Atherosclerosis 241 (2015) 624e633

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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Review

The role of microRNAs in coronary artery disease: From pathophysiology to diagnosis and treatment Evangelos K. Economou 1, Evangelos Oikonomou*, 1, Gerasimos Siasos, Nikolaos Papageorgiou, Sotiris Tsalamandris, Konsantinos Mourouzis, Spyridon Papaioanou, Dimitris Tousoulis 1st Cardiology Department, University of Athens Medical School, “Hippokration” Hospital, Athens, Greece

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2015 Received in revised form 31 May 2015 Accepted 17 June 2015 Available online 18 June 2015

MicroRNAs (miRNAs) are tiny non-coding RNA molecules that regulate gene expression predominantly at the post-transcriptional level. Far from being simple intracellular regulators, miRNAs have recently been involved in intercellular communication and have been shown to circulate in the bloodstream in stable forms. In the past years specific miRNA expression patterns have been linked to the development of atherosclerosis and coronary artery disease, two closely related conditions. The study of miRNAs has promoted our understanding of the processes involved in the pathogenesis of atherosclerosis and innovative diagnostic and therapeutic approaches have emerged. In this review, we present the role of miRNAs in the development of atherosclerosis, on coronary artery disease progression and we assess their role as diagnostic biomarkers. Finally we evaluate the therapeutic and preventive opportunities that arise from the study of miRNAs in coronary artery disease and especially in myocardial infarction. © 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Atherosclerosis Coronary artery disease MicroRNAs Myocardial infarction

1. Introduction Coronary artery disease (CAD) is the leading cause of death in the western world. However, despite the large number of studies that have dealt with the pathogenesis of atherosclerosis and the development of ischemic heart disease several questions remain unanswered. While classic risk factors can explain 70% of the CAD events, another 30% of CAD cases demonstrate no associations with known cardiovascular risk factors. Moreover, it is not always clear why some plaques are stable while others are vulnerable to rupture thus causing acute coronary syndromes. In addition, despite the high sensitivity and specificity of cardiac troponins in diagnosing myocardial infarction, they cannot be used to distinguish chest pain syndrome from stable or unstable angina while they also lack prognostic capabilities in stable patients [1,2]. After the discovery of the first microRNA in Caenorhabditis elegans in 1993 [1], many years passed before the role and mechanisms of action of microRNAs (miRNAs or miRs) were clarified [2].

* Corresponding author. Vasilissis Sofias 114, TK 115 28, Hippokration Hospital, Athens, Greece. E-mail address: [email protected] (E. Oikonomou). 1 Equally contributed. http://dx.doi.org/10.1016/j.atherosclerosis.2015.06.037 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.

The interest in the field of microRNAs was recently reignited by the discovery that miRNAs do not exert their actions exclusively on the intracellular level but they also exist extracellularly and take part in intercellular signaling [3]. MiRNAs were discovered in almost all biological fluids, as reviewed by Turchinov, and exist in stable nuclease-resistant forms [4]. It is now believed that circulating miRNAs are either released by dead cells, secreted by membranebound vesicles or exported by protein-protected-miRNA complexes [5]. The study of miRNAs has also highlighted the delicate regulatory mechanisms of inflammation underlying the formation of fatty streaks and atherosclerotic plaques and the level of interactions among the different cell types involved in this process. More importantly, miRNAs could be utilized to develop new diagnostic tools that will help us to detect atherosclerosis at its earliest stages and thus avoid possible complications. In addition, manipulating miRNAs and thus gene expression may enable us in the future to block or even reverse the progression of coronary artery disease. Finally, miRNAs are a promising field that may inaugurate new ways in the management of atherosclerosis and coronary heart disease. In short, the goal of this review is to provide an insight into the pathophysiologic role of microRNAs in atherogenesis, analyze their

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diagnostic and prognostic capabilities in cases of stable CAD and acute coronary syndromes and finally discuss recent microRNAbased therapeutic applications in atherosclerosis. 2. The biogenesis and function of microRNAs MicroRNAs are small non-coding ribonucleic acid molecules that play a vital role in the regulation of gene expression at the post-transcriptional level by acting either through blockade of messenger RNA (mRNA)-translation or by inducing mRNA degradation [6,7]. MiRNAs are usually transcribed by RNA polymerase II [8] and more rarely by RNA polymerase III [9] and the resulting molecules, called pri-miRNAs, contain a canonical hairpin structure, a 50 cap and a 30 poly-A tail [10]. These precursors are ideal substrates for processing by the Drosha/DGCR8 (Di George Syndrome Critical Region Gene 8) complex, which yields a 70 nucleotide-long hairpin molecule called a pre-miRNA [11]. PremiRNA is then transported out of the nucleus by Exportin 5 [12] and undergoes processing by the RNase III enzyme complex Dicer/TRBP (TAR RNA-binding protein) resulting in a 20e22 nucleotide-long miRNA duplex (the two strands are respectively called miRNA and miRNA* or alternatively miRNA-5p and miRNA3p). One strand is then incorporated into the RNA-induced silencing complex (RISC) which carries out the regulatory actions of miRNAs by binding to the target mRNA and either promoting its degradation or inhibiting its translation [13] (Fig. 1).

Fig. 2. MicroRNAs involved in atherosclerosis progression. MicroRNAs are involved in atherosclerosis by affecting several steps including endothelial repair, nitric oxide synthesis, vessel wall angiogenesis, inflammatory molecule expression, oxidation of low-density lipoprotein and platelet activation. miRNA: MicroRNA.

