Application of microRNAs in diagnosis and treatment of cardiovascular disease.

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Received Date : 08-Sep-2014 Revised Date : 20-Oct-2014 Accepted Date : 24-Oct-2014 Article type

: Review Article

Application of microRNAs in diagnosis and treatment of cardiovascular disease

Anetta Wronska, Iwona Kurkowska-Jastrzebska, Gaetano Santulli

Anetta Wronska Helen and Clyde Wu Center for Molecular Cardiology, Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA

Iwona Kurkowska-Jastrzebska Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Krakowskie Przedmiescie 26/28, 00-927 Warsaw, Poland 2nd Department of Neurology, National Institute of Psychiatry and Neurology, Sobieskiego 9, PL 02-957 Warsaw, Poland

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12416 This article is protected by copyright. All rights reserved.

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Gaetano Santulli Helen and Clyde Wu Center for Molecular Cardiology, Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University Medical Center, New York, NY 10032, USA

Short title: microRNAs in cardiovascular disease

Corresponding author: Anetta Wronska Columbia University, Department of Physiology and Cellular Biophysics 1150 St Nicholas Ave, Russ Berrie Science Pavilion 515 New York, NY 10032, USA tel.: 347-819-7504 fax: 212-851-5346 [email protected]

Abstract Cardiovascular disease is a major cause of morbidity and mortality worldwide. Innovative, more stringent diagnostic and prognostic biomarkers and effective treatment options are needed to lessen its burden. In recent years, microRNAs have emerged as

This article is protected by copyright. All rights reserved.

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progression of CVD (reviewed in (Vickers et al., 2014, Xin et al., 2013c, van Rooij and Olson, 2012, Ono et al., 2011)).

miRNA biology and preclinical studies

First discovered in C. elegans in 1993 (Lee et al., 1993), miRNAs downregulate their target genes through mRNA degradation or translational repression. miRNA genes are transcribed in the nucleus by RNA polymerase II as long primary transcripts (primiRNAs), which may contain more than one miRNA (Figure 1). Pri-miRNAs are processed by the RNase III enzyme (Drosha) into hairpin-like pre-miRNAs, which are exported into the cytosol by exportin-5. Once in the cytosol, pre-miRNAs are processed by RNase III (Dicer) to mature miRNAs, which can then interact with proteins from the Argonaute family to form RNA-induced silencing complexes (RISC). miRNAs then direct RISC to their target mRNAs through complementary sequences, most often located in the 3’-untranslated region (3’UTR) of the target gene. Binding can occur even when the complementarity is limited to the so-called “seed” region of nucleotides 2-8 in the 5’-end of a given miRNA, which increases the number of mRNA targets for a single miRNA and allows for a complicated network of miRNA-mRNA interactions. miRNAs with the same seed sequence constitute miRNA families, and are thought to have redundant functions. It is believed that the mRNA degradation process requires a high degree of complementarity between the miRNA and its target mRNA, while low complementarity (limited to the seed sequence) leads to translational repression (Tuccoli et al., 2006, Lewis et al., 2003). miRNAs are highly conserved across metazoan species, suggesting strong evolutionary pressure due to their important role in essential physiologic processes. Most mammalian


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Multiple target genes of differentially expressed miRNAs are known to be involved in the regulation of inflammation, intima-media thickening, lipid metabolism, oxidation, VSMC phenotype, proliferation, and other processes related to the development of atherosclerosis. Mounting evidence indicates that miRNAs are involved in the homeostasis of atherosclerotic plaques. Buttitta’s group found profound differences between the miRNA profiles of asymptomatic versus unstable plaques obtained from patients who underwent carotid endarterectomy. Five miRNAs (mir-100, -127, -133a, -133b, and 145) were upregulated in symptomatic plaques compared with stable ones (Cipollone et al., 2011). Since atherosclerotic rupture is considered the key factor in the onset of myocardial infarction and ischemic stroke, differentially expressed miRNAs may serve as potential therapeutic targets for plaque stabilization and prevention of ischemic disease.

Inflammation is an important factor underlying the initiation and progression of atherosclerosis. Under normal conditions ECs do not express adhesion molecules. Upon activation by pro-inflammatory cytokines, ECs express VCAM-1 and other adhesion molecules, which mediate the adherence of leukocytes. Leukocytes trigger the process of inflammation, which in turn exacerbates atherosclerosis. On the other hand, ECspecific miR-126 downregulates VCAM-1 expression (Harris et al., 2008, Ono et al., 2011) thus conceivably miR-126 overexpression or its miR-126 mimics would offer a protection against the initiation and progression of inflammation and atherosclerosis (Ono et al., 2011). Inflammation is also an essential player in the pathophysiology of AMI. Necrotic myocytes resulting from AMI activate the innate immune response, which leads to an


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miRNAs are encoded by separate genes with their own promoters; some others are located in introns of other protein-coding genes and released during splicing. Many miRNAs are located in clusters of two or more miRNAs on a single pre-mRNA, and are therefore expressed simultaneously. Processing efficiency of individual miRNAs is regulated by a posttranscriptional mechanism (van Rooij et al., 2006).

A single miRNA can regulate multiple genes (often related functionally) and a single gene can be regulated by multiple miRNAs (Chavali et al., 2013, Brennecke et al., 2005). It is estimated that a miRNA family has on average 300 high- and low-affinity conserved targets. An additional level of expression control is provided by the proteins encoded by mRNA targets – some of which modulate the expression of additional miRNAs (Santoro, 2011). Such complex and intertwined regulatory networks allow a fine modulation of gene expression at the post-transcriptional level such that the regulation of gene expression is the net effect of relative abundance of independent miRNAs (Naga Prasad et al., 2009). This combinatorial regulation by multiple miRNAs has been proposed to compensate for the inherently low specificity and modest effect of individual miRNAs (Friedman et al., 2013). In general, miRNAs usually have a modest effect on the expression of individual target proteins (e.g. 25% decrease). However, the development of CVDs can be significantly influenced by subtle changes in the expression of relevant genes (Nossent et al., 2011). Thus, in contrast to conventional drugs with a single target, miRNA-based drugs targeting a single miRNA molecule could modulate multiple levels of a pathological process, which greatly increases their therapeutic potential (Olson, 2014).


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The general importance of miRNA for the cardiovascular system was demonstrated in mice by cardiomyocyte-specific deletions of Dicer (Thum, 2008) and Dgcr8 (Rao et al., 2009), two critical components of the miRNA maturation machinery. Dicer deletion caused embryonic or early postnatal death, fatal arrhythmias, or severe HF, depending on the timing of the deletion. Perinatal deletion of Dgcr8 led to fatal HF. Since then, miRNAs have been found to play a role in every CVD, and practically in all of its diverse pathophysiological and cellular aspects – angiogenesis, inflammation, fibrosis, proliferation, apoptosis, etc. (Olson, 2014). Each CVD presents a specific signature panel of dysregulated miRNAs. Unfortunately, some studies show contradictory results, but this can result from low sample numbers or different disease stages under investigation. This is still a new, rapidly evolving field, so new in-depth studies are needed to clarify the roles of contentious miRNA players. However, some specific miRNAs are consistently dysregulated in multiple CVDs, and their roles in CV pathophysiology are well established. For instance miR-1, known to be a key player in the maintenance of cardiac function, has been implicated in diverse CVDs; in the adult mouse heart, miR-1 accounts for 40% of total miRNA pool, which suggests it plays a crucial role in the maintenance of cardiac function (Rao et al., 2009). miR-26 plays an important role in cardiovascular repair (Icli et al., 2014, Icli et al., 2013), miR-181 family inhibits vascular inflammation (Sun et al., 2014b), and miR-29 is implicated in various fibrotic disorders resulting from its regulation of multiple extracellular matrix proteins. Leveraging these miRNAs for regulating underlying processes represents a promising new approach to the treatment of CVD. Many miRNAs relevant for CVD are expressed in a tissue- and developmental stagespecific manner. For example, miR-208a and miR-499 are expressed almost exclusively in the heart, while miR-1 and miR-133a are highly expressed in both cardiac and skeletal


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muscle (Chen et al., 2006). miR-1 and miR-133a derive from two common bicistronic miRNA clusters, miR-133a-1/miR-1-2 and miR-133a-2/miR-1-1. Their expression is regulated by myocyte enhancer factor-2 (MEF2) and serum response factor (SRF), two transcriptional factors crucial for muscle-specific gene expression (Liu et al., 2007, Zhao et al., 2005). miR-133b, which differs from miR-133a by only 2 nucleotides at the 3’-end, is expressed from another bicistronic miRNA, together with miR-206, only in skeletal muscle. miR-1 and miR-133 act synergistically to promote mesoderm differentiation in embryonic stem (ES) cells, but act antagonistically to each other later in development – miR-1 promotes, and miR-133 inhibits differentiation of ES cells into cardiac muscle (Ivey et al., 2008). In mice, deletion of the two genes coding for miR-133a leads to late embryonic or neonatal lethality due to ventricular-septal defects in about 50% of animals; the other half died of dilated cardiomyopathy and heart failure (HF) later in life (Liu et al., 2008). The observed effects can be at least partially explained by aberrant expression of two specific targets of miR-133: cyclin D2 and SRF, which regulate genes involved in cardiomyocyte proliferation and differentiation. miR-133 seems to be regulated by a negative feedback loop involving SRF – the opposing effects of the two factors establish a finely tuned balance to control cardiac growth and differentiation (Liu et al., 2008). miR-126 is expressed predominantly in endothelium (Santulli et al., 2014b), where it exerts a pro-angiogenic effect and maintains vascular integrity (Fish et al., 2008), regulating the response of endothelial cells (ECs) to vascular endothelial growth factor (VEGF) (Fish et al., 2008). It is encoded by an intron of the epidermal growth factor (EGF)-like domain gene, which encodes a growth factor involved in cell migration. Direct targets of miR-126 include the vascular cell adhesion molecule 1 (VCAM-1) and negative regulators of VEGF pathway, including the Sprouty-related protein 1 (SPRED1)


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shortening of atrial action potential duration, and upregulation of inward rectifier currents IK1 and IKACh,c (Van Wagoner and Nerbonne, 2000, Ehrlich, 2008).

Muscle-specific miR-1 level was significantly reduced in the left atrium of patients with persistent AF, while the Kir2.1 subunit of inwardly rectifying potassium channel expression and the density of the associated inward rectifier current, IK1, were increased (Girmatsion et al., 2009). An increase in IK1 is associated with stabilization of rotors underlying reentrant arrhythmias in AF. The results of this study suggest a causal relationship between miR-1 and the increase in IK1 density. Another miRNA with a strong arrhythmogenic potential is miR-328, implicated as a causal factor in the downregulation of two subunits of the L-type Ca2+ channel, Cav1.2 (encoded by CACNA1C) and Cavβ1 (encoded by CACNAB1), observed in AF patients (Lu et al., 2010). CACNA1C and CACNAB1 are direct targets of miR-328. miR-328 is highly abundant in human atrial cardiomyocytes compared with CACNA1C transcripts (~4000 vs. ~600 copies per cell, respectively), which supports the role of miR-328 in the suppression of the two L-type channel subunits in vivo.

Other miRNAs were implicated in the pathophysiology of AF through the modulation of fibrosis and apoptosis (reviewed in (Santulli et al., 2014a)), e.g. miR-21 (see HF section); miR-29, downregulated in AF patients (Dawson et al., 2013, Hale and Levis, 2013); and miR-30 and -133 (also downregulated in AF), which directly regulate CTGF, a key profibrotic protein (Duisters et al., 2009).


