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Long non-coding RNAs at the crossroad of vascular smooth muscle cell phenotypic modulation in atherosclerosis and neointimal formation

  • Francesca Fasolo
    Correspondence
    Corresponding author. Experimental Vascular Medicine Unit, Department for Vascular and Endovascular Surgery, Klinikum Rechts der Isar, Technical University Munich, Ismaninger Strasse 22, 81675, Munich, Germany.
    Affiliations
    Department for Vascular and Endovascular Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance (MHA), Berlin, Germany
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  • Valentina Paloschi
    Affiliations
    Department for Vascular and Endovascular Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance (MHA), Berlin, Germany
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  • Lars Maegdefessel
    Affiliations
    Department for Vascular and Endovascular Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance (MHA), Berlin, Germany

    Molecular Vascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
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Open AccessPublished:December 06, 2022DOI:https://doi.org/10.1016/j.atherosclerosis.2022.11.021

      Highlights

      • Vascular smooth muscle cells (VSMCs) are highly plastic cells responsible for vessel architecture and contractility.
      • VSMCs switch to a synthetic and hyperproliferative phenotype is a major hallmark of atherogenesis and plaques buildup.
      • Long non-coding RNAs (lncRNAs) are major contributors of VSMCs phenotypic modulation.
      • LncRNAs may serve as therapeutic targets in atherosclerosis and biomarkers for plaque stability.

      Abstract

      Despite extraordinary advances in the comprehension of the pathophysiology of atherosclerosis and the employment of very effective treatments, cardiovascular diseases are still a major cause of mortality and represent a large share of health expenditure worldwide. Atherosclerosis is a disease affecting the medium and large arteries, which consists of a progressive accumulation of fatty substances, cellular waste products and fibrous elements, which culminates in the buildup of a plaque obstructing the blood flow. Endothelial dysfunction represents an early pathological event, favoring immune cells recruitment and triggering local inflammation. The release of inflammatory cytokines and other signaling molecules stimulates phenotypic modifications in the underlying vascular smooth muscle cells, which, in physiological conditions, are responsible for the maintenance of vessels architecture while regulating vascular tone. Vascular smooth muscle cells are highly plastic and may respond to disease stimuli by de-differentiating and losing their contractility, while increasing their synthetic, proliferative, and migratory capacity. This phenotypic switching is considered a pathological hallmark of atherogenesis and is ruled by the activation of selective gene programs. The advent of genomics and the improvement of sequencing technologies deepened our knowledge of the complex gene expression regulatory networks mediated by non-coding RNAs, and favored the rise of innovative therapeutic approaches targeting the non-coding transcriptome. In the context of atherosclerosis, long non-coding RNAs have received increasing attention as potential translational targets, due to their contribution to the molecular dynamics modulating the expression of vascular smooth muscle cells contractile/synthetic gene programs. In this review, we will focus on the most well-characterized long non-coding RNAs contributing to atherosclerosis by controlling expression of the contractile apparatus and genes activated in perturbed vascular smooth muscle cells.

      Graphical abstract

      Keywords

      1. Role of vascular smooth muscle cells in atherosclerosis

      Atherosclerosis is a chronic inflammatory disease resulting in the thickening or hardening of medium and large arteries caused by a plaque buildup within the arterial wall. The plaques, containing lipids, cells, extracellular matrix (ECM) and apoptotic cells debris, can progressively narrow the blood vessel lumen and plaque rupture, followed by thrombi formation, can obstruct blood flow with fatal consequence to the supplied organ. The most relevant sites for clinically significant consequences of atherosclerosis in humans are the coronary and the carotid arteries, where an atherothrombotic event can lead to myocardial infarction (MI) and stroke, respectively [
      • VanderLaan P.A.
      • Reardon C.A.
      • Getz G.S.
      Site specificity of atherosclerosis: site-selective responses to atherosclerotic modulators.
      ]. Altogether, cardiovascular diseases (CVD) due to atherosclerosis, remain a leading cause of mortality worldwide (https://www.who.int).
      In healthy arteries, the intima layer of the vessel wall is represented by a monolayer of endothelial cells (ECs) that guard vascular homeostasis by regulating permeability, preventing platelet activation and blood clotting, as well as adherence/infiltration of leukocytes [
      • Mussbacher M.
      • Schossleitner K.
      • Kral-Pointner J.B.
      • Salzmann M.
      • Schrammel A.
      • Schmid J.A.
      More than just a monolayer: the multifaceted role of endothelial cells in the pathophysiology of atherosclerosis.
      ]. Below the endothelium, layers of vascular smooth muscle cells (VSMCs), which constitute the major cell type of medium- and large-sized arteries, are responsible for maintaining vessel wall integrity and functionality (Fig. 1A). By driving the contraction of the vascular wall, VSMCs regulate the diameter of the blood vessel lumen and contribute to the regulation of blood pressure [
      • Brozovich F.V.
      • Nicholson C.J.
      • Degen C.V.
      • Gao Y.Z.
      • Aggarwal M.
      • Morgan K.G.
      Mechanisms of vascular smooth muscle contraction and the basis for pharmacologic treatment of smooth muscle disorders.
      ]. VSMCs-derived elastin is essential for ensuring the arterial wall to stretch and expand upon the pressure exerted on the vasculature and the return to its normal size (elastic recoil), that pushes the blood forward. Furthermore, VSMCs produce collagen fibers, which provide tensile forces to resist high blood pressure [
      • Xu J.
      • Shi G.P.
      Vascular wall extracellular matrix proteins and vascular diseases.
      ].
      Early pro-atherogenic events involve perturbances of the vascular ECs homeostasis. By undergoing endothelial to mesenchymal transition (endMT), ECs loose cell-cell contacts and the capacity to act as a first barrier to the circulating leucocytes and lipoproteins. Consequently, in the subendothelial space of the intimal vessel wall layer, blood-derived monocytes begin to engulf atherogenic lipoproteins, differentiate into macrophages or dendritic cells and finally become foam cells, forming early atherosclerotic lesions. VSMCs are key participants throughout all stages of atherosclerosis, as they contribute to the structural modifications of the architecture of arterial walls by interacting with both activated ECs and recruited immune cells [
      • Hu D.
      • Yin C.
      • Luo S.
      • Habenicht A.J.R.
      • Mohanta S.K.
      Vascular smooth muscle cells contribute to atherosclerosis immunity.
      ] (Fig. 1B). These are highly plastic cells that can acquire different properties via phenotypic modulation. Across the different phases of atherosclerosis development, populations of VSMCs can switch from a contractile to a synthetic phenotype, a process accompanied by the loss of contractile markers (e.g., ACTA2, MYH11, SM22) and the acquisition of migratory faculties. At the molecular level, both activated ECs (endMT) and macrophages in the neointima lesion produce platelet-derived growth factor (PDGFBB) [
      • Jawien A.
      • Bowen-Pope D.F.
      • Lindner V.
      • Schwartz S.M.
      • Clowes A.W.
      Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty.
      ], which acts as a chemoattractant for VSMCs, which become highly proliferative and invade the newly formed lesions from the media layer of vessel wall (Fig. 1B) [
      • Newby A.C.
      • Zaltsman A.B.
      Fibrous cap formation or destruction - the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation.
      ].
      VSMCs regulate the progression of early pathological intima thickening into atherosclerotic plaques by acquiring detrimental phenotypic routes. The uptake of modified cholesterol and lipoproteins by VSMCs drive the phenotypic switch to macrophage-like cells expressing macrophage markers (LGALS3, CD68) and producing pro-inflammatory cytokines (MCP1). Studies based on specific labeling of VSMCs in mice, as well as transcriptomic studies of human atherosclerotic plaques, have revealed that the majority of foam cells in atherosclerotic plaques seem to derive from VSMCs [
      • Grootaert M.O.J.
      • Bennett M.R.
      Vascular smooth muscle cells in atherosclerosis: time for a re-assessment.
      ]. While clearance of lipid species may be beneficial at first, the parallel secretion of pro-inflammatory mediators amplifies the recruitment of inflammatory cells and consequently induce VSMCs apoptosis. These events, from a clinical perspective, contribute to the formation of a necrotic core inside the plaque (Fig. 1C). At later disease stages, the different populations of VSMCs are crucial in determining the composition and the stability of the atherosclerotic plaque. The presence of synthetic VSMCs producing a layer of fibrous connective tissue (fibrous cap) on the plaque is regarded a lesion-stabilizing event. Conversely, VSMCs developing an osteochondrogenic phenotype, typically in more advanced atherosclerosis stages, are responsible for the production of calcified deposits triggering vascular calcification and exacerbating local inflammation, which ultimately endangers plaque stability [
      • Durham A.L.
      • Speer M.Y.
      • Scatena M.
      • Giachelli C.M.
      • Shanahan C.M.
      Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness.
      ].

