potential therapeutic role of micrornas in ischemic heart disease

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Contents lists available at SciVerse ScienceDirect

Journal of Cardiology

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otential Therapeutic role of microRNAs in ischemic heart disease

nnalisa Caroli (MD), Maria Teresa Cardillo (MD), Roberto Galea (MD), Luigi M. Biasucci (MD) ∗

nstitute of Cardiology, Catholic University of the Sacred Heart, Rome, Italy

r t i c l e i n f o

rticle history:eceived 20 December 2012eceived in revised form 16 January 2013ccepted 23 January 2013vailable online xxx

eywords:

a b s t r a c t

Cardiovascular disease (CVD) is the most important cause of death and illness in the western world.Atherosclerosis constitutes the single most important contributor to CVD.miRNAs are small ribonucleic acids (RNAs) that negatively regulate gene expression on the post-transcriptional level by inhibiting mRNA translation or promoting mRNA degradation.Several studies demonstrated that miRNAs dysregulation have a key role in the disease process and, focus-ing on atherosclerotic disease, in every step of plaque formation and destabilization. These data suggest

iRNAntimiRimics

schemic heart diseaseherapeutic application

a possible therapeutic application of miRNA modulation, in particular dysregulated miRNAs can be mod-ulated in disease process antagonizing miRNAs up-regulated and increasing miRNAs down-regulated. Inthis review we summarize the miRNA therapeutic techniques (antimiR, mimics, sponges, masking, anderasers) underlining their therapeutic advantages and evaluating their risks and challenges. In particular,the use of miRNA modulators as a therapeutic approach opens a novel and fascinating area of interventionin the therapy of ischemic heart disease.

© 2013 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

ontents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00miRNAs in ischemic heart disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00miRNA in therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Therapeutic advantages of miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00How to modulate miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00AntimiRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00miR mimics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Sponges, masking, and erasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Challenges and risks: delivery, selectivity, and safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

ntroduction

MicroRNAs (miRNAs) are small (approximately 22 nucleotides)on-coding ribonucleic acids (RNAs) that negatively regulate genexpression at the post-transcriptional level by inhibiting mRNAranslation or promoting mRNA degradation [1]. It is estimated that

The biogenesis of the miRNAs is a complex process that involvestwo different RNAse III endonucleases, Dicer and Drosha, and con-sists of two steps, one in the nucleus and one in the cytoplasm.In the nucleus, Drosha processes the primiRNAs, the precursormolecule of the miRNAs, in the premiRNAs that, through exportin

Please cite this article in press as: Caroli A, et al. Potential Therapeuthttp://dx.doi.org/10.1016/j.jjcc.2013.01.012

bout one-third of genes are regulated by miRNAs. Each miRNA canarget several mRNAs and each mRNA can be the target of different

iRNAs.

∗ Corresponding author at: Istituto di Cardiologia – Policlinico A Gemelli, L.goemelli, 8, 00168 Roma, Italy. Tel.: +39 0630154187; fax: +39 063055535.

E-mail address: [email protected] (L.M. Biasucci).

914-5087/$ – see front matter © 2013 Japanese College of Cardiology. Published by Elsettp://dx.doi.org/10.1016/j.jjcc.2013.01.012

5, is exported from the nucleus to the cytoplasm. In the cyto-plasm, Dicer cleaves the premiRNAs into the mature miRNAs. Thisfinal product is incorporated as single-stranded RNA into the RNA-induced silencing complex (RISC) that allows the binding of miRNAto target mRNA leading to the inhibition of mRNA translation or to

ic role of microRNAs in ischemic heart disease. J Cardiol (2013),

mRNA degradation [2].Furthermore, recent studies demonstrated that circulating miR-

NAs are carried by exosomes [3] or microparticles (MP) [4]

vier Ltd. All rights reserved.

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evealing a new mechanism of exchange of genetic informationetween cells.

Many miRNAs have a tissue-specific distribution and play key role in physiological conditions. Specific miRNAs have aey role in various biological processes: control of metabolism,ell-differentiation, cell-proliferation and apoptosis, so they arenvolved in cardiogenesis, stem cell differentiation, neurogene-is, hematopoiesis, insulin and other hormones secretion, andmmune response [5–8]. But miRNAs are also involved in patho-ogical conditions; dysregulation of specific miRNAs could takeart in disease development and progression. miRNAs have been

mplicated in cardiovascular disease (CVD), viral infections, neu-ological and muscular disorders, and in the progression ofancer [9–12].

