acid phosphatase type 2B; PPAR,
TE
Peroxisome proliferator-activated receptor ; PSS, pulsatile shear stress; PTEN,
EP
phosphatase and tensin homolog; RAR, retinoic acid receptor ; s.c.,
AC C
subcutaneous; REST, RE-1-silencing transcription factor; ROS, reactive oxygen species; SELE, selectin E; SEMA, semaphorins; SIRT1, sirtuin1; SMAD2, mothers against decapentaplegic homolog 2; SMC, smooth muscle cell; sSMC, synthetic SMC; SOCS5, suppressor of cytokine signaling 5; SORT1, sortilin-1; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor beta; TGFBR1, TGF-β receptor 1; THBS1, thrombospondin 1;
ACCEPTED MANUSCRIPT TM, thrombomodulin; TNF, tumour necrosis factor ; VCAM-1, vascular cell adhesion molecule-1; WT, wild type.
miRNA-mediated
decreased
in
VSMC
apoptosis
and
phenotype,
including
phenotypic
switching
aberrant to
a
SC
proliferation,
changes
PT
MiRNAs Regulation of Vascular Smooth Muscle Cell Functions
RI
3.
dedifferentiated migratory synthetic state, also contribute significantly to the
NU
initial stages of atherosclerosis [85]. miR-136 is upregulated in atherosclerotic
MA
plaques and targets serine/threonine-protein phosphatase 2 regulatory subunit Balpha (PPP2R2A), leading to reduced inhibition of the extracellular signal-
D
regulated kinase-1/2 (ERK1/2) signaling and subsequently increased VSMC
TE
proliferation [86]. miR-221, miR-26a and miR-146a are other pro-proliferative
EP
miRNAs mediating platelet-derived growth factor (PDGF)-induced VSMC
AC C
proliferation [87–89]. miR-221 mediates this process through inhibiting the anti-proliferation pathway p38/p21/p27 [87], whilst miR-26a does so by decreasing TGF- signaling [88]. miR-146a promotes VSMC proliferation through targeting KLF4 in a negative feedback loop [89]. In addition, miR-26a inhibits VSMC apoptosis and both miR-26a and miR-221 promote VSMC dedifferentiation [87,88]. miR-221’s effect on VSMC dedifferentiation is unrelated to p27 downregulation and instead occurs due to inhibition of c-Kit [87].
ACCEPTED MANUSCRIPT Other miRNAs exert atheroprotective effects on VSMCs. For instance, let-7 family, in addition to their atheroprotective effect on ECs, also ameliorates VSMC inflammatory responses including proliferation, migration, monocyte adhesion and NF-B activation [90]. miR-22-3p and miR-125b too are
PT
downregulated in human atherosclerosis and inhibit proliferation and migration
RI
of VSMCs through targeting high motility group box 1 (HMGB1) and
SC
podocalyxin like protein (PODXL) respectively [91,92]. miR-24, miR-29a, miR-663 and miR-638 have all been shown to reduce VSMC proliferation and
NU
migration, as well as to promote transition to VSMC contractile phenotype via
MA
inhibition of PDGF-BB signaling [93–95]. miR-133 and miR-424 exert similar effects by repressing expression of Sp-1 and cyclin D1, calumenin and stromal
D
interaction molecule 1 (STIM1) respectively [96,97]. miR-22 represses VSMC
TE
dedifferentiation through inhibiting proliferation and migration and enhances
EP
contractile VSMC gene expression via targeting methyl-CpG binding protein 2
AC C
(MECP2), ecotropic virus integration site 1 protein homolog and HDAC4 [98]. miR-22 mimics delivery in wire-injured murine femoral arteries inhibited neointima formation [98].
miR-195 also reduces VSMC proliferation and
migration but additionally decreases VSMC synthesis of the pro-inflammatory cytokines IL-1, IL-6 and IL-8, possibly through targeting cell division cycle 42 (Cdc42), cyclin D1 and fibroblast growth factor 1 [99].
ACCEPTED MANUSCRIPT Finally, maintaining VSMC survival in the fibrous cap in the advanced stage of atherosclerosis is crucial in preventing plaque rupture. miR-181b, in addition to its anti-inflammatory effect on ECs, was recently found to maintain plaque stability in ApoE-/- mice and LdlR-/- mice through downregulation of
PT
macrophage TIMP3 and VSMC elastin synthesis [100]. miR-210, on the other
RI
hand, maintained VSMC survival and phenotypic modulation through targeting
SC
adenomatous polyposis coli (APC)-Wnt signaling, effectively improving fibrous cap stability and decreasing plaque rupture in vivo [101]. miR-21
NU
stabilizes plaque through promoting VSMC proliferation and repair in the
MA
fibrous cap via targeting RE-1-silencing transcription factor in a plaque rupture
MiRNA-mediated
Atherosclerosis
Intercellular
Communication
During
EP
4.
TE
D
atherosclerotic model in vivo [46].
AC C
Shear stress is also able to influence atherogenesis by promoting miRNA as extracellular messengers between vascular cells. This occurs either via incorporation into extracellular vesicles (EVs) including macrovesicles and exosomes, or by association with proteins such as HDL and Ago2 [85]. These interactions are necessary to stabilize miRNA molecules during the period of intercellular transport [102,103]. EVs are taken up by endocytosis or fusion
ACCEPTED MANUSCRIPT with the cell membrane, whilst protein-associated miRNAs are taken up by binding to a specific receptor (Figure 3) [104]. Co-culture of HUVECs with human umbilical artery VSMCs revealed that miR-126-3p is increasingly secreted by ECs exposed to OSS, which mechanism
PT
was recently found to be mediated by soluble N-ethylmaleimide-sensitive factor
RI
attachment protein receptors (SNAREs) activation [52,53]. Its uptake into
SC
VSMCs leads to increased VSMC turnover through inhibition of forkhead box
NU
O3 (FOXO3), BCL-2 and insulin receptor substrate 1 (IRS1) [52], while inhibition of miR-126-3p-mediated communication with rapamycin ameliorates
MA
neointimal formation in ApoE-/- mice [53]. In another study, when HUVECs were exposed to LSS, an increase in the secretion of miR-143/145-containing
TE
D
EVs, which is dependent on KLF2 and Rab7a/Rab27b and protects ApoE-/- mice
EP
from atherosclerosis development, was observed [105,106]. More recently, miRNA-mediated VSMC-to-EC communication has emerged as
AC C
a potential factor in vascular homeostasis. It appears that miR-143/145mediated communication is not unidirectional but reciprocal, with VSMC-EC communication occurring by protrusions of the cell membrane known as tunnelling nanotubes and resulting in EC vessel stabilization [107]. However, it is still unclear if such signaling occurs in the setting of atherosclerosis and whether shear stress patterns influence it.
ACCEPTED MANUSCRIPT Co-culture of dedifferentiated VSMCs and ECs could modulate levels of miR146a in ECs, both under static conditions and LSS, and miR-146a exerts antiinflammatory effect to repress neointima formation in rat and mouse injured carotid arteries by inhibiting NF-κB signalling [82]. Interestingly, vesicle-
PT
mediated miRNA transfer from VSMCs-to-ECs was ruled out as the mechanism
RI
of transfer [82], but other transfer mechanisms remain to be investigated. In
macrophages
[108].
miR-146a-enriched
SC
contrary, miR-146a was found elevated in the EVs of ox-LDL-treated EVs
released
by
atherogenic
NU
macrophages act to transfer miR-146a into recipient naive macrophages and, by
MA
targeting insulin-like growth factor 2s mRNA-binding protein 1 (IGF2BP1) and HuR, limit their migration, which may promote macrophage entrapment in the
D
vessel wall and accelerate atherogenesis [108]. Overall, although the number of
TE
miRNAs confirmed to mediate communication between vascular cells by
EP
extracellular secretion remains small (Table 2), the discovery of this paradigm
AC C
has ensured its importance in atherosclerosis and warrant further researches.
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Figure 3. Vascular cell miRNA-mediated communication. A summary of the
MA
methods of miRNA-mediated intracellular communication suggested by current
D
evidences. The direction of miRNA transfer shown for each method reflects
TE
recent observations, however, it is plausible that further research may prove bi-
EP
directionality. EC, endothelial cell; miRNA, microRNA; SMC, smooth muscle
AC C
cell.
Table 2. miRNAs involved in miRNA-mediated intercellular communication miRNA Transfer
Mechani sm
Experimental
Establis
Stimu
Effects on target
Model
hed
li and
cell
Directio
miRN
n of
A
Commu
level
Ref
ACCEPTED MANUSCRIPT ni-cation chang es Protein-
In vitro shear
126-3p
mediated applied to
EC ->
OSS↑
VSMC
transfer
HUASMC co-
SC NU
LSS (12
MA
dyn/cm2) vs OSS (0.5
TE
D
dyn/cm2)
VSMC
LSS↑
↓ELK1, ↓KLF4,
[10
↓CAMK2d, ↓
5,10
SSH2, ↓PHACTR4
prolonged LSS;
& ↓CFL1 in co-
in vitro co-
cultured HASMCs;
culture of KLF2
reduced
transduced
atherosclerotic
HUVECs with
lesion formation in
HASMCs;
ApoE-/- mice
exposed to
AC C
transfer
EC ->
EP
143/145 mediated HUVECs miRNA
53]
(pro-atherogenic)
flow system,
In vitro
& ↓IRS1 ->
RI
HUVEC-
Vesicle-
[52,
↑VSMC turnover
miRNA
culture by PP
miR-
↓FOXO3, ↓BCL-2
PT
miR-
6]
ACCEPTED MANUSCRIPT induction of
transfected with
KLF2
KLF2 transduced
transduced
ECs (anti-
mouse ECs into
atherogenic)
PT
ApoE-/-
Nanotub
143/145 e-
In vitro murine
VSMC -
EC-VSMC co-
> EC
NU
mediated culture under varying
transfer
conditions; ex-
[10
β↑,
> EC proliferation
7]
vessel
and their capacity
stress
to form vessel-like
↑
structures (effect in atherogenesis
D
vivo mouse
MA
miRNA
↓HKII & ↓ITGβ8 -
TGF-
SC
miR-
RI
mice+HFD
unknown)
TE
aortas treated
EP
with TGF-β; in
AC C
vivo mouse coronary artery stress atheroge
ox-
↓IGF2BP1 & ↓Hur
[10
mediated application of
nic Mϕ -
LDL↑
-> limited
8]
miRNA
EVs derived
> naive
macrophage
transfer
from ox-LDL-
Mϕ
migration ->
miR-
Vesicle-
146a
In vitro
ACCEPTED MANUSCRIPT treated
entrapment in
macrophages to
vessel wall (pro-
naive
atherogenic)
2.
miRNAs
Abbreviations:
in ->,
miRNA-mediated leads
to;
↑,
intercellular
upregulation;
↓,
SC
communication.
involved
RI
Table
PT
macrophages
downregulation; ApoE, apolipoprotein E; BCL, b-cell lymphoma; CAMK2d,
NU
calcium/calmodulin-dependent protein kinase type II delta chain; CFL1, Cofilin
MA
1; EC, endothelial cell; ELK1, E-twenty-six domain containing protein-1; EV, extracellular vesicle; FOXO3, forkhead box O3; HASMC, human aortic smooth
D
muscle cell; HFD, high-fat diet; HKII, mitochondrial hexokinase II; HuR,
TE
human antigen R; HUVEC, human umbilical vein endothelial cell; HUASMC,
EP
human umbilical cell smooth muscle cell; IGF2BP1, insulin-like growth factor
AC C
2 mRNA-binding protein 1; IRS1, insulin receptor substrate 1; ITGβ8, integrin β8; KLF, Kruppel like factor; LSS, laminar shear stress; Mϕ, macrophage; miR, microRNA; OSS, oscillatory shear stress; ox-LDL, oxidized low density lipoprotein; PHACTR4, phosphatase and actin regulator 4; PP, parallel plate; SSH2, slingshot protein phosphatase 2; TGF-β, transforming growth factor β; VSMC, vascular smooth muscle cell.
ACCEPTED MANUSCRIPT 5.
MiRNAs Regulation of Macrophage Functions
Macrophages exist in a polarized state, expressing either a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. The M1 phenotype is predominant in atherogenesis and is associated with the secretion of
PT
inflammatory cytokines [109]. Several miRNAs have been shown to play a role
RI
in regulating macrophage polarisation; miR-33 promotes the M1 phenotype by
SC
encouraging aerobic glycolysis over oxidative phosphorylation [110]. Inhibition of miR-33, in a murine LdlR-/- model fed with HFD, proved to be
NU
atheroprotective [110]. On the other hand, miR-93 [111], miR-124 [112], miR-
MA
223 [113] and let-7c [114] have been shown to tilt the balance of macrophage polarization in favour of the M2 phenotype, through respectively targeting
D
immunoresponsive gene 1 (IRG1), CCAAT/enhancer-binding protein-
TE
(C/EBP-), PBX/knotted 1 homeobox 1 (PKNOX1) and CCAAT/enhancer-
EP
binding protein (C/EBP-) [111,113–115]. Additionally, miR-125a-5p and
AC C
miR-146a exert atheroprotective effects on macrophages by inhibiting the activation and secretion of pro-inflammatory cytokines via suppressing NF- B [116,117]. Notably, expression of miR-146a in hematopoietic cells is crucial in restraining the chronic inflammatory response in vivo [78]. miR-10a is integral to Dicer’s anti-inflammatory effect and promotion of fatty acid oxidative metabolism by binding to ligand-dependent nuclear receptor co-repressor (Lcor), collectively limiting the formation of lipid-filled macrophage-derived
ACCEPTED MANUSCRIPT foam cells and atherosclerotic development in vivo [118]. Increased expression of miR-10a was associated with decreased atherosclerotic progression in humans [118]. Other miRNAs, including miR-147, also limit macrophage activation by
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inhibiting toll-like receptor (TLR) signalling [119]. TLR2, TLR3, and TLR4
RI
engagement induces miR-147 expression, and miR-147 in turn downregulates
SC
TLR-induced inflammatory pathways in a negative feedback loop, resulting in a
NU
decreased release of pro-inflammatory cytokines [119]. miR-124a and miR-150 are both induced by KLF2 and are likely to exert an atheroprotective effect by
MA
inhibiting macrophage secretion of inflammatory chemokines such as chemokine (C–C motif) ligand 2 (CCL2) [120]. CCL2 secretion from
TE
D
macrophages is also inhibited by miR-24 through repression of chitinase 3-like 1 [121]. Finally, apoptosis of macrophages contributes to the development of
EP
the necrotic core found in atherosclerotic plaque [109]. miR-142-5p promotes
AC C
macrophage apoptosis by negative regulation of TGF-2, and as such is likely to have a pro-atherogenic role [122].
6.
MiRNAs Regulation of Cholesterol Homeostasis
Lipoproteins are composed of apolipoproteins, phospholipids, triglycerides and cholesterol. They are responsible for the transport of cholesterol and
ACCEPTED MANUSCRIPT triglycerides in the blood stream, and are key to cholesterol homeostasis. LDL and very low-density lipoprotein (VLDL) carry cholesterol from its production site, the liver, to peripheral tissues where receptors regulate their uptake. In the reverse process, efflux transporters work with high-density lipoprotein (HDL) to
PT
scavenge cholesterol away from tissues back to the liver. An imbalance in these
RI
processes, which are known to be regulated by miRNAs [123], can lead to sub-
SC
endothelial accumulation of cholesterol and promotion of atherogenesis [124].