      2. In the quest for novel anti-atherosclerotic therapeutic approaches exploiting the non-coding transcriptome

      Although conventional therapies in the treatment of atherosclerosis that rely on targeting the cholesterol (mostly enzyme or receptors inhibitors) have shown great effectiveness in reducing the burden of CVD onset, in the last years we assisted to a great effort in researching alternative therapeutic strategies [
      • Libby P.
      • Everett B.M.
      Novel Antiatherosclerotic therapies.
      ]. The ability to ad hoc modulate VSMC phenotypic switch provides a challenging and promising approach to prevent atherosclerosis progression and/or stabilize atherosclerotic lesions already formed.
      A major role in the phenotypic VSMC switching is exerted by transcriptome regulation. In the past decades, advances in sequencing technologies allowed a developing awareness of the central role of RNA in affecting the molecular processes underlying gene expression. Genomic studies revealed that only 1–2% of the transcriptionally active regions in mammalian genomes correspond to protein-coding genes [
      • Carninci P.
      • Kasukawa T.
      • Katayama S.
      • Gough J.
      • Frith M.C.
      • Maeda N.
      • Oyama R.
      • Ravasi T.
      • Lenhard B.
      • Wells C.
      • et al.
      The transcriptional landscape of the mammalian genome.
      ,
      • Feingold E.A.
      • Good P.J.
      • Guyer M.S.
      • Kamholz S.
      • Liefer L.
      • Wetterstrand K.
      • Collins F.S.
      • Gingeras T.R.
      • Kampa D.
      • Sekinger E.A.
      • et al.
      The ENCODE (ENCyclopedia of DNA elements) Project.
      ]. The discovery of a previously underestimated number of non-coding RNAs (ncRNAs), which did not template protein synthesis, challenged the concept of RNA being a bare messenger between DNA and proteins. Besides some previously identified and characterized structural RNA species (i.e. ribosomal RNA -rRNA-, transfer RNA –tRNA-), other regulatory ncRNAs, including small and long non-coding RNAs (lncRNAs), were isolated and studied.
      LncRNAs are defined as transcripts exceeding 200 nucleotides (nts) in length, which are usually not translated into proteins [
      • Rinn J.L.
      • Chang H.Y.
      Genome regulation by long noncoding RNAs.
      ]. Structurally, they share some basic features with mRNAs, like the presence of 5′caps and polyA tails, and they are transcribed by Polimerase II [
      • Rinn J.L.
      • Chang H.Y.
      Genome regulation by long noncoding RNAs.
      ]. When spliced from the antisense (AS) strand to a protein-coding gene, they are referred to as natural antisense transcripts (NATs), which have been extensively acknowledged as key regulators of the host gene expression [
      • Katayama S.
      • Tomaru Y.
      • Kasukawa T.
      • Waki K.
      • Nakanishi M.
      • Nakamura M.
      • Nishida H.
      • Yap C.C.
      • Suzuki M.
      • Kawai J.
      • et al.
      Antisense transcription in the mammalian transcriptome.
      ,
      • Pelechano V.
      • Steinmetz L.M.
      Gene regulation by antisense transcription.
      ]. LncRNAs represent the widest and most heterogeneous class of ncRNAs shaping the genome's architecture and controlling splicing, transcription, translation and RNA/protein degradation [
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • Huarte M.
      Gene regulation by long non-coding RNAs and its biological functions.
      ]. Thanks to an extreme diversity in their modes of action, which are yet to be fully elucidated, they can both repress and enhance the expression of target genes, as well as inhibit or potentiate the activity of protein partners. Regardless of their specific functional features, lncRNAs share a domain organization, exploited to recruit and coordinate the activity of multiple effectors, like protein cofactors or direct-interacting nucleic acids [
      • Guttman M.
      • Rinn J.L.
      Modular regulatory principles of large non-coding RNAs.
      ].
      An increasing number of studies are unveiling cardiovascular disease (CVD)-associated lncRNAs, thus extending the druggable genome to regulatory regions, and not exclusively to protein coding genes [
      • Yeh C.-F.
      • Chang Y.-C.E.
      • Lu C.-Y.
      • Hsuan C.-F.
      • Chang W.-T.
      • Yang K.-C.
      Expedition to the missing link: long noncoding RNAs in cardiovascular diseases.
      ]. A deeper comprehension of their contribution to cardiovascular dysfunction, as well as of their further mechanisms of action represents a crucial point in conceiving novel therapeutic strategies.
      The aim of this review is to describe the state of the art of lncRNAs research applied to the study of atherosclerosis development and progression, with a special focus on their role in orchestrating the distinct phenotypes that VSMCs acquire throughout different disease stages (Table 1 and Fig. 1).
      Table 1Function and mechanism of action of lncRNAs regulating atherosclerosis.
      lncRNAOrganismExperimentally validate functionMechanism of action
      ANRILhumanassociated to the cardiovascular disease locus 9p21.3; up-regulated in atherosclerosisscaffold, mediates TF binding to promoter
      SENCRhumanmaintenance of VSMCs contractile phenotypemicroRNA sponge
      MYMSLhumansignature of VSMCs contractile phenotype; contains MYOCD-binding elementsunknown
      CARMNhuman, mousemaintenance of VSMCs contractile phenotypescaffold, mediates TF binding to promoter
      MYOSLIDhumanmaintenance of VSMCs contractile phenotypea scaffold, mediates TF binding to promoter
      NR2F1-AS1human, mouseinduced upon VSMCs differentiationunknown
      FOXC2-AS1human, mouseinduced upon VSMCs differentiation; regulation of VSMCs survival via Notch pathwayunknown
      GAS5human, mousenegatively regulates VSMCs differentiation; counteracts vascular calcificationprotein sponge; miRNA sponge
      NEAT1humannegatively regulates VSMCs differentiation; triggers proliferation and migrationprotein sponge
      MIAThuman, mouseregulates phenotypic switching by interacting with KLF4; triggers proliferation and migrationscaffold, interacts with TFs
      LncRNA 430945humantriggers proliferationunknown
      LincRNA-p21mousetriggers proliferationscaffold, mediates TF binding to promoter
      KCNQ1OT1mouseprotects from intimal hyperplasiaprotein sponge; miRNA sponge
      Lnc-Ang362humaninduced by angiotensin II; triggers proliferationprovides the host transcript for miR-221 and miR-222
      GIVERhuman, mouse, ratmediates Ang II-induced oxidative stressinteracts with chromatin remodeling complex
      SMILRhumanup-regulated in unstable plaques; triggers proliferationunknown
      Nronmouseup-regulated in unstable plaques; triggers apoptosisprotein sponge
      HAS2-AS1humanstabilizes HAS2 mRNA and increases HA synthesisscaffold; RNA:RNA interactions
      Lrrc75a-as1ratcounteracts vascular calcificationunknown
      Fig. 1
      Fig. 1Summary of the role and mechanism of action of lncRNAs regulating vascular smooth muscle cells (VSMCs) phenotype in different atherosclerosis stages.
      (A) VSMCs are organized in layers forming the tunica media of healthy arteries (top-left). LncRNAs enabling the expression of the contractile apparatus (MYOSLID, CARMN and SENCR) or induced upon VSMCs differentiation (NR2F1-AS1, FOXC2-AS1 and Mimsl) are represented on the top-right. MYOSLID and CARMN act as scaffolds favoring the recruitment and mediating the binding of transcription factors (TF) at promoters of contractile genes, thanks to the presence of TF-binding modules; SENCR, sponges miRNAs targeting genes regulating VSMCs contractility; NR2F1-AS1 and FOXC2-AS1 are conserved lncRNAs induced upon VSMCs differentiation and promoting VSMCs survival; Mimsl represents a non-coding signature contractility in murine VSMCs. (B) Early stages of atherosclerosis are characterized by VSMCs de-differentiation and switch to a hyperproliferative and migratory phenotype, triggering vessel remodeling and plaque formation (middle-left). LncRNAs inhibiting the expression of the contractile apparatus are indicated in blue and include Gas5 and NEAT1, which, respectively, sponge and sequester TFs or chromatin modifiers enabling the expression of VSMCs contractility-mediating genes. ANRIL functions as a scaffold guiding the positioning of Polycomb complex to Alu-containing promoters of target genes (controlling proliferation and apoptosis) by the presence of highly homologous Alu elements within its sequence. LncRNAs MIAT and lincp21 work as scaffolds at proliferation and apoptosis-related gene promoters, respectively. Increased expression of the former and decreased expression of the latter contributes to VSMCs hypeproliferative state. A similar functional output is exerted by lncRNA 430945 via an unknown mechanism. LncRNA Ang362 is induced by cardiovascular (CVD)-mimicking drug Angiotensin II (Ang II) and provides the host transcript for miR-221 and miR-222, which are proposed mediators of VSMCs function. GIVER is also induced by Ang II and interacts with chromatin remodeling complexes, mediating Ang II-induced oxidative stress response. KCNQ1OT1 suppressed VSMCs proliferation, migration and secretion of inflammatory factors by two alternative mechanisms: (i) by sponging miR-221, which targets IκBa mRNA and (ii) by binding IκBa protein and inhibiting its degradation. (C). Advanced plaques are characterized by the presence of a necrotic core, containing fibrous material, calcium deposits, blood clots and apoptotic cell debris, which can rapture and obstruct the blood flow (bottom-left). The presence of the VSMCs-synthesized fibrous cap contributes to lesions' stability. Furthermore, overproduction of hyaluronic acid (HA) may worsen plaques' vulnerability. Targeting lncRNAs up-regulated in advanced atherosclerotic plaques, as SENCR, NRON and HAS2-AS1 (bottom-right), can provide a therapeutic strategy to limit disease progression. SENCR regulates VSMCs proliferation, and its knock down (KD) would limit the detrimental effects of late stent thrombosis. Nron sequesters NFATC3 in the cytosol, thus holding proliferation in murine VSMCs, ultimately leading to plaque instability. HAS2-AS1 triggers HA synthase 2 (HAS2) expression by stabilizing HAS2 mRNA via duplex formation. An alternative therapeutic approach could be increasing the expression of lncRNAs counteracting vascular calcification, like GAS5 and Lrrc75a-as1, both induced upon stimulation with inorganic phosphate. GAS5 sponges miR-26-5p in human VSMCs, determining increase of the phosphatase and tensin homolog (PTEN). Lrrc75a-as1 decreases the expression of osteoblast-related factors [
      • Jeong G.
      • Kwon D.-H.
      • Shin S.
      • Choe N.
      • Ryu J.
      • Lim Y.-H.
      • Kim J.
      • Park W.J.
      • Kook H.
      • Kim Y.-K.
      Long noncoding RNAs in vascular smooth muscle cells regulate vascular calcification.
      ].