Among CVD, there is evidence of miRNA dysregulation inyocardial infarction (MI) [13,14], cardiac hypertrophy [15], con-

estive heart failure (CHF) [16,17], acute hind-limb ischemia, butlso in cardiac development, with defects that can cause embryonicethality [18].

iRNAs in ischemic heart disease

CVD is the most important cause of death and illness in theestern world. Atherosclerosis, characterized by the accumula-

ion of lipids and fibrous elements in the arteries, constitutes theingle most important contributor to CVD. It is a chronic inflam-atory disease characterized by atherosclerotic plaque formation.

laque formation is a long-standing process, initiating with lipidsnd leukocyte infiltration and leading, after the early formation ofatty streak, to a positive remodeling of the artery that tends toilate in order to accommodate the growing plaque. Major actors

n this process are vascular endothelial cells, smooth muscle cells,nd circulating inflammatory cells [19]. Endothelial dysfunction ishe first step in plaque development. Activated endothelial cellsllow leukocyte and monocyte infiltration into the vessel wall. Theonsequential steps are: foam cell formation, plaque angiogene-is, migration and proliferation of vascular smooth muscle cellsVSMC), fibrous cap destabilization and plaque rupture, and even-ually, thrombus formation on destabilized plaque leads to acuteoronary syndromes (ACS). In this scenario miRNAs are involved inll steps [20–22] (Table 1).

Shear stress induces expression of miR-21 in the endothelial


ells; this miRNA influences endothelium by decreasing apoptosisnd activating the nitric oxide (NO) pathway [23] and may exert antherosclerosis-protective role. Apoptosis is also reported to playn important role in progression of left ventricular (LV) remodeling

able 1ole of miRNAs in atherosclerotic plaque development.

Steps of plaque development miRNA Specific

Endothelial cells activation miR-21 Decreas

Monocyte activation andmacrophage maturation

miR-155 MediatmiR-124 MediatmiR-146 Reduce

macrop

Cholesterol biosynthesis miR-122 Raises LmiR-33 Reduce

Plaque angiogenesismiR-126 EnhancmiR-92a ModulamiR-27 Modula

metabo

Fibrous cap stabilization and VSMC proliferationmiR-221/222 Induce

miR-143/145 StabilizmiR-195 Reduce

iRNA, microRNA; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VSMC, v

PRESSology xxx (2013) xxx–xxx

after acute MI [24]. So miR-21 could play a fundamental role alsoin this phase as it is up-regulated in cardiac fibroblast and its up-regulation is related to the post-MI remodeling [25].

Leukocyte adhesion is regulated by miR-126: its down-regulation is related to the up-regulation of vascular cell adhesionmolecule-1 (VCAM-1) enhancing leukocyte adhesion to endothe-lium [26].

In the development of atherosclerotic plaque and in its desta-bilization, inflammation has a key role [27]; macrophages are theprinciple effector cells in the atherosclerotic process.

Several miRNAs are involved in the inflammatory macrophageresponse such as miR-21, miR-210, miR-146a, miR-34a, miR-147,and miR-125a-5p, which are up-regulated in atherosclerotic plaque[28]. In particular miR-155 is specifically expressed in atheroscle-rotic plaque and in pro-inflammatory macrophages; a recent studydemonstrated that the deficiency of miR-155 reduces advancedatherosclerotic plaque formation by up-regulating Bcl6, whichdecreased the expression of chemokine (C–C motif) ligand 2 (CCL2)in macrophages [29].

Also miR-124 is involved in monocyte maturation, in particu-lar it degrades CCAAT/enhancer binding protein (C/EBP) � and itsdownstream target PU.1 involved in monopoiesis [30].

Another miRNA involved in the inflammatory process is miR-146 that targets tumor necrosis factor (TNF) receptor-associatedfactor (TRAF) 6, interleukin-1 receptor-associated kinase (IRAK)1, signal transducers and activators of transcription (STAT) 1,interferon regulatory factor (IRF) 5, therefore reducing pro-inflammatory signaling and cytokine expression by macrophages[31]. Furthermore, miR-146 may prevent activation of theCD40/CD40L axis and downstream pro-atherogenic cytokine pro-duction in macrophages [32].

VSMC are other actors in the atherosclerotic process; theirbalance between differentiation and proliferation differentiate astable from an unstable plaque. miR-145 and miR-143 have animportant role in the regulation of VSMC phenotype; they movethe balance in favor of a condition that stabilizes the plaque[33]. Also miR221/222 induce VSMC proliferation [34], whilemiR-195 negatively modulates VSMC proliferation and migration[35].

