Review
MicroRNAs as therapeutic targets in atherosclerosis Xavier Loyer, Ziad Mallat, Chantal M Boulanger & Alain Tedgui† †
Universit e Paris Descartes, Sorbonne Paris Cit e , Paris Cardiovascular Research Center -- PARCC, INSERM UMR-S 970, Paris, France
1.
Introduction
2.
What are miRNAs?
3.
miRNAs and atherosclerosis
4.
MiRNAs transfer by
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microvesicles in atherosclerosis 5.
Conclusion
6.
Expert opinion
Introduction: Atherosclerosis is a chronic inflammatory disease of the arterial wall. A number of phenotypic cell changes occur during the development and progression of atherosclerosis. MicroRNAs (miRNAs) are key regulators of gene expression that act at the post-transcriptional level. They have been implicated in cardiovascular diseases, including atherosclerosis. Areas covered: This review provides an overview of our knowledge about the expression and the roles of miRNAs in different cell types involved in atherosclerosis, with a focus on experimental strategies to modulate miRNA expression and their therapeutic effects in animal models of atherosclerosis. miRNA expression is regulated by inflammatory conditions and by shear stress in endothelial cells. Therapeutic approaches using antagomiR and miRNA mimic delivery and have been shown potentially effective in atherosclerosis. Moreover, a large body of evidence exists supporting a role for not only intracellular miRNA, but also miRNA carried by extracellular vesicles that are involved in inter-cellular communication through the transfer of miRNA. Expert opinion: Modulation of miRNA expression could represent novel innovative therapeutic approaches to treat atherosclerosis by targeting a single cell type or specific pathways. Future challenges will consist in deciphering the mechanisms involved in miRNA regulation and in improving cell-specific delivery of ‘miR-drugs’ by alternative strategies, including miRNA-enriched micro vesicles. Keywords: atherosclerosis, endothelium, inflammation, microRNAs, therapy Expert Opin. Ther. Targets [Early Online]
1.
Introduction
Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by two main hallmarks. First, atherosclerosis develops preferentially at sites of branching, curvatures, and bifurcations in large arteries where flow conditions are disturbed, with prevalence of low or oscillatory shear stress (SS) [1]. Second, subendothelial accumulation of low-density lipoprotein (LDL) and its subsequent modification at these atheroprone areas lead to further activation of the vascular cells, and initiates innate and adaptive immune responses [2]. Atherosclerosis is also characterized by phenotypic changes in vascular and immuno-inflammatory cells [3], which result from modulation of gene expression. MicroRNAs (miRNAs) have emerged as a novel class of gene regulators, and recent studies have highlighted their crucial role in the setting of atherosclerosis. 2.
What are miRNAs?
miRNAs are short single-strand nucleotide (21 -- 25 nucleotide long), abundantly expressed in all cell types. miRNAs are first transcribed as a primary miRNA (primiRNA) into the nucleus by a mechanism involving RNA polymerase II [4]. Then, pri-miRNAs are processed in the nucleus by the RNase III enzyme Drosha, 10.1517/14728222.2014.989835 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted
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Article highlights. .
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Atherosclerosis is a chronic inflammatory disease characterized by the involvement of several cell types and phenotypic changes. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. They have been shown as crucial mediators in the setting of atherosclerosis in different cell types. Experimental observations have shown that miRNA modulation in vivo (blocking or restoring) is an effective new strategy to modulate atherosclerosis. Targeting miRNAs may provide new therapeutic strategies against the development of atherosclerosis and its clinical complications.
This box summarizes key points contained in the article.
resulting in the generation of precursor-miRNA (pre-miRNA). These pre-miRNAs are then exported to the cytoplasm by a mechanism involving exportin III, and then processed by the RNase III Dicer to become mature miRNAs before they are finally incorporated into the miR-induced silencing complex, that contains an Argonaute protein. MiRNAs act by binding in the 3¢-untranslated region (3¢-UTR) of their target mRNA. Once miRNAs are bound to the 3¢UTR region, they either block translation or promote mRNA degradation [5,6]. 3.
miRNAs and atherosclerosis
Atherosclerosis implicates complex cross-talks between vascular cells (endothelial and smooth muscle cells) and immune cells [7]. Here, we will review current knowledge regarding miRNA expression and function in the different cell types contributing to atherosclerosis (Figure 1). Endothelial cells and miRNAs Atherosclerosis preferentially develops in areas where blood flow is disturbed. This includes curved regions, arterial bifurcations, and branch ostia [1] where endothelial cells (ECs) are exposed to low shear stress (LSS). SS not only regulates endothelial gene expression [8], but also modulates miRNA levels both in vitro and in vivo (for review see [9]). Three subclasses of endothelial miRNAs have been identified as being controlled by SS: 1- antiatherogenic, 2- proatherogenic, also called atheromiR, [10], and 3- bivalent (either pro or anti-atherogenic). Some endothelial miRNAs are also modulated independently of mechanical forces, and have been termed non-mechanosensitive atheromiRs [9]. 3.1
Mechanosensitive endothelial miRNAs Antiatherogenic endothelial miRNAs are 1- increased by laminar or high flow and 2-decreased in response to low or disturbed flow. They act as inhibitors of atherosclerosis development. The role of these miRNAs has been mostly 3.1.1
2
unraveled in vitro in ECs exposed to different conditions of SS, and few of them have been extensively investigated in vivo. miR-10a expression in ECs is decreased in atheroprone areas of the porcine aorta as compared to atheroprotected regions [11], indicating that this miRNA potentially behaves as an antiatherogenic miRNA. Analysis of the functional activity of miR-10a revealed that it exhibits potent anti-inflammatory properties, mediated by the inhibition of NF-kB activity. As a result, miR-10a helps tune down endothelial inflammation and adhesion, suggesting that the shear-dependent down-regulation of miR-10a in athero-susceptible regions promotes in vivo a proinflammatory endothelial phenotype. miR-143/145 have also been reported as atheroprotective mechanosensitive miRNAs. They are regulated by the shearresponsive transcription factor Kru¨ppel-like factor 2 (KLF2), whose expression is enhanced by flow [12]. The mechanosensitivity of miR-143/145 is dependent on the endothelial expression of AMPKa2 [13]. Interestingly, miR-143/145 derived from ECs can be delivered to smooth muscle cells via endothelial extracellular vesicles, promoting an atheroprotective phenotype in smooth muscle cells [12]. In vitro analysis-based on miRNA sensitivity to fluid flow