      3. ANRIL and the discovery of the coronary artery disease risk genotype

      The Chr9p21 locus harbours a cluster of five genes including the tumour suppressors cyclin dependent kinase inhibitor CDKN2A/p16INK4A, CDKN2A/p14ARF, CDKN2B/p15INK4B, the 3.8 kb lncRNA ANRIL (which overlaps the entire CDKN2B/p15INK4B gene in AS orientation and is thus also referred to as CDKN2B antisense RNA, CDKN2B-AS1), and methylthioadenosine phosphorylase (MTAP) [
      • Matarin M.
      • Brown W.M.
      • Singleton A.
      • Hardy J.A.
      • Meschia J.F.
      • investigators I.
      Whole genome analyses suggest ischemic stroke and heart disease share an association with polymorphisms on chromosome 9p21.
      ]. Genome-wide association studies (GWAS), which have significantly contributed to relate coronary artery disease (CAD) risk inheritance to specific genetic profiles, pinpointed a single-nucleotide polymorphism (SNP) in the Chr9p21 locus conferring cardiovascular risk. The SNP rs1333049, in particular, falls within an intron of ANRIL (48). This lncRNA presents more than 20 linear splicing variants, as well as multiple circular isoforms (www.ensembl.org). Linear ANRIL isoforms containing the proximal and distal exons were the most prevalent ones in patients carrying the CAD-risk allele, and, noteworthy, levels of ANRIL in plaques, circulating peripheral blood monocytes (PBMCs) or whole blood correlated with atherosclerosis severity [
      • Holdt L.M.
      • Beutner F.
      • Scholz M.
      • Gielen S.
      • Gabel G.
      • Bergert H.
      • Schuler G.
      • Thiery J.
      • Teupser D.
      ANRIL expression is associated with atherosclerosis risk at chromosome 9p21.
      ,
      • Congrains A.
      • Kamide K.
      • Oguro R.
      • Yasuda O.
      • Miyata K.
      • Yamamoto E.
      • Kawai T.
      • Kusunoki H.
      • Yamamoto H.
      • Takeya Y.
      • et al.
      Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B.
      ,
      • Arslan S.
      • Berkan O.
      • Lalem T.
      • Ozbilum N.
      • Goksel S.
      • Korkmaz O.
      • Cetin N.
      • Devaux Y.
      • Cardiolinc n
      Long non-coding RNAs in the atherosclerotic plaque.
      ]. Jarinova et al. [
      • Jarinova O.
      • Stewart A.F.
      • Roberts R.
      • Wells G.
      • Lau P.
      • Naing T.
      • Buerki C.
      • McLean B.W.
      • Cook R.C.
      • Parker J.S.
      • et al.
      Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus.
      ] reported that PBMCs presenting the aforementioned SNP were characterized by ANRIL expression, and no relevant differences in terms of CDKN2A or CDKN2B mRNA levels could be detected, following comparison with other genotypes. In line with this, a strong genotype/expression correlation was identified for ANRIL and not for CDKN2B in larger patient cohorts [
      • Holdt L.M.
      • Beutner F.
      • Scholz M.
      • Gielen S.
      • Gabel G.
      • Bergert H.
      • Schuler G.
      • Thiery J.
      • Teupser D.
      ANRIL expression is associated with atherosclerosis risk at chromosome 9p21.
      ,
      • Holdt L.M.
      • Hoffmann S.
      • Sass K.
      • Langenberger D.
      • Scholz M.
      • Krohn K.
      • Finstermeier K.
      • Stahringer A.
      • Wilfert W.
      • Beutner F.
      • et al.
      Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.
      ,
      • Holdt L.M.
      • Stahringer A.
      • Sass K.
      • Pichler G.
      • Kulak N.A.
      • Wilfert W.
      • Kohlmaier A.
      • Herbst A.
      • Northoff B.H.
      • Nicolaou A.
      • et al.
      Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans.
      ]. A mouse study by Visel and colleagues [
      • Visel A.
      • Zhu Y.
      • May D.
      • Afzal V.
      • Gong E.
      • Attanasio C.
      • Blow M.J.
      • Cohen J.C.
      • Rubin E.M.
      • Pennacchio L.A.
      Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice.
      ] was determinant in providing direct in vivo evidence of the link between the 9p21 non-coding interval and CAD susceptibility. By knocking out the murine orthologous 70-kb non-coding region on chromosome 4, they demonstrated that, although viable, homozygous mice with the aforementioned deletion showed increased mortality, both during development and as adults. Consistent with accelerated CAD risk phenotypes, the deletion affected cardiac expression of Cdkn2a and Cdkn2b, and triggered proliferation of aortic SMCs.
      Mechanistically, ANRIL acts in trans by contributing to recruit Polycomb complex to Alu-containing promoters of target genes, most likely through RNA: DNA interactions enabled by the presence of highly homologous Alu elements within its sequence [
      • Holdt L.M.
      • Hoffmann S.
      • Sass K.
      • Langenberger D.
      • Scholz M.
      • Krohn K.
      • Finstermeier K.
      • Stahringer A.
      • Wilfert W.
      • Beutner F.
      • et al.
      Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.
      ]. Unlike the linear isoform, circular ANRIL (circANRIL) was down-regulated in patients with the Chr9p21 risk haplotype and inversely correlated with atherosclerotic severity [
      • Holdt L.M.
      • Stahringer A.
      • Sass K.
      • Pichler G.
      • Kulak N.A.
      • Wilfert W.
      • Kohlmaier A.
      • Herbst A.
      • Northoff B.H.
      • Nicolaou A.
      • et al.
      Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans.
      ]. The atheroprotective action of circANRIL has been proposed to be a consequence of its inhibitory action on rRNA maturation and, as a results, the inhibition of cell proliferation.
      ANRIL is expressed in vascular and myocardial tissue, in ECs, VSMCs, and inflammatory cells [
      • Holdt L.M.
      • Teupser D.
      Recent studies of the human chromosome 9p21 locus, which is associated with atherosclerosis in human populations.
      ]. Knock-down (KD) of its linear isoform in human aortic VSMCs, targeting exon 1 or exon 19, affects apoptosis, proliferation, inflammation and ECM remodeling via modulation of BCL2-related protein A1 (BCL2A1), baculoviral IAP repeat containing 3 (BIRC3), cadherin 5 (CDH5) and heparin-binding EGF-like growth factor, suggesting isoform-specific regulatory properties [
      • Congrains A.
      • Kamide K.
      • Katsuya T.
      • Yasuda O.
      • Oguro R.
      • Yamamoto K.
      • Ohishi M.
      • Rakugi H.
      CVD-associated non-coding RNA, ANRIL, modulates expression of atherogenic pathways in VSMC.
      ]. By taking advantage of healthy and CAD-derived cells, Lo Sardo et al. [
      • Lo Sardo V.
      • Chubukov P.
      • Ferguson W.
      • Kumar A.
      • Teng E.L.
      • Duran M.
      • Zhang L.
      • Cost G.
      • Engler A.J.
      • Urnov F.
      • et al.
      Unveiling the role of the most impactful cardiovascular risk locus through haplotype editing.
      ] proposed an in vitro TALEN-based inducible pluripotent stem cell-derived VSMCs model, where the region corresponding to the ∼60 kb risk haplotype (depleted of coding genes) was knocked-out (KO). They compared the transcriptional landscape and some functional phenotypic features (adhesion, contraction and proliferation) of healthy/CAD VSMCs with or without deletion and observed that the KO could rescue a normal phenotype in CAD cells and, conversely, forced expression of the lncRNA ANRIL induced risk phenotypes in non-risk VSMCs.