High levels of low-density lipoprotein (LDL) and low lev-els of high-density lipoprotein (HDL) are established risk factorsfor atherosclerosis. Interestingly, miR-122 participates in the


regulation of cholesterol biosynthetic pathway in particular down-regulating the 3-hydroxy-3-methylglutayl-CoA-reductase [36],and miR-33 reduces HDL formation, targeting ATP-binding cassettetransporter (ABCA)-1 [37].

role of miRNA Reference

es apoptosis of endothelial cells [23]

es inflammatory macrophage response [29]es monocyte maturation [30]s pro-inflammatory signaling and cytokines expression byhage

[31]

DL levels [36]s HDL levels [37]

es leukocyte adhesion to endothelium [27]tes plaque angiogenesis [38]tes plaque angiogenesis, adipogenesis, inflammation, lipidlism, oxidative stress, insulin resistance and type 2 diabetes

[39]

VSMC proliferation [34]e the plaque [33]s VSMC proliferation [35]

ascular smooth muscle cells.

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miR-92a has been associated with plaque angiogenesis, whichs negatively regulated by this miRNA [38]; also miR-27 has beenmplicated in angiogenesis and in other steps of plaque formation,uch as adipogenesis, inflammation, lipid metabolism, oxidativetress, insulin resistance, and type 2 diabetes, representing aotential disease biomarker and a novel therapeutic target intherosclerosis [39].

Several studies have demonstrated dysregulation of miRNA pat-erns in atherosclerotic vessels compared with healthy controlrteries.

Raitoharju et al. [28] demonstrated that miR-21, miR-210,iR-146a, miR-34a, iR-147, and miR-125a-5p are up-regulated

n the atherosclerotic plaque compared with non-atheroscleroticeft internal thoracic arteries. Fichtlscherer et al. evaluated the

iRNA expression of 8 healthy volunteers and 8 patients withtable coronary artery disease (CAD). Interestingly, vascular andeukocyte-derived miRNA, in particular the angiogenesis-related

iRNAs, such as miR-126 and miR-92a, the inflammation asso-iated miR-155, VSMC-enriched miRNAs, such as miR145 andiR-17, are significantly reduced in patients with CAD, whereas

ardiac miR-133a, miR-208 were slightly increased [40]. The mean-ng of this different expression is unclear; a possible explanation forhis reduction might be an uptake of circulating miRNAs into thetherosclerotic lesion.

A recent study compared miRNA levels in aorta with miRNAevel in the coronary sinus; there was a positive transcoronary gra-ient for miR-133a and miR-499 in patients with ACS, suggestinghat these miRNAs are released into the coronary circulation during

yocardial injury. On the contrary, there was a significant nega-ive transcoronary gradient for miR-126 suggesting either uptake oregradation of this miRNA during the passage through the coronaryirculation [41].

iRNA in therapy

The most important implication of these studies is the possi-le therapeutic application of miRNAs that might open a new andascinating area in cardiology.

herapeutic advantages of miRNA

A potential therapeutic advantage of miRNAs is that they targetultiple genes involved in the same pathway process. On the con-

rary, traditional therapies strongly target a single protein, leavinghe others unaffected and potentially able to maintain the patho-ogical mechanism. Even if the power of inhibition may not be theame as for traditional therapy, the final effect of miRNA modula-ion might result in a more effective therapy [42].

On the other hand, miRNAs have a specific and defined tar-et in the pathogenetic mechanism of the disease, resulting in aigh specificity of treatment. Other advantages are the long-lastingffect and the bioavailability, in fact, miRNA modulators can effi-iently act in most tissues in vivo.

It is unclear whether traditional drugs can act regulating miR-As which are dysregulated in disease processes. A recent studyemonstrated that bone marrow mononuclear cell therapy in MIlocked cardiomyocyte apoptosis thanks to a paracrine regula-ion of miR-34a [43]. Munoz-Pacheco et al. studied ezetimibe, a

ember of a new class of lipid-lowering compounds thatelectively inhibit the intestinal absorption of cholesterol, demon-trating that it inhibits monocyte to macrophage differentiationhrough down-regulation of the expression of miR-155, miR-222,


iR-424, and miR-503 [44].Therefore, the use of both therapies, current traditional drugs

nd miRNA-based therapeutics, could improve the therapeuticffect.

PRESSlogy xxx (2013) xxx–xxx 3

How to modulate miRNAs

The modulation of miRNAs is based on antisense technology andgene therapy approaches.