NU
Increased systemic levels of LDL through its dysregulated secretion from the liver are known to be atherogenic. miR-122 promotes hepatic cholesterol
MA
synthesis and release into the circulation [125] whilst miR-223 has an opposing effect, inhibiting hepatic cholesterol synthesis by repressing the sterol enzymes
TE
D
3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGS1) and sterol-C4methyl-oxidase-like gene (SC4MOL) [126]. Similarly, miR-30c reduces
EP
lipogenesis in the liver by targeting microsomal triglyceride transfer protein
AC C
(MTTP) [127]. Overexpression of miR-30c, in a murine model, was found to decrease plasma cholesterol levels and inhibit the formation of atherosclerotic plaques [127]. However, interpretation from findings regarding plasma cholesterol levels in mice require caution, as unlike humans, the predominant fraction of plasma cholesterol in mice is HDL. The increased uptake of lipoprotein-transported cholesterol, by aberrant regulation of receptors, also contributes to cholesterol accumulation in
ACCEPTED MANUSCRIPT peripheral tissues. miR-125a-5p decreases the uptake of cholesterol in macrophages exposed to ox-LDL via targeting oxysterol binding protein-related protein 9 (ORP9), and as such may protect against foam cell formation [116]. Likewise, miR-146a reduces cholesterol uptake in macrophages by inhibiting
PT
TLR4 [128]. The role of miR-146a appears to be context-dependent however, as
RI
miR-146a also acts paradoxically to increase VLDL secretion from the liver via
SC
its action on sortilin-1, as shown by decreased circulating level of VLDL/LDL and reduced atherosclerosis in LdlR-/-Mir146a-/- mice[129]. Its anti-atherogenic
NU
role in dampening EC inflammation [77,78,82] and atherogenic roles in
MA
exacerbating VSMC proliferation [89] and macrophages vascular entrapment [108] further illustrate the complexity of targeting miR-146a as an anti-
TE
D
atherosclerotic therapeutic.
Esterification and efflux of cholesterol from peripheral tissues helps to limit accumulation.
EP
cholesterol
miR-223
upregulates
the
cholesterol
efflux
AC C
transporter ATP binding cassette transporter A1 (ABCA1) in macrophages, by targeting Sp3, and thus is atheroprotective [126]. Conversely, miR-33 [130], miR-144 [131], miR-758 [132], miR-27a/b [133] and miR-9-5p [134] all promote atherogenesis by inhibiting the macrophage ABCA1 to reduce cholesterol efflux and worsen lesion formation. The high degree of regulation this protein is subjected to suggests it is a key determinant in macrophage cholesterol homeostasis. miR-27a/b is also able to inhibit cholesterol efflux by
ACCEPTED MANUSCRIPT up to 45%, via reducing apoA-1 expression, as well as cholesterol esterification, by inhibiting lipoprotein lipase and acetyl-CoA acetyltransferase 1 [133]. The final step required for reverse cholesterol transport is the hepatic uptake of HDL molecules. miR-185, miR-96 and miR-223 have been shown to repress
PT
scavenger receptor class B type 1 (SR-B1) and in turn to downregulate this
RI
process [135]. Subsequently, they contribute to high circulating cholesterol
SC
levels. miR-148a, identified as a key negative regulator of hepatic LDL
NU
clearance, acts by targeting hepatic LDLR and ABCA1, and inhibition of miR148a can substantially restore hepatic LDLR expression and reduces plasma
Emerging Role of lncRNAs in Atherosclerosis
TE
7.
D
MA
LDL/HDL balance in mice [136].
EP
lncRNAs are another class of non-coding RNA defined by a sequence length
AC C
>200 nucleotides [137]. Expression of lncRNA shows considerable spatial and temporal variation with expression occurring in a tissue-specific manner and in response to stimuli [138]. To date very few lncRNAs have been fully characterized, but it is clear that they play a key role in gene expression by a variety of mechanisms [139]. For a comprehensive review on the biosynthesis and functions of lncRNAs, please refer to the review by Quinn & Chang [140].
ACCEPTED MANUSCRIPT A subset of lncRNAs possess atheroprotective effects (Table 3). lincRNA-p21 stimulates apoptosis and supresses proliferation of VSMCs by activating p53 pathway through mouse double minute 2 homolog (MDM2) disinhibition [141]. Its levels are downregulated within murine atherosclerotic plaque and the
PT
coronary arteries of patients with CAD [141]. SENCR also acts on VSMCs,
RI
stabilizing the contractile phenotype and inhibiting migration [142]. RNCR3 is
SC
upregulated in murine and human aortic atherosclerotic lesions and its inhibition, leading to decreased proliferation and increased apoptosis of ECs
NU
and VSMCs, promotes atherosclerosis development [143]. Moreover, several
MA
lncRNAs, such as MALAT1 and MANTIS, are important in angiogenesis [144,145]. MALAT1 is crucial in maintaining EC proliferation during vessel
D
growth and is significantly downregulated in human atherosclerotic plaque
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[144,146]. MANTIS mediates angiogenic sprouting and EC alignment and its
EP
level is found elevated in macaques with atherosclerosis regression [145].
AC C
Finally, it has been noted that HOXC-AS1 is downregulated in atherosclerotic plaques and that overexpression of HOXC-AS1 reduces ox-LDL-induced cholesterol accumulation in macrophages by activating homeobox C6 (HOXC6) [147]. MeXis also acts to reduce peripheral cholesterol accumulation by acting as a mediator to increase liver X receptors (LXRs)-dependent expression of the cholesterol efflux transporter ABCA1 in macrophages [148]. Loss of MeXis was shown to impair ABCA1 expression in mouse bone marrow cells, and these MeXis-/- bone marrow cells accelerate the development of atherosclerosis in
ACCEPTED MANUSCRIPT HFD-fed LdlR-/- mice [148]. LeXis is another lncRNA integral to LXRs’ modulation of cholesterol homeostasis. It not only promotes cholesterol efflux but also inhibits cholesterol biosynthesis in the liver by binding the ribonucleoprotein RALY, a co-factor in the transcription of several
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cholesterogenic genes [149]. Overexpression of LeXis, using an adeno-
RI
associated virus-8 vector with a thyroxine-binding globulin promoter in a
SC
mouse model of familial hypercholesterolemia, resulted in lower total cholesterol and triglyceride levels and a significantly reduced burden of
NU
atherosclerotic disease [150].
MA
Another subset of lncRNAs appear pro-atherogenic (Table 3). Tie-1AS, by inhibition of Tie-1, can lead to disruption of EC junctions and increases
TE
D
propensity of plaque ruptures [151]. GAS5 has been shown to be significantly increased in human atherosclerotic plaque where it promotes macrophage and
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EC apoptosis, the latter occurring secondary to modification of macrophage
AC C
exosome composition [152]. ANRIL and MIAT are elevated lncRNAs that were recently identified in advanced human atherosclerotic plaques [146]. ANRIL is a proven risk factor for CAD, carotid atherosclerosis and peripheral arterial disease [153–155]. Its contribution to atherosclerosis remains unclear, although the circularization of ANRIL (cANRIL) is known to alter its function in a context-dependent manner. cANRIL exerts atheroprotective effect by inducing apoptosis and decreasing VSMC proliferation [156], while it also promotes
ACCEPTED MANUSCRIPT atherogenesis by exacerbating EC inflammation and apoptosis [157]. EC-driven neovessel invasion into the intima is a well-characterized feature in advanced plaques that contribute to plaque destabilization and rupture [85]. The elevated MIAT might play an atherogenic role in advanced plaque, as it is linked to
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pathological angiogenesis in diabetic retinopathy in vivo [158]. SMILR is
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elevated in unstable human atherosclerotic plaque and promotes VSMCs
SC
proliferation by pushing their phenotypic switching to a synthetic state [159]. LINC00305 has similar action but also facilitates lipocalcin-1 interacting
NU
membrane receptor (LIMR) and aryl-hydrocarbon receptor repressor (AHRR)
MA
cooperation in macrophages, leading to NF-B activation and inflammation [160]. ENST00113 promotes VSMC and EC proliferation as well as survival
D
and migration by activating the PI3K/Akt/mTOR signaling pathway [161].
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RP5-833A20.1 reduces cholesterol efflux from peripheral tissues by a RP5-
EP
833A20.1/miR-382-5p/nuclear factor IA (NFIA)-dependent signal transduction
AC C
pathway and so contributes to atherogenesis by attenuating reverse cholesterol transport [162]. The discovery of GAS5 incorporation into EC apoptosispromoting exosomes postulates that lncRNAs, like miRNAs, may be able to function as extracellular messengers [152]. Overall, evidence is emerging to suggest an important role for lncRNAs in addition to legions of miRNAs in mediating several cell types in every stages in the atherosclerosis development (Figure 4),
ACCEPTED MANUSCRIPT
Table 3. lncRNAs involved in endothelial dysfunction and atherosclerosis
li &
o-
A
lncRN
genic
A
Effect
level
s
Affected
Effects on EC
al Systems
Signaling
Dysfunctions
PT
RN
Experiment Targets
RI
Ather
Pathways
SC
Stimu
Ref
and
Atherosclerosis
NU
lnc-
MA
chang es
NA
Anti-
In vitro loss
TE
linc
D
Anti-atherogenic lncRNAs MDM2
↑p300
↓proliferation &
prolife and gain of
interaction ↑apoptosis in
A-
-
-> ↑p53
function in
AC C
p21
EP
RN
rative;
ApoE-/-
Pro-
murine
apop-
VSMCs &
totic
Mϕs; in vivo mouse CA ligation
activity
VSMCs and Mϕs
[14 1]
ACCEPTED MANUSCRIPT model ↑Myocard
↓VSMC
[14
in
in &
migration
2]
HCASMCs
contractile
SEN NA
Anti-
In vitro KD
CR
migratory
NA
PT
genes, ↓
RI
migratory
CR3 LDL↑
Anti-
In vivo KD
miR-
athero- in ApoE-/genic
185-5p
mice; in vitro KD in
3]
levels,
factor release, ↑EC & VSMC
VSMCs
proliferation
EP
TE
1
↓cholesterol
↓CCNA2,
↑EC proliferation,
[14
prolife in HUVECs;
↓CCNB1,
↓EC migration,
4]
-
mouse
↓CCNB2,
↑angiogenesis
rative;
retinal
↓CDK1
Anti-
angiogenesis
↑p21
migra-
model; hind
tory;
limb
hypoxi Pro-
LAT a↑
[14
and human
In vitro KD
AC C
MA
↓atherosclerosis,
↓inflammatory
D
HUVECs
↑KLF2
NU
Ox-
MA
RN
SC
genes
NA
ACCEPTED MANUSCRIPT Pro-
ischaemia
angio-
mouse
genic
model
Pro-
In vitro KD
NTI
angio-
S
genic
↑BRG1
↑EC alignment &
[14
in HUVECs;
ATPase
↑angiogenic
5]
in vitro
activity ->
patients with
efficient
PT
BRG1
IPAH, rats
RNA
MA
e II
D
Macaca
stress
polymeras
monocrotali ne &
response to shear
NU
treated with
sprouting in
RI
NA
SC
MA
machinery binding -> ↑SOX18,
subjected to
↑SMAD6
EP
TE
fasicularis
&
AC C
atherosclero sis
↑COUP-
regression
TFII
diet ↓Ox-LDL
[14
lentivirus-
induced
7]
mediated
cholesterol
HO
Ox-
Anti-
In vitro
XC-
LDL↓
cholesterol
AS1
NA
↑HOXC6
ACCEPTED MANUSCRIPT OE in
accumulation in
human THP-
THP-1 Mϕs
1 Mϕs Anti-
In vivo LXR NA
↑ABCA1
↑cholesterol
[14
is
chole-
activation in
expression efflux from
8]
sterol
WT, Lxr-/-
PT
MeX NA
RI
peripheral tissues
SC
and Lxr-/mice; In
NU
vitro KD in
Mϕs
D
peritoneal
MA
mouse
↓total cholesterol
[14
in a mouse
n of
and triglyceride
9,15
model of
RALY
levels &
FH; HFD-
with DNA
↓atherosclerotic
fed LeXis-/-
-> altered
burden in vivo
mice
transcripti
TE
↓interactio
Anti-
In vivo OE
s
cholesterol
AC C
EP
LeXi NA
RALY
on of cholestero l-genic
0]
ACCEPTED MANUSCRIPT genes ↑apoptosis &
[15
in PASMCs
ase-
↓proliferation in
6]
and Mϕs
mediated
VSMCs and Mϕs
In vitro OE
apoptotic;
PES1
Anti-
pre-rRNA
proli-
processing
ferativ
and
PT
RIL
↓exonucle
Pro-
RI
NA
SC
cAN
e
ribosome
NU
biogenesis
D
MA
->
↑nucleolar stress &
TE
↑p53
EP
activation
TIE- NA 1AS
AC C
Pro-atherogenic lncRNAs
Pro-
In vivo
Tie-1
plaque upregulation mRNA ruptur
in
e
embryonic zebrafish; in vitro
↓Tie-1
Defects in
[15
transcript
endothelial cell
1]
contact junctions
ACCEPTED MANUSCRIPT upregulation in HUVECs
5
LDL↑
↑Caspases
↑ Mϕ apoptosis,
[15
in human
in Mϕs,
↑EC apoptosis
2]
THP-1 Mϕs
altered
Pro-
In vitro OE
apoptotic
NA
PT
GAS Ox-
RI
Mϕ
SC
exosome
compositi
NU
on
AN RIL
Proapop-
In vivo OE
EP
ular
NA
and low
AC C
circ
TE
D
MA
including ↑GAS5 incorporat ion
NA
↑BAX,
↑EC apoptosis,
[15
↑caspase-
↑TC, ↑LDL, ↑TG,
7]
3, ↓bcl-2
↓HDL, ↑IL-1,
totic;
expression
Pro-
in Wistar
↑IL-6, ↑MMP,
chole-
rats injected
↑CRP
sterol;
with a high
Pro-
dose of
inflam
vitamin D3
ACCEPTED MANUSCRIPT -
and fed a
matory high-fat diet MIA NA
Pro-
In vivo KD
miR-
T
patho-
in male
150-5p
logical
Sprague-
angio-
Dawley rats
genesi
with
s
streptozotoc
↑VEGF
↑DM-induced
[15
retinal
8]
PT
microvascular
RI
dysfunction in
KD in
D
HUVECs
MA
DM; in vitro
SMI NA
Pro-
In vitro KD
LR
prolife in IL-1α and
NA
proliferation, migration and tube formation in vitro
↑HAS2
↑VSMC
[15
expression proliferation
9]
↑LIMR &
↑Monocyte
[16
inflammation ->
0]
EP
TE
NU
in-induced
SC
vivo, ↑EC
PDGF
AC C
-rative
stimulated HSVVSMC s
Pro-
In vitro OE
C00
inflam
in human
AHRR
305
-
THP-1 Mϕs
cooperatio HASMC
LIN
NA
NA
ACCEPTED MANUSCRIPT matory with
n ->
switching from
HASMC co-
↑AHRR
contractile to
culture
activation
proliferative
-> ↑NF-
phenotype
PT
B in
↑PI3K/Ak
↑VSMC and EC
[16
t/mTOR
proliferation,
1]
63ignallin
survival and
VSMCs and
g pathway
migration
HUVECs
activation
In vitro
T00
prolife down-
113
-
regulation in
rative; Pro-
D
TE
migra-
LDL↑,
833
Ac-
In vitro OE
AC C
-
Pro-
EP
tory Ox-
NA
NU
Pro-
MA
ENS NA
RP5
SC
RI
monocyte
NA
↑miR-
↑inflammation
[16 2]
inflam
in human
382-5p ->
and disturbance
-
THP-1 Mϕs;
↓NF1A
to normal
A20. LDL↑
matory In vivo
expression cholesterol
1
; Pro-
NF1A OE
->↑IL-1B,
homeostasis in
chole-
in ApoE-/-
↑IL-6,
Mos; NF1A
sterol
mice
↑TNF-α,
overexpression ->
ACCEPTED MANUSCRIPT ↑CRP
↑HDL, ↓LDL, ↓VLDL, decreased inflammatory
PT
cytokines and
RI
promoted
regression in ApoE-/- mice
MA
NU
SC
atherosclerotic
Table 3. lncRNAs involved in endothelial dysfunction and atherosclerosis.