3.1. Endothelial damage and dysfunction 3. The pathophysiologic role of microRNAs in atherosclerosis progression and coronary artery disease development Atherosclerosis of the coronaries arteries is responsible for the development and clinical manifestations of CAD. Atherosclerosis is a relatively complex process involving several steps. Numerous studies have evaluated the role of miRNAs in this process and it is now accepted that miRNAs are involved in almost all steps of atherogenesis, including endothelial damage and dysfunction, monocyte-wall invasion and activation, lipoprotein formation, platelet and vascular smooth muscle cell function exerting either beneficial or detrimental effects [14] (Fig. 2).

Fig. 1. The biosynthesis of microRNAs. Pri-miRNA is synthesized by polymerase II in the nucleus. Drosha (a RNAase III enzyme) cleaves pri-miRNA into pre-miRNA which is subsequently exported to the cytoplasm. This double-stranded molecule is incorporated into the RNA induced silencing complex (RISC) and finally a single stranded mature miRNA is formed. miRNA: microRNA.

The most abundant miRNA in endothelial cells is miR-126, a miRNA involved in vessel development and endothelial cell repair [15e17]. Interestingly, studies with miR-126/ mice have demonstrated an embryonic lethality of almost 50% [17]. On the other hand, with regards to endothelial cell repair, endothelialderived miR-126 is released in microvesicles by apoptotic endothelial cells and triggers the production of CXCL12 in the recipient vascular cells as shown in mouse models. Its experimental delivery in mice has been proven to be beneficial by limiting atherosclerosis, promoting the incorporation of Sca-1þ progenitor cells, and conferring features of plaque stability on different mouse models of atherosclerosis [16]. Moreover, its important role in atherosclerosis relies also on the fact that it inhibits tumor necrosis factor alpha (TNFa)-induced expression of vascular cell adhesion molecule-1 (VCAM1) in the endothelium and thus down-regulates inflammatory cell adhesion [18]. In another study [34] miR-126 secreted by the endothelium was shown to increase vascular smooth muscle cell-turnover in vitro and its release was down-regulated by atheroprotective laminar shear stress. Furthermore, Schober et al. have shown that endothelial miR-126-5p maintains a proliferative reserve in endothelial cells through suppression of the Notch1 inhibitor delta-like 1 homolog (DLK1) thereby preventing atherosclerotic lesion formation. MiR-21, on the other hand, regulates the cellular response that is responsible for neointimal formation by promoting vascular smooth muscle cell (VSMC) de-differentiation and loss of the contractile phenotype [19]. Other studies have shown a relationship between vascular wall-shear stress and miR-21 expression. On the one hand, oscillatory shear stress upregulates activator protein-1 dependent miR-21 expression in cultured human umbilical veinendothelial cells (HUVECs), which in turn targets and downregulates peroxisome proliferator-activated receptor a and thus allows activator protein-1 activation and the binding of monocytes to the vessel-wall [20]. On the other hand, Weber et al. proved that in vitro unidirectional shear stress increased miR-21 expression in human endothelial cells, upregulated endothelial nitric oxide synthase (eNOS) expression and decreased endothelial cell apoptosis

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[21]. MiR-21 is further associated with the effects of oxidative stress on the endothelium. It down-regulates superoxide dismutase 2, a molecule with anti-oxidative properties and is increased in angiogenic progenitor cells treated with asymmetric dimethylarginine, an inhibitor of NOS [22]. There are also other miRNAs, such as miR-92a, miR-19a and miR-663 that are associated with shear stress. More specifically, miR-92a levels are downregulated in response to increased shear stress and the downregulation enhances eNOS expression [23]. It seems that Kruppel-like factor 2 and 4 are intracellular targets of miR-92a and their expression depends on the nature of shearing forces [24] and are partly responsible for the regional atherosusceptibility seen in vivo [25]. Indeed, Loyer et al. [26] have shown that, under low shear stress conditions, miR-92a regulated endothelial cell activation by oxLDL and this was associated with modulation of Kruppel-like factor 2 and Kruppel-like factor 4 (KLF4) expression. In addition to that, blockade of miR-92a expression in in vivo studies with Ldl recepror(/) mice resulted in reduced endothelial inflammation, decreased plaque size and promoted a more stable plaque phenotype. Interestingly, Wang et al. even suggested that miR-92a levels could be used in order to evaluate the effects of statins in the treatment of endothelial dysfunction in coronary heart disease [27]. In addition, two other miRNAs, miR-221 and miR-222, appear to oppose endothelial migration and proliferation, promote apoptosis and upregulate several inflammatory molecules of endothelial cells by targeting Ets-1 transcription factor [28,29]. 3.2. Monocyte-wall invasion and activation Increased miR-155 levels are typical of pro-inflammatory macrophages and atherosclerotic lesions. A study conducted in apoEdeficient mice concluded that miR-155 participates in a program that enables macrophages to sustain and reinforce vascular inflammation, as miR-155 normally binds the mRNA of BCL6 (B-cell lymphoma 6 protein), a transcription factor that inhibits proinflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFkВ) signaling [30]. However, Wei et al. have proposed a stage-specific role of miR-155 in atherosclerosis. These opposite roles of miR-155 are attributable to the inhibition of macrophage proliferation by suppression of the colony-stimulating factor-1 receptor in the early stages of atherosclerosis and decreased efferocytosis by targeting Bcl6 (B-cell lymphoma 6 protein) in advanced atherosclerosis [31]. Moreover, AKT1 (also known as protein kinase B) inhibits macrophage pro-inflammatory phenotype by decreasing the levels of miR-155 [32]. Furthermore, it was found that miR-155 levels are increased in bone marrowederived macrophages in mice after lipopolysaccharide/interferon-g stimulation, an increase mediated via the suppression of AKT1 in classically activated macrophages [33]. Du et al. have also shown that double knockout miR-155(/) and apoE(/) mice developed less atherosclerosis in the aortic root, with reduced neutral lipid and macrophage content while there was also an increased number of regulatory T cells and a reduced numbers of Th17 cells when compared to apoE(/) mice. Furthermore, in bone marrow transplanted mice deficiency of mir-155 effectively suppressed atherogenesis [34]. Therefore miR-155 is currently classified as a miRNA that drives inflammation in atherosclerotic plaques. In M1 pro-inflammatory lipopolysaccharides-stimulated monocytes the inflammatory phenotype is modulated by an increase in miR-9 which down-regulates peroxisome proliferatoractivated receptor d expression [35]. On the other hand, miR467b targets the lipoproteine lipase gene in cultured RAW 264.7 macrophages, inhibits the accumulation of lipids and represses the release of inflammatory cytokines [36]. In addition, down-