Targeting miRNAs with antimiRNAs can reduce the levels ofpathogenic or aberrantly expressed miRNAs leading to the activa-tion of gene expression; on the contrary, the use of miRNA mimicscan elevate the level of miRNAs with a beneficial effect leading tothe suppression of gene expression.

AntimiRs

AntimiRNAs (antimiRs) are modified antisense oligonucleotidescarrying the complementary reverse sequence of a mature miRNA;antimiR may reduce the levels of a pathogenic or aberrantlyexpressed miRNA [42]. As an miRNA reduces the expression of itstarget genes, an antimiR approach results in an increase in expres-sion. An antimiR can act at different levels of the miRNA biogenesis.It can interfere with the export of the pre or primiRNA from thenucleus; it can bind the premiRNA and prevent its processing orentry into the RISC; and it can bind to the mature miRNA withinthe RISC acting as a competitive inhibitor.

A class of antimiR, antagomir, is conjugated to cholesterol tofacilitate cellular uptake.

The silencing of endogenous miRNA with antagomir has beenstudied in vivo, for the first time, by Krützfeldt et al. [36]. Theseauthors used an antagomir to inhibit miR-122, a liver specificmiRNA implicated in cholesterol and lipid metabolism and in hep-atitis C virus (HCV) replication obtaining a significant reduction incholesterol levels in mice [45].

Another study demonstrated that mice treated with antimiR-33, another miRNA involved in lipid metabolism, showed increasedHDL levels and a consequent regression of atherosclerosis [46]. Thistype of treatment combined with statins, raising HDL and loweringLDL levels, could represent a useful therapeutic strategy [47].

Other studies explored the possible use of antagomir as poten-tial therapy. Bonauer et al. investigated, in a mouse model of acuteMI, the effect of the antagomir-92a demonstrating that it mayincrease blood vessel growth and improve recovery of damagedtissue [38].

Other approaches use antimiRs with the locked nucleic acids(LNA) phosphorithioate chemistry.

These antimiRs are chemically modified to ensure in vivo stabil-ity, specificity, and high binding affinity to the miRNA of interest.LNA is a nucleic acid modification that allows a strong duplexformation with oligonucleotides enhancing specificity towardcomplementary RNA or DNA [45].

Recent studies showed LNA-modified oligonucleotide to be effi-cacious in inducing silencing of cardiac expressed miRNAs andmore potent in inhibiting miRNA function than cholesterol con-jugated antagomirs [45]. Intravenous injection of LNA-antimiRresulted in uptake of the LNA-antimiR in cytoplasm of pri-mate hepatocytes and formation of heteroduplexes between theLNA-antimiR and miR-122. Efficient silencing of miR-122 wasachieved in primates by three doses of 10 mg/kg LNA-antimiR,leading to a decrease in total plasma cholesterol that wasdose-dependent, long-lasting, reversible and without any evidencefor LNA-associated toxicities or histopatological changes [45].

Systemic delivery of an antisense oligonucleotide inducespotent and sustained silencing of miR-208a in the heart; ther-apeutic inhibition of miR-208a by subcutaneous delivery ofantimiR-208a during hypertension-induced CHF, in hypertensive


rats, prevented pathological myosin switching and LV remodelingimproving cardiac function and survival [48].

Hullinger et al. [49] showed up-regulation of miR-15 familyin the ischemic region in mice and pigs. miR-15 was induced

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Table 2Effect of miRNA modulation in cardiovascular disease.

miRNA Target AntimiR/mimics Effects Reference

miR-122 3-Hydroxy-3-methylglutayl-CoA-reductase(HMGCR)

Antagomir, LNA-antimiR Reduce plasma cholesterol levels [35,45]

miR-33 ABCA1 AntimiR Increased HDL levels [46]miR-208 THRAP1 LNA-antimiR Prevents cardiac remodeling [48]miR-92a ITGA5 Antagomir Enhanced blood vessel growth post-AMI [38]

miR-15 Bcl2, Arl2 LNA-antimiRReduce infarct size

[49]Inhibits cardiac remodelingEnhances cardiac function in response toischemic damage

miR-101 cFOS Mimics Antifibrotic action [51]miR-133 RhoA, Cdc42, Nelf-A7WHSC2 Sponge Exaggerated hypertrophic response [53]miR-599 Increase cardiomyocyte proliferation

icsReduce infarct size

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miR-199a Homer1, Hopx, Clic5 Mim

iRNA, microRNA; LNA, locked nucleic acids; HDL, high-density lipoprotein; AMI, a

n cardiomyocytes under hypoxia conditions and also by subse-uent reoxygenation. Interestingly, miR-15 inhibition with anNA-mediated antimiR chemistry in mice reduced infarct size,nhibiting cardiac remodeling and enhancing cardiac function inesponse to ischemic damage. Hemodynamic analysis 24 h afterschemia showed a lower LV end-diastolic pressure after infarctionompared with controls. Also echocardiography, 2 weeks afterschemic injury, showed that the antimiR treatment resulted inignificant improvement in ejection fraction, which correspondedith a decrease in both end-systolic and end-diastolic LV volumes