conditions, identified miR-19a as a mechanosensitive miRNA rapidly induced by SS in ECs [14]. MiR-19a targets cyclin D1 and controls cell proliferation. MiR-23b exhibits a similar expression pattern and modulates cell proliferation [15]. However, no data are yet available regarding the in vivo function of these miRNAs. As to proatherogenic miRNAs, atheromiRs, only a few studies examined their in vivo relevance in mouse models of atherosclerosis or in human tissues. miR-92a and miR-712 can be considered as atheromiRs based on their expression pattern in vitro and in vivo: their expression is markedly increased in the atheroprone area and very low in atheroprotected regions of the mouse aorta. The expression of miR-92a, a member of the miR-17/92a cluster, is enhanced by low or oscillatory SS in vitro and in vivo [10,11,15-17]. miR-92a is particularly relevant as a mechanosensitive atheromiR, because its expression increases in ECs exposed to oxidized LDL (oxLDL) under LSS, but not under high shear stress conditions [10]. KLF2 and KLF4 are targets for miR-92a: upregulation of miR-92a decreases their expression, whereas inhibition of miR-92a has an opposite effect, predominantly under LSS conditions when miR-92a is highly expressed [10]. Inhibition of miR-92a also reduces NF-kB activity and promotes the expression of endothelial nitric oxide synthase (NOS3), target of both KLF2 and KLF4 [10,16,17]. Moreover, suppressor of cytokine signaling 5 (SOCS5) is specifically targeted by miR-92a in ECs under LSS conditions and upon exposure to oxLDL. SOCS5 directly protects against endothelial activation and inflammation: inhibition of SOCS5 increases the release of monocyte chemoattractant protein-1 and IL-6 [10]. More importantly, miR-92a appears to play a major role in promoting atherosclerosis in vivo. Ldlr-/- mice treated with an antagomir against miR-92a have
Expert Opin. Ther. Targets (2014) ()
MicroRNAs as therapeutic targets in atherosclerosis
miR-126-3p
Endothelial cells
Immune cells
Non mechanosensitive miRNA
miR-155↑ miR-342-5p↑ miR-24↓ miR-33↓
miR-181b↓ EC
Endothelial cell-derived apoptotic bodies
Mechanosensitive miRNAs miR-92a↑ miR-712↑ miR-143/145↑ miR-126-5p↓
Endothelial cell-derived derived exosomes miR-143/145
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Inflammation cholesterol homeostasis MMP activation
Inflammation/activation
Smooth muscle cells
Transfert
miR-143/145↑
Differentiation
Atherosclerosis development
Atherosclerosis limitation
Figure 1. Synoptic summary of the current knowledge on miRNAs involved in the setting of atherosclerosis. All miRNAs presented in the figure have been shown to be involved in the development of atherosclerosis in vitro, as well as in vivo. These miRNAs have been modulated in vivo using either specific inhibitors or mimic compounds. " indicates increased expression; # indicates decreased expression; EC: Endothelial cell; miRNAs: MicroRNAs.
smaller atherosclerotic lesions, containing less T cells and macrophages. These lesions are also more fibrotic, a phenotype that is reminiscent of a stable human atherosclerotic plaque [10]. miR-712 is another mechanosensitive atheromiR that has been identified in a mouse model of flow-induced atherosclerosis [18]. Disturbed flow increases endothelial miR-712 expression. This miRNA targets tissue inhibitor of metalloproteinase 3, which contributes to activate MMPs and a disintegrin and metalloproteases, which promote endothelial inflammation and pro-atherogenic responses [18]. Specific in vivo inhibition via anti-miR-712 treatment prevents atherosclerosis in ApoE-/- mice. Several miRNAs responding to flow conditions in ECs appear to play a bivalent role in atherosclerosis. For instance, both oscillatory SS [19] and laminar flow [20] increase miR-21 expression in vitro. On the one hand, miR-21 targets PPARa, and therefore promotes inflammatory responses and leukocyte adhesion. On the other hand, endothelial miR-21 targets phosphatase and tensin homolog, which improves NO bioavailability and endothelial survival [20]. The function of miR-126 in atherosclerosis is still controversial. miR-126 is highly expressed in ECs [21], and contradictory results about its expression pattern under flow conditions have been reported in vitro. In response to laminar SS, the expression of miR-126 has been shown to be either increased [22] or unaltered [23]. In vivo, Zhou et al. [23] showed that miR-126 expression is increased in the aortic arch of mouse, an atheroprone region where flow is disturbed, whereas Schober et al. [22] observed a decreased expression in response to LSS in a model of carotid artery flow cessation.
Functional analysis showed that the passenger strand of miR-126, miR-126 -- 5p, facilitates EC proliferation and inhibits atherosclerosis by targeting Dlk1 [22], whereas miR-126 -- 3p is secreted in extracellular vesicles that can act in an autocrine manner [24] on ECs or by paracrine mechanisms on smooth muscle cells [23]. In vivo, systemic delivery of miR-126 -- 5p mimic in miR-126-deficient ApoE-/- reduced atherosclerotic lesion formation in the aorta and aortic sinus [22]. Non-mechanosensitive endothelial miRNAs Endothelial activation and dysfunction play a major role in the development and progression of atherosclerosis by facilitating vascular inflammation. Recent studies have shown that miRNA expression levels and inflammation are mutually influenced by each other in ECs. Notably, miR-181b is a regulator of vascular inflammation by targeting importin-a3 [25] and interfering with the NF-kB activation pathway. Yet, miR-181b is preferentially expressed in healthy endothelium and its expression is decreased by proinflammatory signals. Its expression is decreased in atherosclerotic aorta of ApoE-/mice, and systemic delivery of miR-181b suppresses NF-kB signaling in the endothelium and reduces atherosclerotic lesion formation [26]. miR-146 has also be described as an miRNA regulated by and controlling endothelial inflammation [27]. Endothelial expression of miR-146 is increased by IL-1 b in vitro. miR-146 over expression hampers endothelial activation, whereas its genetic deletion aggravates endothelial inflammation. miR-146a targets human antigen R, an RNA-binding protein that induces 3.1.2
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endothelial activation by inhibiting the expression of endothelial NOS [27]. Vascular smooth muscle cells and miRNAs Limited observations are available regarding the role of miRs in regulating smooth muscle cell function in atherosclerosis. miR-663 has been shown to be involved in the regulation of human smooth muscle cell phenotypic switch and neointimal formation [28]. miR-145, an miRNA specifically enriched in smooth muscle cells [29], regulates smooth muscle cell fate and plasticity. Lentiviral delivery of miR-145 specifically in smooth muscle cells limits atherosclerotic lesion formation in ApoE-/- mice and promotes a more stable phenotype with less macrophages, thick fibrous cap, reduced necrotic core, and increased collagen content [30]. However, surprisingly the genetic deficiency in miR-143/145 protects against atherosclerosis development in Ldlr-/- mice [31].