D
Abbreviations: ->, leads to; ↑, upregulation; ↓, downregulation; ABCA1, ATP-
TE
binding cassette transporter; Ac-LDL, acetylated low-density lipoprotein;
EP
AHRR, aryl-hydrocarbon receptor repressor; Akt, protein kinase B; ApoE,
AC C
apolipoprotein E; BRG1, brahma related gene 1; CA, carotid artery; CAD, coronary artery disease; CCN, cyclin; CDK1, cyclin dependent kinase 1; COUP-TFII, chicken ovalbumin upstream promoter transcription factor 2; CRP, C-reactive protein; diabetes mellitus, DM; EC, endothelial cell; FH, familial hypercholesterolemia; HAS2, hyaluronan synthase 2; HASMC, human arterial smooth muscle cell; HCASMC, human coronary artery smooth muscle cells; HDL, high-density lipoprotein; HFD, high-fat diet; HOXC6, homeobox C6; HUVEC, human umbilical vein endothelial cell; HSVVSMC, human saphenous
ACCEPTED MANUSCRIPT vein vascular smooth muscle cell; IL, interleukin; IPAH, idiopathic pulmonary arterial hypertension; KD, knock down; KLF, Kruppel like factor; LDL, lowdensity lipoprotein; LIMR, lipocalcin-1 interacting membrane receptor; LXR, liver X receptor; Mϕ, macrophage; MDM2, mouse double minute 2; miR,
PT
microRNA; MMP, matrix metalloproteinase; mRNA, messenger RNA; mTOR,
RI
mechanistic target of rapamycin; NA, not assessed; NF1A, nuclear factor 1A;
SC
NF-B, nuclear factor B; OE, over expression; ox-LDL, oxidized low-density lipoprotein; PASMC, primary arterial smooth muscle cell; PDGF, platelet
NU
derived growth factor; PES1, pescadillo homologue 1; PI3K, phosphoinositide 3
MA
kinase; RNA, ribonucleic acid; SOX18, SRY related HMG box 18; TC, total cholesterol; TG, triglyceride; THP-1, Tamm-Horsfall protein 1; Tie-1, tyrosine
D
kinase with immunoglobulin-like and EGF-like domains 1; TNF, tumour
TE
necrosis factor ; VEGF, vascular endothelial growth factor; VLDL- very low-
AC C
EP
density lipoprotein; VSMC, vascular smooth muscle cell.
ACCEPTED MANUSCRIPT EC activation and inflammation miRs-10a*, -30-5p*, -143/145*, 146a*, 181b*, -100, Let-7a -7b -7g miRs-34a*, -92a*, -712/205*, -103, -155, Tie-1AS, circANRIL
Erythrocyte
LUMEN Angiogenesis miRs-126-5p*, MALAT1, MANTIS
miRs -21*
Monocyte
miR-92a*, MIAT
EC apoptosis miRs-21*, -155* miRs-181a, -351, GAS5, cANRIL
Macrophage Macrophage activation miRs-93, -124, -223, -125a, -5p, -146a, -10a, -147, -124a, -150, let-7c
Foam cell Synthetic VSMC VSMC proliferation and migration miRs-22-3p, -125b, -24, -29a, -663, -638, -133, -424, -22, -195, -let-7, lincRNA-p21, SENCR, RNCR3, circANRIL
miRs-126-3p* , -146a VSMC survival in fibrous cap miRs-181b, -210, -21
miRs -122
Blood vessel
INTIMA
Basal lamina
miRs -146a
NU
miRs-136, -221, -26a, -146a, SMILR, LINC00305, ENST00113
Cholesterol accumulation and foam cell formation miRs-10a, -223, -30c, -125a-5p, HOXC-AS1, LeXis, MeXis
Endothelium
MA
Figure 4. Non-coding RNAs (ncRNAs) implicated in the atherosclerotic
D
processes. miRNAs and lncRNAs that participate in various cell types and
TE
atherosclerotic processes are summarized. ncRNAs that are atheroprotective (in green frame), atherogenic (in red frame) or with contradictory evidence (in blue
EP
frame) are shown, while those that are flow-responsive are denoted by *. Disturbed flow leads to endothelial dysfunction and flavours intimal entry of
AC C
Vesicle
PT
Ox-LDL
Cholesterol efflux and reverse transport miR-223 miRs-9-5p, -27a/b, -33, -96, -144, 148a, -185, -223, -758, RP5-883A20.1
RI
miRNA-mediated intracellular communication miR-143/145*
HDL
SC
miRs-33, -142-5p, LINC00305
LDL
oxidized LDL (ox-LDL), which triggers a low-grade inflammatory response including expression of leukocyte adhesion molecules by endothelial cells. The initial stages of atherosclerosis include adhesion of blood monocyte to the activated endothelium, their maturation into macrophage and their activation and uptake of ox-LDL to form macrophage-derived foam cells. Cholesterol efflux mechanisms, aided by HDLs and other factors, help to regress lesion
MEDIA
ACCEPTED MANUSCRIPT development. Plaque progression involves the dedifferentiation, proliferation and migration of VSMCs into the intima where they deposit extracellular matrix protein including collagen. Advanced lesions rely on the survival and proliferation of fibrous cap VSMCs to stabilize the vulnerable plaques from
PT
rupture. LDL, low-density lipoprotein, HDL, high-density lipoprotein; VSMC,
Prospective Clinical Applications of miRNAs in Atherosclerosis
NU
8.
SC
RI
vascular smooth muscle cell.
MA
The changes that occur in miRNA expression, and the functional importance of these changes during atherogenesis, offer enormous clinical potential for
D
monitoring and modulation. miRNAs are measurable in the serum and offer the
TE
benefits of high stability, a wide dynamic range of expression and integration of
EP
biology from the entirety of the vascular system [163]. As such, they make
AC C
promising biomarkers that could facilitate non-invasive early diagnosis, prognosis, guidance of treatments based on risk stratification and follow-up of treatment response. An earlier study found miR-126, miR-17 and miR-92a to be significantly downregulated in the plasma of patients with CAD compared to healthy controls [164]. Others have identified reduced circulating level of 11 miRNAs (miR-19a, -484, -155, -222, -145, -29a, -378, -342, -181d, -150, -30e5p) in patients with angiographically-defined stable CAD and in control subjects with at least 2 cardiac risk factors, implicating that this set of miRNAs
ACCEPTED MANUSCRIPT might be associated with subclinical atherosclerosis [165]. Circulating level of miR-30c-5p were also elevated in subjects with plaques, and is inversely correlated with the LDL level, intimal thickening and plaques severity [28]. Furthermore, the feasibility of combining next generation imaging with
PT
transcoronary miRNA uptakes from blood taken during routine cardiac
RI
catheterization could predict the presence and the severity of vulnerable plaques
SC
in patients with CAD [55]. However, several challenges have hitherto impeded circulating miRNAs as clinical biomarkers for CAD. These include the reliance
NU
of miRNA measurements on PCR with a lack of valid reference miRNA
MA
controls currently available; the difficulty in determining the cell-type origins of many circulating miRNAs; and the effects of current CAD treatments (e.g.
D
platelet inhibitors and heparin) which may disturb accurate miRNA
TE
measurements [166]. To overcome these challenges, large-scale studies using
EP
carefully-stratified patient groups and technical improvements in miRNA
AC C
measurement are needed.
Due to their roles in pre-translational protein suppression, manipulation of miRNA expression could be leveraged for therapeutic benefits. So far two miRNA-based therapies have reached clinical trials: a miR-34 mimic (MRX34) for treatment of advanced liver cancer and a miR-122 antagonist (Miravirsen) for treatment of hepatitis C [167]. Systemic delivery of microRNA-derived agents are mostly taken up in the liver, kidney, and spleen [168], which implies
ACCEPTED MANUSCRIPT targeting the heart or vasculature with miRNA-based therapies might require significantly higher dosing with lower efficiency. Previous efforts focussed on improving antisense oligonucleotides (ASO) bioavailability by linking ASO to cholesterol for enhanced uptake (antagomiRs) or increase linkage within the
PT
RNA nucleotides to augment efficiency (LNA-anti-miRs) [169]. Recent
RI
development strategies have turned to enhance tissue uptakes by cell type-
SC
specific enrichment of miR-mimic/anti-miRs delivery in order to reduce the therapeutic dose and off-target adverse effects. Adenoviral vectors have been
NU
the methods of choice for functional miRNAs/anti-miRNAs delivery, but there
MA
are concerns with immunogenicity against viral vectors leading to toxicity and limited efficacy upon repeat administration [170]. Several technologies have
D
been developed to allay these concerns: targeted vasculature delivery strategies
TE
through local delivery by devices (drug eluting devices and catheter-based
EP
delivery) [171] or light-activated plasmonic nanocarrier [172], specific
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nanoparticle carriers (9 um poly-d, -lactide-co-glycolide) that preferentially target the vasculature [173], and EC-specific aptamer-based direct delivery [174,175]. Aptamers targeting EC-specific transferrin receptor was designed to deliver miR-126 precursors specifically to ECs, which were processed and repressed known miR-126 target VCAM-1 and improved EC-directed angiogenesis in vitro [174]. Notably, a E-Sel-targeting thioaptamer-multistage vector linked with miRNAs-enriched microparticles can specifically deliver EC protective miRNAs (miR146a and miR-181b) to ECs in vivo, successfully
ACCEPTED MANUSCRIPT inhibited endothelial expression of their target CCL2, and repressed inflammation and atherosclerosis in HFD-fed ApoE-/- mice [175]. Paradoxically, the diametric miRNA expression between vascular cells can be leveraged for target gene delivery. Adenoviral vector encoding the anti-proliferation gene
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p27Kip1, which are embedded with target sequences for miR-126-3p (a miRNA
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that is enriched in ECs but absent in VSMCs), can specifically target VSMC
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proliferation and neointimal hyperplasia while maintaining ECs proliferation and reendothelialization (due to adenovirus inactivation by endogenous miR-
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126-3p) in rat carotid artery injury [176,177]. In future, development of novel
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tools in targeting a defined miRNA-mRNA interactions using target-sitespecific rather than miRNA-specific ASOs, will be useful in modulating a
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specific miRNA function without affecting the entire regulatory network. Conversely, strategies in targeting lncRNAs are largely similar to those
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targeting mRNAs, i.e. inhibition by ASO-linked GapmeRs and overexpression
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by viral vectors or synthetic modified RNAs. Notably, GapmeRs targeting lncRNAs in cardiovascular system have been successfully applied in vivo: GapmeR targeting MALAT-1 to inhibit ischemia-induced angiogenesis [144] and GapmeR targeting Chast that affect cardiac remodelling [178]. In conclusion, these novel miRNA-based tests and ncRNA interventions are still in their nascent stages, but they certainly hold considerable promises for
ACCEPTED MANUSCRIPT translation into clinical tools that will aid the early diagnosis and treatment of
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atherosclerosis.
ACCEPTED MANUSCRIPT Funding This work was supported by British Heart Foundation project grant PG13-6330419.
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Disclosures
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None.
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ACCEPTED MANUSCRIPT Highlights Haemodynamic flow patterns influence atherogenesis by regulating endothelial miRNAs Many atherogenic processes are also influenced by non-flow-responsive
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miRNAs
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MiRNAs regulate multiple cell types in all stages of atherosclerotic
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development
Recent evidences suggest a similar role for lncRNAs in atherosclerosis
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MiRNAs offer potentials for prognosis, diagnosis and treatment of
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atherosclerosis
Callum J. Donaldson, Ka Hou Lao, Lingfang Zeng PII: DOI: Reference:
S0022-2828(18)30766-1 doi:10.1016/j.yjmcc.2018.08.004 YJMCC 8782
To appear in:
Journal of Molecular and Cellular Cardiology
Received date: Revised date: Accepted date:
28 January 2018 5 August 2018 6 August 2018
Please cite this article as: Callum J. Donaldson, Ka Hou Lao, Lingfang Zeng , The salient role of microRNAS in atherogenesis. Yjmcc (2018), doi:10.1016/j.yjmcc.2018.08.004
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ACCEPTED MANUSCRIPT The Salient Role of MicroRNAs in Atherogenesis
Callum J. Donaldson 1*, Ka Hou Lao2*, Lingfang Zeng1,2
Department of Pharmacology and Therapeutics, 2Cardiovascular Division,
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1
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Faculty of Life Sciences and Medicine, King’s College London, London, SE5
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9NU UK
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* CJ Donaldson and KH Lao contributed equally to this work
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The correspondence should be addressed to: Dr Ka Hou Lao PhD,
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Cardiovascular Division, Faculty of Life Sciences and Medicine, King’s College London, 125 Coldharbour Lane, London SE5 9NU, United Kingdom. Tel: 44-2078485384, Fax: 44-2078485296. E-mail: [email protected]
ACCEPTED MANUSCRIPT
Abstract
Atherosclerosis, a chronic inflammatory condition that is characterized by the
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accumulation of lipid-loaded macrophages, occurs preferentially at the arterial
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branching points where disturbed flow is prominent. The pathogenesis of
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atherosclerotic lesion formation is a multistage process involving multiple cell types, inflammatory mediators and hemodynamic forces in the vessel wall in
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response to atherogenic stimuli. Researches from the past decade have
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uncovered the critical roles of microRNAs (miRNAs) in regulating multiple pathophysiological effects and signaling pathways in endothelial cells (ECs),
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vascular smooth muscle cells (VSMCs), macrophages and lipid homeostasis,
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which are key in atherosclerotic lesion formation. The expression of these
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miRNAs are either in response to biomechanical (flow-responsive) or
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biochemical (non-flow-responsive) stimuli. Recent evidences also indicate an important role for long non-coding RNAs (lncRNAs) in mediating several atherosclerotic processes. In this review, we provide a detailed summary on the current paradigms in miRNA-dependent regulation, the emerging role of lncRNAs in the initiation and progression of atherosclerosis, and clinical interventions targeting these in an attempt to develop novel diagnostics and treatments for atherosclerosis.
ACCEPTED MANUSCRIPT Keywords: MicroRNA; Long non-coding RNAs; Atherosclerosis; Shear stress;
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Inflammation; Lipoproteins
ACCEPTED MANUSCRIPT 1. Introduction Atherosclerosis is a serious condition that manifests a number of ischemic diseases including ischemic heart disease, stroke and peripheral arterial disease, and is the leading cause of mortality and morbidity in the developed world. The
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pathogenesis of atherosclerotic lesion formation is a multistage process
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involving multiple cell types in the vessel wall. Disturbed flow favours the
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subendothelial entry and modification of cholesterol-loaded low density lipoproteins (LDLs), which triggers a low-grade inflammatory response leading
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to the activation of vascular endothelial cells (ECs) and inflammatory cell
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infiltration. At the lesion site, the monocyte-derived macrophages internalize the retained and oxidised LDLs (ox-LDLs) to form macrophage-derived foam
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cells, while vascular smooth muscle cells (VSMCs) migrate, dedifferentiate and
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proliferate into the neointima to form VSMC-derived foam cells [1,2].
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microRNAs (miRNAs) are evolutionary conserved, small non-coding single-
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stranded RNAs that are produced by multistep processes of transcription, nuclear export and cytoplasmic cleavage [3], and work primarily as posttranscriptional repressors via targeting the 3’-untranslated region (3’UTR) of mRNA to provoke its degradation or its translational repression (Figure 1) [4,5]. Over 2000 miRNAs have hitherto been identified. With each miRNA targeting up to 100 to 200 different RNA strands, they cover an estimated 60% of human protein coding genes and are potentially involved in all physiological and
ACCEPTED MANUSCRIPT pathological processes [6]. Researches from the past decade implicate the importance of miRNAs in regulating multiple signaling pathways in ECs, VSMCs,
inflammatory cells
and
cholesterol homeostasis,
collectively
modulating various key processes of atherosclerotic development [7], while
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recent evidences also indicate an emerging role of long non-coding RNAs
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(lncRNAs) in atherosclerosis [8]. In this review, we provide a detailed summary
SC
on the recent advances in understanding the roles of miRNAs and lncRNAs, and possible clinical interventions targeting these, in the development of novel
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diagnostics and treatments for atherosclerosis.
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EP
TE
D
MA
NU
SC
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ACCEPTED MANUSCRIPT
Figure 1. The production and action of microRNAs. miRNA, microRNA; RNA, ribonucleic acid; Ran, Ras-related nuclear protein; GTP, guanosine triphosphate; TRBP, TAR RNA binding protein; PACT, Protein activator of
ACCEPTED MANUSCRIPT PKR; AGO, Argonaute; RISC, RNA-induced silencing complex; mRNA, messenger RNA.