Table 2).

iR mimics

When a disease process is caused by a down-regulation ofiRNAs, the therapeutic approach is to increase miRNA levels

hrough a mimic approach. Mimics are double-stranded oligonu-leotides composed by 1 strand, identical to the mature miRNAguide strand), complexed with a complementary, or partially com-lementary, strand (passenger strand). Loaded into the RISC, theuide strand can act as the endogenous miRNA of interest and blockargeted gene expression [50].

To improve stability and cellular uptake miRNA mimics arehemically modified into a lipid-based formulation but this formu-ation may increase also the cellular uptake in tissue outside of thearget organ or cell type of interest.

An example of the efficient use of miRNA mimics carried bydenovirus is the in vivo study of Pan et al. [51] that evaluatedhe relation between miR-101 and cardiac fibroblast proliferationn infarcted heart. After coronary artery ligation, the expressionf miR-101 was decreased and fibrosis increased. The anti-fibroticction of miR-101 is mediated by the suppression of cFOS and itsownstream protein TGF�. Moreover in this study, using a miRNA01 mimic it was observed that overexpression of miR-101 reducedeterioration of LV performance, as indicated by the increased ejec-ion fraction and fractional shortening, an alleviated endothelialxtracellular matrix deposition, producing an antifibrotic action. Inddition, miR-101 overexpression reduced apoptosis of cardiomy-cytes in the peri-infarct area of MI hearts and it is not a direct effectut a consequence of the improved cardiac function by miR-101.

More recently Eulalio et al. first demonstrated the possibility ofnducing cardiac regeneration through miRNA mimics. The adult


ardiomyocytes have a limited proliferative capacity, that do notllow an effective myocardial regeneration after MI. The treatmentith miR-590 and miR-199a determined a re-entry of cardiomy-

cytes into cell cycle, leading to the proliferation of differentiated

[52]Improve cardiac function

yocardial infarction.

cardiomyocytes. In particular, the authors delivered these miR-NAs directly into the heart using an adeno-associated virus vectorand demonstrated that the expression of miR-590 and miR-199areduces infarct size and improves cardiac function [52] (Table 2).

Sponges, masking, and erasers

In addition to antimiR and miR mimics there are other interest-ing “technical” approaches that can inhibit miRNA expression andconsequentially therapeutically interfere with the disease process.They are sponges, masking, and erasers.

Therapeutic modulation of miRNA could be established by pre-venting the miRNA action by “adsorbing” the miRNA [42].

The use of sponge technique allows the prevention of bindingof the miRNA with its mRNA target. In particular a vector harbor-ing miRNA target sites bind the miRNA and thereby miRNAs do notbind their mRNA target [53]. The sponge can bind the miRNA withperfect or imperfect complementarity; the miRNAs remain boundlonger to the imperfect target sites so the function of the miRNA isbetter inhibited with imperfect binding sites. Furthermore, spongesare not specific to an miRNA, but they are able to block a whole fam-ily of related miRNAs that have the same target sites. Ebert et al.reported the reduction of miRNA levels with the use of the spongetechnique [53]. Moreover, Carè et al. first demonstrated the silenc-ing of miR-133 with the overexpression of miRNA binding sites [15](Table 2).

Another approach is the use of target masking using oligonu-cleotides with perfect complementarity with the miRNA target[54,55]. An advantage of this approach is its specificity, becauseit modulates a specific miRNA target, whereas inhibiting a miRNAmeans modulating miRNA multiple targets with the possibility ofoff-target effects.

A third approach is the use of erasers. Erasers are oligonu-cleotides in which tandem repeats of a sequence perfectly comple-mentary to the target miRNA inhibits endogenous miRNA function.

Both sponges and erasers scavenge the miRNAs preventing thebinding with the mRNA target but whereas erasers use only 2 copiesof the perfectly complementary antisense sequence of the miRNA,sponges use multiple antisense copies of an miRNA.

Sponges, masking, and erasers are interesting approaches butthere are no in vivo studies that have demonstrated their efficacy.