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3.2
Immune cells and miRNAs Immune cells, including monocytes/macrophages, dendritic cells, as well as T and B lymphocytes, play a key role in atherosclerosis [7]. miRNAs have been shown to control and regulate immune cell functions [32]. In addition, miRNAs can regulate lipid uptake and expression of inflammatory mediators in monocytes/macrophages in the context of atherosclerosis [33]. Besides controlling endothelial NOS3 expression [34], miR-155 acts as a central regulator of macrophage function in atherosclerosis. miR-155 is preferentially expressed and localized in macrophages following partial carotid ligation in ApoE-/- mice [35]. Moreover, miR-155 deletion in leukocytes prevents atherosclerotic lesion formation, whereas its deletion in non-hematopoietic cells does not affect lesion development. These observations have been recently confirmed by Du et al. [36]. However, miR-155-/- or wild-type bone marrow transplantation in Ldlr-/- mice does not corroborate these findings [37]. In this particular model, miR-155-deficient bone marrow transplantation increased lesion formation, which was associated with enhanced inflammation and proinflammatory polarization of macrophages [37]. In macrophages, miR-155 seems to act by repressing B-cell lymphoma 6, which in turn potentiates chemokine ligand 2 expression [35]. Macrophages deficient in miR-155 are characterized by a diminished expression of pro-inflammatory factors (TNFa, IL-1b, IL-6) in response to toll-like receptor 4 activation, via SOCS1 regulation [36]. MiR-155 deficiency increases cholesterol efflux in macrophages, which is independent of ATP-binding cassette transporter 1 (ABCA1) and ATP binding cassette transporter G1 [36]. Altogether these effects can account for the proatherogenic activity of miR-155 in macrophages. However, paradoxically, systemic delivery of miR-155 in ApoE-/- mice does not reduce atherosclerotic lesion formation, but limits immune cell infiltration in plaques [38]. miR-342 -- 5p is highly expressed and upregulated in atherosclerotic lesions, in particular in macrophages [39]. 3.3
4
miR-342 -- 5p-specific inhibition in macrophages impairs the expression of NOS2 induced by pro-inflammatory cytokines. miR-342 -- 5p targets AKT1, which dampens proinflammatory responses [39]. In addition, AKT1 controls the expression of miR-155 [40]. In vivo treatment of ApoE-/mice with specific antagomiRs against miR-342 -- 5p limits atherosclerosis development by increasing AKT 1 and reducing miR-155 expression. A role for miR-24 in macrophage accumulation in atherosclerotic lesions has been recently reported [41]. MiR-24 controls MMP-14 expression and systemic delivery of antagomiR-24 increased plaque size and MMP-14 expression. miR-33 expression is down-regulated in macrophages following cholesterol loading. miR-33 acts via inhibiting ABCA1 expression, which in turn inhibits cholesterol efflux in macrophages [42]. Specific antagomiR-33 treatment promotes reverse cholesterol transport, increases circulating high density lipoprotein and contributes to atherosclerosis diminution in nonhuman primates [43], as well as in ApoE-/- [42] and Ldlr-/mice [44]. However, anti-miR-33 therapy in ApoE-/- mice appears to be ineffective against atherosclerosis progression [45]. These contradictory results can be explained by differences in the antimiR-33 chemistry used and/or by differences in mouse models of atherosclerosis (ApoE-/- vs Ldlr-/- mice). Surprisingly, transplantation of miR-33-deficient bone marrow did not alter plaque size, but reduced lipid content without affecting inflammatory cell infiltration in atherosclerotic plaques, compared with mice transplanted with control ApoE-/- bone marrow [46]. It is noteworthy that most of the studies dealing with the role of miRNAs in atherosclerosis have focused on how miRNAs modulate macrophage functions. So far, no study has addressed the role of miRNAs in T/B cells or dendritic cells in the context of atherosclerosis in vivo.
MiRNAs transfer by microvesicles in atherosclerosis
4.
MiRNA cargos packaged into extracellular microvesicles contribute to cell--cell communication. Interestingly, delivery of miRNA-containing extracellular vesicles is effective in atherosclerosis [24,47]. Zernecke et al. [24] reported for the first time that microvesicles containing miR-126 limit atherosclerosis and stabilize atherosclerotic plaques in different mouse models of atherosclerosis. In addition, miR-145 contained in endothelial microvesicles can be transferred to smooth muscle cells and reduces atherosclerosis in ApoE-/- mice [12]. Recent in vitro findings have shown that miR-145 can be transferred from smooth muscle cells to macrophages [31], suggesting the possibility for local exchanges of miRNAs between different cell types during atherosclerosis development. 5.
Conclusion
Atherosclerosis is a long-term disease that involves inflammatory responses of both vascular and immune cells. MiRNAs
Expert Opin. Ther. Targets (2014) ()
Expert Opin. Ther. Targets (2014) ()
EC EC EC
EC
EC EC EC VSMC
VSMC
Macrophages Macrophages ND
Macrophages Macrophages Lesions macrophages and liver Macrophages
ND
miR-92a miR-712/205 miR-126-3p
miR-126-3p
miR-126-5p miR-181b miR-143/145 miR-143/145
miR-143/145
miR-155 miR-155 miR-155
miR-342-5p miR-24 miR-33
miR-33
Macrophages and BMDM ND
ABCA1, ABCG1, RIP140, CROT ND
Akt1 MMP14 ABCA1
Bcl6 SOCS1 MAP3K10
ND
Dlk1 Importin-3
" In advanced lesions " In advanced lesions " In advanced lesions and apoE-/- vessels " In advanced lesions " In Advanced lesions Infiltrated macrophages in lesions
LSS intima with time LSS lesions ABCA1
In In In In
ND
# # # #
RGS16
KLF2, KLF4, SOCS5 TIMP3 ND
" In " In " In " In ND
LSS and # In HSS LSS HSS aortic arch
Main target(s) involved in atherosclerosis
Modulation
No
ND
Yes Yes ND
Yes Yes Yes
Yes Yes Present in MV Yes
Present in MV
Yes Yes Yes
In vivo validation
Blocking
Genetic deficiency
Blocking Blocking Blocking
AntagomiR AntagomiR miR-126 mimic delivery in miR-126-/- mice miR-126 mimic and MV from miR-126 -/- mice delivery miR-126-5p mimic delivery miR-181b mimic delivery Blocking Lentiviral miR-145 VSMC delivery Genetic deletion in ldlr-/mice Genetic deletion Genetic deletion miR-155 mimic delivery
Modulation strategies
No effect
Prevention
Prevention Prevention Prevention
Prevention Prevention No effect
Prevention
Prevention Prevention Prevention Prevention
Prevention
Prevention Prevention Prevention
Effect on atherosclerosis
[45]
[46]
[39] [41] [43]
[35] [36] [38]
[31]
[22] [26] [12] [30]
[24]
[10] [18] [23]
Ref.