regulation of intracellular miR-125a-5p levels in THP-1 monocytes significantly upregulated the release of inflammatory cytokines, including transforming growth factor b (TGFb), TNFa, Interleukin (IL)2 and IL6 and the expression of macrophage scavenger receptors [37]. 3.3. Low density lipoprotein (LDL) MiR-122 is the most abundant miRNA in the liver corresponding to almost 70% of the hepatic miRNAs [38]. Inhibition of miR-122 in mouse models led to a 30% decrease of plasma cholesterol [39] and the results were reproduced in experiments with non-human primates (African green monkeys) [40]. Another miRNA associated with hyperlipidemia is miR-30c. Contrary to miR-122, high levels of miR-30c have a positive effect on lipid metabolism and decrease hyperlipidemia by downregulating lipid synthesis and lipoprotein secretion. Thus, elevating miR-30c levels could be considered as a novel therapeutic goal to improve hyperlipidemic conditions [41]. Finally, miR-146a has been shown to attenuate the intracellular lipid accumulation driven by oxidized LDL and to down-regulate inflammation by directly inhibiting toll-like receptor 4-expression [42]. 3.4. High density lipoprotein (HDL) The relationship between HDL and miRNAs is dual: not only do miRNAs regulate several pathways involved in HDL metabolism but at the same time HDL molecules offer a vehicle for circulating miRNAs. Notably, the profile of these miRNAs varies between healthy and diseased individuals and is linked to the risk for atherosclerosis [43]. MiR-33 is probably the most important regulator of HDL metabolism. Located in the sterol regulatory element-binding protein genes 1 and 2 (SREBF1 and 2) of mice that are transcribed into transcription factors that control cholesterol and fatty acid synthesis [44], miR-33 coordinates the expression of several sterol transporters associated with HDL metabolism and bile secretion [45] and exerts its actions via repression of genes involved in fatty acid oxidation and cholesterol export. MiR-33 down-regulates also ATP-binding cassette transporter-1 expression and the resulting abnormal polarization in older macrophages promotes an increase in intracellular cholesterol levels [46]. Experimental miR-33 inhibition via miR-33a/b antisense oligonucleotides in non-human primates resulted in a 40% increase in HDL and 50% decrease in very low density lipoprotein within 12 weeks by boosting cholesterol export and fatty acid b-oxidation [47]. 3.5. Platelets Platelets are rich in certain miRNAs and more specifically miR24, miR-197, miR-191 and miR-223. These miRNAs are considered potential biomarkers for assessing the effects of anti-platelet therapy [48]. For example, miR-223 targets purinergic P2Y12 receptor which normally gets activated by ADP and promotes dense granule secretion, inhibition of adenylyl cyclase and production of thromboxane A2 [49,50]. 3.6. Vascular smooth muscle migration and activation MiR-143 and 145 have been described as the «master regulators» of the contractile phenotype of VSMCs via upregulation of contractile protein-expression [51] and decreased miR-143/145 levels are associated with progression to a synthetic and proliferative phenotype and accelerated lesion formation [52,53]. MiR-143/