Challenges and risks: delivery, selectivity, and safety

Delivery is an important challenge of miRNA-based therapy:miRNA modulators must leave the circulatory system to get into the

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arget tissue, enter the cell membrane, and escape from endosomalesicles into the cytoplasm [54]. Another obstacle in the blood-tream is the degradation of miRNA therapeutics by phagocyticmmune cells. And miRNA therapeutics should not be longer than

nm to cross the capillary endothelium. Without an efficient deliv-ry mechanism miRNA therapeutics remain in the bloodstreamntil filtered by the kidneys or degraded by monocytes. One way tovercome this obstacle and increase the bioavailability of miRNAodulators is local delivery by direct injection in target tissue,

ut this approach is limited to eye, skin, mucous membranes, andumors.

Another and consequent challenge is miRNA-based therapyelectivity and safety. Even if certain miRNAs are expressed inpecific tissue, as miR-208 in the heart and miR-122 in the liver,any miRNAs are ubiquitously expressed. MiRNA therapeutics

ould act on targets not involved in the disease process giving,hus, off-target effects. There are two delivery approaches: con-ugation and formulation [50]. Conjugation strategy provides thettachment of targeting molecules such as peptides, antibodies, orther bioactive molecules to the oligonucleotide improving miRNAodulator uptake in the target cell. Alternatively, formulation

pproach provides the package of oligonucleotide into liposomesr nanoparticles. This approach facilitates the endocytosis of theiRNA therapeutics. Another approach achieves cellular uptake

y linking the oligonucleotide with cholesterol. The miRNA mod-lators could be encapsulated into a lipid-based formulation thatnhances cardiac uptake. To date, these delivery technologies haveemonstrated an efficacy in the delivery to the liver. The explana-ion is the permeability of liver cells to molecules up to 200 nm iniameter. For this the liver is the most successful organ for deliv-ring therapeutic miRNAs.

In heart application specific technologies could be exploited. Inarticular, miRNA therapeutics could be delivered during cardiacatheterization improving cardiac uptake. In the context of treatingulnerable plaque, a problem could be the penetration of the fibrousap and to overcome this problem a target could be macrophageshat during their activation would release modified miRNAs [21].

onclusions

To date, many studies demonstrated the key role of the dysreg-lation of miRNAs in the disease process and their involvement inVD. Thus, miRNA modulation could represent a new therapeuticpproach. Therapies addressing miRNA functions are fascinatingnd promising also in ischemic heart disease, but several biologicalnd technical problems (delivery, selectively, safety) still exist andust be better addressed. Available data are relatively weak but

onsistent and suggest that microRNAs might represent in the nearuture a real revolution in the therapy of ischemic heart disease inll its stages.

onflict of interest

None declared.

cknowledgments

The manuscript was supported by grant number 70200755 fromatholic University of Rome and by grant 2009 FJX9KV 003 from the

talian Department of Scientific Research.


eferences

[1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism and function. Cell2004;116:281–97.

[

PRESSlogy xxx (2013) xxx–xxx 5

[2] Urbich C, Kuehbacher A, Dimmeler S. Role of microRNAs in vascular diseases,inflammation, and angiogenesis. Cardiovasc Res 2008;79:581–8.

[3] Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of geneticexchange between cells. Nat Cell Biol 2007;9:654–9.

[4] Chen TS, Lai RC, Lee MM, Choo AB, Lee CN, Lim SK. Mesenchymal stemcells secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res2010;38:215–24.

[5] Krichevsky AM, Sonntag KC, Isacson O, Kosik KS. Specific microRNAs modulateembryonic stem cell-derived neurogenesis. Stem Cells 2006;24:857–64.

[6] Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specificmicroRNA that targets Hand2 during cardiogenesis. Nature 2005;436:214–20.

[7] Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, ConlonFL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscleproliferation and differentiation. Nat Genet 2006;38:228–33.

[8] Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineagedifferentiation. Science 2004;303:83–6.

[9] Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis Cvirus RNA abundance by a liver-specific microRNA. Science 2005;309:1577–81.

10] van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control ofstress-dependent cardiac growth and gene expression by a microRNA. Science2007;316:575–9.

11] Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-CorderoA, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, GolubTR. MicroRNA expression profiles classify human cancers. Nature 2005;435:834–8.

12] Yang B, Lin H, Xiao J, Lu Y, Luo X, Li B, Zhang Y, Xu C, Bai Y, Wang H,Chen G, Wang Z. The muscle-specific microRNA miR-1 regulates cardiacarrhythmogenic potential by targeting GJA1 and KCNJ2. Nat Med 2007;13:486–91.