ABCA1: ATP-binding cassette transporter 1; ABCG1: ATP binding cassette transporter G1; Bcl6: B-cell lymphoma 6; BMDM: Bone marrow-derived macrophages; EC: Endothelial cell; HSS: High shear stress; LSS: Low shear stress; ND: Not determined; SOCS: Suppressor of cytokine signalling; TIMP3: Tissue inhibitor of metalloproteinase 3; VSMC: Vascular smooth muscle cell.
miR-33
Cell expression
microRNA
Table 1. Summary of in vivo based evidence of miR modulation and its impact on atherosclerosis.
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MicroRNAs as therapeutic targets in atherosclerosis
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have recently emerged as powerful regulators of various cellular processes. Expression profile analysis has shown that miRNAs are highly dysregulated in ECs, smooth muscle cells, and macrophages, in the pathological context of atherosclerosis, which contributes to the development and progression of the disease. Experimental evidence from animal models of atherosclerosis suggests that therapeutic approaches using ‘miR-drugs’ (antagomiRs and miR mimics) are novel strategies to combat atherosclerosis.
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6.
Expert opinion
Significant progress in the understanding of miRNA function in atherosclerosis has provided a list of potential targets for therapeutic intervention. Modulating miRNA expression could represent new therapeutic approaches to treat or limit atherosclerosis. Several experimental studies provided strong evidence that specific blockade of miRNA expression modulates atherosclerosis development and progression (Table 1). For instance, antagomiR-based strategies performed in mouse models of atherosclerosis have shown that systemic targeting of specific miRNAs of interest can limit atherosclerosis. [10,24,35,43,44]. Such a therapeutic tool is delivered in the general circulation and likely impacts several cell types such as ECs, monocytes/macrophages. Restoring miRNA levels is quite challenging, but represents also an interesting approach to prevent atherosclerosis progression (Table 1) [22,26,30]. Specific blockade of miR-33 in non-human primates has proved effective in inhibiting atherosclerosis. This could represent a new therapeutic approach against atherosclerosis [48]. Some anti-miRNA strategies such as antimiR-122 are already being tested in liver diseases [49,50]. The importance of miRNAs in the pathophysiological context of atherosclerosis makes them a therapeutic target of choice. A large body of experimental evidence supports the concept that miRNA modulation could be efficient in the setting of this disease. Future challenges consist mainly in understanding molecular mechanisms that lead to miRNA
6
expression and/or dysregulation in pathology, which will help develop new therapeutic approaches. Discovery of therapeutic molecules that block or stimulate miRNA expression represents an exciting area that will likely lead to the development of agents that work in-concert with other drugs affecting atherosclerosis. Deciphering the mechanisms by which miRNA expression is controlled, in the one hand, and in the other hand how to specifically modulate (increase or block miRNA expression) will help in the development of new therapeutic strategies. One limitation concerns the fact that a single miRNA can be expressed by several cell types, but acts on hundreds of target genes. Expanding the number of new targets or targeting known miRNAs in new ways will be critical to improve clinical outcomes in atherosclerosis. It can be anticipated that the next 5 -- 10 years will see much upheaval in anti- and/or promiRNA discovery. Another limitation resides in the fact that, as shown by experimental and clinical experiments with antimiRNAs, the composition of anti-miRNA preparations and the route of administration are major determinants of efficacy [51]. Certainly, innovative anti-miRNA preparations will offer alternatives to inevitable problems of specificity. Especially, miRNA-containing extracellular vesicles [47] represent also a rich site for potential therapeutic intervention. Indeed, the identification and characterization of the specific functions of these miRNA vesicles and their interactions with cells open avenues for future development of specific inhibitors/ activators targeting these components with precise delivery in a pathological context.
Declaration of interest The authors were supported by INSERM. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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MicroRNAs as therapeutic targets in atherosclerosis
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Xin M, Small EM, Sutherland LB, et al. MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury. Genes Dev 2009;23(18):2166-78 Lovren F, Pan Y, Quan A, et al. MicroRNA-145 targeted therapy reduces atherosclerosis. Circulation 2012;126(11 Suppl 1):S81-90 Sala F, Aranda JF, Rotllan N, et al. MiR-143/145 deficiency protects against progression of atherosclerosis in Ldlr-/mice. Thromb Haemost 2014;112(4):796-802 O’Connell RM, Rao DS, Chaudhuri AA, et al. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol 2010;10(2):111-22 Zernecke A. MicroRNAs in the regulation of immune cell functions--implications for atherosclerotic vascular disease. Thromb Haemost 2012;107(4):626-33 Lee KS, Kim J, Kwak SN, et al. Functional role of NF-kappaB in expression of human endothelial nitric oxide synthase. Biochem Biophys Res Commun 2014;448(1):101-7 Nazari-Jahantigh M, Wei Y, Noels H, et al. MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages. J Clin Invest 2012;122(11):4190-202 Du F, Yu F, Wang Y, et al. MicroRNA-155 deficiency results in decreased macrophage inflammation and attenuated atherogenesis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2014;34(4):759-67 Donners MM, Wolfs IM, Stoger LJ, et al. Hematopoietic miR155 deficiency
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Androulidaki A, Iliopoulos D, Arranz A, et al. The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs. Immunity 2009;31(2):220-31
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Di Gregoli K, Jenkins N, Salter R, et al. MicroRNA-24 Regulates Macrophage Behavior and Retards Atherosclerosis. Arterioscler Thromb Vasc Biol 2014;34(9):1990-2000
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Rayner KJ, Suarez Y, Davalos A, et al. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 2010;328(5985):1570-3
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Rayner KJ, Sheedy FJ, Esau CC, et al. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest 2011;121(7):2921-31 This study shows that blocking miR33 affects cholesterol metabolism and atherosclerosis lesion formation.