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145 appear to be released by endothelium in vesicles that are transferred extracellularly to the vascular smooth muscle cells where they exert their atheroprotective actions [54] and could thus be used therapeutically in order to block plaque formation [55] and even prevent plaque rupture or in-stent restenosis [56]. MiR-221 and miR-222 are two other miRNAs with an important role in VSMC function. As aforementioned, miR-221 and miR-222 are molecules with anti-proliferative and pro-apoptotic actions on endothelial cells but they have opposite effects on VSMCs promoting proliferation, migration and inhibiting apoptosis [28,29]. These miRNAs appear to also mediate the effects of Angiotensin II on VSMCs which stimulates their proliferation [57]. In fact, in an interesting study by Tsai et al. that included 67 subjects with ischemic stroke, 66 atherosclerosis subjects with any carotid plaque score and 157 healthy controls, it was found that stroke and atherosclerosis subjects had significantly lower miR-221 serum levels than healthy controls. Therefore, the authors proposed miR221 as an independent predictor of stroke and preclinical atherosclerosis [58]. 3.7. Intraplaque angiogenesis Intraplaque angiogenesis and miRNAs regulating this process (called angiomiRs) are associated with destabilization and increased chance of rupture for an unstable atherosclerotic plaque [59]. The role of miR-92a has been evaluated in ischemia models in mice and it was shown to inhibit angiogenesis, while its blockade via an antagomiR-92a improved recovery of blood flow [60]. In addition, let-7 and the miR-17-92 cluster target a powerful inhibitor of angiogenesis, thrombospondin-1, and thus indirectly promote plaque destabilization [61]. MiR-210 promotes vessel formation in the setting of hypoxia and is involved in vascular endothelial growth factor regulated endothelial cell migration [62], whereas miR-15b, miR-16, miR-20a/b directly target vascular endothelial growth factor and are decreased in hypoxemic circumstances [59]. 4. MicroRNAs as diagnostic tools in coronary artery disease It is evident that microRNAs are involved in and orchestrate every phase of the atherosclerotic plaque development. Therefore the question arises as to whether measurement of their levels in the peripheral blood (circulating microRNAs) can serve diagnostic or prognostic purposes in cases of stable CAD or even in acute coronary syndromes. 4.1. Stable coronary artery disease Fichtlscherer and his team carried out one of the first studies that evaluated the potential role of miRNAs as biomarkers for stable CAD. In their work they analyzed the miRNA profiles of 8 healthy controls and 8 patients with proven stable CAD who all received the indicated pharmacological treatment. The measurement of the levels of several miRNAs in plasma showed that certain miRNAs, such as miR-133 and miR-208a (both cardiomyocyte-enriched) were significantly elevated in the patient group. On the contrary, plasma levels of miR-126, miR-17 and miR-92a (described as endothelial cell-enriched), miR-145 (associated with the vascular smooth muscle cells) and miR-155 (inflammatory cell-enriched) were consistently decreased in the diseased group. Those results were confirmed in a larger study (31 patients, 14 controls) [63]. Weber et al. measured the levels of several miRNAs in whole blood and detected a number of miRNAs (miR-19a, miR-484, miR155, miR-222, miR-145, miR-29a, miR-378, miR-342, miR-181d, miR-150 and miR-30e-5p) that were down-regulated in CAD

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patients in comparison to healthy controls [64]. More specifically, it was also found that miR-155 levels in peripheral blood mononuclear cells were significantly higher in patients with chest pain syndrome, lower in severe CAD and stable angina patient and even lower in the case of acute coronary syndrome [65]. There appears to be an inverse association between the severity of coronary artery lesions as assessed with the Gensini score and miR-155 levels in patients undergoing coronary angiography for suspected coronary artery disease [66]. Another study by D'Alessandra et al. examined the miRNA profile in the plasma of coronary patients and showed a positive modulation of miR-337-5p, miR-433 and miR-485-3p in coronary artery disease. Furthermore, miR-1, miR-122, miR-126, miR-133a/b, miR-199a, miR-485-3p and miR-337-5p were elevated in stable angina and the triad of miR-1, miR-126 and miR-485-3p correctly classified individuals as stable angina-patients versus controls in more than 87% of cases [67]. MiRNA profiles can be also analyzed in samples other than plasma or whole blood. MiRNAs measured in peripheral blood mononuclear cells in CAD versus non-CAD patients showed a fivefold increase in miR-135a and fourfold decrease in miR-147 in the patient group. As a result, the researchers proposed that the miR-135a/147 ratio could be used as a biomarker for CAD [68]. In two other studies, down-regulated expression of miR-181a in monocytes of obese patients was associated with an increased incidence of CAD [69], while a relative up-regulation of miR-146a/b in CAD versus the control group was linked to a higher risk of future cardiac events [70]. Li et al. investigated the relative expression of miRNAs in intima samples of peripheral artery disease patients and found certain biomarkers for early atherosclerosis (atherosclerosis obliterans) that could be used in the diagnosis of coronary artery disease. More specifically, miR-21, miR-27b, miR-130a, miR-210 and let-7f were upregulated and miR-221 and miR-22 were decreased in the studied intima samples. In addition, miR-27b, miR-210 and miR130a were increased in serum samples and could be considered as serum biomarkers for early atherosclerosis [71]. Table 1 summarizes the aforementioned information on the role of miRNAs as biomarkers for stable coronary artery disease. 4.2. Unstable angina To date, there is no ideal test to distinguish stable from unstable angina pectoris based solely on the miRNA profile of a patient. D'Alessandra et al. concluded that miR-1, miR-122, miR-126, miR133a/b, miR-199a, miR-485-3p were all upregulated both in stable and unstable angina, while miR-145 was significantly elevated only in unstable angina. Even though the triad of miR-1, miR-126 and miR-133a correctly classified more than 87% of cases as unstable angina patients, no combination was able to differentiate between unstable and stable angina [67]. Hoekstra et al. stated that increased peripheral blood mononuclear cell levels of miR-134, miR-198 and miR-370 in unstable versus stable angina pectoris patients may provide a biomarker for increased risk of progression to acute coronary syndromes [68]. Finally, Ren et al. detected certain miRNAs that are upregulated in unstable angina versus other non-cardiac causes of chest pain. These miRNAs (miR-106b/ 25 and miR-17/92a cluster, miR-21/590-5p family miR-126* and miR-451) could be further evaluated as markers for vulnerable coronary artery disease [75]. 4.3. Acute myocardial infarction 4.3.1. MiR-208a/b MiR-208 is encoded in the region of myosin genes [76]. There