      4. The non-coding signatures of VSMC contractility

      4.1 SENCR

      By carrying out RNA sequencing of human coronary artery VSMCs, Bell and colleagues [
      • Bell R.D.
      • Long X.
      • Lin M.
      • Bergmann J.H.
      • Nanda V.
      • Cowan S.L.
      • Zhou Q.
      • Han Y.
      • Spector D.L.
      • Zheng D.
      • et al.
      Identification and initial functional characterization of a human vascular cell-enriched long noncoding RNA.
      ] identified several unannotated lncRNAs and characterized a vascular cells-enriched one, termed smooth muscle and endothelial cell–enriched migration/differentiation-associated lncRNA (SENCR). SENCR is transcribed from the AS strand and partially overlaps an intron of the neighbour friend leukaemia virus integration 1 (FLI1) protein-coding gene. Although SENCR and FLI1 are co-expressed in various tissues, KD studies showed that they do not affect each other's expression. SENCR shows cell type- and tissue-specific transcript variants, mostly residing in the cytosolic compartment. Interestingly, the VSMCs-specific isoform is exclusively expressed in contractile cells and its silencing triggers the loss of contractile gene signature and their de-differentiation, alongside with the induction of a migratory phenotype. Although the molecular mechanisms and cell pathways underlying SENCR regulatory activity are yet to be elucidated, the authors speculate that this lncRNA could sponge a low abundant microRNA targeting VSMCs contractile gene program. A major objection to this model lies however in the very limited number of SENCR copies in cells (0.8/cell) [
      • Thum T.
      • Kumarswamy R.
      The smooth long noncoding RNA SENCR.
      ]. More recently, a study conducted in ECs subjected to laminar shear stress [
      • Lyu Q.
      • Xu S.
      • Lyu Y.
      • Choi M.
      • Christie C.K.
      • Slivano O.J.
      • Rahman A.
      • Jin Z.G.
      • Long X.
      • Xu Y.
      • et al.
      SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4.
      ] reported a protective function of SENCR in response to flow variations. In particular, the lncRNA binds the cytoskeleton associated protein 4 (CKAP4) RNA-binding protein and prevents it from triggering Cadherin 5 (CDH5) internalization. In summary, SENCR contributes to keep vessel wall integrity and function by maintaining membrane homeostasis in ECs and by regulating contraction in VSMCs.

      4.2 MYMSL

      Choi and colleagues [
      • Choi M.
      • Lu Y.W.
      • Zhao J.
      • Wu M.
      • Zhang W.
      • Long X.
      Transcriptional control of a novel long noncoding RNA Mymsl in smooth muscle cells by a single Cis-element and its initial functional characterization in vessels.
      ] identified a Myocardin-induced muscle specific lncRNA (Mymsl), expressed by contractile VSMCs and down-regulated upon switching to a synthetic phenotype. lncRNAs were profiled from primary mouse aortic VSMCs isolated from serum response factor (Srf) KO or wild type (WT) mice. Mymsl was highly detected in murine aorta, with a 60-fold higher expression in VSMCs compared to ECs. Phenotypic switch triggers, such as vascular injury, resulted in decreased Mymsl expression, and cultured VSMCs were characterized by sharply lower Mymsl levels compared to medial layer contractile aortic VSMCs. Mymsl contains the consensus sequence (CArG Box) for Myocd binding within its promoter and its transcription is triggered in response to stimuli governing VSMC differentiation in a Myocd-dependent fashion. Hence, similarly to the protein coding Myocd, Mymsl can be regarded as a reliable non-coding marker of VSMC contractile phenotype.

      4.3 CARMN

      In search for lncRNAs differentially expressed throughout the development of atherosclerosis and modulating VSMCs plasticity, Ni et al. [
      • Ni H.
      • Haemmig S.
      • Deng Y.
      • Chen J.
      • Simion V.
      • Yang D.
      • Sukhova G.
      • Shvartz E.
      • Wara A.
      • Cheng H.S.
      • et al.
      A smooth muscle cell-enriched long noncoding RNA regulates cell plasticity and atherosclerosis by interacting with serum response factor.
      ] sequenced murine atherosclerotic plaques isolated from Ldlr−/−mice fed on high cholesterol diet (HCD). They observed that the expression of the Cardiac mesoderm enhancer-associated (Carmn ncRNA, a conserved VSMC-enriched lncRNA, was significantly decreased after 12 weeks, but interestingly restored following 6 further weeks of normal diet. CARMN is conserved in mouse and human and is highly expressed in the aortic media, with a predominant localization within the nucleus of VSMCs. In human, CARMN expression was decreased in atherosclerotic lesions compared with non-atherosclerotic arteries. Gapmer-mediated in vivo KD suppressed VSMCs proliferation and held up plaques formation. Results relative to in vitro experiments with both murine and human VSMCs confirmed in vivo findings, with CARMN silencing inhibiting VSMC proliferation and migration, and OE having opposing effects. A crucial consequence of CARMN KD was a remarkable inhibition of VSMCs differentiation in vivo, as proved by quantification of contractile VSMC markers such as ACTA2, SM22α, MYH11, and CNN1 in the aortic intima and media from control and CARMN knockdown HCD-fed Ldlr−/− mice. In line with this, the lncRNA was induced by transforming growth factor β (TGF-β) type 1 treatment in both murine and human VSMC, in a time- and dose-dependent manner and through the mediation of SMAD2 and SMAD3 proteins. TGF-β represents an important anti-inflammatory stimulus ensuring VSMCs contractility and contributing to the maintenance of normal blood vessel wall architecture [
      • Grainger D.J.
      TGF-beta and atherosclerosis in man.
      ]. In vitro studies have shown that TGF-β1 stimulates ECM production [
      • Penttinen R.P.
      • Kobayashi S.
      • Bornstein P.
      Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability.
      ], thereby contributing to plaque stabilization. Moreover, TGF-β can be involved in the molecular dynamics underlying plaque remodeling: via a mechanism dependent on SRF, TGF-β induces the trans-differentiation and recruitment of mesenchymal cells to the plaque site [
      • Abedin M.
      • Tintut Y.
      • Demer L.L.
      Mesenchymal stem cells and the artery wall.
      ], where they fully differentiate to VSMCs [
      • Hirschi K.K.
      • Lai L.
      • Belaguli N.S.
      • Dean D.A.
      • Schwartz R.J.
      • Zimmer W.E.
      Transforming growth factor-beta induction of smooth muscle cell phenotpye requires transcriptional and post-transcriptional control of serum response factor.
      ]. Combined pull-down and chromatin IP analysis showed that CARMN, in synergy with TGF-β, interacts with SRF, a transcription factor that regulates VSMC contractile gene expression program, and facilitates its binding to the promoters of targets, including ACTA2, SM22a, MYH11 and CNN1.
      In their study, Dong and colleagues [
      • Dong K.
      • Shen J.
      • He X.
      • Hu G.
      • Wang L.
      • Osman I.
      • Bunting K.M.
      • Dixon-Melvin R.
      • Zheng Z.
      • Xin H.
      • et al.
      CARMN is an evolutionarily conserved smooth muscle cell-specific LncRNA that maintains contractile phenotype by binding myocardin.
      ] confirmed that CARMN was significantly decreased in patients' specimens and murine vascular disease models and strengthen an association between CARMN expression and the maintenance of the contractile phenotype of VSMCs in vitro. At molecular level, Carmn interacts with myocardin, facilitating its activity and thereby ensuring the contractile phenotype of VSMCs. In vivo, VSMC-specific deletion of Carmn significantly exacerbated, while overexpression of Carmn considerably inhibited, injury-induced neointima formation in mouse and rat, respectively. These findings are slightly in contrast with results by Ni et al., which could be due to the lack of any putative additional cis-regulation as a consequence of the genetic KO model. Vacante et al. agree that the loss of Carmn represents a primary event behind the phenotypic switch towards pro-atherogenic VSMC phenotype and triggers plaque formation [
      • Vacante F.
      • Rodor J.
      • Lalwani M.K.
      • Mahmoud A.D.
      • Bennett M.
      • De Pace A.L.
      • Miller E.
      • Van Kuijk K.
      • de Bruijn J.
      • Gijbels M.
      • et al.
      CARMN loss regulates smooth muscle cells and accelerates atherosclerosis in mice.
      ], in both mouse and human. However, major discrepancies with Ni et al. exist, when the results of CARMN silencing in proliferation and migration are assessed. Indeed, although KD strategies and cell types are similar, Vacante and colleagues report augmented proliferation and migration of human coronary artery SMCs upon silencing. Furthermore, they claim that only the effects of KD on proliferation, and not the ones on migration and de-differentiation, are independent of the miR143/145 cluster, located directly downstream of the CARMN locus and previously shown to be involved in VSMCs’ phenotypic switch [
      • Cordes K.R.
      • Sheehy N.T.
      • White M.P.
      • Berry E.C.
      • Morton S.U.
      • Muth A.N.
      • Lee T.H.
      • Miano J.M.
      • Ivey K.N.
      • Srivastava D.
      miR-145 and miR-143 regulate smooth muscle cell fate and plasticity.
      ]. This would slightly scale back the mechanistic contribution of the lncRNA CARMN as a direct molecular mediator in gene expression regulation of VSMC fate.