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Ehrlich, J. R. 2008. Inward rectifier potassium currents as a target for atrial fibrillation therapy. J Cardiovasc Pharmacol, 52, 129-35. Ellis, K. L., Cameron, V. A., Troughton, R. W., Frampton, C. M., Ellmers, L. J. & Richards, A. M. 2013. Circulating microRNAs as candidate markers to distinguish heart failure in breathless patients. Eur J Heart Fail, 15, 1138-47. Eskildsen, T. V., Jeppesen, P. L., Schneider, M., Nossent, A. Y., Sandberg, M. B., Hansen, P. B., Jensen, C. H., Hansen, M. L., Marcussen, N., Rasmussen, L. M., Bie, P., Andersen, D. C. & Sheikh, S. P. 2013. Angiotensin II Regulates microRNA-132/-212 in Hypertensive Rats and Humans. Int J Mol Sci, 14, 11190-207. Fichtlscherer, S., De Rosa, S., Fox, H., Schwietz, T., Fischer, A., Liebetrau, C., Weber, M., Hamm, C. W., Roxe, T., Muller-Ardogan, M., Bonauer, A., Zeiher, A. M. & Dimmeler, S. 2010. Circulating microRNAs in patients with coronary artery disease. Circ Res, 107, 677-84. Figueira, M. F., Monnerat-Cahli, G., Medei, E., Carvalho, A. B., Morales, M. M., Lamas, M. E., da Fonseca, R. N. & Souza-Menezes, J. 2014. MicroRNAs: potential therapeutic targets in diabetic complications of the cardiovascular and renal systems. Acta Physiol (Oxf), 211, 491-500. Fish, J. E., Santoro, M. M., Morton, S. U., Yu, S., Yeh, R. F., Wythe, J. D., Ivey, K. N., Bruneau, B. G., Stainier, D. Y. & Srivastava, D. 2008. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell, 15, 272-84. Friedman, Y., Balaga, O. & Linial, M. 2013. Working together: combinatorial regulation by microRNAs. Adv Exp Med Biol, 774, 317-37. Frost, R. J. & van Rooij, E. 2010. miRNAs as therapeutic targets in ischemic heart disease. J Cardiovasc Transl Res, 3, 280-9. Gaziano, T. A., Bitton, A., Anand, S., Abrahams-Gessel, S. & Murphy, A. 2010. Growing epidemic of coronary heart disease in low- and middle-income countries. Curr Probl Cardiol, 35, 72-115. Girmatsion, Z., Biliczki, P., Bonauer, A., Wimmer-Greinecker, G., Scherer, M., Moritz, A., Bukowska, A., Goette, A., Nattel, S., Hohnloser, S. H. & Ehrlich, J. R. 2009. Changes in microRNA-1 expression and IK1 up-regulation in human atrial fibrillation. Heart Rhythm, 6, 1802-9. Goldraich, L. A., Martinelli, N. C., Matte, U., Cohen, C., Andrades, M., Pimentel, M., Biolo, A., Clausell, N. & Rohde, L. E. 2014. Transcoronary gradient of plasma microRNA 423-5p in heart failure: evidence of altered myocardial expression. Biomarkers, 19, 135-41.


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al., 2007). miR-1 levels correlated well with QRS widening in AMI, and the increase in QRS duration positively correlates with the size of infarct area (Holland and Brooks, 1976), and returned to normal values at the time of discharge of AMI patients following standard medical care (Ai et al., 2010). The usefulness of miR-1 as a biomarker for acute coronary syndrome (ACS) has been questioned in a study performed in 444 patients. Plasma levels of miR-1, miR-133a, and miR-208b were increased in patients with AMI compared with patients with unstable angina (Widera et al., 2011). However, these and other three miRNAs (miR-133b, miR-208a, and miR-499) exhibited a large overlap between patients with unstable angina and MI, suggesting that factors other than acute myocardial necrosis may impact the circulatory level of these miRNAs, making their diagnostic usefulness in discriminating between MI and unstable angina limited (Widera et al., 2011). Interestingly, levels of miR-133a and miR-208b in the circulation correlated with all-cause mortality at six months after ACS. However, when they were adjusted for the levels of high-sensitivity troponin T (hsTnT), a standard sensitive myonecrosis marker, at admission, these miRNAs did not provide any additional prognostic information. On the other side, in a seminal study examining miRNAs in AMI, among a total of ~100 miRNAs identified in the plasma of healthy subjects, cardiacspecific miRNAs were either detected at very low levels or were altogether absent. The expression levels of miR-1, -133a, -208a, and -499 were increased in AMI, but not in other CVD patients. Strikingly, two months after AMI, in patients who received percutaneous coronary intervention (PCI) and conventional pharmacological treatment, the plasma levels of the heart-specific miRNAs went down; especially miR-208a was reduced to undetectable level (Wang et al., 2010). These findings point to potential usefulness of miR-1, -133a, -499, and/or miR-208a as biomarkers in the clinical diagnosis of AMI. miR-208a seems to be especially amenable to this role, as the other three miRs are also expressed in skeletal muscle and may be released into the


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circulation after skeletal muscle injury (e.g. after surgery or strenuous physical exercise), while the release of miR-208a is specific for cardiac injury. Interestingly, miR-208a may be a better biomarker than cTnI in initial stages of cardiac injury, as it was detectable after 1-4 hours of chest pain in AMI patients, while cTnI levels increase after 4-8 hours (Wang et al., 2010). Schroen’s group demonstrated that the levels of miR-208a and miR499 were elevated 1600-fold and 100-fold, respectively, in AMI patients compared with healthy controls (Corsten et al., 2010). A milder, but still significant, increase of the two miRNAs was observed in viral myocarditis. Interestingly, circulating miRNA levels were not affected by such clinical confounders as age, sex, and renal function (Corsten et al., 2010), which increases their value as potential biomarkers. Circulating levels of miR-133a and miR-208a were also increased in CAD patients compared with healthy controls (Fichtlscherer et al., 2010). In contrast, the levels of vascular miRNAs (vasculoprotective miR-126 and highly expressed in ECs miR-17 and miR-92a), inflammation-associated miR-155, and VSMC-enriched miR-145 were significantly decreased. A markedly increased expression of miR-134, miR-370, and especially miR-198, found in patients with unstable versus stable angina pectoris, has been proposed as a clinical biomarker for CAD patients at risk of acute coronary events (Xu et al., 2009).

Multiple miRNAs have been demonstrated to be functionally involved in the pathophysiology of atherosclerosis in humans. miR-21, -34, 146a, 146b-5p, and -210 were found to be significantly upregulated in atherosclerotic plaques from peripheral arteries compared with non-atherosclerotic arteries (Raitoharju et al., 2011). Intriguingly, there was a corresponding downregulation of many of these miRNAs’ predicted targets.


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Multiple target genes of differentially expressed miRNAs are known to be involved in the regulation of inflammation, intima-media thickening, lipid metabolism, oxidation, VSMC phenotype, proliferation, and other processes related to the development of atherosclerosis. Mounting evidence indicates that miRNAs are involved in the homeostasis of atherosclerotic plaques. Buttitta’s group found profound differences between the miRNA profiles of asymptomatic versus unstable plaques obtained from patients who underwent carotid endarterectomy. Five miRNAs (mir-100, -127, -133a, -133b, and 145) were upregulated in symptomatic plaques compared with stable ones (Cipollone et al., 2011). Since atherosclerotic rupture is considered the key factor in the onset of myocardial infarction and ischemic stroke, differentially expressed miRNAs may serve as potential therapeutic targets for plaque stabilization and prevention of ischemic disease.

Inflammation is an important factor underlying the initiation and progression of atherosclerosis. Under normal conditions ECs do not express adhesion molecules. Upon activation by pro-inflammatory cytokines, ECs express VCAM-1 and other adhesion molecules, which mediate the adherence of leukocytes. Leukocytes trigger the process of inflammation, which in turn exacerbates atherosclerosis. On the other hand, ECspecific miR-126 downregulates VCAM-1 expression (Harris et al., 2008, Ono et al., 2011) thus conceivably miR-126 overexpression or its miR-126 mimics would offer a protection against the initiation and progression of inflammation and atherosclerosis (Ono et al., 2011). Inflammation is also an essential player in the pathophysiology of AMI. Necrotic myocytes resulting from AMI activate the innate immune response, which leads to an


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acute inflammatory reaction that plays an important role in early repair process after AMI, but can turn deleterious if it gets too intense (van der Pouw Kraan et al., 2014). Bostjancic et al. analyzed the role of miRNAs in ventricular rupture, which usually occurs 2-6 days after AMI and is associated with the regional and systemic inflammatory response (Zidar et al., 2011). All three tested miRNAs known to be involved in the inflammatory response, miR-146a, -150, and -155, were dysregulated in autopsy samples from infarcted heart tissue from AMI patients 2 to 7 days after the onset of infarction. There was significantly increased interstitial neutrophil infiltration of the infarcted myocardium in patients with ventricular rupture. At least one of those miRNAs, miR-150, was significantly downregulated in patients with compared to those without rupture. These findings strongly support the role of inflammation and its regulation by miRNAs in cardiac rupture and point to miRNAs as promising new therapeutic targets. Given that miR-21, -146a, and -155 have been implicated in multiple inflammatory diseases (Ranjha and Paul, 2013), their role in atherosclerosis, AMI, CAD, and stroke (Kishore et al., 2014) is not surprising.

A small proof-of-principle study on the miRNA signature of peripheral blood mononuclear cells (PBMCs) shed a light on a mechanism underlying the regulation of inflammation in CAD. Out of 129 tested miRNAs, only two, miR-135a and miR-147, were significantly altered in patients with CAD compared with healthy controls (Hoekstra et al., 2010). The miR-135a/miR-147 expression ratio was increased 19-fold in CAD patients. The changed miRNA profile of PBMCs may reflect alterations in the cadherin/Wnt-mediated cellular signaling. miR-135a downregulates JAK2 expression and increases apoptosis in lymphoma cell lines (Navarro et al., 2009). In murine macrophages, the expression of miR-147 is stimulated by toll-like receptor 4 (TLR4), leading to downregulation of


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proinflammatory cytokines (Liu et al., 2009). The increased miR-135a/miR-147 ratio may lead to a more pro-inflammatory phenotype, which is an established risk factor for the development of atherosclerotic lesions (Hoekstra et al., 2010). TLR4 is an important player in the initiation and progression of atherosclerotic disease: it is activated in response to tissue injury, is involved in the release of inflammatory cytokines in circulating monocytes in patients with CAD, and is an upstream regulator in the VEGF signaling pathway (Satoh et al., 2006). VEGF is a crucial regulator of vascular formation; its plasma levels are increased in patients with CAD (Blann et al., 2002), which probably indicates the presence of critical coronary lesions (Kucukardali et al., 2008). In addition, TLR4 expressed in macrophages infiltrating coronary arteries is believed to play an important role in coronary plaque destabilization and rupture. Interestingly, TLR4 is a direct target of miR-526b, which was found to be upregulated in patients with CAD (Li et al., 2014a).

On the other hand, mir-146 family (miR-146a/b) is believed to regulate downstream TLR4 signaling molecules (including nuclear factor NF-κB and activating protein 1 (AP1)) via a negative-feedback loop (Taganov et al., 2006). Interestingly, in patients with CAD a combined treatment with RAS inhibitors and a statin (atorvastatin) reduced monocytic levels of miR-146a/b and TLR4. The 12-month follow-up study showed that high levels of miR-146a, TLR4 mRNA, and protein levels at baseline were potent independent predictors of cardiac events in CAD patients (Takahashi et al., 2010). miR-146a was also increased in PBMCs from patients with ACS. Pro-atherosclerotic properties of miR-146a are believed to be exerted through its regulation of type 1 T helper cells (Th1) differentiation through the enhancement of the T-bet pathway in


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PBMCs (Guo et al., 2011). Th1 activation is believed to play a key role in the development of atherosclerosis (Nilsson et al., 2009).