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Rotllan N, Ramirez CM, Aryal B, et al. Therapeutic silencing of microRNA-33 inhibits the progression of atherosclerosis in Ldlr-/- mice--brief report. Arterioscler Thromb Vasc Biol 2013;33(8):1973-7
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Marquart TJ, Wu J, Lusis AJ, et al. Anti-miR-33 therapy does not alter the progression of atherosclerosis in lowdensity lipoprotein receptor-deficient
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mice. Arterioscler Thromb Vasc Biol 2013;33(3):455-8
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van der Ree MH, van der Meer AJ, de Bruijne J, et al. Long-term safety and efficacy of microRNA-targeted therapy in chronic hepatitis C patients. Antiviral Res 2014;111:53-9
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van Rooij E, Olson EN. MicroRNA therapeutics for cardiovascular disease: opportunities and obstacles. Nat Rev Drug Discov 2012;11(11):860-72
Affiliation Xavier Loyer1 PhD, Ziad Mallat1,2 MD PhD, Chantal M Boulanger1 PharmD PhD & Alain Tedgui†1 PhD † Author for correspondence 1 Universite Paris Descartes, Sorbonne Paris Cite, Paris Cardiovascular Research Center -- PARCC, INSERM UMR-S 970, 56 rue Leblanc, 75015 Paris, France Tel: +33 1 53 98 80 06; Fax: +33 1 53 98 79 52; E-mail: [email protected] 2 University of Cambridge, Department of Medicine, Cambridge, UK
MicroRNAs as therapeutic targets in atherosclerosis Xavier Loyer, Ziad Mallat, Chantal M Boulanger & Alain Tedgui† †
Universit e Paris Descartes, Sorbonne Paris Cit e , Paris Cardiovascular Research Center -- PARCC, INSERM UMR-S 970, Paris, France
1.
Introduction
2.
What are miRNAs?
3.
miRNAs and atherosclerosis
4.
MiRNAs transfer by
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microvesicles in atherosclerosis 5.
Conclusion
6.
Expert opinion
Introduction: Atherosclerosis is a chronic inflammatory disease of the arterial wall. A number of phenotypic cell changes occur during the development and progression of atherosclerosis. MicroRNAs (miRNAs) are key regulators of gene expression that act at the post-transcriptional level. They have been implicated in cardiovascular diseases, including atherosclerosis. Areas covered: This review provides an overview of our knowledge about the expression and the roles of miRNAs in different cell types involved in atherosclerosis, with a focus on experimental strategies to modulate miRNA expression and their therapeutic effects in animal models of atherosclerosis. miRNA expression is regulated by inflammatory conditions and by shear stress in endothelial cells. Therapeutic approaches using antagomiR and miRNA mimic delivery and have been shown potentially effective in atherosclerosis. Moreover, a large body of evidence exists supporting a role for not only intracellular miRNA, but also miRNA carried by extracellular vesicles that are involved in inter-cellular communication through the transfer of miRNA. Expert opinion: Modulation of miRNA expression could represent novel innovative therapeutic approaches to treat atherosclerosis by targeting a single cell type or specific pathways. Future challenges will consist in deciphering the mechanisms involved in miRNA regulation and in improving cell-specific delivery of ‘miR-drugs’ by alternative strategies, including miRNA-enriched micro vesicles. Keywords: atherosclerosis, endothelium, inflammation, microRNAs, therapy Expert Opin. Ther. Targets [Early Online]
1.
Introduction
Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by two main hallmarks. First, atherosclerosis develops preferentially at sites of branching, curvatures, and bifurcations in large arteries where flow conditions are disturbed, with prevalence of low or oscillatory shear stress (SS) [1]. Second, subendothelial accumulation of low-density lipoprotein (LDL) and its subsequent modification at these atheroprone areas lead to further activation of the vascular cells, and initiates innate and adaptive immune responses [2]. Atherosclerosis is also characterized by phenotypic changes in vascular and immuno-inflammatory cells [3], which result from modulation of gene expression. MicroRNAs (miRNAs) have emerged as a novel class of gene regulators, and recent studies have highlighted their crucial role in the setting of atherosclerosis. 2.
What are miRNAs?
miRNAs are short single-strand nucleotide (21 -- 25 nucleotide long), abundantly expressed in all cell types. miRNAs are first transcribed as a primary miRNA (primiRNA) into the nucleus by a mechanism involving RNA polymerase II [4]. Then, pri-miRNAs are processed in the nucleus by the RNase III enzyme Drosha, 10.1517/14728222.2014.989835 © 2014 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted
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Article highlights. .
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.
Atherosclerosis is a chronic inflammatory disease characterized by the involvement of several cell types and phenotypic changes. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. They have been shown as crucial mediators in the setting of atherosclerosis in different cell types. Experimental observations have shown that miRNA modulation in vivo (blocking or restoring) is an effective new strategy to modulate atherosclerosis. Targeting miRNAs may provide new therapeutic strategies against the development of atherosclerosis and its clinical complications.
This box summarizes key points contained in the article.
resulting in the generation of precursor-miRNA (pre-miRNA). These pre-miRNAs are then exported to the cytoplasm by a mechanism involving exportin III, and then processed by the RNase III Dicer to become mature miRNAs before they are finally incorporated into the miR-induced silencing complex, that contains an Argonaute protein. MiRNAs act by binding in the 3¢-untranslated region (3¢-UTR) of their target mRNA. Once miRNAs are bound to the 3¢UTR region, they either block translation or promote mRNA degradation [5,6]. 3.