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2. Roles of Endothelial miRNAs in Atherosclerosis
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Atherosclerotic plaques distribute discontinuously along the arterial tree,
SC
occurring preferentially at sites of arterial branch points, bifurcations and the lesser curvature of large and medium-sized arteries in mice and human subjects
NU
where flows are disturbed with low oscillatory shear stress (OSS) (Figure 2)
MA
[9,10]. ECs in these regions are prone to endothelial dysfunction that are hallmarked by increases in apoptosis, hyperpermeability (e.g. altered expression localization
of
intracellular
junctional
proteins
and
matrix
D
and
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metalloproteinases (MMPs)) and inflammation (e.g. nuclear factor kappa b (NF-
EP
κB) activation and adhesion molecules upregulation), processes which are
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largely contributed by the lack of vasoprotective nitric oxide (NO) from impaired endothelial NO synthase (eNOS) expression and aberrant reactive oxygen species (ROS) production [11–13]. These collectively lead to disrupted monolayer integrity, inflammation and intimal extracellular matrix composition remodelling,
which
encourage oxLDL and
leukocytes
sub-endothelial
accumulation that characterizes the initial stage of atherosclerosis [1,2]. Chronic exposure to disturbed flow also promotes coordinated process of EC deaths and repair attempts involving proliferation and angiogenesis [9,14]. In contrary,
ACCEPTED MANUSCRIPT streamlined blood flow with high laminar shear stress (LSS) experienced at the straightened parts of arteries (Figure 2) confers anti-inflammatory, anti-adhesive and anti-thrombotic effects on the vessel wall via upregulation of flowresponsive master regulators Krüppel-like factor-2 and 4 (KLF2 and KLF4).
PT
Both factors are critical in maintaining vascular tone, EC permeability and
RI
metabolism, and inhibit NF-κB signaling through induction of eNOS and NO
SC
production [15–19].
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Over 200 miRNAs are expressed in human ECs, some of which have been found to be differentially regulated in response to different hemodynamic flow
MA
patterns (flow-responsive) and endothelial insults including inflammatory cytokines and hyperlipidaemia (non-flow-responsive) which target key
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EP
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signalling pathways critical in atherosclerotic development [7,20].
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EP
TE
D
MA
NU
SC
RI
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ACCEPTED MANUSCRIPT
Figure 2. Flow patterns at atherosclerotic predilection sites. The flow is high shear and uniform in the obtuse angle of the aortic arch and in the descending aorta. The flow is disturbed in the acute angles of the aortic arch and at bifurcations, predisposing these areas to atherosclerotic plaque formation.
ACCEPTED MANUSCRIPT
2.1
Role of flow-responsive endothelial miRNAs
Shear stress plays a prominent role in regulating endothelial functions, where ECs sense mechanical forces through their surface integrins and transduce into
PT
signaling cascades via a network of cytoskeletal filaments and flow-responsive
RI
miRNAs to regulate gene expression [21,22]. These miRNAs have been largely
SC
identified and characterized in vitro to modulate gene expression mediating
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endothelial dysfunction and atherogenesis, using human ECs subjected to flows with uniform pattern or the more physiologically-relevant pulsatile flow (flow
MA
with periodic variation) in a high LSS or low OSS pattern. Many of these miRNAs were subsequently characterized and validated through either gain-of-
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D
function or loss-of-function approaches in vivo in genetic models of atherosclerosis and/or with partial carotid ligation to recreate disrupted flow at
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branched arterial sections (summarized in Table 1). Some of these flow-
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responsive endothelial miRNAs (e.g. miR-10, -19a, -21, -23b, 17-92a, -101, 126, -143/145, -155, -663, -712/205) have been previously discussed [20,23], therefore the following sections focus on the more recent findings on this miRNA class in atherogenesis.
2.1.1 Laminar flow-responsive anti-atherogenic endothelial miRNAs miR-10a
ACCEPTED MANUSCRIPT One of the first identified flow-responsive endothelial miRNAs, diminished expression of miR-10a was found in the atheroprone, lesser curvature region of the porcine aortic arch [24–26]. LSS-regulated KLF2 was recently identified as the master switch to regulate the binding of hormone receptor retinoic acid
PT
receptor-α (RARα) onto the miR-10a enhancer RA-responsive element (RARE)
RI
in ECs [26]. Therefore LSS increases, while OSS inhibits, endothelial miR-10a
SC
expression respectively through the effects of KLF2-mediated “binding enabler” retinoid X receptor-α or “binding inhibitor” HDAC3/5/7 on RARα-RARE
NU
interaction [26]. miR-10a exerts anti-inflammatory effects through inhibiting
MA
NF-κB signaling by either targeting both mitogen-activated protein (MAP) kinase kinase kinase 7 (MAP3K7) and β-transducin repeat-containing gene, or
D
targeting GATA6/vascular cell adhesion protein-1 (VCAM-1) signalling
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leading to downregulation of adhesion molecules [24,26]. Its systemic anti-
EP
inflammatory role has been validated by ameliorating atherosclerotic lesions
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development in atheroprone ApoE−/− mice [26].
miR-30-5p family
All miR-30-5p family members (miR-30a, -30b, -30c, -30d and -30e) are upregulated in ECs by uniform flows, as examined in human umbilical vein ECs (HUVECs) exposed to prolonged LSS in vitro and in perfused mouse femoral arteries subjected to uniform or pulsatile laminar flows ex vivo [27]. Like miR-10a, KLF2 was also found to be the upstream regulator of LSS-
ACCEPTED MANUSCRIPT mediated miR-30-5p family. miR-30-5p exerts anti-inflammatory effects through targeting angiopoietin 2, thus downregulating adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), VCAM-1 and Eselectin, and thus impairing leukocyte adhesion to HUVECs [27]. Importantly,
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circulating miR30c-5p was recently identified to be inversely correlated with
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severity of human atherosclerotic plaques, and was found to restrain ox-LDL-
SC
induced proatherogenic events (IL-1β release, caspase-3 expression and apoptosis) in macrophages in vitro and is responsible for endothelial healing
MA
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after carotid injury in vivo [28].
miR-181b
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miR-181b, upregulated by LSS and downregulated by hyperlipidaemia in ECs,
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is reduced in circulation in humans with coronary artery diseases (CAD)
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[25,29]. Mechanistically, it confers anti-inflammatory effects on arterial ECs at
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atheroprotective regions through targeting importin-α3, a protein that is critical in the nuclear translocation of NF-κB for activating downstream inflammatory cascades [29]. Systemic delivery of miR-181b mimics in ApoE-/- mice fed with high fat diet (HFD) reduces endothelial NF-κB activation in the atheroprone aortic arch, an effect that is sufficient to downregulate a range of proinflammatory genes and markedly suppress leukocyte recruitments in the vessel wall, thence ameliorating atherosclerosis development [29].
ACCEPTED MANUSCRIPT miR-143/145 The survival of ECs is paramount in maintaining endothelial integrity to protect against lesion formation. LSS-mediated miR-143/145 cluster maintains EC integrity and survival through targeting angiotensin-converting enzyme, thus angiotensin-2
that
is
largely
responsible
for
pathological
PT
reducing
RI
vasoconstriction and endothelial damages [30]. Notably, endothelial miR-145
SC
seems to inhibit monocyte recruitment to the arterial wall and lesion formation
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in HFD-fed ApoE-/- mice via targeting junctional adhesion molecule-A [31].
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2.1.2 Turbulent flow-responsive pro-atherogenic endothelial miRNAs miR-92a is upregulated by turbulent flows in human ECs in vitro [32,33], and
D
found at sites prone to atherosclerosis in vivo [24,33,34] and in human
components
KLF2,
KLF4,
and
Phosphatidic
Acid
EP
anti-inflammatory
TE
atherosclerotic plaques [35]. Mechanistically, miR-92a targets flow-mediated,
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Phosphatase type 2B, aggravating ECs through reducing NO production and directing ECs towards a pro-inflammatory phenotype through upregulated NFκB-mediated gene profile, enhanced leukocyte adhesions and compromised monolayer integrity [32–34,36]. In vivo blockade of miR-92a expression in an experimental atherosclerosis model like HFD-fed Ldlr-/- mice (LDL receptor (LDLR)-deficiency) reduces endothelial inflammation, curtails atherosclerotic plaque development and promotes stable plaques through rescuing its target suppressor of cytokine signaling 5 [33,37]. On the other hand, miR-92a also
ACCEPTED MANUSCRIPT impairs endothelial repair through inhibiting EC proliferation and migration both in vitro and in vivo, an effect that has been associated with its targeting of the pro-proliferative integrin-α5 and sirtuin-1 (SIRT1) [38]. Therefore miR-92a might act in multiple fronts, inducing inflammatory activation together with
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impairing EC regeneration, collectively contribute to EC dysfunction and
RI
atherogenesis. Other OSS-induced miRNAs include miR-663 and miR-
SC
712/miR-205 worked by exacerbating EC hyperpermeability and inflammation [39,40], and treatment with anti-miR-712 derepresses metalloproteinase
NU
inhibitor 3 (TIMP3) expression and mitigates atherosclerosis progression in
MA
mice [40].
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2.1.3 Flow-responsive endothelial miRNAs with conflicting pleiotropic
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actions in atherosclerosis
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There are several miRNA candidates with discrepancies reported in response to
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different flow patterns and in their roles in atherosclerosis. This might due to the multivalent nature of miRNAs, where a single miRNA can be regulated similarly by different flow patterns, and can target a set of different genes and participate in different knots in the network depending on cell types, conditions or environment.
miR-21
ACCEPTED MANUSCRIPT miR-21 was reported to confer a pro-inflammatory EC phenotype through suppressing peroxisome-proliferator-activated receptor-α (PPARα), and its endothelial expression is elevated by disturbed flow produced by a parallel plate flow chamber [41], and in the inner curvature of porcine aortic arch [24], human
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atherosclerotic plaques [35,42] and porcine, murine and human vein graft
RI
neointimas where turbulent flows prevail [43]. However, its pro-atherogenic
SC
role is complicated by a study that links elevated endothelial miR-21 expression with LSS generated by a different shear system (cone-and-plate system),
NU
enhancing EC survival and NO production [44]. Importantly, miR-21 was found
MA
to exert anti-atherogenic effects through downregulating the dual specificity of MAP2K3 to enhance apoptosis and cholesterol efflux of macrophages during
D
the early stage of atherosclerosis in HFD-fed LdlR-/- mice [45], while miR-21
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helps to stabilize plaque in advanced atherosclerosis through restraining
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macrophage activation and improving VSMC proliferation in the fibrous cap via
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targeting RE1-silencing transcription factor in plaque rupture model of HFD-fed ApoE-/- mice [46]. miR-21 was also shown to protect against arterial dysfunction and remodelling by inhibition of PTEN, with levels of miR-21 decreased in hypertension [47]. These indicate that miR-21 might play opposite roles in atherogenesis depending on cell-types and stages of atherosclerosis.
miR-126
ACCEPTED MANUSCRIPT miR-126 is among the most highly expressed endothelial miRNAs that has been shown to regulate both angiogenesis and inflammation in a flow-dependent manner [48–50]. Schober et al. have elegantly identified that the passenger strand miR-126-5p, and not the guide strand miR-126-3p, as the functional
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strand upregulated in LSS-exposed HUVECs in vitro [51]. Treatment with miR-
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126-5p mimics rescued EC proliferation in repairing damaged endothelium at
SC
the predilection sites by targeting Notch 1 inhibitor delta-like 1 homolog and limits atherosclerotic features in HFD-fed miR-126-/-ApoE-/- mice with partial
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carotid ligation [51]. Conversely, others have shown that OSS-induced
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endothelial secretion of miR-126-3p increased VSMCs turnover and reversed the atheroprotective effects of miR-126-deficiency in mice with complete
D
carotid ligation-induced atherosclerosis [52,53]. This is in contrast with another
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study demonstrating the therapeutic effects of endothelial-derived apoptotic
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bodies of miR-126-3p in limiting atherosclerotic lesions in vivo [54]. In relation
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to human atherosclerosis, higher uptakes of both miR-126 strands by coronary plaques are observed in patients with more vulnerable plaques [55], while levels of miR-126-5p within human atherosclerotic plaque are correlated with increased luminal EC proliferation and decreased foam cell formation [51]. These demonstrate that both strands might play a protective role against plaque formation or being uptaken to stabilise vulnerable plaques. Overall, these conflicting findings indicate that further studies will be necessary to determine
ACCEPTED MANUSCRIPT the relative role of both miR-126 strands in response to differential biochemical and biomechanical stimuli and in different stages of atherosclerosis.
miR-155
PT
miR-155 is upregulated by LSS in HUVECs and in the intima of the
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atheroprotective thoracic aorta that is naturally exposed to atheroprotective
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unidirectional blood flow [56]. Mechanistically, miR-155 exerts differential functional effects on ECs, for instance, suppressing EC proliferation, migration
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and apoptosis through targeting myosin light chain kinase [56], and promoting
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angiogenesis by targeting E2F2 [57]. In contrary, miR-155 has been demonstrated to enhance monocyte adhesion to HAECs [58] and impairs
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endothelium relaxation in human arteries through targeting eNOS [59]. Studies
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for miR-155 in mouse models of atherosclerosis also suggest conflicting effects
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depending on the contexts. For instance, systemic delivery of miR-155 reduced
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atherosclerotic lesion formation in ApoE-/- mice [60], and hematapoietic deficiency of miR-155 increased atherosclerosis with reduced plaque stability in LdlR-/- mice [61]. Other studies, however, have demonstrated that both global and macrophage-specific miR-155-deficiency suppressed atherogenesis in ApoE-/- mice through reduced macrophage recruitment and inflammatory response, and increased cholesterol efflux [58,62,63].
ACCEPTED MANUSCRIPT 2.2
Role of non-flow-responsive endothelial miRNAs
Several EC-derived miRNAs are regulated by inflammatory insults such as cytokines and hyperlipidaemia rather than disturbed flow pattern to modulate atherosclerosis (Table 1) [64,65]. Ox-LDL particles modulate endothelial
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hyperlipidaemic stress through binding to scavenger lectin-like ox-LDL
RI
receptor 1 (LOX-1) [66], leading to exacerbation in endothelial permeability,
Ox-LDL-mediated
upregulation
of
LOX-1
is
obtained
through
NU
69].
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inflammation and apoptosis, while limiting EC regenerative proliferation [67–
downregulation of let-7a, let-7b and let-7g in ECs in vitro [70,71]. These three
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let-7 family members were found to be downregulated in hyperlipidaemic conditions and exert atheroprotective effects on ECs [70–72]. For instance,
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D
overexpression of let-7a and let-7b inhibited ox-LDL-induced EC apoptosis and repressed activation of NF-B and MAPK pathways in ox-LDL-treated
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HUVECs [70]. Moreover, let-7g inhibited endothelial ox-LDL uptake, and
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reduced inflammation and neointimal lesions in HFD-fed ApoE-/- mice through targeting three components in TGF-β signalling, and decreased senescence by indirectly upregulating SIRT1 [71,72]. Recently, endothelial miR-100 was also found to be repressed in response to pro-inflammatory cytokine tumor necrosis factor- (TNF-) in a NF-B-dependent manner, and inversely correlated with inflammatory cells and cholesterol contents in human atherosclerotic plaques [73]. miR-100 represses inflammation and atherosclerotic plaque formation
ACCEPTED MANUSCRIPT through directly targeting components of rapamycin complex 1-signalling (rapamycin and raptor), which stimulates EC autophagy, downregulated adhesion molecules (E-Selectin, ICAM-1 and VCAM-1) and attenuated NF- B signalling both in vitro in HUVECs and in vivo in HFD-fed LdlR-/- mice [73].
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miR-103, maintained by endothelial Dicer expression, is highly upregulated in
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ECs within atherosclerotic plaque, and has been found to supresses endothelial
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KLF4 signalling with an associated increase in NF-B mediated chemokines
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expression (CXCL-1, CX3CL1 and CCL2) [74]. In HFD-fed ApoE-/- mice, inhibiting the interaction between miR-103 and KLF4 led to a decrease in
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macrophage accumulation and atherosclerotic plaque formation, suggesting a
D
pro-inflammatory and pro-atherogenic role of miR-103 by targeting KLF4 [74].