The renin-angiotensin-aldosterone system (RAAS) is known to play a significant role in the pathogenesis of human atherosclerosis and hypertension. Deiuliis at al. assessed miRNA expression in peripheral mononuclear cells (given their central role in atherosclerotic plaque progression) in subjects treated with direct renin antagonist (Aliskiren). They observed increased plaque progression in Aliskiren-treated patients. Surprisingly, out of 734 miRNAs tested on microarrays, none seemed to be upregulated in the Aliskiren group, and only three were downregulated (at least 1.5-fold) compared with the placebo group – miR-106b-5p, -27a-3p, and -18b-5p. The baseline expression of two of them, miR-18b and miR-106b, correlated with plaque progression as expressed in terms of percent and total wall volume. Thus, these two miRs could serve as prognostic biomarkers in patients with atherosclerosis. miR-27a was also implicated in the pathogenesis of metabolic syndrome (see below).

miRNAs are also involved in the pathophysiology of CVD through the regulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a). SERCA2a is a regulator of intracellular Ca2+ homeostasis in cardiomyocytes, where it participates in the process of excitation-contraction coupling. It pumps Ca2+ from the cytosol into the lumen of the sarcoplasmic reticulum (SR), enabling the replenishment of Ca2+ ions released from the SR via the ryanodine receptor channel during each contraction. SERCA2a is downregulated in HF and AMI (Periasamy et al., 2008). miR-574-3p was upregulated in infarcted compared with healthy cardiac tissue in humans, and in silico analysis


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identified SERCA2a as its potential target (Bostjancic et al., 2012). In addition to miR574-3p, there were 42 differentially expressed miRNAs in infarcted tissue compared with remote myocardium. Only half of the miRNAs differentially expressed in infarcted tissue compared with remote myocardium overlapped with the set differentially expressed compared to healthy adult hearts (Bostjancic et al., 2012). Such difference supports the notion that some miRNAs in the non-infarcted area might also participate in the pathophysiology of AMI (Dong et al., 2009).

miRNAs could also facilitate differential diagnosis of AMI patients. The classification of MI, ST-elevation MI (STEMI) versus non-STEMI (NSTEMI), is an important factor informing therapeutic management: STEMI implies a thrombus completely and permanently occluding a coronary artery, calling for thrombolysis and immediate revascularization, while NSTEMI suggests an incomplete occlusion, usually requiring antiplatelet and antithrombotic drugs instead (Mauric and Oreto, 2008). Ward et al. identified unique circulating miRNA profiles in STEMI versus NSTEMI patients. In addition to miR-30d-5p, which was upregulated in plasma, platelets and leukocytes of both STEMI and NSTEMI patients, miR-221-3p and -483-5p were increased in plasma and platelets only in NSTEMI patients (Ward et al., 2013). Equally important, miR-499 represents a promising biomarker for acute NSTEMI in geriatric patients – its level was increased more than 80-fold in acute NSTEMI patients compared with healthy controls (Olivieri et al., 2013). This aspect is particularly relevant since the diagnosis of NSTEMI can often be difficult in the elderly due to atypical symptoms, and the currently used marker, cTnT, can be unreliable in differentiating NSTEMI from other cardiac conditions. Diagnostic accuracy (in terms of sensitivity and


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specificity) of miR-499 was higher than that of conventional cTnT or hsTnT in differentiating between NSTEMI and acute HF in patients with modest cTnT elevation at presentation.

Therapeutic potential of miRNAs in atherosclerosis and CAD has been recently supported in a study on apolipoprotein E-deficient (ApoE-/-) mouse model (Sun et al., 2014a). ApoE-/- mice fed a high-fat diet (HFD) exhibit a reduced expression of miR-181b in the aortic intima and plasma, mirroring significantly lower levels of circulating miR181b in patients with CAD. Systemic delivery of miR181b mimics inhibited atherosclerotic lesion formation in ApoE-/- mice fed HFD and was associated with decreased expression of inflammatory markers and reduced influx of inflammatory cells (macrophages and CD+ T cells). These beneficial effects were mediated by the inhibition of NF-κB nuclear translocation via a direct target of miR-181b, importin-α3. Interestingly, hepatic NF-κB was not affected and the infusion did not cause liver toxicity, further supporting the therapeutic potential of miR-181b.

miR-26a represents a promising therapeutic target for the treatment of AMI: it was elevated in ACS patients, as well as in a mouse model of AMI (Icli et al., 2013). Mechanistically, miR-26a inhibits EC proliferation and proangiogenic functions of EC through bone morphogenic protein (BMP)/SMAD1 signaling. Intravenous administration of a miR-26a inhibitor (LNA antimiR-26a) led to robust myocardial angiogenesis, reduced infarct size, and improved LV function in mice after AMI.


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miRNAs in ischemic stroke Stroke is a leading cause of death and disability worldwide. Cerebral ischemia triggers pathological events such as excitotoxicity (within minutes of stroke onset), inflammatory response (within hours), and apoptosis (hours and days). In preclinical studies, several miRNAs have been implicated in the pathophysiology of stroke (Rink and Khanna, 2011). Currently, the only proven treatment for acute ischemic stroke is tissue plasminogen activator (t-PA) (Sandercock et al., 2011). Multiple endovascular strategies have proven unsuccessful in clinical trials, probably due to the complexity of the interplay of signaling pathways involved in stroke and short therapeutic window for acute neuroprotection (Ouyang and Giffard, 2013). miRNAs seem to be able to overcome these difficulties and have great therapeutic potential, since they are known to be master regulators of multiple interrelated pathways and to be early responders in many pathophysiological processes relevant to stroke. miRNA-based therapeutic strategies for stroke are promising due to their potential for rapid onset and for targeting neurons and astrocytes in a coordinated and complementary manner (Stary and Giffard, 2014). Indeed, in a transient middle cerebral artery occlusion rat model, 20 miRNAs were dysregulated, 11 of them as early as 3 hours after reperfusion. Some of these miRNAs’ potential targets were gene promoters, strongly indicating a possibility of miRNA-induced activation of gene expression. Other targets included genes known to mediate inflammation, neuroprotection, receptor function, and ionic homeostasis (Dharap et al., 2009). Similarly, in a study in young (18-49 year old) ischemic stroke patients, most of the dysregulated miRNAs have been implicated in endothelial/vascular function, inflammation, erythropoiesis, neural function, or hypoxia-related processes. Interestingly, different subtypes of stroke (large-artery atherosclerosis, small-vessel occlusion, cardioembolism, and undetermined etiology) exhibited unique miRNA dysregulation


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profiles, which indicates that peripheral blood miRNA profiles can be useful in diagnosis and/or prognosis of ischemic stroke (Tan et al., 2013). In humans, miR-21 and miR-221 levels are significant predictors for stroke, even after adjusting for traditional risk factors (Tsai et al., 2013). Five miRNAs (miR-125b, -27a, 422a, -488, and -627) were upregulated at the onset of ischemic stroke and a panel of 32 miRNAs was differentially expressed depending on the etiology of stroke (Sepramaniam et al., 2014).

A novel encouraging approach to the treatment of stroke is the transplantation of bonemarrow-derived mesenchymal stem cells (BMSCs), which was successful in improving post-stroke neurological deficits in preclinical studies (Chen et al., 2001). This beneficial effect is at least partially attributable to miRNAs located in exosomes released from BMSCs. Indeed, miR-133b, the main miRNA transferred from BMSCs to astrocytes and neurons, was shown to promote neural plasticity (Xin et al., 2013b), while miR-210 and miR-107 regulate apoptosis (Olson, 2014). BMSCs are strong therapeutic candidates, since they are, unlike embryonic stem cells, easily isolated from humans and expanded in culture without ethical and technical issues. In addition, in rodents BMSCs evade the immune system and migrate to the ischemic boundary zone after transplantation (Li et al., 2014b). Instead of the whole cells, BMSC-generated exosomes could be used in the treatment of stroke. In a rat stroke model intravenous administration of cell-free exosomes derived from multipotent mesenchymal stromal cells significantly improved neurologic outcome and contributed to neurovascular remodeling (Xin et al., 2013a). The outcomes could be even further improved by using exosomes engineered to contain specific neuroprotective miRNAs or antimiRs against deleterious miRNAs.


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Role of miRNAs in hypertension Hypertension is a key predictor of morbidity and mortality for diverse CVD, such as myocardial hypertrophy, AMI, HF, and stroke (Doris, 2002, Santulli, 2012). It has a strong genetic component. The role of many of the single nucleotide polymorphisms (SNPs) associated with hypertension is unknown (Kumar et al., 2014, Santulli et al., 2013). SNP are often located in areas called not so long ago “junk DNA”, as it was believed to not contain any genetic information. Recently it has become clear that this “junk” is a treasure trove of diverse regulatory sequences, including miRNAs and their targeting sequences. SNPs in miRNA binding sites in some RAAS genes influence arterial blood pressure and the risk of AMI. Increased levels of type-1 angiotensin II (AngII) receptor (ATR1) are widely acknowledged to contribute to CVD, and ATR1 antagonists are commonly used to treat hypertension. In multiple studies, there was a strong association between the 1166C allele of ATR1 gene (AGTR1) and hypertension, cardiac hypertrophy, and AMI (Poirier et al., 1998, Hindorff et al., 2002). The 1166C SNP abrogates the binding of miR-155 to the AGTR1 3’UTR. miR-155 and ATR1 are coexpressed in the spleen and kidney; given the key role of kidney in blood pressure regulation (Santulli et al., 2012, Trimarco et al., 1985), the effect of the miR-155 binding abrogation is probably exerted mostly through this organ (Sethupathy et al., 2007). In PBMCs isolated from young hypertensive subjects with the mutant allele the level of ATR1 correlated positively with blood pressure and negatively with miR-155 expression level (Ceolotto et al., 2011). Thus, the hypertensive effect of the 1166C mutation seems to result from the defective regulation of ATR1 expression by miR-155. The mutation is also an example of rodent model inadequacy, due to genetic differences between humans and mice and rats, as in the rodents only one of either miR-155 or its AGTR1 target site is preserved.


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Eskildsen et al. observed reduced expression levels of miR-132 and miR-212 in internal mammary arteries from bypass-operated patients treated with ATR1 blockers, but not in patients treated with β-blockers (Eskildsen et al., 2013). These results are consistent with the notion that not all blood pressure-reducing agents downregulate miR-132/-212 expression and suggest that the miR-132/-212 cluster in humans may be part of the response to Gαq-vasopressors such as AngII. The results in humans were consistent with observations in an AngII-induced hypertension rat model.

Two new miRNAs were recently implicated in hypertension in the large population-based case control Study of Myocardial Infarctions LEiden (SMILE) (Nossent et al., 2011). The SNP that had the strongest association with increased blood pressure was found on arginine vasopressin 1A receptor (AVPR1A), in the region complementary to the seed sequences of miR-526b and miR-578. Two rare SNPs in the bradykinin 2 receptor gene (BDKRB2) were associated with reduced systolic pressure. Since the two SNPs are linked, it is possible that only one of them is responsible for the observed association. One of the SNPs (rs5225) is located in a potential seed recognition site for miR-34a, miR-34b, and miR-449, while the other is in the recognition sequence for miR-151-3p. This miRNA’s gene is located in an intron of the PTK2B gene, the expression of which is stimulated by vasopressin. Thus vasopressin could potentially inhibit the expression of bradykinin 2 receptor by upregulating PTK2B and hence miR-151-3p. Arterial blood pressure was also decreased in carriers of the T-allele of the thromboxane A2 (a known vasoconstrictor) receptor (TBXA2R). The SNP is located in the seed recognition site for miR-571 and miR-765 (Nossent et al., 2011), thus offering an