miRNAs and atherosclerosis
Atherosclerosis implicates complex cross-talks between vascular cells (endothelial and smooth muscle cells) and immune cells [7]. Here, we will review current knowledge regarding miRNA expression and function in the different cell types contributing to atherosclerosis (Figure 1). Endothelial cells and miRNAs Atherosclerosis preferentially develops in areas where blood flow is disturbed. This includes curved regions, arterial bifurcations, and branch ostia [1] where endothelial cells (ECs) are exposed to low shear stress (LSS). SS not only regulates endothelial gene expression [8], but also modulates miRNA levels both in vitro and in vivo (for review see [9]). Three subclasses of endothelial miRNAs have been identified as being controlled by SS: 1- antiatherogenic, 2- proatherogenic, also called atheromiR, [10], and 3- bivalent (either pro or anti-atherogenic). Some endothelial miRNAs are also modulated independently of mechanical forces, and have been termed non-mechanosensitive atheromiRs [9]. 3.1
Mechanosensitive endothelial miRNAs Antiatherogenic endothelial miRNAs are 1- increased by laminar or high flow and 2-decreased in response to low or disturbed flow. They act as inhibitors of atherosclerosis development. The role of these miRNAs has been mostly 3.1.1
2
unraveled in vitro in ECs exposed to different conditions of SS, and few of them have been extensively investigated in vivo. miR-10a expression in ECs is decreased in atheroprone areas of the porcine aorta as compared to atheroprotected regions [11], indicating that this miRNA potentially behaves as an antiatherogenic miRNA. Analysis of the functional activity of miR-10a revealed that it exhibits potent anti-inflammatory properties, mediated by the inhibition of NF-kB activity. As a result, miR-10a helps tune down endothelial inflammation and adhesion, suggesting that the shear-dependent down-regulation of miR-10a in athero-susceptible regions promotes in vivo a proinflammatory endothelial phenotype. miR-143/145 have also been reported as atheroprotective mechanosensitive miRNAs. They are regulated by the shearresponsive transcription factor Kru¨ppel-like factor 2 (KLF2), whose expression is enhanced by flow [12]. The mechanosensitivity of miR-143/145 is dependent on the endothelial expression of AMPKa2 [13]. Interestingly, miR-143/145 derived from ECs can be delivered to smooth muscle cells via endothelial extracellular vesicles, promoting an atheroprotective phenotype in smooth muscle cells [12]. In vitro analysis-based on miRNA sensitivity to fluid flow conditions, identified miR-19a as a mechanosensitive miRNA rapidly induced by SS in ECs [14]. MiR-19a targets cyclin D1 and controls cell proliferation. MiR-23b exhibits a similar expression pattern and modulates cell proliferation [15]. However, no data are yet available regarding the in vivo function of these miRNAs. As to proatherogenic miRNAs, atheromiRs, only a few studies examined their in vivo relevance in mouse models of atherosclerosis or in human tissues. miR-92a and miR-712 can be considered as atheromiRs based on their expression pattern in vitro and in vivo: their expression is markedly increased in the atheroprone area and very low in atheroprotected regions of the mouse aorta. The expression of miR-92a, a member of the miR-17/92a cluster, is enhanced by low or oscillatory SS in vitro and in vivo [10,11,15-17]. miR-92a is particularly relevant as a mechanosensitive atheromiR, because its expression increases in ECs exposed to oxidized LDL (oxLDL) under LSS, but not under high shear stress conditions [10]. KLF2 and KLF4 are targets for miR-92a: upregulation of miR-92a decreases their expression, whereas inhibition of miR-92a has an opposite effect, predominantly under LSS conditions when miR-92a is highly expressed [10]. Inhibition of miR-92a also reduces NF-kB activity and promotes the expression of endothelial nitric oxide synthase (NOS3), target of both KLF2 and KLF4 [10,16,17]. Moreover, suppressor of cytokine signaling 5 (SOCS5) is specifically targeted by miR-92a in ECs under LSS conditions and upon exposure to oxLDL. SOCS5 directly protects against endothelial activation and inflammation: inhibition of SOCS5 increases the release of monocyte chemoattractant protein-1 and IL-6 [10]. More importantly, miR-92a appears to play a major role in promoting atherosclerosis in vivo. Ldlr-/- mice treated with an antagomir against miR-92a have
Expert Opin. Ther. Targets (2014) ()
MicroRNAs as therapeutic targets in atherosclerosis
miR-126-3p
Endothelial cells
Immune cells
Non mechanosensitive miRNA
miR-155↑ miR-342-5p↑ miR-24↓ miR-33↓
miR-181b↓ EC
Endothelial cell-derived apoptotic bodies
Mechanosensitive miRNAs miR-92a↑ miR-712↑ miR-143/145↑ miR-126-5p↓
Endothelial cell-derived derived exosomes miR-143/145
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Inflammation cholesterol homeostasis MMP activation
Inflammation/activation
Smooth muscle cells
Transfert
miR-143/145↑
Differentiation
Atherosclerosis development
Atherosclerosis limitation
Figure 1. Synoptic summary of the current knowledge on miRNAs involved in the setting of atherosclerosis. All miRNAs presented in the figure have been shown to be involved in the development of atherosclerosis in vitro, as well as in vivo. These miRNAs have been modulated in vivo using either specific inhibitors or mimic compounds. " indicates increased expression; # indicates decreased expression; EC: Endothelial cell; miRNAs: MicroRNAs.
smaller atherosclerotic lesions, containing less T cells and macrophages. These lesions are also more fibrotic, a phenotype that is reminiscent of a stable human atherosclerotic plaque [10]. miR-712 is another mechanosensitive atheromiR that has been identified in a mouse model of flow-induced atherosclerosis [18]. Disturbed flow increases endothelial miR-712 expression. This miRNA targets tissue inhibitor of metalloproteinase 3, which contributes to activate MMPs and a disintegrin and metalloproteases, which promote endothelial inflammation and pro-atherogenic responses [18]. Specific in vivo inhibition via anti-miR-712 treatment prevents atherosclerosis in ApoE-/- mice. Several miRNAs responding to flow conditions in ECs appear to play a bivalent role in atherosclerosis. For instance, both oscillatory SS [19] and laminar flow [20] increase miR-21 expression in vitro. On the one hand, miR-21 targets PPARa, and therefore promotes inflammatory responses and leukocyte adhesion. On the other hand, endothelial miR-21 targets phosphatase and tensin homolog, which improves NO bioavailability and endothelial survival [20]. The function of miR-126 in atherosclerosis is still controversial. miR-126 is highly expressed in ECs [21], and contradictory results about its expression pattern under flow conditions have been reported in vitro. In response to laminar SS, the expression of miR-126 has been shown to be either increased [22] or unaltered [23]. In vivo, Zhou et al. [23] showed that miR-126 expression is increased in the aortic arch of mouse, an atheroprone region where flow is disturbed, whereas Schober et al. [22] observed a decreased expression in response to LSS in a model of carotid artery flow cessation.