13] van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, MarshallWS, Hill JA, Olson EN. Dysregulation of microRNAs after myocardial infarc-tion reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci U S A2008;105:13027–32.

14] Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES, ZhangC. MicroRNA expression signature and the role of microRNA-21 in the earlyphase of acute myocardial infarction. J Biol Chem 2009;284:29514–25.

15] Carè A, Catalucci D, Felicetti F, Bonci D, Addario A, Gallo P, Bang ML, SegnaliniP, Gu Y, Dalton ND, Elia L, Latronico MV, Høydal M, Autore C, Russo MA, et al.MicroRNA-133 controls cardiac hypertrophy. Nat Med 2007;13:613–8.

16] Divakaran V, Mann DL. The emerging role of microRNAs in cardiac remodelingand heart failure. Circ Res 2008;103:1072–83.

17] Funahashi H, Izawa H, Hirashiki A, Cheng XW, Inden Y, Nomura M, Muro-hara T. Altered microRNA expression associated with reduced catecholaminesensitivity in patients with chronic heart failure. J Cardiol 2011;57:338–44.

18] Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, TsuchihashiT, McManus MT, Schwartz RJ, Srivastava D. Dysregulation of cardiogen-esis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell2007;129:303–17.

19] Libby P. Inflammation in atherosclerosis. Nature 2002;420:868–74.20] Haver VG, Slart RH, Zeebregts CJ, Peppelenbosch MP, Tio RA. Rupture of vul-

nerable atherosclerotic plaques: microRNAs conducting the orchestra? TrendsCardiovasc Med 2010;20:65–71.

21] Martin K, O’Sullivan JF, Caplice NM. New therapeutic potential of microRNAtreatment to target vulnerable atherosclerotic lesions and plaque rupture. CurrOpin Cardiol 2011;26:569–75.

22] D’Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, RubinoM, Carena MC, Spazzafumo L, De Simone M, Micheli B, Biglioli P, Achilli F,Martelli F, Maggiolini S, et al. Circulating microRNAs are new and sensitivebiomarkers of myocardial infarction. Eur Heart J 2010;31:2765–73.

23] Weber M, Baker MB, Moore JP, Searles CD. miR-21 is induced in endothelial cellsby shear stress and modulates apoptosis and eNOS activity. Biochem BiophysRes Commun 2010;393:643–8.

24] Hojo Y, Saito T, Kondo H. Role of apoptosis in left ventricular remodeling afteracute myocardial infarction. J Cardiol 2012;60:91–2.

25] Roy S, Khanna S, Hussain SR, Biswas S, Azad A, Rink C, Gnyawali S, Shilo S,Nuovo GJ, Sen CK. MicroRNA expression in response to murine myocardialinfarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase andtensin homologue. Cardiovasc Res 2009;82:21–9.

26] Harris TA, Yamakuchi M, Ferlito M, Mendell JT, Lowenstein CJ. MicroRNA-126regulates endothelial expression of vascular cell adhesion molecule 1. Proc NatlAcad Sci USA 2008;105:1516–21.

27] Liuzzo G, Biasucci LM, Gallimore JR, Grillo RL, Rebuzzi AG, Pepys MB, MaseriA. The prognostic value of C-reactive protein and serum amyloid a protein insevere unstable angina. N Engl J Med 1994;331:417–24.

28] Raitoharju E, Lyytikäinen LP, Levula M, Oksala N, Mennander A, Tarkka M, KloppN, Illig T, Kähönen M, Karhunen PJ, Laaksonen R, Lehtimäki T. miR-21, miR-210,miR-34a, and miR-146a/b are up-regulated in human atherosclerotic plaquesin the Tampere Vascular Study. Atherosclerosis 2011;219:211–7.

29] Nazari-Jahantigh M, Wei Y, Noels H, Akhtar S, Zhou Z, Koenen RR, HeyllK, Gremse F, Kiessling F, Grommes J, Weber C, Schober A. MicroRNA-155promotes atherosclerosis by repressing Bcl6 in macrophages. J Clin Invest


2012;122:4190–202.30] Ponomarev ED, Veremeyko T, Barteneva N, Krichevsky AM, Weiner HL.

MicroRNA-124 promotes microglia quiescence and suppresses EAE by deacti-vating macrophages via the C/EBP-�-PU.1 pathway. Nat Med 2011;17:64–70.

dx.doi.org/10.1016/j.jjcc.2013.01.012

ING ModelJ

6 Cardi

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31] Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependent induc-tion of microRNA miR-146, an inhibitor targeted to signaling proteins of innateimmune responses. Proc Natl Acad Sci USA 2006;103:12481–6.