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Table 1 MicroRNAs as biomarkers of atherosclerosis and stable coronary artery disease. Author-year

microRNAs

Sample

Main findings

Fichtlscherer et al., 2010 [63] Li et al., 2011 [71] D'Allessandra et al., 2013 [67] D'Allessandra et al., 2013 [67] Li et al., 2013 [72] Hoekstra et al., 2010 [68] Takahashi et al., 2010 [70] Minami et al., 2009 [73] Sondermeijer et al., 2011 [74] Fichtlscherer et al., 2010 [63] Hoekstra et al., 2010 [68] Hulsmans et al., 2012 [69] Sondermeijer et al., 2011 [74] Weber et al., 2011 [64]

miR-133, -208a miR-27b, -130a, -210 miR-1, -122, -126, -133a/b, -199a, miR-337-5p, 485-3p miR-208, -499 miR-135a miR-146a/b miR-221, -222 miR-340*,-624* miR-126, -17, -92a, -145, -155 miR-147 miR-181a miR-1280 miR-19a, -484, -155, -222, -145, -29a, -378, -342, -181d,-150, -30e-5p

Plasma Serum Plasma Plasma Serum PBMCs PBMCs EPCs Platelets Plasma PBMCs PBMCs Platelets Whole blood

Increase in patients with CAD. Cardiac muscle-enriched microRNAs Increased in patients with early atherosclerosis Increased in both stable angina or unstable angina Increased in CAD compared to control Increased in patients with angina pectoris compared patients with AMI. Increased in stable CAD Increased in CAD patients. Treatment can decrease the levels of miR-146a/b Increased in CAD patients compared to control Increased in patients with CAD compared to control Decreased in patients with CAD compared to control Decreased (4-fold) in CAD patients compared to control Decreased in obesed patients. A putative marker of CAD Decreased in patients with CAD compared to control Decreased in patients with CAD compared to control

CAD: Coronary artery disease; AMI: Acute myocardial infarction; PBMCs: peripheral blood mononuclear cells; EPCs: Endothelial progenitor cells.

are two subtypes (miR-208a and miR-208b) and both have been linked to myocardial infarction, even though most papers on humans have shown better results for the miR-208b subtype. Salic et al. believe that this is due to differences in the time of sampling and the fact that expression of myosin isoforms may vary between mice and humans. One of the first studies was done in a rat model where Ji et al. induced myocardial infarction (MI) via isoproterenolstimulation and noticed that miR-208a levels in plasma highly correlated with cardiac troponin (cTn) I levels [77]. MiR-208 is indeed a highly cardiac-specific miRNA [78] and it appears in the bloodstream 3 h after the onset of an acute MI and is rapidly elevated after 12 h [79]. Zile et al. examined the time-dependent change in plasma levels and concluded that miR-208a is significantly increased after 5 days and remains upregulated up to 90 days after the acute myocardial infarction (AMI) event [80]. Wang et al. showed its superiority versus cTnI in the early diagnosis of myocardial infarction (MI). In their study miR-208 levels were elevated in 100% of patients 4 h after the onset of MI contrary to only 85% for cTnI. Moreover, miR-208a had an overall better receiver operating characteristic curve in comparison to miR-1, miR-133a and miR-499 in distinguishing AMI from other cardiovascular disorders [81]. Similar results were found for miR-208b [82] and miR-208b levels were correlated with disease severity [83]. Furthermore, Li et al. suggested that a panel of six microRNAs (miR-1/134/186/208/233/499) has an increased sensitivity and specificity in detecting MI. In addition, levels of miR-208 and miR499 in their study were surprisingly higher in angina pectoris-cases than in MI [72]. Tijsen et al. on the other hand evaluated another triad's diagnostic value (miR-1, miR-208 and miR-133) [84]. Despite the encouraging results mentioned above, it seems that even though miR-208 as well as miR-499 were both increased in MI cases, none offered additional diagnostic value when compared to cTnI and cTnT nor predicted long-term mortality [85]. 4.3.2. MiR-499-5p Another myosin gene-regulated miRNA [76] of cardiac origin [86] is miR-499-5p. It peaks at 12 h after MI onset, can still be detected in plasma after 48 h and then returns back to normal values after 72 h. Given its kinetics, Tijsen suggested that miR-4995p might be useful in the later stages of myocardial infarction [84]. It appears that miR-499-5p may have a comparable diagnostic accuracy to that of troponins and even greater than that of miR-208b [83]. In cases of non-ST elevation myocardial infarction (NSTEMI) with modest elevation at presentation, the sensitivity of miR-4995p was superior to that of cTnT [87]. It has been described as the

most consistent marker of heart damage and can effectively separate troponin-positive from troponin-negative events [67]. Indeed, Corsten et al. identified miR-208b and miR-499 as the most upregulated circulating miRNAs after MI [82] and moreover, the combination of miR-1, miR-499 and miR-21 increased the diagnostic value of high sensitivity cTnT in suspected AMI cases. 4.3.3. MiR-1 and miR-133 MiR-1-1/133a-2 and miR-1-2/133a-1 are two bicistronic miRNA clusters that are not only expressed in cardiomyocytes, where they seem to exert antihypertrophic actions [88], but also in skeletal muscle. On the contrary, miR-133b is solely present in skeletal muscle [89]. It is therefore necessary to exclude skeletal muscle disease prior to interpreting miR-1 or miR-133 levels [89]. Both miR-1 and miR-133a peak 2.5 h after the onset of a myocardial infarction [78] and return to normal after 24e48 h [87,90] indicating myocardial damage [91]. As a result, MI can be effectively distinguished from unstable angina based on miR-1 and miR-133a up-regulation, whereas miR-133b levels remain unchanged [92]. It is possible that determining miR-1 and miR-133a levels may also be of prognostic value. Both miRNAs correlate with cTnT levels [92]. In addition, miR-1 is also positively correlated with creatine kinase myocardial brain subtypes (CK-MB) and cell damage in vitro [93] and is associated with post-MI QRS widening [94], whereas higher levels of miR-133a are correlated with decreased myocardial salvage, increased infarct size and enhanced reperfusion injury. Despite the promising results concerning miR-208b, miR-4995p, miR-1 and miR-133a, it is still unknown whether these miRNAs can offer additional information when compared to traditional biomarkers. Li et al. concluded that the combination of the aforementioned four miRNAs may be useful but is not superior to cTnT for AMI diagnosis [95]. Surprisingly, miR-208a and miR-499a do not belong to the 20 most enriched miRNAs in the blood of MI-patients, as measured by Meder et al. in whole blood-microarrays. This study showed that the miRNAs measured in plasma are nothing more than a fraction of the total miRNAs present in whole blood. Meder suggested an alternative signature of 20 miRNAs with a predicted area under the curve value (in a receiver operating characteristic curve) of 0.99. Among these miRNAs, miR-663b and miR-1291 had the greatest positive predictive value for MI and miR-30c and miR145 were positively upregulated in STEMI and correlated with cTnT and thus infarct size. However, these miRNAs are not cardiacspecific and could be increased in various other conditions [96]. At this point, it is worth commenting that the inability of microRNAs to increase the diagnostic accuracy in myocardial infarction patients may be due to the already high accuracy of cardiac