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opportunity to use these miRNAs as therapeutic targets in the treatment of hypertension. None of the described SNPs was associated with the risk of AMI. The risk of AMI was instead increased in homozygous carriers of the A-allele of the aldosterone receptor (NR3C2), especially in men below 50 years of age. The SNP of this allele is located in the binding site for miR-383 (Nossent et al., 2011). The aldosterone receptor is involved in the regulation of Von Willebrand factor, whose elevated levels are associated with an increased risk of AMI (Chion et al., 2007). Notably, all associations were observed only in homozygous carriers, with no changes in phenotype observed in heterozygous individuals, consistent with the modulatory, fine-tuning mode of action of most miRNAs. Some miRNAs could serve as novel prognostic biomarkers and therapeutic targets in essential hypertension. miR-9 and miR-126 have been found to be downregulated in PBMCs from hypertensive patients compared with those from healthy controls (Kontaraki et al., 2014b). Moreover, the levels of these two miRNAs positively correlated with the 24-hour mean pulse pressure, an established predictor of advanced targetorgan damage (Carpinella et al., 2014). One may speculate that decreased levels of miR-126 lead to impaired endothelial function and vascular remodeling, which are predictive of cardiovascular events in hypertensive patients with target-organ damage. On the other hand, miR-9 acts through the negative regulation of myocardin, which triggers hypertension-related left ventricular hypertrophy. miRNAs which modulate VSMCs, whose phenotypic plasticity has a fundamental role in the development of hypertension, represent other noteworthy potential biomarkers and therapeutic targets in essential hypertension. Indeed, miR-143 and miR-145 levels were found to be lower in PBMCs from hypertensive patients compared with those from healthy controls and negatively correlated with 24-hour mean pulse pressure (Kontaraki et al., 2014a). One of the direct targets of miR-143 and miR-145 is angiotensin-


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converting enzyme, a critical factor in hypertension, and the two miRNAs are regulated by myocardin via feed-forward feedback mechanism. The elucidation of regulatory networks through which miR-9, miR-126, miR-143, and miR-145 are involved in the pathophysiology of hypertension underscores their usefulness as potential biomarkers of hypertension-related cardiovascular events and target-organ damage, and therapeutic targets in the treatment of hypertension and its complications.

miRNA dysregulation in left ventricular hypertrophy and heart failure Cardiac hypertrophy is associated with diverse types of CVD, including hypertension, ischemic disease, aortic stenosis, valvular dysfunctions, cardiomyopathies. Sustained pathological hypertrophy is the major clinical predictor of HF and sudden cardiac death (Dirkx et al., 2013, Meijs et al., 2007, Towbin and Bowles, 2002, Knoll et al., 2011). Several animal models of HF showed differential expression of multiple miRNAs in affected versus control animals (Latronico and Condorelli, 2011). The group led by Eric N. Olson was the first to show a dysregulation of miRNAs in human HF (van Rooij et al., 2006). In idiopathic end-stage failing human hearts there was an increased expression of miR-24, -125b, -195, -199a, and miR-214. The altered expression pattern of miRNAs in the failing human heart showed an overlap with the pattern found in the hypertrophic mouse heart, suggesting that the differentially expressed miRNAs are real molecular signatures of adverse cardiac remodeling. Interestingly, a transgenic mouse model indicated that cardiac overexpression of miR-195 was sufficient to evoke cardiac hypertrophy (van Rooij et al., 2006).


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The first extensive genome-wide miRNA expression profiling in left ventricular myocardium of patients with different cardiac disorders (ischemic or dilated cardiomyopathy, aortic stenosis) versus non-failing controls revealed a unique miRNA expression pattern for each of the diagnostic groups, suggesting that the pattern of miRNA alterations differs by underlying disease etiology (Ikeda et al., 2007). Similar conclusions were reached by Sucharov and colleagues, who found distinct miRNA patterns in two types of HF, ischemic and idiopathic dilated cardiomyopathy (ISC and IDC, respectively). For instance, miR-222 was downregulated only in ISC, not IDC, while miR-382 was upregulated and miR-10b and -139 were downregulated only in IDC (Sucharov et al., 2008). The common subset of miRNAs differentially expressed in both types of HF included miR-100 and miR-195 (upregulated in HF), and miR-133a, -133b, 150, -221, -422b, -486, -594, and -92 (downregulated).

The expression of two miRNAs specific for cardiac and skeletal muscle – miR-1 and miR-133 was downregulated in the hearts of patients with hypertrophic cardiomyopathy and atrial dilation (Care et al., 2007). The findings in humans were consistent with the results of an extensive research in murine models, which showed that the suppression of miR-133 (obtained through an infusion of antimiR-133) was sufficient to induce a significant and sustained cardiac hypertrophy, and its upregulation reduced or prevented adrenergic-mediated induction of hypertrophic gene program, while inhibition of miR-100 prevented downregulation of adult gene component associated with fetal gene reprogramming, a hallmark of HF (Sucharov et al., 2008, Care et al., 2007). Activation of fetal cardiac genes, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), or β-myosin heavy chain (β-MHC), which leads to the recapitulation of the


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neonatal growth phenotype, is a molecular signature of cardiac hypertrophy. A striking similarity in miRNA expression patterns has been found between ventricles from endstage failing hearts and fetal cardiac tissue (Thum et al., 2007). In particular, miR-133 seems to have a critical role in establishing and sustaining the fetal gene reprogramming (Bostjancic et al., 2010), by targeting at least three genes relevant to cardiac hypertrophy development: Rhoa, Cdc42, and NELFA/Whsc2. RhoA and Cdc42 are members of the Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, and are associated with the rearrangement of cytoskeleton and myofibrillae during hypertrophy (Nagai et al., 2003, Brown et al., 2006). NELF-A/Whsc2 is a negative regulator of RNA polymerase II and is linked to Wolf-Hirschhorn syndrome, which is characterized, among other abnormalities, by cardiac dysgenesis. In animal studies, Whsc2 has been shown to upregulate fetal gene expression in the heart (Care et al., 2007). All these results point to miR-133 as a global regulator of cardiomyocyte hypertrophy and seem to indicate a modulation of miR-133 as a potential therapeutic approach in the treatment of cardiac hypertrophy.

Other miRNAs with therapeutic potential in HF include miR-21, miR-129, and miR-212. Transfection of isolated adult rat cardiomyocytes with this set of miRNAs (which are upregulated in both human failing and fetal hearts) led to cardiomyocyte hypertrophy and activation of a fetal gene program, thus confirming the causal role of miRNAs in the induction of fetal reprogramming (Thum et al., 2007). Notably, transfection with only one of these miRNAs had only minor effects, which is in agreement with the notion that miRNAs act mostly in concerted networks of often overlapping and redundant factors rather than as singular on/off switches. miR-21 was also identified as an important regulator of fibrotic remodeling in HF and in atrial fibrillation (AF). Left ventricular cardiac


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tissue from end-stage HF patients and left atria of AF patients exhibited increased levels of miR-21 and decreased level of its direct target Sprouty homolog 1 (SPRY1). Both miR-21 and SPRY1 are expressed in cardiac fibroblasts and not in cardiomyocytes. Their imbalance found in AF and HF stimulates fibrosis, hypertrophy, and left ventricular dysfunction through activation of the Ras/MEK/ERK pathway (Thum et al., 2008, Adam et al., 2012). Studies in rodents indicate that miR-21 is regulated by AngII, via Drosha and Dicer (Adam et al., 2012). Actually, miR-21 was the target of the first miRNA-based therapeutics in a CVD model, where the injection of a chemically modified antimiR-21 was shown to improve structural and functional cardiac characteristics in two different murine models, offering the first in vivo confirmation of the great therapeutic potential of miRNA modulation (Thum et al., 2008).

In HF there is also an imbalance in MHC isoforms expression – a decrease in α-MHC and an increase in β-MHC levels. In patients with dilated cardiomyopathy (DCM), the improvement in left ventricular ejection fraction (LVEF) after chronic β-blocker treatment has been associated with normalization of MHC isoforms levels (Abraham et al., 2002). Corroborating these data, the level of miR-208a in endomyocardial tissue from DCM patients positively correlated with β-MHC expression and with myocardial fibrosis, and negatively with α-MHC expression (Satoh et al., 2010). The levels of the other myomiRs (miR-208b and –499) were not correlated with β-MHC. Interestingly, high levels of miR208a were associated with poor clinical outcomes in DCM, suggesting a key functional role of miR-208a in the progression of human DCM and its potential use as a biomarker for cardiac death and HF progression.


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In another clinical study eight miRNAs were found to be differentially expressed in endstage HF compared with nonfailing human hearts: miR-1, -29b, -7, -378 were downregulated and miR-214, -342, -125b, and -181b were upregulated in left ventricle samples from patients with dilated cardiomyopathy (Naga Prasad et al., 2009). Bioinformatic analysis identified several nodal molecules on the global signaling networks, including epidermal growth factor receptor 2 (ERBB2) and collagen 1 (Col1A), as potential targets of these miRNAs. As these proteins play essential roles in the corresponding signaling networks, the combinatorial effect of the altered miRNAs could explain the observed global changes in cardiac dysfunction and HF. The results were in agreement with the corresponding up- or downregulation of multiple key proteins encoded by the potential targets of the dysregulated miRNAs. Interestingly, most of the differentially expressed miRNAs directly target some members of the NFκB network NFκB is an established mediator in cardiac dysfunction (Sorriento et al., 2008, Valen et al., 2001). miRNA and mRNA profiling in HF revealed that most of the miRNAs are upregulated and, accordingly, mRNA transcripts are downregulated, indicative of a mechanism to suppress the steady state levels of mRNA transcripts (Olson, 2014, Matkovich et al., 2009). These results support the notion that miRNAs, through their antagonistic action towards their target mRNAs, may indeed play a role of master regulators in HF initiation and progression. This also seems to indicate that antimiRs rather than miRNA mimics may be more promising as therapeutic agents in the treatment of CVD. Overall, levels of miRNAs upregulated in HF either normalized or significantly decreased in HF patients treated with left ventricular assist devices (LVAD). In contrast, mRNA profile changes did not reflect functional improvements after treatment with LVAD treatment, as well as with β-blockers and synchronization therapy (Wright et al., 2007,


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Lowes et al., 2002, Margulies et al., 2005). These results suggest that miRNA may be more reactive to acute alterations in pathophysiological status than mRNA. A combined profiling of miRNA and mRNA may have superior potential as a diagnostic and prognostic test in end-stage cardiomyopathy. To discern whether the observed miRNA expression changes were a cause or an effect of HF, miRNA expression analysis was performed on the transverse aortic constriction mouse model. Four miRs (miR-7, -378, 214, and -181b) were found to be altered at the initial stages of cardiac dysfunction, indicating their mechanistic role in the dysregulation of signaling networks at the initial phases of the disease. It is worth noting, however, that most of the cited studies were performed in end-stage HF patients, and may have missed important indicators and/or determinants of HF progression.

miRNAs could also serve as novel diagnostic tools in HF. miR-423-5p seems to have such a potential, especially in HF affecting LV (Tijsen et al., 2012). Circulating level of miR-423-5p was increased in HF patients but not in patients with non-HF-related dyspnea and correlated well with the level of currently used biomarker N-terminal probrain natriuretic peptide (NT-proBNP) (Tijsen et al., 2010, Goren et al., 2012, Ellis et al., 2013). It was also robustly increased in two rodent models of HF (Dickinson et al., 2013). Crucially, miR423-5p may be specific for HF, as its level was increased in HF patients in a study where subjects with recent cardiac ischemia or infarction were excluded to minimize the effect of recent cardiac damage on circulating microRNA profile. In that case mir-423-5p level was still increased even when the levels of miR-1 and miR-208, two cardiomyocyte damage markers, were unchanged (Tijsen et al., 2010). However, in


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a larger human study the level of miR-423-5p in HF patients was similar to that in nonHF-related dyspnea patients, but still the addition of miR-423-5p levels to NT-proBNP significantly increased the diagnostic power of the current standard biomarker. The discrepancy between the studies may result from small sample sizes, different diagnostic criteria, HF etiologies and/or treatment received before and/or during the study (Bauters et al., 2013). Clearly, more studies are needed to validate the usefulness of miR-423-5p in HF diagnosis. Interestingly, miR-423-5p has also been found to be upregulated in human failing myocardium (Thum et al., 2007), and the difference in its transcoronary gradients seem to confirm a cardiac origin of circulating miR-423-5p (Goldraich et al., 2014). There are indications based on animal models that miRNAs also play a role in cardiac aging (Dimmeler and Nicotera, 2013). miR-34a downregulates protein phosphatase

1 nuclear targeting subunit (PNUTS) mRNA, which regulates telomere maintenance and DNA damage responses and is reduced in aging, thus offering a potential therapeutic strategy to improve cardiac contractility after AMI (Boon and Vickers, 2013). The functional role of miRNA-34a and its downstream effectors needs to be confirmed in humans.

miRNAs in atrial fibrillation Atrial fibrillation (AF) is a complex arrhythmia whose molecular mechanisms are not completely understood (D'Ascia et al., 2011, Xie et al., 2013). It is characterized by an electrical and structural remodeling of the atrial tissue leading to persistent arrhythmia (Nattel, 2002). In AF there is a significant reduction in L-type Ca2+ current (ICaL) density,


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shortening of atrial action potential duration, and upregulation of inward rectifier currents IK1 and IKACh,c (Van Wagoner and Nerbonne, 2000, Ehrlich, 2008).