Functional analysis showed that the passenger strand of miR-126, miR-126 -- 5p, facilitates EC proliferation and inhibits atherosclerosis by targeting Dlk1 [22], whereas miR-126 -- 3p is secreted in extracellular vesicles that can act in an autocrine manner [24] on ECs or by paracrine mechanisms on smooth muscle cells [23]. In vivo, systemic delivery of miR-126 -- 5p mimic in miR-126-deficient ApoE-/- reduced atherosclerotic lesion formation in the aorta and aortic sinus [22]. Non-mechanosensitive endothelial miRNAs Endothelial activation and dysfunction play a major role in the development and progression of atherosclerosis by facilitating vascular inflammation. Recent studies have shown that miRNA expression levels and inflammation are mutually influenced by each other in ECs. Notably, miR-181b is a regulator of vascular inflammation by targeting importin-a3 [25] and interfering with the NF-kB activation pathway. Yet, miR-181b is preferentially expressed in healthy endothelium and its expression is decreased by proinflammatory signals. Its expression is decreased in atherosclerotic aorta of ApoE-/mice, and systemic delivery of miR-181b suppresses NF-kB signaling in the endothelium and reduces atherosclerotic lesion formation [26]. miR-146 has also be described as an miRNA regulated by and controlling endothelial inflammation [27]. Endothelial expression of miR-146 is increased by IL-1 b in vitro. miR-146 over expression hampers endothelial activation, whereas its genetic deletion aggravates endothelial inflammation. miR-146a targets human antigen R, an RNA-binding protein that induces 3.1.2
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endothelial activation by inhibiting the expression of endothelial NOS [27]. Vascular smooth muscle cells and miRNAs Limited observations are available regarding the role of miRs in regulating smooth muscle cell function in atherosclerosis. miR-663 has been shown to be involved in the regulation of human smooth muscle cell phenotypic switch and neointimal formation [28]. miR-145, an miRNA specifically enriched in smooth muscle cells [29], regulates smooth muscle cell fate and plasticity. Lentiviral delivery of miR-145 specifically in smooth muscle cells limits atherosclerotic lesion formation in ApoE-/- mice and promotes a more stable phenotype with less macrophages, thick fibrous cap, reduced necrotic core, and increased collagen content [30]. However, surprisingly the genetic deficiency in miR-143/145 protects against atherosclerosis development in Ldlr-/- mice [31].
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3.2
Immune cells and miRNAs Immune cells, including monocytes/macrophages, dendritic cells, as well as T and B lymphocytes, play a key role in atherosclerosis [7]. miRNAs have been shown to control and regulate immune cell functions [32]. In addition, miRNAs can regulate lipid uptake and expression of inflammatory mediators in monocytes/macrophages in the context of atherosclerosis [33]. Besides controlling endothelial NOS3 expression [34], miR-155 acts as a central regulator of macrophage function in atherosclerosis. miR-155 is preferentially expressed and localized in macrophages following partial carotid ligation in ApoE-/- mice [35]. Moreover, miR-155 deletion in leukocytes prevents atherosclerotic lesion formation, whereas its deletion in non-hematopoietic cells does not affect lesion development. These observations have been recently confirmed by Du et al. [36]. However, miR-155-/- or wild-type bone marrow transplantation in Ldlr-/- mice does not corroborate these findings [37]. In this particular model, miR-155-deficient bone marrow transplantation increased lesion formation, which was associated with enhanced inflammation and proinflammatory polarization of macrophages [37]. In macrophages, miR-155 seems to act by repressing B-cell lymphoma 6, which in turn potentiates chemokine ligand 2 expression [35]. Macrophages deficient in miR-155 are characterized by a diminished expression of pro-inflammatory factors (TNFa, IL-1b, IL-6) in response to toll-like receptor 4 activation, via SOCS1 regulation [36]. MiR-155 deficiency increases cholesterol efflux in macrophages, which is independent of ATP-binding cassette transporter 1 (ABCA1) and ATP binding cassette transporter G1 [36]. Altogether these effects can account for the proatherogenic activity of miR-155 in macrophages. However, paradoxically, systemic delivery of miR-155 in ApoE-/- mice does not reduce atherosclerotic lesion formation, but limits immune cell infiltration in plaques [38]. miR-342 -- 5p is highly expressed and upregulated in atherosclerotic lesions, in particular in macrophages [39]. 3.3
4
miR-342 -- 5p-specific inhibition in macrophages impairs the expression of NOS2 induced by pro-inflammatory cytokines. miR-342 -- 5p targets AKT1, which dampens proinflammatory responses [39]. In addition, AKT1 controls the expression of miR-155 [40]. In vivo treatment of ApoE-/mice with specific antagomiRs against miR-342 -- 5p limits atherosclerosis development by increasing AKT 1 and reducing miR-155 expression. A role for miR-24 in macrophage accumulation in atherosclerotic lesions has been recently reported [41]. MiR-24 controls MMP-14 expression and systemic delivery of antagomiR-24 increased plaque size and MMP-14 expression. miR-33 expression is down-regulated in macrophages following cholesterol loading. miR-33 acts via inhibiting ABCA1 expression, which in turn inhibits cholesterol efflux in macrophages [42]. Specific antagomiR-33 treatment promotes reverse cholesterol transport, increases circulating high density lipoprotein and contributes to atherosclerosis diminution in nonhuman primates [43], as well as in ApoE-/- [42] and Ldlr-/mice [44]. However, anti-miR-33 therapy in ApoE-/- mice appears to be ineffective against atherosclerosis progression [45]. These contradictory results can be explained by differences in the antimiR-33 chemistry used and/or by differences in mouse models of atherosclerosis (ApoE-/- vs Ldlr-/- mice). Surprisingly, transplantation of miR-33-deficient bone marrow did not alter plaque size, but reduced lipid content without affecting inflammatory cell infiltration in atherosclerotic plaques, compared with mice transplanted with control ApoE-/- bone marrow [46]. It is noteworthy that most of the studies dealing with the role of miRNAs in atherosclerosis have focused on how miRNAs modulate macrophage functions. So far, no study has addressed the role of miRNAs in T/B cells or dendritic cells in the context of atherosclerosis in vivo.
MiRNAs transfer by microvesicles in atherosclerosis
4.
MiRNA cargos packaged into extracellular microvesicles contribute to cell--cell communication. Interestingly, delivery of miRNA-containing extracellular vesicles is effective in atherosclerosis [24,47]. Zernecke et al. [24] reported for the first time that microvesicles containing miR-126 limit atherosclerosis and stabilize atherosclerotic plaques in different mouse models of atherosclerosis. In addition, miR-145 contained in endothelial microvesicles can be transferred to smooth muscle cells and reduces atherosclerosis in ApoE-/- mice [12]. Recent in vitro findings have shown that miR-145 can be transferred from smooth muscle cells to macrophages [31], suggesting the possibility for local exchanges of miRNAs between different cell types during atherosclerosis development. 5.