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troponins. Therefore studies should focus on the potential of microRNAs to diagnose vulnerable plaques with minimal erosions that do not cause myocardial necrosis and do not result in a raise of the cardiac troponin levels. One more recent study from Wang et al., in 2014 showed that miR-21-5p and miR-361-5p plasma levels were significantly upregulated in AMI patients when compared to healthy subjects, whereas plasma levels of miR-519e-5p were significantly lower in this group. Among the 3 miRNAs studied in this study, only miR519e-5p showed significant difference between the AMI group and a group composed of patients with ischemic stroke and pulmonary embolism, therefore suggesting a potential value for miR519e-5p in differentiating AMI from other ischemic conditions [97]. The studies examining the potential diagnostic role of microRNAs in MI are summarized in Table 2. 4.4. Difficulties and challenges in microRNA quantification Since the diagnostic capabilities of circulating microRNAs were first demonstrated, different methods have been applied for their identification and quantification. Microarray technology has been utilized to provide a comprehensive microRNA expression profile. However, the current commercially available microRNA microarray systems fail to show good inter-platform concordance, probably due to a lack of an adequate normalization method and severe divergence in the stringency of detection call criteria among different platforms [102]. Next generation sequencing has also several limitations. Real-time quantitative PCR is a simple tool that can efficiently determine the amount of a gene transcript in a given sample and is currently regarded as the method of choice. However, the simplicity of this methodology can itself be problematic, as one tends to overlook critical factors that make this technique work [103]. For example, there is no consensus whether plasma or serum is a more reliable substrate for measuring circulating miRNAs. Hemolysis during sample preparation or even due to physiologic processes (i.e. dysfunctional mechanical valves) can also affect the levels of circulating microRNAs [104]. Moreover, antiplatelet treatment may affect circulating microRNAs in plasma and serum samples and may act as a confounding factor in case-control studies relating plasma miRNAs to cardiovascular disease [48]. The aforementioned difficulties may explain the observed differences in the diagnostic ability of microRNAs between studies and why they have failed to increase diagnostic accuracy until now.

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by several microRNAs [105,106]. Moreover, polymorphisms in microRNA which are called miRSNPs are associated with different disease phenotypes and even with cancer development [107]. Furthermore, inhibition of a specific microRNA may be beneficial concerning atherosclerosis progression but may adversely affects other organ systems causing immunosuppression, liver damage or even oncogenesis. Therefore careful monitoring and studying of these interactions is essential in order to guarantee a safe application in humans. 5.1. Therapeutic strategies 5.1.1. Inhibition of microRNAs-antisense miRNA oligonucleotides In cases where specific microRNAs have a well demonstrated role in disease development, inhibition of their activity can be achieved by antisense oligonucleotides (ASOs). These usually represent a single strand of synthesized nucleotide sequences and can affect miRNA expression through multiple mechanisms such as (1) inhibiting the export of the pre-microRNA from the nucleus; (2) inhibiting the transformation of pre-microRNAs into active microRNAs; or (3) by competitive inhibition through complementary binding to specific microRNAs [108]. 5.1.2. MicroRNAs mimics In cases where a microRNA exerts beneficial effects, these effects can be increased through microRNA mimics. These are usually double-stranded synthesized oligonucleotides where one strand is identical to the target microRNA. However, in such cases special caution is required since the complimentary strand may have the potential to act as a separate microRNA with unpredictable effects [109]. 5.1.3. Other approaches used to modulate microRNAs levels Sharp and colleagues described an approach in which microRNA function was blocked by scavenging away the miRNAs with what they called “miRNA sponges” [110]. Possessing multiple binding sites, miRNA sponges can bind to a programmed RNA-induced silencing complex reducing its availability and effectively inhibit the function of entire microRNA families [110]. Alternatively, the socalled “erasers” are exact complementary sequences to the target endogenous microRNAs and can therefore effectively inhibit their function [111]. 5.2. Delivery strategies for microRNAs