      4.4 MYOSLID

      The MYOcardin-induced Smooth muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) provides another example of contractile VSMCs-specific lncRNA marker. MYOSLID was found to be up-regulated in MYOCD-overexpressing human coronary artery VSMCs, in response to the presence of CArG elements within its promoter [
      • Zhao J.
      • Zhang W.
      • Lin M.
      • Wu W.
      • Jiang P.
      • Tou E.
      • Xue M.
      • Richards A.
      • Jourd'heuil D.
      • Asif A.
      • et al.
      MYOSLID is a novel serum response factor-dependent long noncoding RNA that amplifies the vascular smooth muscle differentiation program.
      ]. Similarly to CARMN, MYOSLID promoter also contains SMAD-binding elements, allowing its induction upon stimulation with TGF-β. Proliferation-triggering (as PDGFBB) and inflammatory stimuli (like TNFα and IL1β), on the other hand, strongly inhibited its expression. In line with a role of positive regulator of the VSMC differentiation program, MYOSLID was shown to be depleted under pathological conditions promoting VSMCs de-differentiation, like human arteriovenous fistula (AVF). Mechanistically, the expression of MYOSLID enables F-actin assembly and triggers MKL1 nuclear translocation and the subsequent transcriptional activation of downstream VSMC contractile genes.

      4.5 NR2F1-AS1 and FOXC2-AS1

      To fish out lncRNAs regulated upon VSMCs differentiation, Lim et al. [
      • Lim Y.H.
      • Ryu J.
      • Kook H.
      • Kim Y.K.
      Identification of long noncoding RNAs involved in differentiation and survival of vascular smooth muscle cells.
      ] sequenced human coronary artery VSMCs and mouse MOVAS cells which had been cultured and differentiated in vitro by utilizing serum-deprived/growth factors (as PDGFBB and TGF-β)-implemented medium. They identified two conserved lncRNAs, NR2F1-AS1 and FOXC2-AS1, which were similarly induced upon differentiation in murine and human VSMCs and expressed in both the nuclear and cytosolic compartment. If silenced when differentiation stimuli were applied, a strong down-regulation in the expression of contractile genes, along with impaired contractility were observed. No effects on proliferation and apoptosis were detected for NR2F1-AS1 in human cells while, almost counterintuitively, FOXC2-AS1 KD decreased proliferation and increased apoptosis. Regulation of VSMCs survival by FOXC2-AS1 KD relied on Akt/mTOR signaling, as phosphorylation of both kinases was reduced upon its silencing. A regulatory loop between FOXC2-AS1 lncRNA and its neighbouring gene Forkhead Box Protein C2 (FOXC2) was suggested due to a remarkable down-regulation of FOXC2 mRNA when its AS lncRNA was KD. As FOXC2 was previously shown to participate to VSMCs differentiation via regulating Notch signaling [
      • Lagha M.
      • Brunelli S.
      • Messina G.
      • Cumano A.
      • Kume T.
      • Relaix F.
      • Buckingham M.E.
      Pax3:Foxc2 reciprocal repression in the somite modulates muscular versus vascular cell fate choice in multipotent progenitors.
      ], the authors eventually propose that FOXC2-AS plays an important role in the survival and differentiation of VSMCs, possibly in cooperation with FOXC2, by affecting the Notch and Akt/mTOR signaling pathways.

      5. LncRNAs induced upon VSMC phenotypic switching

      5.1 Gas5

      Alongside with lncRNAs which contribute to switch contractility gene programs on, other large regulatory transcripts exerting an opposite functional output have been reported. The growth arrest-specific 5 (Gas5) provides an example of lncRNA negatively regulating VSMCs differentiation by suppressing TGF-β/Smad3 signaling [
      • Tang R.
      • Zhang G.
      • Wang Y.C.
      • Mei X.
      • Chen S.Y.
      The long non-coding RNA GAS5 regulates transforming growth factor beta (TGF-beta)-induced smooth muscle cell differentiation via RNA Smad-binding elements.
      ]. Infection with Gas5 adenovirus resulted in inhibited expression of contractile genes in TGF-β-stimulated C3H10T1/2, a murine fibroblast cell line. Smad–binding elements (Sbes) contained within Gas5 sequence are required to bind and retain Smad3 protein, which is ultimately unable to reach DNA Sbes within TGF-β-responsive VSMC gene promoters. Interestingly, when subcloned into other natural or artificially synthesized lncRNAs, Gas5 Smad–binding modules are sufficient to confer a TGF-β-inhibiting action. Gas5 thus represents an example of lncRNA with a domain organization working as a flexible modular scaffold [
      • Guttman M.
      • Rinn J.L.
      Modular regulatory principles of large non-coding RNAs.
      ].

      5.2 NEAT1

      NEAT1 is extensively known for its structural role in the formation of nuclear paraspeckles and plays a critical role in tumorigenesis, thanks to its ability to trigger proliferation and migration [
      • Pisani G.
      • Baron B.
      NEAT1 and paraspeckles in cancer development and chemoresistance.
      ]. Its expression was found to be induced in PDGFBB−treated rat aortic primary VSMCs, as well as upon arterial injury (a stimulus triggering phenotypic switching and neointima formation) in in vivo models [
      • Ahmed A.S.I.
      • Dong K.
      • Liu J.
      • Wen T.
      • Yu L.
      • Xu F.
      • Kang X.
      • Osman I.
      • Hu G.
      • Bunting K.M.
      • et al.
      Long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1) is critical for phenotypic switching of vascular smooth muscle cells.
      ,
      • Lim Y.H.
      • Kwon D.H.
      • Kim J.
      • Park W.J.
      • Kook H.
      • Kim Y.K.
      Identification of long noncoding RNAs involved in muscle differentiation.
      ]. In line with this, upon silencing of NEAT1 in human coronary artery VSMCs, an enhanced expression of SM-specific genes was accompanied by attenuated proliferation and migration, while a reverted cell phenotype was observed upon the lncRNA overexpression. Furthermore, Neat1 knockout (KO) mice were characterized by significantly decreased neointima formation in response to vascular injury, as a result of reduced VSMCs proliferation. At molecular level, NEAT1 acts via sequestration of the key chromatin modifier WD Repeat Domain 5 (WDR5) from SM-specific gene loci, thus promoting an epigenetic “off” state and the repression of SM-specific gene programs. Hence, targeting NEAT1 could provide a potential therapeutic strategy for early treatment of occlusive vascular diseases.