Muscle-specific miR-1 level was significantly reduced in the left atrium of patients with persistent AF, while the Kir2.1 subunit of inwardly rectifying potassium channel expression and the density of the associated inward rectifier current, IK1, were increased (Girmatsion et al., 2009). An increase in IK1 is associated with stabilization of rotors underlying reentrant arrhythmias in AF. The results of this study suggest a causal relationship between miR-1 and the increase in IK1 density. Another miRNA with a strong arrhythmogenic potential is miR-328, implicated as a causal factor in the downregulation of two subunits of the L-type Ca2+ channel, Cav1.2 (encoded by CACNA1C) and Cavβ1 (encoded by CACNAB1), observed in AF patients (Lu et al., 2010). CACNA1C and CACNAB1 are direct targets of miR-328. miR-328 is highly abundant in human atrial cardiomyocytes compared with CACNA1C transcripts (~4000 vs. ~600 copies per cell, respectively), which supports the role of miR-328 in the suppression of the two L-type channel subunits in vivo.

Other miRNAs were implicated in the pathophysiology of AF through the modulation of fibrosis and apoptosis (reviewed in (Santulli et al., 2014a)), e.g. miR-21 (see HF section); miR-29, downregulated in AF patients (Dawson et al., 2013, Hale and Levis, 2013); and miR-30 and -133 (also downregulated in AF), which directly regulate CTGF, a key profibrotic protein (Duisters et al., 2009).


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miRNAs in diabetic cardiovascular complications and in metabolic syndrome Complications of type 1 and type 2 diabetes mellitus encompass microvascular complications, including retinopathy, nephropathy, and neuropathy; and macrovascular ones, such as CAD, atherosclerosis, hypertension, HF, and stroke (Kato et al., 2013). Here, we focus on the macrovascular effects of diabetes. A study comparing miRNA expression profiles in left ventricle tissue from diabetic and non-diabetic patients with non-end stage ischemic cardiomyopathy who had similar hemodynamic impairment indicated a dysregulation of the hypoxia response mechanisms in diabetic HF (Greco et al., 2012). In addition to several miRNAs differentially expressed in diabetic and non-diabetic HF patients compared with non-HF control subjects, there was a set of six miRNAs specifically dysregulated in diabetic HF patients: miR-34b, miR-34c, miR-199b, miR-210, miR-650, and miR-223. miR-210 is considered a master miRNA of hypoxic response (Devlin et al., 2011), which is in agreement with the evidence that diabetic hearts have a compromised hypoxia signaling pathway (Heather and Clarke, 2011). mir-199b was implicated in cardiac fibrosis and HF in mice, probably via the calcineurin/nuclear factor of activated T-cell (NFAT) signaling (da Costa Martins et al., 2010). NFAT is a glucose sensor in smooth muscle cells, which may provide a mechanism for the involvement of mir-199b in HF in diabetic patients. Such mechanisms (oxidation, high-glucose response) may at least in part explain the increased risk of ischemic HF after AMI observed in diabetic versus non-diabetic patients, even when other HF risk factors (infarct size, left ventricular ejection fraction, or infarct artery patency) are lower than in non-diabetic patients (Stone et al., 1989, Mak et al., 1997, Figueira et al., 2014).


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Metabolic syndrome is characterized by the presence of multiple risk factors for CVD, including abdominal obesity, lipid abnormalities, elevated blood pressure and/or abnormal fasting glucose levels (Schivo et al., 2013). Analyses of miRNAs isolated from the whole blood and exosomes indicated that miRNAs are dysregulated in metabolic syndrome as well as in its individual metabolic disorders (Karolina et al., 2012). The miRNAs dysregulated in metabolic syndrome can be divided into 3 clusters – each involved in different aspect of its pathogenesis: cluster A (comprising of miR-103, -17, 183, -197, -23a, -509-5p, -584, and -652) is associated with hypercholesterolemia, cluster B (miR-130a, -195, and 92a) – with hypertension, and cluster C (miR-150, -192, 27a, and 320a) – with hyperglycemia. Predicted targets of miR-27a and other miRNAs dysregulated in metabolic syndrome include genes involved in pathways related to sphingolipid and fatty acid metabolism, and ATR1 signaling in addition to those implicated in diabetes. These pathways are implicated in lipid metabolism and vascular signaling, which suggests their important role in the maintenance of the metabolic homeostasis and offers an opportunity to develop a miRNA-based therapy and/or biomarkers for metabolic syndrome or its individual components.

Translational aspects and clinical perspectives In recent years miRNAs have emerged as critical factors in cardiovascular system. They are known to be involved in normal development of cardiovascular system and in the initiation and progression of CVD. miRNAs are promising candidates for therapeutic targets, diagnostic and prognostic biomarkers, or possible indicators for stratification of patients in clinical trials.


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By regulating miRNAs, it is possible to dramatically influence signal-dependent tissue remodeling without introducing negative effects upon tissue function, which suggests miRNAs can play a role as potentially powerful disease modifiers and therapeutic targets. However, most of the data on the role of miRNAs in cardiovascular physiology and pathophysiology are still preliminary. Target genes of specific miRNAs are often not validated and the mechanism of their action is unclear. Many target genes predicted in silico or through overexpression of specific miRNAs may be physiologically irrelevant at lower, physiological concentrations, or due to localization discrepancies between miRs and their potential targets. There is also a lack of universally accepted analytical methods for relative miRNA quantification, which may lead to many false positives in quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Due to the cooperative nature of miRNA regulation and multiple targets of particular miRNAs, unwanted or even harmful off-target effects are of concern when designing miRNA-based therapeutics. Thus, a better understanding of mechanism of action of potential miRNA targets or therapeutic miRNAs and systems biology approach (to appreciate the whole range of cellular changes triggered by a specific therapeutic agent), and tests on larger, more relevant animals are needed before effective and safe miRNA-based drugs can be designed for and tested in humans. This is especially important for antimiRs, given their delayed but sustained action (it takes time to rebalance the proteome through the actions of multiple miRNAs targeted by antimiR and, at the other end, antimiRs are very stable and can be slowly released from intracellular deposits) (Olson, 2014). While such sustained action can be beneficial for treatment of chronic diseases such as CVD, it also poses a challenge of lingering toxicity, which can be alleviated by targeted delivery.


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The perennial problem of any RNA-interference (RNAi)-based therapy, including miRNAs, is to achieve effective and stable targeted delivery. Therapeutic agents in the form of oligonucleotides such as miRNA mimics (mimicking the sequence and action of dysregulated beneficial miRNAs), antimiRs, or miRNA sponges (neutralizing detrimental miRNAs) can be chemically modified to increase their stability and/or affinity (e.g. locked nucleic acids (LNA) show enhanced binding to miRNAs (van Rooij et al., 2012)). Specificity and/or targeting can be enhanced by using adeno-associated virus vectors (AAV), especially AAV9, which shows tropism to the heart (Olson, 2014), without affecting the liver (Piras et al., 2013). Other possible solutions include delivery directly to the injured heart through a catheter in case of AMI, or stents coated with miRNAmodifying drugs (Natarajan et al., 2012, Frost and van Rooij, 2010). Cell-based therapy may provide another means of miRNA-based CVD treatment – BMSCs delivered intravenously release miRNA-enriched microvesicles (Chen et al., 2014). BMSC infusions proved to be safe and beneficial in several stroke clinical trials (Bang et al., 2005, Suarez-Monteagudo et al., 2009, Lee et al., 2010, Moniche et al., 2012). Transplantation of BMSCs engineered to overexpress miR-126 enhanced functional angiogenesis in the ischemic myocardium and improved cardiac function in mice (Huang et al., 2013). Similar therapeutic effects could be achieved by using exosomes derived from such BMSCs (Xin et al., 2013a). Cardiac transplantation of angiogenic early outgrowth cells (EOCs) or CD34+ cells represents another promising cell-based approach in the treatment of CVD (Kocher et al., 2001, Kawamoto et al., 2001). Administration of EOCs has been shown to enhance myocardial neovascularization and improve cardiac function after ischemic injury (Kocher et al., 2001). A recent study ascribed this beneficial effect to angiogenic miRNAs, miR-126 and miR-130a, expressed in and secreted from these cells (Jakob et al., 2012). EOCs from chronic HF patients significantly diminished angiogenic and cardiac repair capacity compared with EOCs


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from healthy subjects; this capacity was rescued by transfection of HF-patients-derived EOCs with miR-130a (in vitro) or miR-126 (in vitro and in vivo). This finding offers new therapeutic strategies for miRNA-based therapy of chronic HF based on genetic manipulation of EOCs or CD34+ cells. Administration of antimiRs and, to a lesser degree, miRNA mimics, proved to be very efficient at protecting cardiovascular system against induced deleterious effects in multiple experimental models, suggesting new therapeutic targets and approaches for treatment of CVD in humans. For example, miR-328 is known to be upregulated in AF. Injection of antimiR-328 into WT mice diminished AF vulnerability in the presence of carbachol (yielding similar phenotype as miR-328 knock-out mice) – the effect most probably brought about by two direct targets of miR-328, Cav1.2 and Cavβ1 (see AF section) (Lu et al., 2010). miR-199b, known to be upregulated in HF, is activated by calcineurin/NFAT and itself directly targets the nuclear NFAT kinase dual-specificity tyrosine (Y)-phosphorylation regulated kinase 1a (Dyrk1a), creating a feed-forward mechanism underlying HF effected through calcineurin-responsive gene expression. Intraperitoneal injections of antimiR-199b in TAC and calcineurin HF mouse models prevented Dyrk1a repression, fetal gene reprogramming, and NFAT activity, thus improving morphologic (fibrosis, hypertrophy, LV dilation) and functional (fractional shortening, contractility) properties of the heart (da Costa Martins et al., 2010). A whole miRNA family regulating a set of functionally related genes can be inhibited by a single antimiR. Inhibition of miR-34 family (miR-34a, -34b, and -34c) by subcutaneous delivery of antimiR-34 consisting of LNA-modified 8-mer complementary to miR-34 seed region protected the heart against pathological cardiac remodeling and improved cardiac function in TAC and MI murine models (Bernardo et al., 2012). The resulting upregulation of direct targets of miR-34, such as VEGF, vinculin, Notch1, and


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semaphorin 4B (all implicated in CV patho/physiology) may account for the observed attenuation. Importantly, even though miR-34 family members are known tumor suppressors, their inhibition does not seem to promote tumorigenesis (Concepcion et al., 2012, Bernardo et al., 2012), but targeting antimiR-34 to the heart would still be prudent when designing therapeutic approaches for humans. On the other hand, cardiac delivery (facilitated by cardiotropic AAV9) of miR-1 mimic to rats with pressure overload-induced cardiac hypertrophy normalized miR-1 expression, reversed the hypertrophy and prevented deterioration of cardiac function by modifying the expression and activity of major players in pathophysiology of cardiac hypertrophy and HF, such as SERCA2a, miR-1 direct targets calmodulin, insulin growth factor 1, Ncx1 (calcium homeostasis regulators), and Bcl-2/Bax and fibullin-2 (apoptosis and fibrosis regulators, respectively) (Karakikes et al., 2013). Another miRNA-based therapeutic approach could be the application of miRNA targeting sequences to specifically regulate transgene expression. Such strategy was used for targeted overexpression of p27 to reduce restenosis after PCI without affecting reendothelialization by inserting targeting sequences for EC-specific miR-126-3p at the 3’ end of the p27 transgene – such design allowed the expression of p27 in VSMC, but blocked it in EC in a rat balloon injury model (Santulli et al., 2014b). Conceivably, such approach could be applied in the treatment of other conditions involving vessel wall injury, such as vascular graft failure or coronary artery bypass graft surgery.