5. MicroRNA modulation as a therapeutic approach: opportunities and challenges After realizing the important role of microRNAs in cellular biology and in cardiovascular disease, a series of studies started focusing on the potential therapeutic modulation of microRNA expression. However, several limitations exist which must be taken into consideration. Most importantly, microRNAs have an inhibitory effect in mRNA and consequently in gene expression. In other words, inhibition of microRNAs releases gene expression while addition or enhancement of a microRNA has the opposite results. The biggest challenge here lies in the ability to predict the exact effects of miRNA modulation in the human body. While delivery of a specific miRNA may downregulate a crucial pathway in atherogenesis, it may also affect a wide number of other pathways and cells therefore resulting in unpredictable and often unwanted effects. It is currently accepted that microRNAs are not specific for the regulation of a single gene. Instead, they can modulate hundreds of target genes. In addition, they demonstrate concentrationdependent effects whereas the same targets are often regulated

Nucleic acids are rapidly degraded by a variety of nucleases and phosphodiesterases in the body. Therefore specific chemical modifications are required to alter the biophysical properties of synthetic nucleotides. Overall, viral vectors represent the most widely applied method of experimental miRNA delivery. However, in a review article by Zhang et al. that focuses on delivery strategies for microRNAs, the authors state that their use is limited by toxicity and immunogenicity [112]. Therefore, newer approaches have been proposed and developed. Conjugation of synthetic nucleotides with peptides, antibodies or other biologically active and stable molecules is one approach which has been tested in several animal studies. Another approach is to combine synthetic nucleotides with liposomes or polymers of nanoparticles with the ability to passively cross the cell membrane. When compared to the viral vectors, these methods have several advantages, including lower immunogenicity and control of their molecular composition but appear to have lower efficiency. On the other hand liposomes are most commonly used in in vitro studies but are rarely used in vivo due to their toxicity

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Table 2 MicroRNAs as biomarkers in myocardial infraction. Author-year

microRNAs

Study sample

Sample Main findings

Wang et al., 2010 [81]

miR-208a

33 AMI subjects vs. 33 non-AMI subjects

Plasma

Corsten et al., 2010 [82]

miR-208b, -499

Plasma

D'Allessandra et al., 2010 [78] Li et al., 2013 [72]

miR-1, -133, -499-5p

32 AMI patients vs. 15 patients with viral myocarditis vs. 36 subjects with chest pain and normal coronography vs. 20 healthy subjects 33 patients with AMI vs. 17 healthy subjects

miR-1, -134, -186, 117 AMI patients vs. 182 patients with -208, -223, -499 angina and 100 control subjects Devaux et al., 2013 [85] miR-208b, -499, -320a 1155 patients with chest pain. 224 have been diagnosed with AMI Oerlemans et al., 2012 [98] miR-1, -499 -21 332 patients with suspected ACS. 106 patients have been finally diagnosed with ACS Wang et al., 2011 [99] miR-92a 82 patients with STEMI vs. 116 with stable angina Meder et al., 2011 [96] miR-1291, -663b, 20 patients with AMI vs. 20 control subjects -30c, -145 Olivieri et al., 2013 [87] miR-1, -21 -133a, 92 non-STEMI patients vs. 81 acute heart -423-5p, -499-5p failure patients vs. 99 control subjects Yao et al., 2011 [100]

miR-21, 146a

D'Allessandra et al., 2010 [78] Yao et al., 2011 [100]

miR-122, -375

Li et al., 2014 [101]

miR-1

Wang et al., 2014 [97]

miR-21-5p, 361-5p, 519e-5p

miR-155

31 ACS patients vs. 15 stable angina patients and 16 patients with chest pain syndrome 33 patients with AMI vs. 17 healthy subjects

Increase in patients with AMI. High sensitivity and specificity for the diagnosis of AMI Increased in patients with AMI. (miR-208b is 1600-fold elevated and mir-499 100-fold)

Plasma

Increased both in human and mice after AMI

Serum

A profile of six miRNAs increased in AMI

Plasma

PBMCs

Increased in AMI with no incremental to TnT diagnostic value Increased in ACS. The combination of these three miRs resulted in a higher diagnostic ability than TnT Increased in STEMI patients miR-1291, -663b increase in AMI. miR-30c, -145 are correlated with infract size miR-1, -21 -133a, -423-5p showed a 3- to 10-fold increase and miR-499-5p 80-fold increase in acute non-STEMI patients Increased in ACS two-fold

Plasma

Decreased in STEMI patients

Plasma Plasma Whole Blood Plasma

31 ACS patients vs. 15 stable angina patients and 16 patients with chest pain syndrome 56 AMI patients vs. 28 non-AMI control subjects

PBMCs

Decreased in ACS by 60%

Plasma

17 AMI patients vs. 28 healthy controls

Plasma

Increased in AMI, highly sensitive and specific, though not superior to cardiac TnT MiR-21-5p and 361-5p levels were increased, while 519e-5p levels were decreased in AMI versus non-AMI patients.

CAD: Coronary artery disease; AMI: Acute myocardial infarction; vs.: Versus; PBMCs: peripheral blood mononuclear cells; PE: pulmonary embolism; STEMI: ST elevation myocardial infarction; TnT: Troponin-T; ACS: Acute coronary syndrome.