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Atherogenesis is exacerbated by disrupted EC integrity caused by dysregulated
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level of apoptosis, and several miRNAs participating in this process have been found elevated in atherosclerotic areas. One of these candidates, miR-181a,
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aggravates the rate of EC apoptosis by downregulating the pro-survival signalling B-Cell lymphoma 2 (BCL-2) [75]. miR-351, on the other hand, increased EC apoptosis by targeting the pro-survival signal transducer and activator of transcription 3 (STAT3) [76]. Not all endothelial atheroprotective miRNAs are downregulated in human atherosclerotic plaques however, and could be upregulated to prevent atherosclerosis in a negative feedback loop. For instance, miR-146a, found
ACCEPTED MANUSCRIPT elevated in human atherosclerotic plaques [42], are induced in ECs by proinflammatory cytokines TNF- and IL-1β, but restraining endothelial proinflammatory milieu by repressing NF-B and MAPK pathways both in vitro (in HUVECs) and in vivo (HFD-fed Ldlr-/- mice), while upregulating EC-
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protective eNOS through targeting RNA binding protein HuR [77,78].
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Similarly, both miR-31 and miR-17-3p can also be induced by TNF- in a
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feedback control of inflammation, and respectively targeting TNF--induced
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EC expression of E-selectin and ICAM-1 [79].
Table 1. Endothelial microRNAs involved in endothelial dysfunction and
Athe
RN
li &
ro-
l Systems
miRN genic
level
Effec
Effects on EC Re
Signaling
Dysfunctions
Pathways
and
f
Atherosclerosi
AC C
A
Affected
EP
A
Experimenta Targets
D
Stimu
TE
mi-
MA
atherosclerosis
ts
s
chang es
Section 2.1.1: Laminar flow-responsive anti-atherogenic endothelial miRNAs mi
d-
Anti-
Porcine AA;
MAP3
↓ E-Sel,
Inhibits EC
[2
R-
flow↑
infla
In vitro
K7
VCAM-1 and
NF-κB
4]
ACCEPTED MANUSCRIPT m-
10a
βTRC
HAECs
mato
NF-κB
inflammatory
signaling
activation
ry LSS↑
Anti-
In vitro shear
R-
OSS↓
infla
by PP flow
Rα/RXRα &
m-
system on
↑miR10-a ->
mato
HAECs for 24
↓GATA6/VCA
ry
h; LSS (12
RI
SC
10a
GATA6 LSS↑KLF2/RA
M-1;
dyn/cm2) vs
prolif by PP flow
atherosclerosis in ApoE-/- mice
↓miR10-a -> ↑GATA6/VCA M-1
Cyclin
Cell cycle
Inhibits EC
[2
D1
arrest (G1/S)
proliferation
5]
E2F1
Cell cycle
Inhibits EC
[8
arrest (G1/S)
proliferation
0]
AC C
R-
6]
NU MA
D TE
In vitro shear
EP
Anti-
inhibits
5/7/RARα &
dyn/cm2)
LSS↑
[2
OSS↑HDAC3/
OSS (0.5
mi
PreR-10a
PT
mi
19a
e-
system on
rativ
HUVECs for
e
12 h; LSS (12 dyn/cm2)
mi R-
LSS↑
Anti-
In vitro shear
prolif by PP flow
ACCEPTED MANUSCRIPT 23b
e-
system on
and ↑Rb hypo-
rativ
HUVECs for
phosphorylatio
e
24 h; LSS (12
n
dyn/cm2) Anti-
In vitro shear
R-
High
infla
by ibidi pump
30-
PSS↑
m-
system on
mato
HUVECs for
ry
72 h; LSS (20
↓E-sel, ICAM1 & VCAM-1
SC
leukocyte
[2 7]
adhesion to ECs
MA
dyn/cm2); ex
Inhibits
NU
5p
Ang2
PT
LSS↑
RI
mi
vivo shear by
D
syringe pump
TE
on mouse
EP
femoral artery
AC C
explant for 6 h; high PSS (76 dyn/cm2) vs low PSS (7.6 dyn/cm2)
mi R-
LSS↑
Anti-
In vitro shear
prolif by PP flow
mTOR
Cell cycle
Inhibits EC
[8
arrest (G1/S)
proliferation
1]
ACCEPTED MANUSCRIPT 101
e-
system on
rativ
HUVECs for
e
12 h; LSS (12 dyn/cm2) In vitro shear
ACE
R-
ather
by cone-plate
↑AMPKα2/
143/
o-
viscometer on
pp53/miR-
145
genic HUVECs for
D
In vitro shear
R-
infla
by ibidi pump
143/
mma tory
145
RI
miR-143/145 -
[3 0]
> impaired vasodilator
↓ACE in ECs
response in murine hindlimb
EP
JAM-A
system on
JAM-A-/-
[3
↓monocytes
1]
recruitment and
AC C
LSS↑
TE
Anti-
mi
Low level of
143/145 ->
MA
dyn/cm2)
SC
72 h; LSS (12
LSS->
NU
LSS↑
PT
Anti-
mi
HAECs for 48
↓
h; LSS (5 or
atherosclerosis
20 dyn/cm2);
in
in vivo
ApoE−/− mice
ApoE−/− mice mi
LSS+
Anti-
In vitro shear
IRAK
↓NF-kB
miR-146a
[8
ACCEPTED MANUSCRIPT EC/sS
infla
by PP flow
(miR-
pathway
↓neointima
146
MC
m-
system on
146);
activation
formation in
a,
co-
mato
HUVECs for
IKK-γ
mouse CA
708, culture ry
6-24 h; LSS
(miR-
ligation model
451, ↑
(12 dyn/cm2);
708);
in vivo mouse IL-6R
flow↓
partial CA
RI
d-
(miR-
ligation model 451);
NU
CHUK
SC
98
2]
PT
R-
MA
(miR98)
LSS↑
Anti-
ApoE-/- mice
R-
HLD↓
infla
with HFD
mato
TE
b
EP
m-
AC C
181
D
mi
ry
IPOA3
↓E-sel, ICAM-
i.v. injection of
[2
1 & VCAM-1
miR-181b
5,2
& NF-kB
mimics
9]
signaling in
↓inflammation,
AA of ApoE-/-
leukocyte
mice; ↓IPOA3
recruitment &
& NF-kB-p65
atherosclerosis
translocation in in ApoE-/- mice lesional ECs in ApoE-/- mice +
+ HFD
ACCEPTED MANUSCRIPT HFD Section 2.1.2: Turbulent flow-responsive pro-atherogenic endothelial miRNAs ↓eNOS, NO
s.c. treatment
[4
by PP flow
production,
with pre-92a
1]
m-
system on
TM
↓eNOS-
mato
HUVECs for
ry
8 h; LSS (12
In vitro shear
R-
PSS↓
infla
92a
OSS↑
KLF2
PT
Pro-
dependent
RI
LSS↓
dyn/cm2),
NU
PSS (12±4
D
dyn/cm2) d-
Pro-
Ex vivo
R-
flow↑
infla
Porcine AA,
KLF2
↑E-sel,
miR-92a KD
[3
KLF4
VCAM-1 &
↓leukocyte
4]
MCP-1 ↓eNOS
adhesion to
EP
m-
TE
mi
in vitro
AC C
92a
MA
dyn/cm2) OSS (4±4
mato
vasodilatation
SC
mi
HAECs
HAECs
ry
Pro-
In vitro
ITGA5
pre-miR-92a
[3
R-
infla
HCAECs; in
SIRT1
↓EC
8]
92a
m-
vivo Tie2-
proliferation &
mato
Cre;miR-
migration;
mi
NA
ACCEPTED MANUSCRIPT ry &
92a(fl/fl)-
anti-miR-92a
anti-
mice
& ECconditional KO
-
of miR-92a
genic
↑re-
PT
angio
D
MA
NU
SC
RI
endothelization
Low
Pro-
In vitro shear
R-
LSS+
infla
by PP flow
92a
HLD↑
m-
flow↑
EP
system on
AC C
d-
TE
mi
SOCS5
& ↓leucocyte recruitment & neointima formation in mice wireinjured artery
↓NOS3, KLF2,
AntagomiR-
[3
KLF4;
92a ↓EC
3]
↑NF-κB
inflammation
signaling
& monocyte
mato
HUVECs for
ry
24 h; high
adhesion in
LSS (15
vitro;
dyn/cm2), low
↓atherosclerosi
LSS+HLD (4
s & ↑stable
dyn/cm2+ox-
plaque in LdlR-
ACCEPTED MANUSCRIPT /-
LDL);
mice+HFD
AA of LdlR-/mice+HFD mi
OSS↑
Pro-
In vitro shear
PPAP2
miR-92a ↓LSS- PPAP2B KD -
R-
d-
infla
by cone-plate
B
induced
92a
flow↑
m-
viscometer on
KLF2/PPAP2B
mato
HAECs for 24
-> ↑LPA
ry
or 72 h; LSS
PT
RI
SC
↓EC alignment & monolayer
&↑MCP-1,
integrity &
VCAM-1 &
↑leukocyte
SELE
adhesion
D
dyn/cm2); in
MA
dyn/cm2),
6]
inflammation:
signalling
NU
(12-40
OSS (5
> ↑EC
[3
TE
vivo mouse
EP
partial CA
mi R663
OSS↑
AC C
ligation model
Pro-
In vitro shear
infla
NA
EC
[3
by cone-plate
inflammation:
9]
m-
viscometer on
↑monocyte
mato
HUVECs for
adhesion &
ry
24 h; LSS (15
inflammatory
dyn/cm2),
markers in ECs
ACCEPTED MANUSCRIPT OSS (5 dyn/cm2) mi
d-
Pro-
In vivo mouse TIMP3
↑MMPs
EC
[4
R-
flow↑
infla
partial CA
activity
inflammation:
0]
712/ OSS↑
m-
ligation
mi
mato
model;
R-
ry
In vitro shear
PT
↑monocyte
SC
RI
adhesion,
viscometer on
MA
iMAECs &
NU
by cone-plate
205
↑TNFα release. anti-miR712 ↓EC inflammation &
h; LSS (15
atherosclerosis
D
HAECs for 24
dyn/cm2),
TE
in vivo
EP
OSS (5
AC C
dyn/cm2)
Section 2.1.3: Flow-responsive endothelial miRNAs with conflicting pleiotropic
↑Akt & eNOS
↑EC survival
[4
by cone-plate
phosphorylatio
&NO
4]
viscometer on
n& NO
bioavailability
HUVECs for
production
Anti-
In vitro shear
R-
apo-
21
totic
mi
LSS↑
actions in atherosclerosis PTEN
ACCEPTED MANUSCRIPT 24 h; LSS (15 dyn/cm2) LdlR-/-
MAP2
↓p38-CHOP &
miR-21-/- BM
[4
R-
infla
mice+HFD
K3
JNK signaling
↑macrophage
5]
21
m-
with BM
-> ↑ABCG1
arterial
mato
transplanted
degradation
ry
from WT or
RI SC
NA
PT
Anti-
mi
Inducible
R-
infla
plaque rupture
21
m-
EP
TE
Anti-
model of miR-
AC C
mi
NA
D
MA
NU
miR-21-/- mice
REST
infiltration, cytokine releases, & apoptosis-> ↑foam cell formation & atherosclerosis miR-21-/-ApoE-
[4
/-
6]
mice +HFD:
↓VSMC
mato
21-/-ApoE-/-
proliferation
ry
mice+HFD
and ↑plaque rupture, lesion size, foam cell formation and arterial
ACCEPTED MANUSCRIPT macrophage infiltration in an inducible plaque rupture
PT
model.
plaque rupture phenotype.
-response
inflammation:
1]
system on
element->
↑monocyte
HUVECs for
↑AP1
adhesion &
activation,
inflammatory
(12 dyn/cm2),
MCP-1,
markers in ECs
OSS (0.5
VCAM-1
infla
by PP flow
21
mmato
PPARα
TE
D
R-
6-24 h; LSS
AC C
ry
vulnerable
[4
In vitro shear
EP
OSS↑
rescues this
↓PPARα/PPAR ↑EC
Pro-
mi
MA
NU
SC
RI
miR-21 mimic
dyn/cm2) ↓STAT3,
miR-21-/-
[4
prolif murine and
PTEN &
↓mouse vein
3]
e-
BMPR2
graft
mi
Vein
Pro-
R-
graft↑
21
porcine,
human vein
NA
ACCEPTED MANUSCRIPT rativ
graft models
neointimal
e Pro-
In vitro shear
FOXJ2
R-
differ by PP flow
34a
e-
system on
ntiati
eEPCs for 6-
on
24 h; LSS (15
↑EC markers,
↑EC
[8
↓SMC markers
differentiation
3]
PT
of eEPCs
RI
LSS↑
dyn/cm2)
↑NF-kB
EC
[8
signaling,
inflammation:
4]
↑ICAM-1 &
↑monocyte
VCAM-1
adhesion and
LSS↓
Pro-
In vitro shear
R-
OSS↑
infla
by PP flow
m-
system or
mato
ibidi pump
ry
system on
inflammatory
HUVECs for
markers
EP
TE
D
MA
NU
mi
34a
SIRT1
SC
mi
formation
AC C
24 h; LSS (15 dyn/cm2), OSS (5 dyn/cm2) ↑EC repair &
i.v. injection of
[5
angio partial CA
cell cycle
miR-126-5p
1]
-
progression
mimics rescues
mi
d-
Pro-
R-
flow↓
126
LSS↑
In vivo mouse Dlk1
ligation
ACCEPTED MANUSCRIPT genic model; In
(G2/M)
EC
;
vitro shear by
proliferation &
Anti-
ibidi pump
↓atherosclerosi
ather
system on
s in miR126-/-
o-
HUVECs for
ApoE-/-
PT
-5p
mice+HFD
RI
genic 72 h; LSS (10
Anti-
R-
OSS
prolif by ibidi pump
155
↔
e-
system on
rativ
HUVECs for
e;
0.5-4 h; LSS
anti-
(15 dyn/cm2),
migr
OSS (5
MYLK
↑RhoA activity
↓EC
[5
cytoskeleton
6]
rearrangement -> ↓migration,
D
proliferation &
EP
TE
apoptosis
dyn/cm2); ex
AC C
a-
In vitro shear
NU
LSS↑
MA
mi
SC
dyn/cm2)
tory
vivo mouse AA
Pro-
In vivo mouse BCL6
↑NF-kB
↑atherosclerosi
[6
R-
infla
partial CA
signaling
s
2]
155
m-
ligation model
mi
NA
mato
in vivo
ACCEPTED MANUSCRIPT ry HLD↑
In vitro
R-
infla
155
NA
miR-151
[5
HAECs
mimics
8]
m-
exposed to
↑monocyte
mato
HLD
adhesion to
PT
Pro-
mi
HAECs;
MA
NU
SC
RI
ry
miR-151-/↓atherosclerosi s in ApoE-/mice
Section 2.2: Non-flow-responsive endothelial miRNAs in atherosclerosis In vitro
infla
HUVECs
Let-
m-
exposed to
7b
mato
TE
LDL↓
EP
7a,
ry
D
Anti-
ox-LDL
AC C
Let- ox-
LOX-1
↓NF-κB
Overexpression
[7
signaling and
of Let-7a and -
0]
p38MAPK
7b inhibits oxLDL-induced EC apoptosis, NO deficiency, ROS overproduction and inflammation
ACCEPTED MANUSCRIPT in HUVECs ↓TGF-β
Let-7g mimics
[7
infla
HUVECs
THBS1
signalling
↓HUVEC
1,7
m-
exposed to
TGFBR (pSmad2/PAI-
TGF-β-
2]
mato
ox-LDL; In
1
1)
dependent
ry
vivo ApoE-/-
SMAD
↑SIRT1
mice+HFD
2
RI SC NU
MA D
PT
LOX-1
TE
LDL↓
In vitro
EP
7g
Anti-
AC C
Let- ox-
cytokines secretions (VCAM-1, MCP-1, IL-6) & SIRT1dependent senescence in vitro. Let-7g lentiviral overexpression ↓macrophage infiltration, TGF-β signalling & lesion size in
ACCEPTED MANUSCRIPT ApoE-/mice+HFD TNF
Anti-
In vitro
Rapam
↓NF-κB
miR-100
[7
R-
↓
infla
HUVECs
ycin
signaling &
mimics ↓
3]
m-
treated with
Raptor
↓E-Sel, ICAM-
macrophages
mato
TNF; In
ry
vivo LdlR-/-
RI
1, VCAM-1
SC
100
PT
mi
↑EC autophagy in HUVECs; miR-100 mimcs ↓plaque size in LdlR-/mice+HFD
mi
TNF
Anti-
In vitro
E-SEL
miR-31or miR-
[7
R-
↑
infla
EP
TE
D
MA
NU
mice+HFD
infiltration &
(miR-
17-3p mimics
9]
AC C
HUVECs
31, mi
m-
treated with
31);
↓neutrophil
mato
TNF
ICAM-
adhesion to
1 (miR-
TNF-treated
17-3p)
HUVECs
HuR on ↓NF-κB
miR-146a
ry
R173p mi
IL-1β
Anti-
In vitro
[7
ACCEPTED MANUSCRIPT HUVECs
ECs;
signaling and
mimics ↓NF-
7,7
146
m-
treated with
SORT1
MAPK
κB and
8]
a
mato
TNF or IL-
in liver
↓ IL-1β -
monocyte
ry
1β; In vivo
induced E-Sel,
adhesion to IL-
LdlR-/-
ICAM-1,
1β -treated
mice+HFD &
VCAM-1,
MiR-146a-/-
MCP-1
RI
SC
LdlR-/-
EP
TE
D
MA
NU
mice+HFD
AC C
↑
PT
infla
R-
HUVECs in vitro; MiR146a-/-LdlR-/mice + WT BM:↑EC activation & plaque burden MiR-146a-/LdlR-/- mice + MiR-146a-/LdlR-/- mice BM: impaired hematopoietic cells & ↓circulating LDL
ACCEPTED MANUSCRIPT ↑NF-κB
miR-103
[7
HAECs;
signalling;
mimics
4]
m-
In vivo ApoE-
↑CXCL1, CX3
↑monocyte
mato
/-
CL1 & CCL2
adhesion to
mi
Dicer↓ Pro-
In vitro
R-
Ox-
infla
103
LDL↑
KLF4
mice+HFD
HAECs; i.v.