      5.3 MIAT

      The Myocardial infarction associated transcript (MIAT) was found to have aberrant expression in various diseases (reviewed in [
      • Sun C.
      • Huang L.
      • Li Z.
      • Leng K.
      • Xu Y.
      • Jiang X.
      • Cui Y.
      Long non-coding RNA MIAT in development and disease: a new player in an old game.
      ]). Its discovery was initially reported in a large-scale case-control association study [
      • Ishii N.
      • Ozaki K.
      • Sato H.
      • Mizuno H.
      • Susumu S.
      • Takahashi A.
      • Miyamoto Y.
      • Ikegawa S.
      • Kamatani N.
      • Hori M.
      • et al.
      Identification of a novel non-coding RNA, MIAT, that confers risk of myocardial infarction.
      ], where Ishii et al. identified a susceptible locus for myocardial infarction and isolated the full-length transcript spliced from a novel gene, designated MIAT. A SNP in MIAT (SNP rs2301523) resulted in altered expression of the lncRNA, which had a strong association with the pathogenesis of MI. MIAT up-regulation co-existed with other cardiovascular risk (CVR) factors, such as hypertension and smoking, and provided a significant predictor of left ventricular dysfunction [
      • Vausort M.
      • Wagner D.R.
      • Devaux Y.
      Long noncoding RNAs in patients with acute myocardial infarction.
      ]. Its expression was remarkably increased also in heart tissue of a murine model of MI [
      • Qu X.
      • Du Y.
      • Shu Y.
      • Gao M.
      • Sun F.
      • Luo S.
      • Yang T.
      • Zhan L.
      • Yuan Y.
      • Chu W.
      • et al.
      MIAT is a pro-fibrotic long non-coding RNA governing cardiac fibrosis in post-infarct myocardium.
      ], further supporting a role of MIAT as disease biomarker and therapeutic target. In our study [
      • Fasolo F.
      • Jin H.
      • Winski G.
      • Chernogubova E.
      • Pauli J.
      • Winter H.
      • Li D.Y.
      • Glukha N.
      • Bauer S.
      • Metschl S.
      • et al.
      Long noncoding RNA MIAT controls advanced atherosclerotic lesion formation and plaque destabilization.
      ], MIAT up-regulation was detected in human carotid plaques compared with non-atherosclerotic control arteries, and similar expression patterns were also observed in ApoE−/− mice and Yucatan LDLR−/− mini-pigs disease models. MIAT-associated CVR has been linked to its ability to influence VSMC dynamics. Ma and colleagues [
      • Ma G.
      • Bi S.
      • Zhang P.
      Long non-coding RNA MIAT regulates ox-LDL-induced cell proliferation, migration and invasion by miR-641/STIM1 axis in human vascular smooth muscle cells.
      ] demonstrated that the transcript is responsible for OxLDL-induced increased proliferation, migration and invasion of human VSMCs, via a sponge mechanism (MIAT/miR-641/STIM1). By knocking down MIAT with site-specific AS oligonucleotides (LNA-GapmeRs), our group confirmed that the lncRNA affected proliferation and migration rates of cultured VSMCs, similarly to what was previously described [
      • Ma G.
      • Bi S.
      • Zhang P.
      Long non-coding RNA MIAT regulates ox-LDL-induced cell proliferation, migration and invasion by miR-641/STIM1 axis in human vascular smooth muscle cells.
      ], and, in addition to this, triggered their apoptosis [
      • Fasolo F.
      • Jin H.
      • Winski G.
      • Chernogubova E.
      • Pauli J.
      • Winter H.
      • Li D.Y.
      • Glukha N.
      • Bauer S.
      • Metschl S.
      • et al.
      Long noncoding RNA MIAT controls advanced atherosclerotic lesion formation and plaque destabilization.
      ]. We proposed an alternative (or complementary) mechanism through which MIAT exerts its regulation on VSMC proliferation, involving the Early Growth Response 1 (EGR1)- ETS-like Transcription Factor 1 (ELK1)-Extracellular Signal-Regulated Kinase (ERK) pathway. By interacting with ELK1, MIAT facilitates its positioning to the EGR1 promoter, augmenting its transcription rate. Furthermore, our work supports an involvement of MIAT in SMC phenotypic transition to proinflammatory macrophage-like cells. Luciferase reporter assays pointed out that MIAT enhanced the transcription of KLF4, which has been previously described as pivotal in mediating VSMC phenotypic switch and atherosclerosis development [
      • Shankman L.S.
      • Gomez D.
      • Cherepanova O.A.
      • Salmon M.
      • Alencar G.F.
      • Haskins R.M.
      • Swiatlowska P.
      • Newman A.A.
      • Greene E.S.
      • Straub A.C.
      • et al.
      KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis.
      ]. Studies using Miat−/− and Miat−/−ApoE−/− mice, and Yucatan LDLR−/− mini-pigs confirmed the regulatory role of this lncRNA in VSMCs de- and trans-differentiation and advanced atherosclerotic lesion formation [
      • Fasolo F.
      • Jin H.
      • Winski G.
      • Chernogubova E.
      • Pauli J.
      • Winter H.
      • Li D.Y.
      • Glukha N.
      • Bauer S.
      • Metschl S.
      • et al.
      Long noncoding RNA MIAT controls advanced atherosclerotic lesion formation and plaque destabilization.
      ].

      5.4 LncRNA 430945

      LncRNA EN ST00000430945 (lncRNA 430945) provides another example of transcript contributing to atherosclerosis dynamics through the regulation of proliferation and migration of VSMCs. As reported by Cui et al. [
      • Cui C.
      • Wang X.
      • Shang X.M.
      • Li L.
      • Ma Y.
      • Zhao G.Y.
      • Song Y.X.
      • Geng X.B.
      • Zhao B.Q.
      • Tian M.R.
      • et al.
      lncRNA 430945 promotes the proliferation and migration of vascular smooth muscle cells via the ROR2/RhoA signaling pathway in atherosclerosis.
      ], its up-regulation in human tissues was interestingly accompanied by increased expression of Receptor tyrosine kinase-like orphan receptor 2 (ROR 2), a member of the tyrosine kinase receptor family. ROR 2 engagement by Wnt5a is part of a signaling cascade regulating cell proliferation and motility. Manipulation of lncRNA 430945 expression in either direction caused similar de-regulation of ROR 2, with silencing negatively affecting Ang II-induced murine VSMC proliferation and migration in vitro. The therapeutic potential of IncRNA 430945 inhibition was tested in a mouse model of carotid artery ligation-induced intimal thickening. By acting on cell proliferation, the KD significantly reduced vascular intimal hyperplasia, compared to mock treatment.

      5.5 LincRNA-p21

      LincRNA-p21 expression was found to be decreased in Apoe-deficient murine model of atherosclerosis, as well as in specimens from patients with CAD [
      • Wu G.
      • Cai J.
      • Han Y.
      • Chen J.
      • Huang Z.P.
      • Chen C.
      • Cai Y.
      • Huang H.
      • Yang Y.
      • Liu Y.
      • et al.
      LincRNA-p21 regulates neointima formation, vascular smooth muscle cell proliferation, apoptosis, and atherosclerosis by enhancing p53 activity.
      ]. The lncRNA exerts its action by physically interacting with p53 repressive complex and contributes to switch a subset of p53 target genes off [
      • Huarte M.
      • Guttman M.
      • Feldser D.
      • Garber M.
      • Koziol M.J.
      • Kenzelmann-Broz D.
      • Khalil A.M.
      • Zuk O.
      • Amit I.
      • Rabani M.
      • et al.
      A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response.
      ]. As a consequence of an enhancement of p53 transcriptional activity, VSMCs and mouse macrophages proliferation was observed to be reduced and apoptosis triggered [
      • Wu G.
      • Cai J.
      • Han Y.
      • Chen J.
      • Huang Z.P.
      • Chen C.
      • Cai Y.
      • Huang H.
      • Yang Y.
      • Liu Y.
      • et al.
      LincRNA-p21 regulates neointima formation, vascular smooth muscle cell proliferation, apoptosis, and atherosclerosis by enhancing p53 activity.
      ]. Lentivirus-mediated lincRNA-p21 silencing in the mouse carotid artery injury model favored neointimal hyperplasia [
      • Wu G.
      • Cai J.
      • Han Y.
      • Chen J.
      • Huang Z.P.
      • Chen C.
      • Cai Y.
      • Huang H.
      • Yang Y.
      • Liu Y.
      • et al.
      LincRNA-p21 regulates neointima formation, vascular smooth muscle cell proliferation, apoptosis, and atherosclerosis by enhancing p53 activity.
      ]. Reducing lincRNA-p21 levels would be a suitable therapeutic strategy against VSMC hyperproliferative response observed during atherogenesis or (re)stenosis; on the other side of the coin, the potentiation of its expression in later stages could improve plaque stability and reduce the risk of rupture.

      5.6 KCNQ1OT1

      Intimal hyperplasia is characterized by the migration of VSMCs from the vessel media to the intimal layer, where they acquire a hyperproliferative and inflammatory cell-like phenotype. The lncRNA KCNQ1OT1 has been shown to be down-regulated in a vein graft (VG) mouse model, displaying intimal hyperplasia [
      • Ye B.
      • Wu Z.H.
      • Tsui T.Y.
      • Zhang B.F.
      • Su X.
      • Qiu Y.H.
      • Zheng X.T.
      lncRNA KCNQ1OT1 suppresses the inflammation and proliferation of vascular smooth muscle cells through IkappaBa in intimal hyperplasia.
      ] and in VSMCs isolated from a normal mice stimulated with PDGFBB. In both cases, the KCNQ1OT1 promoter was found to be hypermethylated, suggesting a link between this lncRNA and the proliferative capacity of VSMCs. When overexpressed, KCNQ1OT1 suppressed VSMC proliferation, migration, and secretion of inflammatory factors, as a result of increased IκBa, the inhibitory subunit of NF-κB. IκBa blocks NF-κB nuclear translocation and the subsequent activation of proliferation and inflammatory gene networks. The KCNQ1OT1 functional output is achieved through sponging miR-221, which targets the IκBa mRNA, as well as via binding to the IκBa protein and inhibiting its degradation.