which most commonly affects the liver [113,114]. Interestingly, administration of synthetic nucleotides in the heart and coronary arteries may be facilitated by catheterization of the coronaries arteries which allows more local delivery close to the targeted areas and at higher concentration levels. 5.3. Specific therapeutic approaches in myocardial infarction 5.3.1. Angiogenesis Local angiogenesis and neovascularization is a mechanism that protects cardiac tissue from hypoxia and ischemia. One of the most important miRNAs that is upregulated in hypoxia is miR-210 (described as a master «hypoxamiR» by Chen et al.). MiR-210 does not only promote angiogenesis but it also stalls DNA repair, represses mitochondrial metabolism, supports stem cell survival and cell differentiation and has overall versatile functions [115]. Therapeutic miR-210 delivery has positive effects and decreases the infarct size, improves angiogenesis and cardiac function [116]. 5.3.2. Ischemia-reperfusion injury and apoptosis HypoxamiR-210 is involved in ischemic preconditioning of myocardial cells after multiple ischemia-reperfusion episodes via FLASH (FLICE associated huge protein)/Casp8ap2 (caspase 8 associated protein 2) suppression and leads to prolonged survival [117], while also stalling DNA repair, repressing mitochondrial metabolism and inhibiting apoptosis [115]. Another, miRNA involved in ischemic preconditioning is miR-21 which acts through the phosphatase and tensin homolog/AKT pathway in vivo [118] and experimental delivery of miR-21 via an adenovirus inhibits cell apoptosis in rat hearts [119]. Furthermore, miRNA expression can be manipulated to mimic hypoxia. Such is the case of the therapeutic knock-down of miR-

199a during normoxia that can protect heart cells against future hypoxic episodes [120]. In addition, deletion of the miR-144/451 cluster decreases the protection which is induced by ischemic preconditioning by targeting Ras-related C3 botulinum toxin substrate 1 [121]. Mitochondrial fission is an important step in the process of apoptotic death. MiR-761 blocks mitochondrial fission and thus apoptosis of cardiomyocytes by inhibiting mitochondrial fission factor, which is upregulated in ischemia/reperfusion injury [122]. Alternatively, miR-484 down-regulation in apoptosis allows mitochondiral fission 1 protein to become upregulated and promote mitochondrial fission [123]. On the other hand, miR-30 [124] and miR-499 [125] cause inhibition of the dynamin-related-protein-1 which ultimately blocks the fission of mitochondria. 5.3.3. Fibrosis and remodeling MiR-133 and miR-30 down-regulate proteins involved in profibrotic events [126] and more specifically, decreased miR-133a expression in fibroblasts of the left ventricle in pigs promotes the up-regulation of membrane type-1 matrix metalloproteinase [127]. Other miRNAs that promote fibrosis when down-regulated are miR-29 which has been targeted with antagomiRs both in vivo and in vitro [109] and miR-24 [128] which was also reported to function as an anti-apoptotic and anti-angiogenic molecule. 5.3.4. Stem cells, progenitor cells and therapy Several miRNAs have successfully promoted mitosis of cardiomyocytes when injected into the diseased myocardium. Administering antagomiRs of miR-15 family members increased the number of mitotic cardiomyocytes [129] and overexpression of miR-17-92 cluster also activates cardiomyocytes to proliferate [130]. Another strategy involves manipulation of stem or progenitor

E.K. Economou et al. / Atherosclerosis 241 (2015) 624e633

cells so that they develop into functional cardiomyocytes or support regeneration of cardiomyocytes by secreting proproliferative molecules. MiR-499 expressed in human cardiac progenitor cells acts by inhibiting transcription factors (Sox-6) and RNA binding protein (Rod) and promotes cardiogenesis. Tests in animal models showed indeed increased cardiac regeneration induced by miR-499 overexpression in progenitor cells [131]. Further research into this topic showed that the therapeutic engraftment of transplanted cardiac progenitor cells in infarcted tissue could be boosted by a miRNA cocktail containing miR-21, miR-24 and miR-221 [132]. Mesenchymal stem cells can be further modified to improve clinical outcome. Mesenchymal stem cells over expressing miR-126 promote angiogenesis and cell survival through secretion of angiogenic factors and activation of Notch-ligand Delta-like-4 factor [133]. HypoxamiR-210 delivery via stem cells also promotes survival of mesenchymal stem cells and appears to be transferred to cardiomyocytes of the infracted tissue via gap junctions [117]. Moreover, injection of mesenchymal stem cells that overexpress miR-23a appears to improve cardiac function in rat models of myocardial infarction by targeting caspase-7 and inhibiting TNF-a induced apoptosis [134], whereas miR-1 transfection of mesenchymal stem cells increased their survival and differentiation into mature cardiomyocytes [135]. 6. Conclusion MicroRNAs have emerged as useful biomarkers in the diagnosis and therapeutics management of atherosclerosis and CAD. Nevertheless, a significant number of studies were based on animal models. Even those that were done in humans were based on a small sample, used different protocols and often resulted in contradictory conclusions. New studies are needed on a larger scale in order to better evaluate the potential uses of miRNAs either as biomarkers or therapeutic molecules. Secondly, the current method-of-choice for analyzing miRNAs is real-time quantitative PCR that is neither fast nor cost-effective. Integrating the use of miRNAs in everyday clinical practice will require the development of rapid and inexpensive techniques. Thirdly, most of the studies agreed on the fact that miRNAs were usually inferior or equal and only rarely superior to traditional biomarkers in terms of diagnostic accuracy. It can thus be inferred, that miRNAs may be useful as adjunctive and supplementary rather than independent diagnostic tests. However, they may offer important additional help in the early stages of a myocardial infarction, when enzyme levels in the bloodstream are still low and undetectable. Last but not least, the study of miRNAs and their importance in the field of cardiovascular medicine serves as herald of the increasing involvement of genetics and molecular biology in future medicine and points towards new, innovative and above all more effective molecular therapies that will most likely substitute the traditional drug regimens and therapeutic interventions. Disclosure None. References [1] K. Thygesen, J.S. Alpert, A.S. Jaffe, et al., Third universal definition of myocardial infarction, Eur. Heart J. 33 (2012) 2551e2567. [2] J. Perk, G. De Backer, H. Gohlke, et al., European guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts), Eur. Heart J. 33 (2012)

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