PT
ry
mi
H2O2
Pro-
In vitro
R-
↑
apop
EP
TE
D
MA
NU
SC
RI
injection of
BCL-2
HUVECs
AC C
a
blockers in ApoE-/mice+HFD ↓lesion size & lesional macrophages
-totic
181
miR-103/KLF4
Anti-miR-181a
[7
↓H2O2-
5]
induced apoptosis in HUVECs
Pro-
In vitro
R-
apop
mouse
351
-totic primary
mi
HLD↑
STAT3
miR-351
[7
mimics ↑
6]
H2O2-induced
ACCEPTED MANUSCRIPT CAECs from
apoptosis in
ApoE-/- mice
mouse CAECs
Table 1. Endothelial microRNAs involved in endothelial dysfunction and
PT
atherosclerosis. Abbreviations: ->, leads to; ↑, upregulation; ↓, downregulation;
RI
↔, no change; AA, aortic arch; ACE, angiotensin-converting enzyme;
SC
AMPK2, 5'-AMP-activated protein kinase catalytic subunit 2; Ang2, angiopoieting-2; ApoE, apolipoprotein E; TRC, -transducin repeat-containing
NU
gene; BCL, b-cell lymphoma; BMPR2, bone morphogenetic protein receptor 2;
MA
CA, carotid artery; CAECs, coronary artery endothelial cells; CCL2, chemokine (C–C motif) ligand 2; CHUK, conserved helix-loop-helix ubiquitous kinase; d-
D
flow, disturbed flow; CXCL1, chemokine C–X–C motif chemokine 1;
TE
CX3CX1, chemokine (C–X3–C) ligand 1; Dlk1, delta-like 1 homolog; E-sel, E-
EP
selectin; EC, endothelial cell; eEPCs, early endothelial progenitor cells; FOXJ2,
AC C
forkhead box J2; GATA6, GATA-binding factor 6; H2O2, hydrogen peroxide; HAECs, human aortic ECs; HBVPs, human brain vascular pericytes; HCAEC, human coronary artery ECs; HDAC, histone deacetylase; HFD, high fat diet; HLD, hyperlipidemia; HuR, ELAV-like RNA binding protein 1; HUVECs, human umbilical cord ECs; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1 beta; IL-6R, IL-6 receptor; IKK-, inhibitor of nuclear factor kappa-B kinase subunit ; iMAECs, immortalized mouse aortic ECs; i.p.,
ACCEPTED MANUSCRIPT intraperitoneal; IPOA3, importin-a3; IRAK, interleukin-1 receptor-associated kinase 1; ITGA5, integrin subunit alpha 5; JAM-A, junctional adhesion molecule A; JNK, c-jun N-terminal kinase; KD, knockdown; KLF, Kruppel like factor; KO, knockout; LdlR, low-density lipoprotein receptor; LOX-1, lectin-
PT
like oxidized LDL receptor-1; LSS, laminar shear stress; MAP2K3, mitogen-
RI
activated protein kinase kinase 3; MAP3K7, MAP kinase kinase kinase 7;
SC
MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; miR, microRNA; mTOR, rapamycin; MYLK, myosin light chain
NU
kinase; NA, not assessed; NF-B, nuclear factor B; NO, nitric oxide; NOS3,
MA
nitric oxide synthase 3; OSS, oscillatory shear stress; ox-LDL, oxidized lowdensity lipoprotein; p38-CHOP, p38 MAP kinase-C/EBP homologous protein;
D
PP, parallel plate; PPAP2B, phosphatidic acid phosphatase type 2B; PPAR,
TE
Peroxisome proliferator-activated receptor ; PSS, pulsatile shear stress; PTEN,
EP
phosphatase and tensin homolog; RAR, retinoic acid receptor ; s.c.,
AC C
subcutaneous; REST, RE-1-silencing transcription factor; ROS, reactive oxygen species; SELE, selectin E; SEMA, semaphorins; SIRT1, sirtuin1; SMAD2, mothers against decapentaplegic homolog 2; SMC, smooth muscle cell; sSMC, synthetic SMC; SOCS5, suppressor of cytokine signaling 5; SORT1, sortilin-1; STAT3, signal transducer and activator of transcription 3; TGF-β, transforming growth factor beta; TGFBR1, TGF-β receptor 1; THBS1, thrombospondin 1;
ACCEPTED MANUSCRIPT TM, thrombomodulin; TNF, tumour necrosis factor ; VCAM-1, vascular cell adhesion molecule-1; WT, wild type.
miRNA-mediated
decreased
in
VSMC
apoptosis
and
phenotype,
including
phenotypic
switching
aberrant to
a
SC
proliferation,
changes
PT
MiRNAs Regulation of Vascular Smooth Muscle Cell Functions
RI
3.
dedifferentiated migratory synthetic state, also contribute significantly to the
NU
initial stages of atherosclerosis [85]. miR-136 is upregulated in atherosclerotic
MA
plaques and targets serine/threonine-protein phosphatase 2 regulatory subunit Balpha (PPP2R2A), leading to reduced inhibition of the extracellular signal-
D
regulated kinase-1/2 (ERK1/2) signaling and subsequently increased VSMC
TE
proliferation [86]. miR-221, miR-26a and miR-146a are other pro-proliferative
EP
miRNAs mediating platelet-derived growth factor (PDGF)-induced VSMC
AC C
proliferation [87–89]. miR-221 mediates this process through inhibiting the anti-proliferation pathway p38/p21/p27 [87], whilst miR-26a does so by decreasing TGF- signaling [88]. miR-146a promotes VSMC proliferation through targeting KLF4 in a negative feedback loop [89]. In addition, miR-26a inhibits VSMC apoptosis and both miR-26a and miR-221 promote VSMC dedifferentiation [87,88]. miR-221’s effect on VSMC dedifferentiation is unrelated to p27 downregulation and instead occurs due to inhibition of c-Kit [87].
ACCEPTED MANUSCRIPT Other miRNAs exert atheroprotective effects on VSMCs. For instance, let-7 family, in addition to their atheroprotective effect on ECs, also ameliorates VSMC inflammatory responses including proliferation, migration, monocyte adhesion and NF-B activation [90]. miR-22-3p and miR-125b too are
PT
downregulated in human atherosclerosis and inhibit proliferation and migration
RI
of VSMCs through targeting high motility group box 1 (HMGB1) and
SC
podocalyxin like protein (PODXL) respectively [91,92]. miR-24, miR-29a, miR-663 and miR-638 have all been shown to reduce VSMC proliferation and
NU
migration, as well as to promote transition to VSMC contractile phenotype via
MA
inhibition of PDGF-BB signaling [93–95]. miR-133 and miR-424 exert similar effects by repressing expression of Sp-1 and cyclin D1, calumenin and stromal
D
interaction molecule 1 (STIM1) respectively [96,97]. miR-22 represses VSMC
TE
dedifferentiation through inhibiting proliferation and migration and enhances
EP
contractile VSMC gene expression via targeting methyl-CpG binding protein 2
AC C
(MECP2), ecotropic virus integration site 1 protein homolog and HDAC4 [98]. miR-22 mimics delivery in wire-injured murine femoral arteries inhibited neointima formation [98].
miR-195 also reduces VSMC proliferation and
migration but additionally decreases VSMC synthesis of the pro-inflammatory cytokines IL-1, IL-6 and IL-8, possibly through targeting cell division cycle 42 (Cdc42), cyclin D1 and fibroblast growth factor 1 [99].
ACCEPTED MANUSCRIPT Finally, maintaining VSMC survival in the fibrous cap in the advanced stage of atherosclerosis is crucial in preventing plaque rupture. miR-181b, in addition to its anti-inflammatory effect on ECs, was recently found to maintain plaque stability in ApoE-/- mice and LdlR-/- mice through downregulation of
PT
macrophage TIMP3 and VSMC elastin synthesis [100]. miR-210, on the other
RI
hand, maintained VSMC survival and phenotypic modulation through targeting
SC
adenomatous polyposis coli (APC)-Wnt signaling, effectively improving fibrous cap stability and decreasing plaque rupture in vivo [101]. miR-21
NU
stabilizes plaque through promoting VSMC proliferation and repair in the
MA
fibrous cap via targeting RE-1-silencing transcription factor in a plaque rupture
MiRNA-mediated
Atherosclerosis
Intercellular
Communication
During
EP
4.
TE
D
atherosclerotic model in vivo [46].
AC C
Shear stress is also able to influence atherogenesis by promoting miRNA as extracellular messengers between vascular cells. This occurs either via incorporation into extracellular vesicles (EVs) including macrovesicles and exosomes, or by association with proteins such as HDL and Ago2 [85]. These interactions are necessary to stabilize miRNA molecules during the period of intercellular transport [102,103]. EVs are taken up by endocytosis or fusion
ACCEPTED MANUSCRIPT with the cell membrane, whilst protein-associated miRNAs are taken up by binding to a specific receptor (Figure 3) [104]. Co-culture of HUVECs with human umbilical artery VSMCs revealed that miR-126-3p is increasingly secreted by ECs exposed to OSS, which mechanism
PT
was recently found to be mediated by soluble N-ethylmaleimide-sensitive factor
RI
attachment protein receptors (SNAREs) activation [52,53]. Its uptake into
SC
VSMCs leads to increased VSMC turnover through inhibition of forkhead box
NU
O3 (FOXO3), BCL-2 and insulin receptor substrate 1 (IRS1) [52], while inhibition of miR-126-3p-mediated communication with rapamycin ameliorates
MA
neointimal formation in ApoE-/- mice [53]. In another study, when HUVECs were exposed to LSS, an increase in the secretion of miR-143/145-containing
TE
D
EVs, which is dependent on KLF2 and Rab7a/Rab27b and protects ApoE-/- mice
EP
from atherosclerosis development, was observed [105,106]. More recently, miRNA-mediated VSMC-to-EC communication has emerged as
AC C
a potential factor in vascular homeostasis. It appears that miR-143/145mediated communication is not unidirectional but reciprocal, with VSMC-EC communication occurring by protrusions of the cell membrane known as tunnelling nanotubes and resulting in EC vessel stabilization [107]. However, it is still unclear if such signaling occurs in the setting of atherosclerosis and whether shear stress patterns influence it.
ACCEPTED MANUSCRIPT Co-culture of dedifferentiated VSMCs and ECs could modulate levels of miR146a in ECs, both under static conditions and LSS, and miR-146a exerts antiinflammatory effect to repress neointima formation in rat and mouse injured carotid arteries by inhibiting NF-κB signalling [82]. Interestingly, vesicle-
PT
mediated miRNA transfer from VSMCs-to-ECs was ruled out as the mechanism
RI
of transfer [82], but other transfer mechanisms remain to be investigated. In
macrophages
[108].
miR-146a-enriched
SC
contrary, miR-146a was found elevated in the EVs of ox-LDL-treated EVs
released
by
atherogenic
NU
macrophages act to transfer miR-146a into recipient naive macrophages and, by
MA
targeting insulin-like growth factor 2s mRNA-binding protein 1 (IGF2BP1) and HuR, limit their migration, which may promote macrophage entrapment in the
D
vessel wall and accelerate atherogenesis [108]. Overall, although the number of
TE
miRNAs confirmed to mediate communication between vascular cells by
EP
extracellular secretion remains small (Table 2), the discovery of this paradigm
AC C
has ensured its importance in atherosclerosis and warrant further researches.
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
Figure 3. Vascular cell miRNA-mediated communication. A summary of the
MA
methods of miRNA-mediated intracellular communication suggested by current
D
evidences. The direction of miRNA transfer shown for each method reflects
TE
recent observations, however, it is plausible that further research may prove bi-
EP
directionality. EC, endothelial cell; miRNA, microRNA; SMC, smooth muscle
AC C
cell.
Table 2. miRNAs involved in miRNA-mediated intercellular communication miRNA Transfer
Mechani sm
Experimental
Establis
Stimu
Effects on target
Model
hed
li and
cell
Directio
miRN
n of
A
Commu
level
Ref
ACCEPTED MANUSCRIPT ni-cation chang es Protein-
In vitro shear
126-3p
mediated applied to
EC ->
OSS↑
VSMC
transfer
HUASMC co-
SC NU
LSS (12
MA
dyn/cm2) vs OSS (0.5
TE
D
dyn/cm2)
VSMC
LSS↑
↓ELK1, ↓KLF4,
[10
↓CAMK2d, ↓
5,10
SSH2, ↓PHACTR4
prolonged LSS;
& ↓CFL1 in co-
in vitro co-
cultured HASMCs;
culture of KLF2
reduced
transduced
atherosclerotic
HUVECs with
lesion formation in
HASMCs;
ApoE-/- mice
exposed to
AC C
transfer
EC ->
EP
143/145 mediated HUVECs miRNA
53]
(pro-atherogenic)
flow system,
In vitro
& ↓IRS1 ->
RI
HUVEC-
Vesicle-
[52,
↑VSMC turnover
miRNA
culture by PP
miR-
↓FOXO3, ↓BCL-2
PT
miR-
6]
ACCEPTED MANUSCRIPT induction of
transfected with
KLF2
KLF2 transduced
transduced
ECs (anti-
mouse ECs into
atherogenic)
PT
ApoE-/-
Nanotub
143/145 e-
In vitro murine
VSMC -
EC-VSMC co-
> EC
NU
mediated culture under varying
transfer
conditions; ex-
[10
β↑,
> EC proliferation
7]
vessel
and their capacity
stress
to form vessel-like
↑
structures (effect in atherogenesis
D
vivo mouse
MA
miRNA
↓HKII & ↓ITGβ8 -
TGF-
SC
miR-
RI
mice+HFD
unknown)
TE
aortas treated
EP
with TGF-β; in
AC C
vivo mouse coronary artery stress atheroge
ox-
↓IGF2BP1 & ↓Hur
[10
mediated application of
nic Mϕ -
LDL↑
-> limited
8]
miRNA
EVs derived
> naive
macrophage
transfer
from ox-LDL-
Mϕ
migration ->
miR-
Vesicle-
146a
In vitro
ACCEPTED MANUSCRIPT treated
entrapment in
macrophages to
vessel wall (pro-
naive
atherogenic)
2.
miRNAs
Abbreviations:
in ->,
miRNA-mediated leads
to;
↑,
intercellular
upregulation;
↓,
SC
communication.
involved
RI
Table
PT
macrophages
downregulation; ApoE, apolipoprotein E; BCL, b-cell lymphoma; CAMK2d,
NU
calcium/calmodulin-dependent protein kinase type II delta chain; CFL1, Cofilin
MA
1; EC, endothelial cell; ELK1, E-twenty-six domain containing protein-1; EV, extracellular vesicle; FOXO3, forkhead box O3; HASMC, human aortic smooth
D
muscle cell; HFD, high-fat diet; HKII, mitochondrial hexokinase II; HuR,
TE
human antigen R; HUVEC, human umbilical vein endothelial cell; HUASMC,
EP
human umbilical cell smooth muscle cell; IGF2BP1, insulin-like growth factor
AC C
2 mRNA-binding protein 1; IRS1, insulin receptor substrate 1; ITGβ8, integrin β8; KLF, Kruppel like factor; LSS, laminar shear stress; Mϕ, macrophage; miR, microRNA; OSS, oscillatory shear stress; ox-LDL, oxidized low density lipoprotein; PHACTR4, phosphatase and actin regulator 4; PP, parallel plate; SSH2, slingshot protein phosphatase 2; TGF-β, transforming growth factor β; VSMC, vascular smooth muscle cell.