      5.7 LncRNAs induced upon Angiotensin II stimulation

      Angiotensin II (Ang II) regulates diverse crucial processes throughout the cardiovascular system including vascular tone, hormone and radical oxygen species (ROS) production, cardiac hypertrophy and VSMCs proliferation [
      • Brasier A.R.
      • Recinos 3rd, A.
      • Eledrisi M.S.
      Vascular inflammation and the renin-angiotensin system.
      ]. Although the exact mechanisms underlying the effects of Ang II treatment are not fully elucidated, this drug has been extensively used to mimic CVD [
      • Daugherty A.
      • Manning M.W.
      • Cassis L.A.
      Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.
      ] Ang II administration results in dysregulated proliferation and hypertrophy of cultured VSMCs and is potently proatherogenic in hypercholesterolemic animals [
      • Brasier A.R.
      • Recinos 3rd, A.
      • Eledrisi M.S.
      Vascular inflammation and the renin-angiotensin system.
      ]. Proatherogenic responses include altering the structural integrity of the vessels by affecting extracellular matrix production and degradation, enhancing lipid oxidation and triggering inflammation. To isolate lncRNAs regulated by Ang II, Leung et al. [
      • Leung A.
      • Trac C.
      • Jin W.
      • Lanting L.
      • Akbany A.
      • Saetrom P.
      • Schones D.E.
      • Natarajan R.
      Novel long noncoding RNAs are regulated by angiotensin II in vascular smooth muscle cells.
      ] performed transcriptome and epigenome profiling of rat VSMCs in response to Ang II pharmacological treatment. An Ang II-induced lncRNA, named lnc-Ang362, was found to provide the host transcript for miR-221 and miR-222, which are proposed mediators of VSMCs function. Lnc-Ang362 KD reduced miR-221 and miR-222 expression and suppressed VSMCs proliferation. In a similar study, Das and colleagues [
      • Das S.
      • Zhang E.
      • Senapati P.
      • Amaram V.
      • Reddy M.A.
      • Stapleton K.
      • Leung A.
      • Lanting L.
      • Wang M.
      • Chen Z.
      • et al.
      A novel angiotensin II-induced long noncoding RNA giver regulates oxidative stress, inflammation, and proliferation in vascular smooth muscle cells.
      ] identified increased levels of the lncRNA growth factor– and proinflammatory cytokine–induced vascular cell-expressed RNA (Giver) in rat VSMCs and in rat and mouse aortas treated ex vivo with Ang II. Giver knockdown mitigated the effects of Ang II-induced oxidative stress via reducing the expression of oxidative stress- and inflammation related genes, suggesting its role in mediating the cellular response to such triggers. Mechanistically, Giver interacts with chromatin remodeling complexes. Upregulated GIVER expression was also detected in human VSMCs upon Ang II stimulation, as well as in arteries from hypertensive patients, but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or Angiotensin II receptor blockers. The results argue for the possibility of investigating the therapeutic potential Ang II-modulated ncRNAs for treatment of cardiovascular pathologies associated to AngII de-regulation.

      6. Targeting lncRNAs to modulate plaque stability in late stages of atherosclerosis

      6.1 SMILR

      In search for lncRNAs that are regulated upon the induction of proliferative and inflammatory pathways, Ballantyne and co-authors [
      • Ballantyne M.D.
      • Pinel K.
      • Dakin R.
      • Vesey A.T.
      • Diver L.
      • Mackenzie R.
      • Garcia R.
      • Welsh P.
      • Sattar N.
      • Hamilton G.
      • et al.
      Smooth muscle enriched long noncoding RNA (SMILR) regulates cell proliferation.
      ] sequenced RNA of human saphenous vein vascular smooth muscle cells (HSVSMCs) treated/untreated with IL1α or PDGFBB, and identified a novel differentially expressed lncRNA, referred to as smooth muscle–induced lncRNA enhances replication (SMILR). SMILR was enriched in VSMCs of either venous or arterial lineage (HSVSMCs and human coronary artery SMCs), while it failed in being detected in primary human saphenous vein endothelial cells (HSVECs). Inhibition of the Mitogen-Activated Protein Kinase Kinase Kinase 1 (MEKK1) pathway prevented SMILR induction consequent to PDGFBB and IL1α treatment. Interestingly, knockdown of SMILR markedly reduced proliferation, while its overexpression increased it. To further explore the role of SMILR in human vascular pathologies, its expression was monitored in human unstable atherosclerotic plaques. By taking advantage of established inflammatory ([18F]fluorodeoxyglucose [FDG]) and calcification ([18F]fluoride) positron emission tomography radiotracers, dissection of areas of high-risk plaque (showing higher uptake of both [18F]FDG and [18F]fluoride) and non-disease adjacent sections was carried out from individual patients, thus ensuring to profile non-coding RNA expression from within each micro environment. Alongside with an upregulation of a panel of miRNAs associated to atherosclerosis, they detected augmented levels of SMILR in high-risk plaques, in comparison with adjacent stable regions of the carotid artery. SMILR VSMCs-restricted expression make it a potential suitable candidate to improve selectivity of antiproliferative therapies, ultimately reducing the risk of late stent thrombosis [
      • Lemesle G.
      • Maluenda G.
      • Collins S.D.
      • Waksman R.
      Drug-eluting stents: issues of late stent thrombosis.
      ].

      6.2 Nron

      The lncRNA “Noncoding Repressor of Nuclear factor of activated T cells NFAT)” (NRON) was first reported as part of an RNA-protein scaffold complex regulating the activity of NFAT [
      • Sharma S.
      • Findlay G.M.
      • Bandukwala H.S.
      • Oberdoerffer S.
      • Baust B.
      • Li Z.
      • Schmidt V.
      • Hogan P.G.
      • Sacks D.B.
      • Rao A.
      Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.
      ], an important family of transcription factors in immune response. Du and colleagues [
      • Du M.
      • Wang C.
      • Yang L.
      • Liu B.
      • Zheng Z.
      • Yang L.
      • Zhang F.
      • Peng J.
      • Huang D.
      • Huang K.
      The role of long noncoding RNA Nron in atherosclerosis development and plaque stability.
      ] investigated NRON in human atherosclerosis and found a strong depletion in carotid atherosclerotic plaques compared to healthy arteries. Coherently, analysis of the dynamic changes of Nron in the progression of atherosclerosis in ApoE−/−mice showed an inverse trend between the expression of this lncRNA and disease severity. Nron OE in ApoE−/− induced a highly characteristic architecture of unstable plaques, with thinner fibrous cap, loose collagen, VSMCs-deprived and immune infiltrate, lipid-rich necrotic cores. Conversely, adenovirus-mediated Nron KD was beneficial in terms of decreased area and increased stability of plaques. Functional studies in VSMCs highlighted that Nron contributes to the vulnerability of atherosclerotic lesions and its regulation relies on binding to NFATC3 within the cytosolic compartment of VSMCs, which prevents the transcription factor from accessing the nucleus and switching proliferation-controlling gene sets on, ultimately enabling the activation of apoptotic pathways. Furthermore, Nron triggers the production and release of VEGFA by mouse VSMCs, in turn stimulating intra-plaque angiogenesis, which plays a crucial contribution to the switch of an asymptomatic fibroatheromatous plaque into a vulnerable lesion.

      6.3 GAS5 and Lrrc75a-as1: counteracting vascular calcification

      Vascular calcification is a process characterizing the most advanced stages of atherosclerosis, and is the result of an impaired calcium phosphate metabolism [
      • Massy Z.A.
      • Drueke T.B.
      Magnesium and outcomes in patients with chronic kidney disease: focus on vascular calcification, atherosclerosis and survival.
      ]. It impacts vessel integrity and elasticity, thus increasing the risk of cardiovascular diseases [
      • Giachelli C.M.
      Vascular calcification mechanisms.
      ]. In a human study [
      • Chang Z.
      • Yan G.
      • Zheng J.
      • Liu Z.
      The lncRNA GAS5 inhibits the osteogenic differentiation and calcification of human vascular smooth muscle cells.
      ], the aforementioned GAS5 was found down-regulated in aortic VSMCs, where vascular calcification was induced in vitro and ex vivo by treatment with inorganic phosphate. Although, the study does not provide an in vivo model of vessel calcification, as induced in later atherosclerosis stages, the authors proved that this lncRNA combats calcification by sponging miR-26-5p, which subsequently increases phosphatase and tensin homolog (PTEN). In a similar setting, Jeong and co-authors identified Lrrc75a-as1 as a negative regulator of vascular calcification in rat VSMCs stimulated with inorganic phosphate. His could be attributed to its ability to decrease the expression of osteoblast-related factors [
      • Jeong G.
      • Kwon D.-H.
      • Shin S.
      • Choe N.
      • Ryu J.
      • Lim Y.-H.
      • Kim J.
      • Park W.J.
      • Kook H.
      • Kim Y.-K.
      Long noncoding RNAs in vascular smooth muscle cells regulate vascular calcification.
      ].