To date there is only one miRNA-based drug tested in humans – for the treatment of hepatitis C virus (HCV) infection (Janssen et al., 2013). One clinical trial is in phase I, recruiting patients for the treatment of liver cancer, and around 300 clinical studies have


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been approved to evaluate the diagnostic value of miRNAs (http://clinicaltrials.gov), many of them in cardiovascular disease (e.g one study is set to examine circulating miR-126 as a novel biomarker for post myocardial infarction remodeling).

miRNAs as biomarkers

Recently miRNAs have emerged as promising candidates for diagnostic and prognostic biomarkers for diverse CVDs. In contrast to mRNAs, circulating miRNAs are surprisingly stable and are enclosed in exosomes, microvesicles, and apoptotic bodies and in complexes with RNA-binding proteins such as nucleophosmin or proteins of Argonaute family (mostly Ago2), and finally, some miRNA fractions are transported in high-density lipoprotein (Norata et al., 2013, Vickers et al., 2011, Turchinovich et al., 2011, Arroyo et al., 2011, Wang et al., 2010). All these means of transportation facilitate intercellular signaling by miRNAs and offer protection against degradation by RNases. From a practical perspective, they render miRNAs able to withstand multiple freezing/thawing cycles (Mitchell et al., 2008). Karolina et al. demonstrated that the expression patterns of miRNAs in exosomes mirrored those in the whole blood (Karolina et al., 2012). However, as of today, it is unclear how miRNAs are released into the circulation and what triggers their release. Interestingly, some miRNAs found to be upregulated in diseased tissue were downregulated in the circulation. This has been observed in some malignancies (nonHodgkin’s lymphoma (Lwin et al., 2013) and hepatocellular carcinoma (Shigoka et al., 2010)), but also in CVD; for example, multiple miRNAs in abdominal aortic aneurism were downregulated in the circulation but upregulated in the aneurismatic tissue (Kin et al., 2012). Similarly, VSMC-derived miR-145 was downregulated in the plasma of CAD


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patients (Fichtlscherer et al., 2010) and upregulated in unstable atherosclerotic plaques (Cipollone et al., 2011). It is possible that diseased tissue preferentially uptakes exosomes containing those specific miRNAs, thus lowering their levels in the blood. Other explanations might include increased degradation of circulating miRNAs or their epigenetic silencing (Han et al., 2007). In atherosclerosis such regulation constitutes a protective mechanism where EC-derived apoptotic bodies, rich in miR-126, are delivered to atherosclerotic lesions, ameliorating atherosclerosis through a CXC-chemokinedependent process (Zernecke et al., 2009). One of the advantages of using miRNAs as biomarkers over currently used peptide/protein markers is the potential for quantification of specific miRNAs using qRTPCR. Specific antimiRs, miRNA mimics, and target site blockers enable in vitro and in vivo experiments to be performed aimed at elucidating the role of specific miRNAs and their potential application as diagnostic and/or prognostic biomarkers. miRNAs have also proved helpful in studying CV physiology and elucidating mechanism of CVD, which may lead to discovering new therapeutic targets not directly related to miRNAs.

Research on miRNAs as biomarkers is still in its early stages. Different studies sometimes yield contradicting or at least non-overlapping results. Often these discrepancies can be ascribed to using different experimental methods, testing predetermined panels of miRNAs or testing patients at different stages of CVD. Before those promising biomarker candidates could be used in clinical settings, to guide and inform diagnostic and/or therapeutic decisions, trials with greater statistical power are needed, as well as better insight into the role of particular miRNAs in the development and/or progression of specific CVDs. Given the cooperative mechanism of action of


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miRNAs it seems highly probable that panels of dysregulated miRNAs rather than singular molecules will serve as biomarkers. They probably will not replace current diagnostic tools, such as imaging, protein biomarkers, etc., but rather would complement them to make diagnoses/prognoses/stratification more precise and accurate.

Final conclusions

The positive results from the HCV trial (which uses LNA-modified antimiR against miR122, which is required for replication of HCV) and recent translational research offer a very promising perspective on the usefulness of miRNAs as therapeutic targets in CVD. Given the advances in our understanding of the role of miRNAs in cardiovascular physiology and diseases, it seems just a matter of time before new miRNA-based therapeutic approaches are proposed and tested in clinical settings and, hopefully, found effective in the treatment of CVD.

Conflict of interest None.

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Accepted Article

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Accepted Article

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Accepted Article

Meijs, M. F., de Windt, L. J., de Jonge, N., Cramer, M. J., Bots, M. L., Mali, W. P. & Doevendans, P. A. 2007. Left ventricular hypertrophy: a shift in paradigm. Curr Med Chem, 14, 157-71. Minami, Y., Satoh, M., Maesawa, C., Takahashi, Y., Tabuchi, T., Itoh, T. & Nakamura, M. 2009. Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease. Eur J Clin Invest, 39, 359-67. Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., Peterson, A., Noteboom, J., O'Briant, K. C., Allen, A., Lin, D. W., Urban, N., Drescher, C. W., Knudsen, B. S., Stirewalt, D. L., Gentleman, R., et al. 2008. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A, 105, 10513-8. Moniche, F., Gonzalez, A., Gonzalez-Marcos, J. R., Carmona, M., Pinero, P., Espigado, I., GarciaSolis, D., Cayuela, A., Montaner, J., Boada, C., Rosell, A., Jimenez, M. D., Mayol, A. & GilPeralta, A. 2012. Intra-arterial bone marrow mononuclear cells in ischemic stroke: a pilot clinical trial. Stroke, 43, 2242-4. Naga Prasad, S. V., Duan, Z. H., Gupta, M. K., Surampudi, V. S., Volinia, S., Calin, G. A., Liu, C. G., Kotwal, A., Moravec, C. S., Starling, R. C., Perez, D. M., Sen, S., Wu, Q., Plow, E. F., Croce, C. M. & Karnik, S. 2009. Unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks. J Biol Chem, 284, 27487-99. Nagai, R., Shindo, T., Manabe, I., Suzuki, T. & Kurabayashi, M. 2003. KLF5/BTEB2, a Kruppel-like zinc-finger type transcription factor, mediates both smooth muscle cell activation and cardiac hypertrophy. Adv Exp Med Biol, 538, 57-65; discussion 66. Natarajan, R., Putta, S. & Kato, M. 2012. MicroRNAs and diabetic complications. J Cardiovasc Transl Res, 5, 413-22. Nattel, S. 2002. Therapeutic implications of atrial fibrillation mechanisms: can mechanistic insights be used to improve AF management? Cardiovasc Res, 54, 347-60. Navarro, A., Diaz, T., Martinez, A., Gaya, A., Pons, A., Gel, B., Codony, C., Ferrer, G., Martinez, C., Montserrat, E. & Monzo, M. 2009. Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin lymphoma. Blood, 114, 2945-51. Nilsson, J., Wigren, M. & Shah, P. K. 2009. Regulatory T cells and the control of modified lipoprotein autoimmunity-driven atherosclerosis. Trends Cardiovasc Med, 19, 272-6. Norata, G. D., Sala, F., Catapano, A. L. & Fernandez-Hernando, C. 2013. MicroRNAs and lipoproteins: a connection beyond atherosclerosis? Atherosclerosis, 227, 209-15. Nossent, A. Y., Hansen, J. L., Doggen, C., Quax, P. H., Sheikh, S. P. & Rosendaal, F. R. 2011. SNPs in microRNA binding sites in 3'-UTRs of RAAS genes influence arterial blood pressure and risk of myocardial infarction. Am J Hypertens, 24, 999-1006.


Accepted Article

Olivieri, F., Antonicelli, R., Lorenzi, M., D'Alessandra, Y., Lazzarini, R., Santini, G., Spazzafumo, L., Lisa, R., La Sala, L., Galeazzi, R., Recchioni, R., Testa, R., Pompilio, G., Capogrossi, M. C. & Procopio, A. D. 2013. Diagnostic potential of circulating miR-499-5p in elderly patients with acute non ST-elevation myocardial infarction. Int J Cardiol, 167, 531-6. Olson, E. N. 2014. MicroRNAs as therapeutic targets and biomarkers of cardiovascular disease. Sci Transl Med, 6, 239ps3. Ono, K., Kuwabara, Y. & Han, J. 2011. MicroRNAs and cardiovascular diseases. FEBS J, 278, 161933. Ouyang, Y. B. & Giffard, R. G. 2013. microRNAs affect BCL-2 family proteins in the setting of cerebral ischemia. Neurochem Int. Parving, H. H., Brenner, B. M., McMurray, J. J., de Zeeuw, D., Haffner, S. M., Solomon, S. D., Chaturvedi, N., Persson, F., Desai, A. S., Nicolaides, M., Richard, A., Xiang, Z., Brunel, P. & Pfeffer, M. A. 2012. Cardiorenal end points in a trial of aliskiren for type 2 diabetes. N Engl J Med, 367, 2204-13. Periasamy, M., Bhupathy, P. & Babu, G. J. 2008. Regulation of sarcoplasmic reticulum Ca2+ ATPase pump expression and its relevance to cardiac muscle physiology and pathology. Cardiovasc Res, 77, 265-73. Piras, B. A., O'Connor, D. M. & French, B. A. 2013. Systemic delivery of shRNA by AAV9 provides highly efficient knockdown of ubiquitously expressed GFP in mouse heart, but not liver. PLoS One, 8, e75894. Poirier, O., Georges, J. L., Ricard, S., Arveiler, D., Ruidavets, J. B., Luc, G., Evans, A., Cambien, F. & Tiret, L. 1998. New polymorphisms of the angiotensin II type 1 receptor gene and their associations with myocardial infarction and blood pressure: the ECTIM study. Etude CasTemoin de l'Infarctus du Myocarde. J Hypertens, 16, 1443-7. Raitoharju, E., Lyytikainen, L. P., Levula, M., Oksala, N., Mennander, A., Tarkka, M., Klopp, N., Illig, T., Kahonen, M., Karhunen, P. J., Laaksonen, R. & Lehtimaki, T. 2011. miR-21, miR210, miR-34a, and miR-146a/b are up-regulated in human atherosclerotic plaques in the Tampere Vascular Study. Atherosclerosis, 219, 211-7. Ranjha, R. & Paul, J. 2013. Micro-RNAs in inflammatory diseases and as a link between inflammation and cancer. Inflamm Res, 62, 343-55. Rao, P. K., Toyama, Y., Chiang, H. R., Gupta, S., Bauer, M., Medvid, R., Reinhardt, F., Liao, R., Krieger, M., Jaenisch, R., Lodish, H. F. & Blelloch, R. 2009. Loss of cardiac microRNAmediated regulation leads to dilated cardiomyopathy and heart failure. Circ Res, 105, 585-94.