ACCEPTED MANUSCRIPT 5.
MiRNAs Regulation of Macrophage Functions
Macrophages exist in a polarized state, expressing either a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. The M1 phenotype is predominant in atherogenesis and is associated with the secretion of
PT
inflammatory cytokines [109]. Several miRNAs have been shown to play a role
RI
in regulating macrophage polarisation; miR-33 promotes the M1 phenotype by
SC
encouraging aerobic glycolysis over oxidative phosphorylation [110]. Inhibition of miR-33, in a murine LdlR-/- model fed with HFD, proved to be
NU
atheroprotective [110]. On the other hand, miR-93 [111], miR-124 [112], miR-
MA
223 [113] and let-7c [114] have been shown to tilt the balance of macrophage polarization in favour of the M2 phenotype, through respectively targeting
D
immunoresponsive gene 1 (IRG1), CCAAT/enhancer-binding protein-
TE
(C/EBP-), PBX/knotted 1 homeobox 1 (PKNOX1) and CCAAT/enhancer-
EP
binding protein (C/EBP-) [111,113–115]. Additionally, miR-125a-5p and
AC C
miR-146a exert atheroprotective effects on macrophages by inhibiting the activation and secretion of pro-inflammatory cytokines via suppressing NF- B [116,117]. Notably, expression of miR-146a in hematopoietic cells is crucial in restraining the chronic inflammatory response in vivo [78]. miR-10a is integral to Dicer’s anti-inflammatory effect and promotion of fatty acid oxidative metabolism by binding to ligand-dependent nuclear receptor co-repressor (Lcor), collectively limiting the formation of lipid-filled macrophage-derived
ACCEPTED MANUSCRIPT foam cells and atherosclerotic development in vivo [118]. Increased expression of miR-10a was associated with decreased atherosclerotic progression in humans [118]. Other miRNAs, including miR-147, also limit macrophage activation by
PT
inhibiting toll-like receptor (TLR) signalling [119]. TLR2, TLR3, and TLR4
RI
engagement induces miR-147 expression, and miR-147 in turn downregulates
SC
TLR-induced inflammatory pathways in a negative feedback loop, resulting in a
NU
decreased release of pro-inflammatory cytokines [119]. miR-124a and miR-150 are both induced by KLF2 and are likely to exert an atheroprotective effect by
MA
inhibiting macrophage secretion of inflammatory chemokines such as chemokine (C–C motif) ligand 2 (CCL2) [120]. CCL2 secretion from
TE
D
macrophages is also inhibited by miR-24 through repression of chitinase 3-like 1 [121]. Finally, apoptosis of macrophages contributes to the development of
EP
the necrotic core found in atherosclerotic plaque [109]. miR-142-5p promotes
AC C
macrophage apoptosis by negative regulation of TGF-2, and as such is likely to have a pro-atherogenic role [122].
6.
MiRNAs Regulation of Cholesterol Homeostasis
Lipoproteins are composed of apolipoproteins, phospholipids, triglycerides and cholesterol. They are responsible for the transport of cholesterol and
ACCEPTED MANUSCRIPT triglycerides in the blood stream, and are key to cholesterol homeostasis. LDL and very low-density lipoprotein (VLDL) carry cholesterol from its production site, the liver, to peripheral tissues where receptors regulate their uptake. In the reverse process, efflux transporters work with high-density lipoprotein (HDL) to
PT
scavenge cholesterol away from tissues back to the liver. An imbalance in these
RI
processes, which are known to be regulated by miRNAs [123], can lead to sub-
SC
endothelial accumulation of cholesterol and promotion of atherogenesis [124].
NU
Increased systemic levels of LDL through its dysregulated secretion from the liver are known to be atherogenic. miR-122 promotes hepatic cholesterol
MA
synthesis and release into the circulation [125] whilst miR-223 has an opposing effect, inhibiting hepatic cholesterol synthesis by repressing the sterol enzymes
TE
D
3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGS1) and sterol-C4methyl-oxidase-like gene (SC4MOL) [126]. Similarly, miR-30c reduces
EP
lipogenesis in the liver by targeting microsomal triglyceride transfer protein
AC C
(MTTP) [127]. Overexpression of miR-30c, in a murine model, was found to decrease plasma cholesterol levels and inhibit the formation of atherosclerotic plaques [127]. However, interpretation from findings regarding plasma cholesterol levels in mice require caution, as unlike humans, the predominant fraction of plasma cholesterol in mice is HDL. The increased uptake of lipoprotein-transported cholesterol, by aberrant regulation of receptors, also contributes to cholesterol accumulation in
ACCEPTED MANUSCRIPT peripheral tissues. miR-125a-5p decreases the uptake of cholesterol in macrophages exposed to ox-LDL via targeting oxysterol binding protein-related protein 9 (ORP9), and as such may protect against foam cell formation [116]. Likewise, miR-146a reduces cholesterol uptake in macrophages by inhibiting
PT
TLR4 [128]. The role of miR-146a appears to be context-dependent however, as
RI
miR-146a also acts paradoxically to increase VLDL secretion from the liver via
SC
its action on sortilin-1, as shown by decreased circulating level of VLDL/LDL and reduced atherosclerosis in LdlR-/-Mir146a-/- mice[129]. Its anti-atherogenic
NU
role in dampening EC inflammation [77,78,82] and atherogenic roles in
MA
exacerbating VSMC proliferation [89] and macrophages vascular entrapment [108] further illustrate the complexity of targeting miR-146a as an anti-
TE
D
atherosclerotic therapeutic.
Esterification and efflux of cholesterol from peripheral tissues helps to limit accumulation.
EP
cholesterol
miR-223
upregulates
the
cholesterol
efflux
AC C
transporter ATP binding cassette transporter A1 (ABCA1) in macrophages, by targeting Sp3, and thus is atheroprotective [126]. Conversely, miR-33 [130], miR-144 [131], miR-758 [132], miR-27a/b [133] and miR-9-5p [134] all promote atherogenesis by inhibiting the macrophage ABCA1 to reduce cholesterol efflux and worsen lesion formation. The high degree of regulation this protein is subjected to suggests it is a key determinant in macrophage cholesterol homeostasis. miR-27a/b is also able to inhibit cholesterol efflux by
ACCEPTED MANUSCRIPT up to 45%, via reducing apoA-1 expression, as well as cholesterol esterification, by inhibiting lipoprotein lipase and acetyl-CoA acetyltransferase 1 [133]. The final step required for reverse cholesterol transport is the hepatic uptake of HDL molecules. miR-185, miR-96 and miR-223 have been shown to repress
PT
scavenger receptor class B type 1 (SR-B1) and in turn to downregulate this
RI
process [135]. Subsequently, they contribute to high circulating cholesterol
SC
levels. miR-148a, identified as a key negative regulator of hepatic LDL
NU
clearance, acts by targeting hepatic LDLR and ABCA1, and inhibition of miR148a can substantially restore hepatic LDLR expression and reduces plasma
Emerging Role of lncRNAs in Atherosclerosis
TE
7.
D
MA
LDL/HDL balance in mice [136].
EP
lncRNAs are another class of non-coding RNA defined by a sequence length
AC C
>200 nucleotides [137]. Expression of lncRNA shows considerable spatial and temporal variation with expression occurring in a tissue-specific manner and in response to stimuli [138]. To date very few lncRNAs have been fully characterized, but it is clear that they play a key role in gene expression by a variety of mechanisms [139]. For a comprehensive review on the biosynthesis and functions of lncRNAs, please refer to the review by Quinn & Chang [140].
ACCEPTED MANUSCRIPT A subset of lncRNAs possess atheroprotective effects (Table 3). lincRNA-p21 stimulates apoptosis and supresses proliferation of VSMCs by activating p53 pathway through mouse double minute 2 homolog (MDM2) disinhibition [141]. Its levels are downregulated within murine atherosclerotic plaque and the
PT
coronary arteries of patients with CAD [141]. SENCR also acts on VSMCs,
RI
stabilizing the contractile phenotype and inhibiting migration [142]. RNCR3 is
SC
upregulated in murine and human aortic atherosclerotic lesions and its inhibition, leading to decreased proliferation and increased apoptosis of ECs
NU
and VSMCs, promotes atherosclerosis development [143]. Moreover, several
MA
lncRNAs, such as MALAT1 and MANTIS, are important in angiogenesis [144,145]. MALAT1 is crucial in maintaining EC proliferation during vessel
D
growth and is significantly downregulated in human atherosclerotic plaque
TE
[144,146]. MANTIS mediates angiogenic sprouting and EC alignment and its
EP
level is found elevated in macaques with atherosclerosis regression [145].
AC C
Finally, it has been noted that HOXC-AS1 is downregulated in atherosclerotic plaques and that overexpression of HOXC-AS1 reduces ox-LDL-induced cholesterol accumulation in macrophages by activating homeobox C6 (HOXC6) [147]. MeXis also acts to reduce peripheral cholesterol accumulation by acting as a mediator to increase liver X receptors (LXRs)-dependent expression of the cholesterol efflux transporter ABCA1 in macrophages [148]. Loss of MeXis was shown to impair ABCA1 expression in mouse bone marrow cells, and these MeXis-/- bone marrow cells accelerate the development of atherosclerosis in
ACCEPTED MANUSCRIPT HFD-fed LdlR-/- mice [148]. LeXis is another lncRNA integral to LXRs’ modulation of cholesterol homeostasis. It not only promotes cholesterol efflux but also inhibits cholesterol biosynthesis in the liver by binding the ribonucleoprotein RALY, a co-factor in the transcription of several
PT
cholesterogenic genes [149]. Overexpression of LeXis, using an adeno-
RI
associated virus-8 vector with a thyroxine-binding globulin promoter in a
SC
mouse model of familial hypercholesterolemia, resulted in lower total cholesterol and triglyceride levels and a significantly reduced burden of
NU
atherosclerotic disease [150].
MA
Another subset of lncRNAs appear pro-atherogenic (Table 3). Tie-1AS, by inhibition of Tie-1, can lead to disruption of EC junctions and increases
TE
D
propensity of plaque ruptures [151]. GAS5 has been shown to be significantly increased in human atherosclerotic plaque where it promotes macrophage and
EP
EC apoptosis, the latter occurring secondary to modification of macrophage
AC C
exosome composition [152]. ANRIL and MIAT are elevated lncRNAs that were recently identified in advanced human atherosclerotic plaques [146]. ANRIL is a proven risk factor for CAD, carotid atherosclerosis and peripheral arterial disease [153–155]. Its contribution to atherosclerosis remains unclear, although the circularization of ANRIL (cANRIL) is known to alter its function in a context-dependent manner. cANRIL exerts atheroprotective effect by inducing apoptosis and decreasing VSMC proliferation [156], while it also promotes
ACCEPTED MANUSCRIPT atherogenesis by exacerbating EC inflammation and apoptosis [157]. EC-driven neovessel invasion into the intima is a well-characterized feature in advanced plaques that contribute to plaque destabilization and rupture [85]. The elevated MIAT might play an atherogenic role in advanced plaque, as it is linked to
PT
pathological angiogenesis in diabetic retinopathy in vivo [158]. SMILR is
RI
elevated in unstable human atherosclerotic plaque and promotes VSMCs
SC
proliferation by pushing their phenotypic switching to a synthetic state [159]. LINC00305 has similar action but also facilitates lipocalcin-1 interacting
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membrane receptor (LIMR) and aryl-hydrocarbon receptor repressor (AHRR)
MA
cooperation in macrophages, leading to NF-B activation and inflammation [160]. ENST00113 promotes VSMC and EC proliferation as well as survival
D
and migration by activating the PI3K/Akt/mTOR signaling pathway [161].