Conclusion
Atherosclerosis is a long-term disease that involves inflammatory responses of both vascular and immune cells. MiRNAs
Expert Opin. Ther. Targets (2014) ()
Expert Opin. Ther. Targets (2014) ()
EC EC EC
EC
EC EC EC VSMC
VSMC
Macrophages Macrophages ND
Macrophages Macrophages Lesions macrophages and liver Macrophages
ND
miR-92a miR-712/205 miR-126-3p
miR-126-3p
miR-126-5p miR-181b miR-143/145 miR-143/145
miR-143/145
miR-155 miR-155 miR-155
miR-342-5p miR-24 miR-33
miR-33
Macrophages and BMDM ND
ABCA1, ABCG1, RIP140, CROT ND
Akt1 MMP14 ABCA1
Bcl6 SOCS1 MAP3K10
ND
Dlk1 Importin-3
" In advanced lesions " In advanced lesions " In advanced lesions and apoE-/- vessels " In advanced lesions " In Advanced lesions Infiltrated macrophages in lesions
LSS intima with time LSS lesions ABCA1
In In In In
ND
# # # #
RGS16
KLF2, KLF4, SOCS5 TIMP3 ND
" In " In " In " In ND
LSS and # In HSS LSS HSS aortic arch
Main target(s) involved in atherosclerosis
Modulation
No
ND
Yes Yes ND
Yes Yes Yes
Yes Yes Present in MV Yes
Present in MV
Yes Yes Yes
In vivo validation
Blocking
Genetic deficiency
Blocking Blocking Blocking
AntagomiR AntagomiR miR-126 mimic delivery in miR-126-/- mice miR-126 mimic and MV from miR-126 -/- mice delivery miR-126-5p mimic delivery miR-181b mimic delivery Blocking Lentiviral miR-145 VSMC delivery Genetic deletion in ldlr-/mice Genetic deletion Genetic deletion miR-155 mimic delivery
Modulation strategies
No effect
Prevention
Prevention Prevention Prevention
Prevention Prevention No effect
Prevention
Prevention Prevention Prevention Prevention
Prevention
Prevention Prevention Prevention
Effect on atherosclerosis
[45]
[46]
[39] [41] [43]
[35] [36] [38]
[31]
[22] [26] [12] [30]
[24]
[10] [18] [23]
Ref.
ABCA1: ATP-binding cassette transporter 1; ABCG1: ATP binding cassette transporter G1; Bcl6: B-cell lymphoma 6; BMDM: Bone marrow-derived macrophages; EC: Endothelial cell; HSS: High shear stress; LSS: Low shear stress; ND: Not determined; SOCS: Suppressor of cytokine signalling; TIMP3: Tissue inhibitor of metalloproteinase 3; VSMC: Vascular smooth muscle cell.
miR-33
Cell expression
microRNA
Table 1. Summary of in vivo based evidence of miR modulation and its impact on atherosclerosis.
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MicroRNAs as therapeutic targets in atherosclerosis
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have recently emerged as powerful regulators of various cellular processes. Expression profile analysis has shown that miRNAs are highly dysregulated in ECs, smooth muscle cells, and macrophages, in the pathological context of atherosclerosis, which contributes to the development and progression of the disease. Experimental evidence from animal models of atherosclerosis suggests that therapeutic approaches using ‘miR-drugs’ (antagomiRs and miR mimics) are novel strategies to combat atherosclerosis.
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6.
Expert opinion
Significant progress in the understanding of miRNA function in atherosclerosis has provided a list of potential targets for therapeutic intervention. Modulating miRNA expression could represent new therapeutic approaches to treat or limit atherosclerosis. Several experimental studies provided strong evidence that specific blockade of miRNA expression modulates atherosclerosis development and progression (Table 1). For instance, antagomiR-based strategies performed in mouse models of atherosclerosis have shown that systemic targeting of specific miRNAs of interest can limit atherosclerosis. [10,24,35,43,44]. Such a therapeutic tool is delivered in the general circulation and likely impacts several cell types such as ECs, monocytes/macrophages. Restoring miRNA levels is quite challenging, but represents also an interesting approach to prevent atherosclerosis progression (Table 1) [22,26,30]. Specific blockade of miR-33 in non-human primates has proved effective in inhibiting atherosclerosis. This could represent a new therapeutic approach against atherosclerosis [48]. Some anti-miRNA strategies such as antimiR-122 are already being tested in liver diseases [49,50]. The importance of miRNAs in the pathophysiological context of atherosclerosis makes them a therapeutic target of choice. A large body of experimental evidence supports the concept that miRNA modulation could be efficient in the setting of this disease. Future challenges consist mainly in understanding molecular mechanisms that lead to miRNA
6
expression and/or dysregulation in pathology, which will help develop new therapeutic approaches. Discovery of therapeutic molecules that block or stimulate miRNA expression represents an exciting area that will likely lead to the development of agents that work in-concert with other drugs affecting atherosclerosis. Deciphering the mechanisms by which miRNA expression is controlled, in the one hand, and in the other hand how to specifically modulate (increase or block miRNA expression) will help in the development of new therapeutic strategies. One limitation concerns the fact that a single miRNA can be expressed by several cell types, but acts on hundreds of target genes. Expanding the number of new targets or targeting known miRNAs in new ways will be critical to improve clinical outcomes in atherosclerosis. It can be anticipated that the next 5 -- 10 years will see much upheaval in anti- and/or promiRNA discovery. Another limitation resides in the fact that, as shown by experimental and clinical experiments with antimiRNAs, the composition of anti-miRNA preparations and the route of administration are major determinants of efficacy [51]. Certainly, innovative anti-miRNA preparations will offer alternatives to inevitable problems of specificity. Especially, miRNA-containing extracellular vesicles [47] represent also a rich site for potential therapeutic intervention. Indeed, the identification and characterization of the specific functions of these miRNA vesicles and their interactions with cells open avenues for future development of specific inhibitors/ activators targeting these components with precise delivery in a pathological context.
Declaration of interest The authors were supported by INSERM. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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MicroRNAs as therapeutic targets in atherosclerosis
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Affiliation Xavier Loyer1 PhD, Ziad Mallat1,2 MD PhD, Chantal M Boulanger1 PharmD PhD & Alain Tedgui†1 PhD † Author for correspondence 1 Universite Paris Descartes, Sorbonne Paris Cite, Paris Cardiovascular Research Center -- PARCC, INSERM UMR-S 970, 56 rue Leblanc, 75015 Paris, France Tel: +33 1 53 98 80 06; Fax: +33 1 53 98 79 52; E-mail: [email protected] 2 University of Cambridge, Department of Medicine, Cambridge, UK