      6.4 HAS2-AS1

      Hyaluronic acid (HA) is a multifunctional matrix protein having a critical role in vascular injury and atherogenesis. When accumulated, it may favour vessel wall thickening and neointima formation [
      • Cuff C.A.
      • Kothapalli D.
      • Azonobi I.
      • Chun S.
      • Zhang Y.
      • Belkin R.
      • Yeh C.
      • Secreto A.
      • Assoian R.K.
      • Rader D.J.
      • et al.
      The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.
      ,
      • Chai S.
      • Chai Q.
      • Danielsen C.C.
      • Hjorth P.
      • Nyengaard J.R.
      • Ledet T.
      • Yamaguchi Y.
      • Rasmussen L.M.
      • Wogensen L.
      Overexpression of hyaluronan in the tunica media promotes the development of atherosclerosis.
      ,
      • Vigetti D.
      • Viola M.
      • Karousou E.
      • Genasetti A.
      • Rizzi M.
      • Clerici M.
      • Bartolini B.
      • Moretto P.
      • De Luca G.
      • Passi A.
      Vascular pathology and the role of hyaluronan.
      ], as well as it triggers the expression of adhesion molecules by VSMCs, ultimately initiating the immune cascade. The potential proatherosclerotic effect of HA could be limited by the inhibition of HA synthases (HAS), providing a possible strategy to limit the burden of vascular pathologies [
      • Vigetti D.
      • Rizzi M.
      • Viola M.
      • Karousou E.
      • Genasetti A.
      • Clerici M.
      • Bartolini B.
      • Hascall V.C.
      • De Luca G.
      • Passi A.
      The effects of 4-methylumbelliferone on hyaluronan synthesis, MMP2 activity, proliferation, and motility of human aortic smooth muscle cells.
      ,
      • Vigetti D.
      • Rizzi M.
      • Moretto P.
      • Deleonibus S.
      • Dreyfuss J.M.
      • Karousou E.
      • Viola M.
      • Clerici M.
      • Hascall V.C.
      • Ramoni M.F.
      • et al.
      Glycosaminoglycans and glucose prevent apoptosis in 4-methylumbelliferone-treated human aortic smooth muscle cells.
      ]. Mammalian HA is synthesized at the cell membrane by three HAS, with the HAS2 isoform being mainly responsible for HA synthesis in adult mammalian tissues. HAS2 possesses a NAT, termed HAS2-AS1 [
      • Spicer A.P.
      • Seldin M.F.
      • Olsen A.S.
      • Brown N.
      • Wells D.E.
      • Doggett N.A.
      • Itano N.
      • Kimata K.
      • Inazawa J.
      • McDonald J.A.
      Chromosomal localization of the human and mouse hyaluronan synthase genes.
      ], transcribed from the opposite strand of the HAS2 gene and partially overlapping its first exon. This complementarity allows a HAS2 mRNA:HAS2-AS1 duplex formation, which results in the stabilization of HAS2 [
      • Michael D.R.
      • Phillips A.O.
      • Krupa A.
      • Martin J.
      • Redman J.E.
      • Altaher A.
      • Neville R.D.
      • Webber J.
      • Kim M.Y.
      • Bowen T.
      The human hyaluronan synthase 2 (HAS2) gene and its natural antisense RNA exhibit coordinated expression in the renal proximal tubular epithelial cell.
      ]. In their in vitro study in aortic VSMCs, Vigetti and colleagues [
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      • Deleonibus S.
      • Moretto P.
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      • Hanover J.A.
      • Cinquetti R.
      • et al.
      Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation.
      ] found that HAS-AS1 was able to induce chromatin remodeling (O-GlcNAcylation) in the HAS2 proximal promoter, thus enabling transcription. Interestingly, the expression of HAS2-AS1 was increased in human plaques collected from atherectomy of carotid arteries with a higher grade of severity and upregulation of both Has2 and Has-As1 was also detected in a murine model of atherosclerosis induced by high cholesterol.

      7. Exploiting lncRNAs in RNA therapy

      The most recent advances in the biomedical field have led to envision ncRNAs as powerful templates for the development of nucleotide-based biotherapeutics. The utilization of siRNA and antisense oligonucleotides (ASOs) is in fact not limited to investigational interventions in animal models, but has rather extended to the design of RNA drugs, some of which already undergoing Phase I-III clinical trials [
      • Pham T.P.
      • Kremer V.
      • Boon R.A.
      RNA-based therapeutics in cardiovascular disease.
      ]. Both siRNAs and ASOs exploit endogenous RNA processing machinery and proved to be efficient in down-regulating the expression of target genes, including lncRNAs. The choice of lncRNAs' silencing strategies is strictly linked to their subcellular localization: RNA-induced silencing complex (RISC)-mediated KD employing siRNAs is more suitable for cytoplasmic lncRNAs, while ASOs, triggering RNAseH-based degradation, represent the preferred strategy for nuclear lncRNAs [
      • Crooke S.T.
      • Liang X.H.
      • Baker B.F.
      • Crooke R.M.
      Antisense technology: a review.
      ]. It is widely acknowledged that chemical modifications to the nucleotides incorporated into siRNAs and ASOs can largely improve affinity for target RNAs, make them less vulnerable to nuclease degradation, and lower their immunogenicity [
      • Dowdy S.F.
      Overcoming cellular barriers for RNA therapeutics.
      ]. These are usually inserted at either the 2′ carbon position on the ribose sugar or the phosphate linkage between nucleotides. Furthermore, RNA-conjugated nanoparticles can be exploited to increase stability and nuclear localization of ASOs [
      • Gong N.
      • Teng X.
      • Li J.
      • Liang X.J.
      Antisense oligonucleotide-conjugated nanostructure-targeting lncRNA MALAT1 inhibits cancer metastasis.
      ] or to improve their delivery to privileged sites like the brain [
      • Min H.S.
      • Kim H.J.
      • Naito M.
      • Ogura S.
      • Toh K.
      • Hayashi K.
      • Kim B.S.
      • Fukushima S.
      • Anraku Y.
      • Miyata K.
      • et al.
      Systemic brain delivery of antisense oligonucleotides across the blood-brain barrier with a glucose-coated polymeric nanocarrier.
      ]. Recently, it has been shown that lncRNAs can be directly targeted by small chemotypes, which selectively bind to structural elements contained in lncRNAs, ultimately altering their expression profiles [
      • Abulwerdi F.A.
      • Xu W.
      • Ageeli A.A.
      • Yonkunas M.J.
      • Arun G.
      • Nam H.
      • Schneekloth Jr., J.S.
      • Dayie T.K.
      • Spector D.
      • Baird N.
      • et al.
      Selective small-molecule targeting of a triple helix encoded by the long noncoding RNA, MALAT1.
      ].
      Unlike their “small relatives”, the employment of lncRNAs as templates for engineering novel RNA therapeutics is still in its infancy, mostly due to their functional heterogeneity, the current poor mechanistic insight, compared to small RNAs, and the challenges deriving from delivery of large molecules, usually requiring viral vectors [
      • Dowdy S.F.
      Overcoming cellular barriers for RNA therapeutics.
      ]. On the other hand, current research suggests that lncRNAs, thanks to their tissue specific expression patterns, could become appealing druggable targets especially for the treatment of orphan diseases.

      8. Conclusions and perspectives

      Across the different stages of plaques build up, VSMCs are exposed to a variety of signaling molecules producing phenotypic changes in terms of their contractile, proliferative and migratory capacity, as well as vulnerability to apoptosis. Progressive loss of VSMCs contractility and their de-differentiation to a highly proliferative and synthetic phenotype represent major early pathological hallmarks of atherogenesis. However, at later disease stages, the ability to keep dividing, peculiar to these “modulated” VSMCs, turns to be essential to cope with the massive cell death characterizing the necrotic core of advanced plaques and to build a fibrous cap. Besides other strategies, targeting lncRNAs has revealed to be an appealing and promising approach to ad libitum modulate VSMCs phenotype in the effort to meet therapeutic needs. LncRNAs represent potent regulators of reprogramming gene expression and have been shown to actively contribute to regulate the molecular dynamics underlying vascular remodeling. Understanding the grammar behind the formation of clinically relevant atherosclerotic plaques and unveiling the role played by lncRNAs represent major points in conceiving break-through therapies for early intervention and re-definition of diagnostic and prognostic criteria. Exploiting lncRNAs as targets or templates for a new generation of therapeutics is not exempt from major challenges. These are usually represented by immunogenicity of delivery vectors, size, delivery specificity and efficiency. Continuous advances in nanotechnology are contributing to boost the success of RNA drug technologies, and could pave the way of lncRNAs as therapeutics of the future.

      Financial support

      Research in LM's laboratories in Munich and Stockholm are supported by the Swedish Heart-Lung-Foundation (20210450), the Swedish Research Council (Vetenkapsrådet, 2019-01577), a DZHK Translational Research Project on microRNA modulation in aortic aneurysms, the CRC1123 and TRR267 of the German Research Council (DFG), the National Institutes of Health (NIH; 1R011HL150359-01), and the Bavarian State Ministry of Health and Care through the research project DigiMed Bayern.

      Declaration of competing interest

      LM is a scientific consultant and adviser for Novo Nordisk (Malov, Denmark), DrugFarm (Shanghai, China), and Angiolutions (Hannover, Germany), and received research funds from Roche Diagnostics (Rotkreuz, Switzerland). The other authors have nothing to disclose.

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