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RP5-833A20.1 reduces cholesterol efflux from peripheral tissues by a RP5-
EP
833A20.1/miR-382-5p/nuclear factor IA (NFIA)-dependent signal transduction
AC C
pathway and so contributes to atherogenesis by attenuating reverse cholesterol transport [162]. The discovery of GAS5 incorporation into EC apoptosispromoting exosomes postulates that lncRNAs, like miRNAs, may be able to function as extracellular messengers [152]. Overall, evidence is emerging to suggest an important role for lncRNAs in addition to legions of miRNAs in mediating several cell types in every stages in the atherosclerosis development (Figure 4),
ACCEPTED MANUSCRIPT
Table 3. lncRNAs involved in endothelial dysfunction and atherosclerosis
li &
o-
A
lncRN
genic
A
Effect
level
s
Affected
Effects on EC
al Systems
Signaling
Dysfunctions
PT
RN
Experiment Targets
RI
Ather
Pathways
SC
Stimu
Ref
and
Atherosclerosis
NU
lnc-
MA
chang es
NA
Anti-
In vitro loss
TE
linc
D
Anti-atherogenic lncRNAs MDM2
↑p300
↓proliferation &
prolife and gain of
interaction ↑apoptosis in
A-
-
-> ↑p53
function in
AC C
p21
EP
RN
rative;
ApoE-/-
Pro-
murine
apop-
VSMCs &
totic
Mϕs; in vivo mouse CA ligation
activity
VSMCs and Mϕs
[14 1]
ACCEPTED MANUSCRIPT model ↑Myocard
↓VSMC
[14
in
in &
migration
2]
HCASMCs
contractile
SEN NA
Anti-
In vitro KD
CR
migratory
NA
PT
genes, ↓
RI
migratory
CR3 LDL↑
Anti-
In vivo KD
miR-
athero- in ApoE-/genic
185-5p
mice; in vitro KD in
3]
levels,
factor release, ↑EC & VSMC
VSMCs
proliferation
EP
TE
1
↓cholesterol
↓CCNA2,
↑EC proliferation,
[14
prolife in HUVECs;
↓CCNB1,
↓EC migration,
4]
-
mouse
↓CCNB2,
↑angiogenesis
rative;
retinal
↓CDK1
Anti-
angiogenesis
↑p21
migra-
model; hind
tory;
limb
hypoxi Pro-
LAT a↑
[14
and human
In vitro KD
AC C
MA
↓atherosclerosis,
↓inflammatory
D
HUVECs
↑KLF2
NU
Ox-
MA
RN
SC
genes
NA
ACCEPTED MANUSCRIPT Pro-
ischaemia
angio-
mouse
genic
model
Pro-
In vitro KD
NTI
angio-
S
genic
↑BRG1
↑EC alignment &
[14
in HUVECs;
ATPase
↑angiogenic
5]
in vitro
activity ->
patients with
efficient
PT
BRG1
IPAH, rats
RNA
MA
e II
D
Macaca
stress
polymeras
monocrotali ne &
response to shear
NU
treated with
sprouting in
RI
NA
SC
MA
machinery binding -> ↑SOX18,
subjected to
↑SMAD6
EP
TE
fasicularis
&
AC C
atherosclero sis
↑COUP-
regression
TFII
diet ↓Ox-LDL
[14
lentivirus-
induced
7]
mediated
cholesterol
HO
Ox-
Anti-
In vitro
XC-
LDL↓
cholesterol
AS1
NA
↑HOXC6
ACCEPTED MANUSCRIPT OE in
accumulation in
human THP-
THP-1 Mϕs
1 Mϕs Anti-
In vivo LXR NA
↑ABCA1
↑cholesterol
[14
is
chole-
activation in
expression efflux from
8]
sterol
WT, Lxr-/-
PT
MeX NA
RI
peripheral tissues
SC
and Lxr-/mice; In
NU
vitro KD in
Mϕs
D
peritoneal
MA
mouse
↓total cholesterol
[14
in a mouse
n of
and triglyceride
9,15
model of
RALY
levels &
FH; HFD-
with DNA
↓atherosclerotic
fed LeXis-/-
-> altered
burden in vivo
mice
transcripti
TE
↓interactio
Anti-
In vivo OE
s
cholesterol
AC C
EP
LeXi NA
RALY
on of cholestero l-genic
0]
ACCEPTED MANUSCRIPT genes ↑apoptosis &
[15
in PASMCs
ase-
↓proliferation in
6]
and Mϕs
mediated
VSMCs and Mϕs
In vitro OE
apoptotic;
PES1
Anti-
pre-rRNA
proli-
processing
ferativ
and
PT
RIL
↓exonucle
Pro-
RI
NA
SC
cAN
e
ribosome
NU
biogenesis
D
MA
->
↑nucleolar stress &
TE
↑p53
EP
activation
TIE- NA 1AS
AC C
Pro-atherogenic lncRNAs
Pro-
In vivo
Tie-1
plaque upregulation mRNA ruptur
in
e
embryonic zebrafish; in vitro
↓Tie-1
Defects in
[15
transcript
endothelial cell
1]
contact junctions
ACCEPTED MANUSCRIPT upregulation in HUVECs
5
LDL↑
↑Caspases
↑ Mϕ apoptosis,
[15
in human
in Mϕs,
↑EC apoptosis
2]
THP-1 Mϕs
altered
Pro-
In vitro OE
apoptotic
NA
PT
GAS Ox-
RI
Mϕ
SC
exosome
compositi
NU
on
AN RIL
Proapop-
In vivo OE
EP
ular
NA
and low
AC C
circ
TE
D
MA
including ↑GAS5 incorporat ion
NA
↑BAX,
↑EC apoptosis,
[15
↑caspase-
↑TC, ↑LDL, ↑TG,
7]
3, ↓bcl-2
↓HDL, ↑IL-1,
totic;
expression
Pro-
in Wistar
↑IL-6, ↑MMP,
chole-
rats injected
↑CRP
sterol;
with a high
Pro-
dose of
inflam
vitamin D3
ACCEPTED MANUSCRIPT -
and fed a
matory high-fat diet MIA NA
Pro-
In vivo KD
miR-
T
patho-
in male
150-5p
logical
Sprague-
angio-
Dawley rats
genesi
with
s
streptozotoc
↑VEGF
↑DM-induced
[15
retinal
8]
PT
microvascular
RI
dysfunction in
KD in
D
HUVECs
MA
DM; in vitro
SMI NA
Pro-
In vitro KD
LR
prolife in IL-1α and
NA
proliferation, migration and tube formation in vitro
↑HAS2
↑VSMC
[15
expression proliferation
9]
↑LIMR &
↑Monocyte
[16
inflammation ->
0]
EP
TE
NU
in-induced
SC
vivo, ↑EC
PDGF
AC C
-rative
stimulated HSVVSMC s
Pro-
In vitro OE
C00
inflam
in human
AHRR
305
-
THP-1 Mϕs
cooperatio HASMC
LIN
NA
NA
ACCEPTED MANUSCRIPT matory with
n ->
switching from
HASMC co-
↑AHRR
contractile to
culture
activation
proliferative
-> ↑NF-
phenotype
PT
B in
↑PI3K/Ak
↑VSMC and EC
[16
t/mTOR
proliferation,
1]
63ignallin
survival and
VSMCs and
g pathway
migration
HUVECs
activation
In vitro
T00
prolife down-
113
-
regulation in
rative; Pro-
D
TE
migra-
LDL↑,
833
Ac-
In vitro OE
AC C
-
Pro-
EP
tory Ox-
NA
NU
Pro-
MA
ENS NA
RP5
SC
RI
monocyte
NA
↑miR-
↑inflammation
[16 2]
inflam
in human
382-5p ->
and disturbance
-
THP-1 Mϕs;
↓NF1A
to normal
A20. LDL↑
matory In vivo
expression cholesterol
1
; Pro-
NF1A OE
->↑IL-1B,
homeostasis in
chole-
in ApoE-/-
↑IL-6,
Mos; NF1A
sterol
mice
↑TNF-α,
overexpression ->
ACCEPTED MANUSCRIPT ↑CRP
↑HDL, ↓LDL, ↓VLDL, decreased inflammatory
PT
cytokines and
RI
promoted
regression in ApoE-/- mice
MA
NU
SC
atherosclerotic
Table 3. lncRNAs involved in endothelial dysfunction and atherosclerosis.
D
Abbreviations: ->, leads to; ↑, upregulation; ↓, downregulation; ABCA1, ATP-
TE
binding cassette transporter; Ac-LDL, acetylated low-density lipoprotein;
EP
AHRR, aryl-hydrocarbon receptor repressor; Akt, protein kinase B; ApoE,
AC C
apolipoprotein E; BRG1, brahma related gene 1; CA, carotid artery; CAD, coronary artery disease; CCN, cyclin; CDK1, cyclin dependent kinase 1; COUP-TFII, chicken ovalbumin upstream promoter transcription factor 2; CRP, C-reactive protein; diabetes mellitus, DM; EC, endothelial cell; FH, familial hypercholesterolemia; HAS2, hyaluronan synthase 2; HASMC, human arterial smooth muscle cell; HCASMC, human coronary artery smooth muscle cells; HDL, high-density lipoprotein; HFD, high-fat diet; HOXC6, homeobox C6; HUVEC, human umbilical vein endothelial cell; HSVVSMC, human saphenous
ACCEPTED MANUSCRIPT vein vascular smooth muscle cell; IL, interleukin; IPAH, idiopathic pulmonary arterial hypertension; KD, knock down; KLF, Kruppel like factor; LDL, lowdensity lipoprotein; LIMR, lipocalcin-1 interacting membrane receptor; LXR, liver X receptor; Mϕ, macrophage; MDM2, mouse double minute 2; miR,
PT
microRNA; MMP, matrix metalloproteinase; mRNA, messenger RNA; mTOR,
RI
mechanistic target of rapamycin; NA, not assessed; NF1A, nuclear factor 1A;
SC
NF-B, nuclear factor B; OE, over expression; ox-LDL, oxidized low-density lipoprotein; PASMC, primary arterial smooth muscle cell; PDGF, platelet
NU
derived growth factor; PES1, pescadillo homologue 1; PI3K, phosphoinositide 3
MA
kinase; RNA, ribonucleic acid; SOX18, SRY related HMG box 18; TC, total cholesterol; TG, triglyceride; THP-1, Tamm-Horsfall protein 1; Tie-1, tyrosine
D
kinase with immunoglobulin-like and EGF-like domains 1; TNF, tumour
TE
necrosis factor ; VEGF, vascular endothelial growth factor; VLDL- very low-
AC C
EP
density lipoprotein; VSMC, vascular smooth muscle cell.
ACCEPTED MANUSCRIPT EC activation and inflammation miRs-10a*, -30-5p*, -143/145*, 146a*, 181b*, -100, Let-7a -7b -7g miRs-34a*, -92a*, -712/205*, -103, -155, Tie-1AS, circANRIL
Erythrocyte
LUMEN Angiogenesis miRs-126-5p*, MALAT1, MANTIS
miRs -21*
Monocyte
miR-92a*, MIAT
EC apoptosis miRs-21*, -155* miRs-181a, -351, GAS5, cANRIL
Macrophage Macrophage activation miRs-93, -124, -223, -125a, -5p, -146a, -10a, -147, -124a, -150, let-7c
Foam cell Synthetic VSMC VSMC proliferation and migration miRs-22-3p, -125b, -24, -29a, -663, -638, -133, -424, -22, -195, -let-7, lincRNA-p21, SENCR, RNCR3, circANRIL
miRs-126-3p* , -146a VSMC survival in fibrous cap miRs-181b, -210, -21
miRs -122
Blood vessel
INTIMA
Basal lamina
miRs -146a
NU
miRs-136, -221, -26a, -146a, SMILR, LINC00305, ENST00113
Cholesterol accumulation and foam cell formation miRs-10a, -223, -30c, -125a-5p, HOXC-AS1, LeXis, MeXis
Endothelium
MA
Figure 4. Non-coding RNAs (ncRNAs) implicated in the atherosclerotic
D
processes. miRNAs and lncRNAs that participate in various cell types and
TE
atherosclerotic processes are summarized. ncRNAs that are atheroprotective (in green frame), atherogenic (in red frame) or with contradictory evidence (in blue
EP
frame) are shown, while those that are flow-responsive are denoted by *. Disturbed flow leads to endothelial dysfunction and flavours intimal entry of
AC C
Vesicle
PT
Ox-LDL
Cholesterol efflux and reverse transport miR-223 miRs-9-5p, -27a/b, -33, -96, -144, 148a, -185, -223, -758, RP5-883A20.1
RI
miRNA-mediated intracellular communication miR-143/145*
HDL
SC
miRs-33, -142-5p, LINC00305
LDL
oxidized LDL (ox-LDL), which triggers a low-grade inflammatory response including expression of leukocyte adhesion molecules by endothelial cells. The initial stages of atherosclerosis include adhesion of blood monocyte to the activated endothelium, their maturation into macrophage and their activation and uptake of ox-LDL to form macrophage-derived foam cells. Cholesterol efflux mechanisms, aided by HDLs and other factors, help to regress lesion
MEDIA
ACCEPTED MANUSCRIPT development. Plaque progression involves the dedifferentiation, proliferation and migration of VSMCs into the intima where they deposit extracellular matrix protein including collagen. Advanced lesions rely on the survival and proliferation of fibrous cap VSMCs to stabilize the vulnerable plaques from
PT
rupture. LDL, low-density lipoprotein, HDL, high-density lipoprotein; VSMC,
Prospective Clinical Applications of miRNAs in Atherosclerosis
NU
8.
SC
RI
vascular smooth muscle cell.
MA
The changes that occur in miRNA expression, and the functional importance of these changes during atherogenesis, offer enormous clinical potential for
D
monitoring and modulation. miRNAs are measurable in the serum and offer the
TE
benefits of high stability, a wide dynamic range of expression and integration of
EP
biology from the entirety of the vascular system [163]. As such, they make
AC C
promising biomarkers that could facilitate non-invasive early diagnosis, prognosis, guidance of treatments based on risk stratification and follow-up of treatment response. An earlier study found miR-126, miR-17 and miR-92a to be significantly downregulated in the plasma of patients with CAD compared to healthy controls [164]. Others have identified reduced circulating level of 11 miRNAs (miR-19a, -484, -155, -222, -145, -29a, -378, -342, -181d, -150, -30e5p) in patients with angiographically-defined stable CAD and in control subjects with at least 2 cardiac risk factors, implicating that this set of miRNAs
ACCEPTED MANUSCRIPT might be associated with subclinical atherosclerosis [165]. Circulating level of miR-30c-5p were also elevated in subjects with plaques, and is inversely correlated with the LDL level, intimal thickening and plaques severity [28]. Furthermore, the feasibility of combining next generation imaging with
PT
transcoronary miRNA uptakes from blood taken during routine cardiac
RI
catheterization could predict the presence and the severity of vulnerable plaques
SC
in patients with CAD [55]. However, several challenges have hitherto impeded circulating miRNAs as clinical biomarkers for CAD. These include the reliance
NU
of miRNA measurements on PCR with a lack of valid reference miRNA
MA
controls currently available; the difficulty in determining the cell-type origins of many circulating miRNAs; and the effects of current CAD treatments (e.g.
D
platelet inhibitors and heparin) which may disturb accurate miRNA
TE
measurements [166]. To overcome these challenges, large-scale studies using
EP
carefully-stratified patient groups and technical improvements in miRNA
AC C
measurement are needed.
Due to their roles in pre-translational protein suppression, manipulation of miRNA expression could be leveraged for therapeutic benefits. So far two miRNA-based therapies have reached clinical trials: a miR-34 mimic (MRX34) for treatment of advanced liver cancer and a miR-122 antagonist (Miravirsen) for treatment of hepatitis C [167]. Systemic delivery of microRNA-derived agents are mostly taken up in the liver, kidney, and spleen [168], which implies
ACCEPTED MANUSCRIPT targeting the heart or vasculature with miRNA-based therapies might require significantly higher dosing with lower efficiency. Previous efforts focussed on improving antisense oligonucleotides (ASO) bioavailability by linking ASO to cholesterol for enhanced uptake (antagomiRs) or increase linkage within the
PT
RNA nucleotides to augment efficiency (LNA-anti-miRs) [169]. Recent
RI
development strategies have turned to enhance tissue uptakes by cell type-
SC
specific enrichment of miR-mimic/anti-miRs delivery in order to reduce the therapeutic dose and off-target adverse effects. Adenoviral vectors have been
NU
the methods of choice for functional miRNAs/anti-miRNAs delivery, but there
MA
are concerns with immunogenicity against viral vectors leading to toxicity and limited efficacy upon repeat administration [170]. Several technologies have
D
been developed to allay these concerns: targeted vasculature delivery strategies
TE
through local delivery by devices (drug eluting devices and catheter-based
EP
delivery) [171] or light-activated plasmonic nanocarrier [172], specific
AC C
nanoparticle carriers (9 um poly-d, -lactide-co-glycolide) that preferentially target the vasculature [173], and EC-specific aptamer-based direct delivery [174,175]. Aptamers targeting EC-specific transferrin receptor was designed to deliver miR-126 precursors specifically to ECs, which were processed and repressed known miR-126 target VCAM-1 and improved EC-directed angiogenesis in vitro [174]. Notably, a E-Sel-targeting thioaptamer-multistage vector linked with miRNAs-enriched microparticles can specifically deliver EC protective miRNAs (miR146a and miR-181b) to ECs in vivo, successfully
ACCEPTED MANUSCRIPT inhibited endothelial expression of their target CCL2, and repressed inflammation and atherosclerosis in HFD-fed ApoE-/- mice [175]. Paradoxically, the diametric miRNA expression between vascular cells can be leveraged for target gene delivery. Adenoviral vector encoding the anti-proliferation gene
PT
p27Kip1, which are embedded with target sequences for miR-126-3p (a miRNA
RI
that is enriched in ECs but absent in VSMCs), can specifically target VSMC
SC
proliferation and neointimal hyperplasia while maintaining ECs proliferation and reendothelialization (due to adenovirus inactivation by endogenous miR-
NU
126-3p) in rat carotid artery injury [176,177]. In future, development of novel
MA
tools in targeting a defined miRNA-mRNA interactions using target-sitespecific rather than miRNA-specific ASOs, will be useful in modulating a
TE
D
specific miRNA function without affecting the entire regulatory network. Conversely, strategies in targeting lncRNAs are largely similar to those
EP
targeting mRNAs, i.e. inhibition by ASO-linked GapmeRs and overexpression
AC C
by viral vectors or synthetic modified RNAs. Notably, GapmeRs targeting lncRNAs in cardiovascular system have been successfully applied in vivo: GapmeR targeting MALAT-1 to inhibit ischemia-induced angiogenesis [144] and GapmeR targeting Chast that affect cardiac remodelling [178]. In conclusion, these novel miRNA-based tests and ncRNA interventions are still in their nascent stages, but they certainly hold considerable promises for
ACCEPTED MANUSCRIPT translation into clinical tools that will aid the early diagnosis and treatment of
AC C
EP
TE
D
MA
NU
SC
RI
PT
atherosclerosis.
ACCEPTED MANUSCRIPT Funding This work was supported by British Heart Foundation project grant PG13-6330419.
PT
Disclosures
AC C
EP
TE
D
MA
NU
SC
RI
None.
ACCEPTED MANUSCRIPT References [1]
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EP
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TE
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AC C
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