Accepted Article

Rink, C. & Khanna, S. 2011. MicroRNA in ischemic stroke etiology and pathology. Physiol Genomics, 43, 521-8. Robbins, C. S., Hilgendorf, I., Weber, G. F., Theurl, I., Iwamoto, Y., Figueiredo, J. L., Gorbatov, R., Sukhova, G. K., Gerhardt, L. M., Smyth, D., Zavitz, C. C., Shikatani, E. A., Parsons, M., van Rooijen, N., Lin, H. Y., Husain, M., et al. 2013. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat Med, 19, 1166-72. Sandercock, P., Lindley, R., Wardlaw, J., Dennis, M., Innes, K., Cohen, G., Whiteley, W., Perry, D., Soosay, V., Buchanan, D., Venables, G., Czlonkowska, A., Kobayashi, A., Berge, E., Slot, K. B., Murray, V., et al. 2011. Update on the third international stroke trial (IST-3) of thrombolysis for acute ischaemic stroke and baseline features of the 3035 patients recruited. Trials, 12, 252. Santoro, M. M. 2011. "Fishing" for endothelial microRNA functions and dysfunction. Vascul Pharmacol, 55, 60-8. Santulli, G. 2012. Coronary heart disease risk factors and mortality. JAMA, 307, 1137; author reply 1138. Santulli, G., Cipolletta, E., Sorriento, D., Del Giudice, C., Anastasio, A., Monaco, S., Maione, A. S., Condorelli, G., Puca, A., Trimarco, B., Illario, M. & Iaccarino, G. 2012. CaMK4 Gene Deletion Induces Hypertension. J Am Heart Assoc, 1, e001081. Santulli, G., Iaccarino, G., De Luca, N., Trimarco, B. & Condorelli, G. 2014a. Atrial fibrillation and microRNAs. Front Physiol, 5, 15. Santulli, G., Trimarco, B. & Iaccarino, G. 2013. G-protein-coupled receptor kinase 2 and hypertension: molecular insights and pathophysiological mechanisms. High Blood Press Cardiovasc Prev, 20, 5-12. Santulli, G., Wronska, A., Kunihiro, U., Diacovo, T., Gao, M., Marx, S., Kitajewski, J., Chilton, J., Kemal, M., Tuschl, T., Marks, A. R. & Totary Jain, H. 2014b. A selective microRNA-based strategy inhibits restenosis while preserving endothelial function. J Clin Invest, 124, 4102-4114. Satoh, M., Minami, Y., Takahashi, Y., Tabuchi, T. & Nakamura, M. 2010. Expression of microRNA208 is associated with adverse clinical outcomes in human dilated cardiomyopathy. J Card Fail, 16, 404-10. Satoh, M., Shimoda, Y., Akatsu, T., Ishikawa, Y., Minami, Y. & Nakamura, M. 2006. Elevated circulating levels of heat shock protein 70 are related to systemic inflammatory reaction through monocyte Toll signal in patients with heart failure after acute myocardial infarction. Eur J Heart Fail, 8, 810-5.


Accepted Article

Schivo, M., Aksenov, A. A., Yeates, L. C., Pasamontes, A. & Davis, C. E. 2013. Diabetes and the Metabolic Syndrome: Possibilities of a New Breath Test in a Dolphin Model. Front Endocrinol (Lausanne), 4, 163. Sepramaniam, S., Tan, J. R., Tan, K. S., Desilva, D. A., Tavintharan, S., Woon, F. P., Wang, C. W., Yong, F. L., Karolina, D. S., Kaur, P., Liu, F. J., Lim, K. Y., Armugam, A. & Jeyaseelan, K. 2014. Circulating MicroRNAs as Biomarkers of Acute Stroke. Int J Mol Sci, 15, 1418-32. Sethupathy, P., Borel, C., Gagnebin, M., Grant, G. R., Deutsch, S., Elton, T. S., Hatzigeorgiou, A. G. & Antonarakis, S. E. 2007. Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes. Am J Hum Genet, 81, 405-13. Sharma, S., Jackson, P. G. & Makan, J. 2004. Cardiac troponins. J Clin Pathol, 57, 1025-6. Shigoka, M., Tsuchida, A., Matsudo, T., Nagakawa, Y., Saito, H., Suzuki, Y., Aoki, T., Murakami, Y., Toyoda, H., Kumada, T., Bartenschlager, R., Kato, N., Ikeda, M., Takashina, T., Tanaka, M., Suzuki, R., et al. 2010. Deregulation of miR-92a expression is implicated in hepatocellular carcinoma development. Pathol Int, 60, 351-7. Sorriento, D., Ciccarelli, M., Santulli, G., Campanile, A., Altobelli, G. G., Cimini, V., Galasso, G., Astone, D., Piscione, F., Pastore, L., Trimarco, B. & Iaccarino, G. 2008. The G-proteincoupled receptor kinase 5 inhibits NFkappaB transcriptional activity by inducing nuclear accumulation of IkappaB alpha. Proc Natl Acad Sci U S A, 105, 17818-23. Stary, C. M. & Giffard, R. G. 2014. Advances in Astrocyte-targeted Approaches for Stroke Therapy: An Emerging Role for Mitochondria and microRNAS. Neurochem Res. Stone, P. H., Muller, J. E., Hartwell, T., York, B. J., Rutherford, J. D., Parker, C. B., Turi, Z. G., Strauss, H. W., Willerson, J. T., Robertson, T. & et al. 1989. The effect of diabetes mellitus on prognosis and serial left ventricular function after acute myocardial infarction: contribution of both coronary disease and diastolic left ventricular dysfunction to the adverse prognosis. The MILIS Study Group. J Am Coll Cardiol, 14, 4957. Suarez-Monteagudo, C., Hernandez-Ramirez, P., Alvarez-Gonzalez, L., Garcia-Maeso, I., de la Cuetara-Bernal, K., Castillo-Diaz, L., Bringas-Vega, M. L., Martinez-Aching, G., MoralesChacon, L. M., Baez-Martin, M. M., Sanchez-Catasus, C., Carballo-Barreda, M., Rodriguez-Rojas, R., Gomez-Fernandez, L., Alberti-Amador, E., Macias-Abraham, C., et al. 2009. Autologous bone marrow stem cell neurotransplantation in stroke patients. An open study. Restor Neurol Neurosci, 27, 151-61. Sucharov, C., Bristow, M. R. & Port, J. D. 2008. miRNA expression in the failing human heart: functional correlates. J Mol Cell Cardiol, 45, 185-92.


Accepted Article

Sun, X., He, S., Wara, A. K., Icli, B., Shvartz, E., Tesmenitsky, Y., Belkin, N., Li, D., Blackwell, T. S., Sukhova, G. K., Croce, K. & Feinberg, M. W. 2014a. Systemic delivery of microRNA-181b inhibits nuclear factor-kappaB activation, vascular inflammation, and atherosclerosis in apolipoprotein E-deficient mice. Circ Res, 114, 32-40. Sun, X., Sit, A. & Feinberg, M. W. 2014b. Role of miR-181 family in regulating vascular inflammation and immunity. Trends Cardiovasc Med, 24, 105-12. Taganov, K. D., Boldin, M. P., Chang, K. J. & Baltimore, D. 2006. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A, 103, 12481-6. Takahashi, Y., Satoh, M., Minami, Y., Tabuchi, T., Itoh, T. & Nakamura, M. 2010. Expression of miR-146a/b is associated with the Toll-like receptor 4 signal in coronary artery disease: effect of renin-angiotensin system blockade and statins on miRNA-146a/b and Toll-like receptor 4 levels. Clin Sci (Lond), 119, 395-405. Tan, J. R., Tan, K. S., Koo, Y. X., Yong, F. L., Wang, C. W., Armugam, A. & Jeyaseelan, K. 2013. Blood microRNAs in Low or No Risk Ischemic Stroke Patients. Int J Mol Sci, 14, 2072-84. Thum, T. 2008. Cardiac dissonance without conductors: how dicer depletion provokes chaos in the heart. Circulation, 118, 1524-7. Thum, T., Galuppo, P., Wolf, C., Fiedler, J., Kneitz, S., van Laake, L. W., Doevendans, P. A., Mummery, C. L., Borlak, J., Haverich, A., Gross, C., Engelhardt, S., Ertl, G. & Bauersachs, J. 2007. MicroRNAs in the human heart: a clue to fetal gene reprogramming in heart failure. Circulation, 116, 258-67. Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz, S., Castoldi, M., Soutschek, J., Koteliansky, V., Rosenwald, A., Basson, M. A., Licht, J. D., et al. 2008. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature, 456, 980-4. Thygesen, K., Alpert, J. S. & White, H. D. 2007. Universal definition of myocardial infarction. Eur Heart J, 28, 2525-38. Tijsen, A. J., Creemers, E. E., Moerland, P. D., de Windt, L. J., van der Wal, A. C., Kok, W. E. & Pinto, Y. M. 2010. MiR423-5p as a circulating biomarker for heart failure. Circ Res, 106, 1035-9. Tijsen, A. J., Pinto, Y. M. & Creemers, E. E. 2012. Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases. Am J Physiol Heart Circ Physiol, 303, H1085-95. Towbin, J. A. & Bowles, N. E. 2002. The failing heart. Nature, 415, 227-33.


Accepted Article

Trimarco, B., De Simone, A., Cuocolo, A., Ricciardelli, B., Volpe, M., Patrignani, P., Sacca, L. & Condorelli, M. 1985. Role of prostaglandins in the renal handling of a salt load in essential hypertension. Am J Cardiol, 55, 116-21. Tsai, P. C., Liao, Y. C., Wang, Y. S., Lin, H. F., Lin, R. T. & Juo, S. H. 2013. Serum microRNA-21 and microRNA-221 as potential biomarkers for cerebrovascular disease. J Vasc Res, 50, 34654. Tuccoli, A., Poliseno, L. & Rainaldi, G. 2006. miRNAs regulate miRNAs: coordinated transcriptional and post-transcriptional regulation. Cell Cycle, 5, 2473-6. Turchinovich, A., Weiz, L., Langheinz, A. & Burwinkel, B. 2011. Characterization of extracellular circulating microRNA. Nucleic Acids Res, 39, 7223-33. Valen, G., Yan, Z. Q. & Hansson, G. K. 2001. Nuclear factor kappa-B and the heart. J Am Coll Cardiol, 38, 307-14. van der Pouw Kraan, T. C., Bernink, F. J., Yildirim, C., Koolwijk, P., Baggen, J. M., Timmers, L., Beek, A. M., Diamant, M., Chen, W. J., van Rossum, A. C., van Royen, N., Horrevoets, A. J. & Appelman, Y. E. 2014. Systemic toll-like receptor and interleukin-18 pathway activation in patients with acute ST elevation myocardial infarction. J Mol Cell Cardiol, 67, 94-102. van Rooij, E. & Olson, E. N. 2012. MicroRNA therapeutics for cardiovascular disease: opportunities and obstacles. Nat Rev Drug Discov, 11, 860-72. van Rooij, E., Purcell, A. L. & Levin, A. A. 2012. Developing microRNA therapeutics. Circ Res, 110, 496-507. van Rooij, E., Quiat, D., Johnson, B. A., Sutherland, L. B., Qi, X., Richardson, J. A., Kelm, R. J., Jr. & Olson, E. N. 2009. A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance. Dev Cell, 17, 662-73. van Rooij, E., Sutherland, L. B., Liu, N., Williams, A. H., McAnally, J., Gerard, R. D., Richardson, J. A. & Olson, E. N. 2006. A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc Natl Acad Sci U S A, 103, 18255-60. Van Wagoner, D. R. & Nerbonne, J. M. 2000. Molecular basis of electrical remodeling in atrial fibrillation. Journal of Molecular and Cellular Cardiology, 32, 1101-1117. Vickers, K. C., Palmisano, B. T., Shoucri, B. M., Shamburek, R. D. & Remaley, A. T. 2011. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol, 13, 423-33. Vickers, K. C., Rye, K. A. & Tabet, F. 2014. MicroRNAs in the onset and development of cardiovascular disease. Clin Sci (Lond), 126, 183-94.


Accepted Article

Wang, G. K., Zhu, J. Q., Zhang, J. T., Li, Q., Li, Y., He, J., Qin, Y. W. & Jing, Q. 2010. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J, 31, 659-66. Ward, J. A., Esa, N., Pidikiti, R., Freedman, J. E., Keaney, J. F., Tanriverdi, K., Vitseva, O., Ambros, V., Lee, R. & McManus, D. D. 2013. Circulating Cell and Plasma microRNA Profiles Differ between Non-ST-Segment and ST-Segment-Elevation Myocardial Infarction. Fam Med Med Sci Res, 2, 108. Widera, C., Gupta, S. K., Lorenzen, J. M., Bang, C., Bauersachs, J., Bethmann, K., Kempf, T., Wollert, K. C. & Thum, T. 2011. Diagnostic and prognostic impact of six circulating microRNAs in acute coronary syndrome. J Mol Cell Cardiol, 51, 872-5. Wright, J. T., Jr., Bakris, G. L., Bell, D. S., Fonseca, V., Katholi, R. E., McGill, J. B., Messerli, F. H., Phillips, R. A., Raskin, P., Holdbrook, F. K., Lukas, M. A. & Iyengar, M. 2007. Lowering blood pressure with beta-blockers in combination with other renin-angiotensin system blockers in patients with hypertension and type 2 diabetes: results from the GEMINI Trial. J Clin Hypertens (Greenwich), 9, 842-9. Xie, W., Santulli, G., Guo, X., Gao, M., Chen, B. X. & Marks, A. R. 2013. Imaging atrial arrhythmic intracellular calcium in intact heart. J Mol Cell Cardiol, 64, 120-3. Xin, H., Li, Y., Cui, Y., Yang, J. J., Zhang, Z. G. & Chopp, M. 2013a. Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J Cereb Blood Flow Metab, 33, 1711-5. Xin, H. Q., Li, Y., Liu, Z. W., Wang, X. L., Shang, X., Cui, Y. S., Zhang, Z. G. & Chopp, M. 2013b. MiR133b Promotes Neural Plasticity and Functional Recovery After Treatment of Stroke with Multipotent Mesenchymal Stromal Cells in Rats Via Transfer of Exosome-Enriched Extracellular Particles. Stem Cells, 31, 2737-2746. Xin, M., Olson, E. N. & Bassel-Duby, R. 2013c. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Rev Mol Cell Biol, 14, 529-41. Xu, C. F., Yu, C. H. & Li, Y. M. 2009. Regulation of hepatic microRNA expression in response to ischemic preconditioning following ischemia/reperfusion injury in mice. OMICS, 13, 51320. Yang, B., Lin, H., Xiao, J., Lu, Y., Luo, X., Li, B., Zhang, Y., Xu, C., Bai, Y., Wang, H., Chen, G. & Wang, Z. 2007. The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2. Nat Med, 13, 486-91. Zernecke, A., Bidzhekov, K., Noels, H., Shagdarsuren, E., Gan, L., Denecke, B., Hristov, M., Koppel, T., Jahantigh, M. N., Lutgens, E., Wang, S., Olson, E. N., Schober, A. & Weber, C.


Accepted Article

2009. Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal, 2, ra81. Zhao, Y., Samal, E. & Srivastava, D. 2005. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature, 436, 214-20. Zidar, N., Bostjancic, E., Glavac, D. & Stajer, D. 2011. MicroRNAs, innate immunity and ventricular rupture in human myocardial infarction. Dis Markers, 31, 259-65.

miRNA

Role

Localization

References

miR-1

maintenance of cardiac function; arrhythmogenic; proarrhythmic; AMI (↑c); CAD (↑); hypertrophic cardiomyopathy (↓); atrial dilation (↓); HF-DCM (↓); AF (↓)

heart; skeletal muscle

(Chen et al., 2006), (Rao et al., 2009), (Wang et al., 2010), (Ai et al., 2010), (Widera et al., 2011), (Yang et al., 2007), (Care et al., 2007), (Naga Prasad et al., 2009), (Girmatsion et al., 2009)

miR-7

HF-DCM (↓)

(Naga Prasad et al., 2009)

miR-9

hypertension (↓PBMC)

(Kontaraki et al., 2014b)

miR-10b

HF-IDC (↓)

(Sucharov et al., 2008)

miR-17

CAD (↓c); metabolic syndrome (hypercholesterolemia) (↑c)

(Fichtlscherer et al., 2010), (Karolina et al., 2012)

miR-18b-5p

atherosclerosis (through RAAS)

(Parving et al., 2012)

miR-21

atherosclerosis (↑); stroke (↑c); HF (↑); cardiac aging (↑); AF (↑)

(Raitoharju et al., 2011), (Tsai et al., 2013), (Thum et al.,


Accepted Article

2007), (Thum et al., 2008), (Adam et al., 2012) miR-23a

metabolic syndrome (hypercholesterolemia) (↑c)

(Karolina et al., 2012)

miR-24

HF (↑)

(van Rooij et al., 2006)

miR-26a

cardiovascular repair; MI (↑)

(Icli et al., 2014), (Icli et al., 2013)

miR-27a-3p

atherosclerosis (through RAAS); ischemic stroke (↑c); metabolic syndrome (hyperglycemia) (↑c)

(Parving et al., 2012), (Sepramaniam et al., 2014), (Karolina et al., 2012)

miR-29

AF (↓); HF (↓)

(Dawson et al., 2013), (Hale and Levis, 2013)

miR-29b

HF-DCM (↓)


miR-30

AF (↓)

(Duisters et al., 2009)

miR-30d-5p

MI (↑c)

(Ward et al., 2013)

miR-34

atherosclerosis (↑); hypertension

(Raitoharju et al., 2011), (Nossent et al., 2011)

miR-34b

diabetic HF

(Greco et al., 2012)

miR-34c

diabetic HF


miR-92

HF (↓)


miR-92a

CAD (↓c); metabolic syndrome (hypertension) (↑c)

(Fichtlscherer et al., 2010), (Karolina et al., 2012)

miR-100

atherosclerosis-unstable plaques (↑); HF (↑)

(Cipollone et al., 2011), (Sucharov et


Accepted Article

al., 2008) miR-103

metabolic syndrome (hypercholesterolemia) (↓c)


miR-106b-5p

atherosclerosis (through RAAS)

(Parving et al., 2012)

miR-125b

ischemic stroke (↑c); HF (↑); HF-DCM (↑)

(Sepramaniam et al., 2014), (van Rooij et al., 2006), (Naga Prasad et al., 2009)

miR-126

pro-angiogenic; vascular integrity; response to VEGF; CAD (↓c); hypertension (↓PBMC)

miR-127

atherosclerosis-unstable plaques (↑)

(Cipollone et al., 2011)

miR-129

HF (↑)

(Thum et al., 2007)

miR-130a

metabolic syndrome (hypertension) (↑c)


miR-132

hypertension

(Eskildsen et al., 2013)

miR-133

AF (↓)

(Duisters et al., 2009)

miR-133a

AMI (↑c); CAD (↑c); HF (↓); hypertrophic cardiomyopathy (↓); atrial dilation (↓); fetal gene reprogramming

miR-133b

atherosclerosis-unstable plaques (↑); HF (↓)

endothelium



(Fish et al., 2008), (Fichtlscherer et al., 2010), (Kontaraki et al., 2014b)

(Chen et al., 2006), (Wang et al., 2010), (Widera et al., 2011), (Fichtlscherer et al., 2010), (Sucharov et al., 2008), (Care et al., 2007), (Nagai et al., 2003), (Bostjancic et al., 2010) (Cipollone et al., 2011), (Sucharov et al., 2008)

Accepted Article

miR-134

unstable angina (↑)

(Xu et al., 2009)

miR-135a

CAD (↑PBMC)

(Hoekstra et al., 2010)

miR-139

HF-IDC (↓)


miR-143

hypertension (↓PBMC)

(Kontaraki et al., 2014a)

miR-145

atherosclerosis-unstable plaques (↑); CAD (↓c); hypertension

miR-146a

atherosclerosis (↑); AMI; CAD (↑PBMC); ACS (↑PBMC)

(Raitoharju et al., 2011), (Zidar et al., 2011), (Takahashi et al., 2010), (Nilsson et al., 2009)

miR-146b-5p

atherosclerosis (↑); CAD (↑PBMC)

(Raitoharju et al., 2011), (Takahashi et al., 2010)

miR-147

CAD (↓PBMC)

(Hoekstra et al., 2010)

miR-150

AMI; ventricular rupture (↑); HF (↓); metabolic syndrome (hyperglycemia) (↑c)

(Zidar et al., 2011), (Sucharov et al., 2008), (Karolina et al., 2012)

miR-151-3p

hypertension

(Nossent et al., 2011)

miR-155

AMI; CAD (↓c); hypertension (↓PBMC)

(Zidar et al., 2011), (Fichtlscherer et al., 2010), (Ceolotto et al., 2011)

miR-181b

vascular inflammation; HFDCM (↑); CAD (↓c)

(Naga Prasad et al., 2009, Sun et al., 2014b), (Sun et al., 2014a)

VSMC


(Cipollone et al., 2011), (Fichtlscherer et al., 2010), (Kontaraki et al., 2014a)

Accepted Article

miR-183



miR-192

metabolic syndrome (hyperglycemia) (↑c)


miR-195

HF (↑); metabolic syndrome (hypertension) (↑c)

(van Rooij et al., 2006), (Sucharov et al., 2008), (Karolina et al., 2012)

miR-197



miR-198


(Xu et al., 2009)

miR-199a

HF (↑)

(van Rooij et al., 2006)

miR-199b

diabetic HF


miR-208a

myomiR; AMI (↑c); viral myocarditis (↑); CAD (↑c); DCM (↑)

miR-208b

myomiR; AMI (↑c)

(van Rooij et al., 2009), (Widera et al., 2011)

miR-210

atherosclerosis (↑); diabetic HF

(Raitoharju et al., 2011), (Greco et al., 2012)

miR-212

hypertension; HF (↑)

(Eskildsen et al., 2013), (Thum et al., 2007)

miR-214

HF (↑); HF-DCM (↑)

(van Rooij et al., 2006), (Naga Prasad

heart


(van Rooij et al., 2009), (Wang et al., 2010), (Corsten et al., 2010), (Fichtlscherer et al., 2010), (Satoh et al., 2010)

Accepted Article

et al., 2009) miR-221-3p

NSTEMI (↑c); CAD (↑); stroke (↓c); HF (↓)

(Ward et al., 2013), (Minami et al., 2009), (Tsai et al., 2013), (Sucharov et al., 2008)

miR-222

CAD (↑); HF-ISC (↓)

(Minami et al., 2009), (Sucharov et al., 2008)

miR-223

diabetic HF


miR-320a

metabolic syndrome (hyperglycemia) (↑c)


miR-328

AF (↑)

(Lu et al., 2010)

miR-342

HF-DCM (↑)


miR-370


(Xu et al., 2009)

miR-378

HF-DCM (↓)


miR-382

HF-IDC (↑)


miR-383

MI


miR-422a

ischemic stroke (↑c)

(Sepramaniam et al., 2014)

miR-422b

HF (↓)


miR-423-5p

HF (↑) (↑c)

(Thum et al., 2007, Goldraich et al., 2014, Tijsen et al., 2010, Ellis et al., 2013, Goren et al., 2012)

miR-449

hypertension


miR-483p

NSTEMI (↑c)

(Ward et al., 2013)

miR-486

HF (↓)



Accepted Article


miR-488


miR-499

myomiR; AMI (↑c); viral myocarditis (↑); geriatric NSTEMI (↑c)

miR-509-5p



miR-526b

CAD (↑c); hypertension

(Li et al., 2014a), (Nossent et al., 2011)

miR-571

hypertension


miR-574-3p

MI (↑) (through SERCA2a)

(Bostjancic et al., 2012)

miR-578

hypertension


miR-584



miR-594

HF (↓)


miR-627



miR-650

diabetic HF


miR-652



miR-765

hypertension



(Wang et al., 2010), (van Rooij et al., 2009), (Corsten et al., 2010), (Olivieri et al., 2013)

Table 1. miRNAs in human cardiovascular disease. Localization specified if relevant. (↑) denotes up- and (↓) downregulation; c next to an arrow indicates circulating miR levels.


Accepted Article

Legends to figures: Figure 1. miRNA biogenesis and mechanism of action. Figure 2. An overlapping network of miRNA-regulated pathways implicated in CVD; denotes direct miRNA targeting;


inhibition.

Accepted Article This article is protected by copyright. All rights reserved.

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