32] Chen T, Li Z, Jing T, Zhu W, Ge J, Zheng X, Pan X, Yan H, Zhu J. MicroRNA-146aregulates the maturation process and pro-inflammatory cytokine secre-tion by targeting CD40L in oxLDL-stimulated dendritic cells. FEBS Lett2011;585:567–73.

33] Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, Muth AN, Lee TH, MianoJM, Ivey KN, Srivastava D. miR-145 and miR-143 regulate smooth muscle cellfate and plasticity. Nature 2009;460:705–10.

34] Liu X, Cheng Y, Zhang S, Lin Y, Yang J, Zhang C. A necessary role of miR-221 andmiR-222 in vascular smooth muscle cell proliferation and neointimal hyper-plasia. Circ Res 2009;104:476–87.

35] Wang YS, Wang HY, Liao YC, Tsai PC, Chen KC, Cheng HY, Lin RT, Juo SH.MicroRNA-195 regulates vascular smooth muscle cell phenotype and preventsneointimal formation. Cardiovasc Res 2012;95:517–26.

36] Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, StoffelM. Silencing of microRNAs in vivo with ‘antagomirs’. Nature 2005;438:685–9.

37] Rayner KJ, Suárez Y, Dávalos A, Parathath S, Fitzgerald ML, Tamehiro N, FisherEA, Moore KJ, Fernández-Hernando C. miR-33 contributes to the regulation ofcholesterol homeostasis. Science 2010;328:1570–3.

38] Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A, Burchfield J,Fox H, Doebele C, Ohtani K, Chavakis E, Potente M, Tjwa M, Urbich C, Zeiher AM,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemictissues in mice. Science 2009;324:1710–3.

39] Chen WJ, Yin K, Zhao GJ, Fu YC, Tang CK. The magic and mystery of microRNA-27in atherosclerosis. Atherosclerosis 2012;222:314–23.

40] Fichtlscherer S, De Rosa S, Fox H, Schwietz T, Fischer A, Liebetrau C, WeberM, Hamm CW, Röxe T, Müller-Ardogan M, Bonauer A, Zeiher AM, DimmelerS. Circulating microRNAs in patients with coronary artery disease. Circ Res2010;107:677–84.

41] De Rosa S, Fichtlscherer S, Lehmann R, Assmus B, Dimmeler S, Zeiher AM.Transcoronary concentration gradients of circulating microRNAs. Circulation2011;124:1936–44.


42] Montgomery RL, van Rooij E. Therapeutic advances in MicroRNA targeting.J Cardiovasc Pharmacol 2011;57:1–7.

43] Iekushi K, Seeger F, Assmus B, Zeiher AM, Dimmeler S. Regulation of cardiacmicroRNAs by bone marrow mononuclear cell therapy in myocardial infarction.Circulation 2012;125:1765–73 [S1–7].

[[

PRESSology xxx (2013) xxx–xxx

44] Munoz-Pacheco P, Ortega-Hernández A, Miana M, Cachofeiro V, Fernández-Cruz A, Gómez-Garre D. Ezetimibe inhibits PMA-induced monocyte/macrophage differentiation by altering microRNA expression: a novel anti-atherosclerotic mechanism. Pharmacol Res 2012;66:536–43.

45] Elmén J, Lindow M, Schütz S, Lawrence M, Petri A, Obad S, Lindholm M, HedtjärnM, Hansen HF, Berger U, Gullans S, Kearney P, Sarnow P, Straarup EM, Kaup-pinen S. LNA-mediated microRNA silencing in non-human primates. Nature2008;452:896–9.

46] Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van GilsJM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA, MooreKJ. Antagonism of miR-33 in mice promotes reverse cholesterol transport andregression of atherosclerosis. J Clin Invest 2011;121:2921–31.

47] Ono K. Current concept of reverse cholesterol transport and novel strategy foratheroprotection. J Cardiol 2012;60:339–43.

48] Montgomery RL, Hullinger TG, Semus HM, Dickinson BA, Seto AG, Lynch JM,Stack C, Latimer PA, Olson EN, van Rooij E. Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure. Circulation2011;124:1537–47.

49] Hullinger TG, Montgomery RL, Seto AG, Dickinson BA, Semus HM, Lynch JM,Dalby CM, Robinson K, Stack C, Latimer PA, Hare JM, Olson EN, van RooijE. Inhibition of miR-15 protects against cardiac ischemic injury. Circ Res2012;110:71–81.

50] van Rooij E, Marshall WS, Olson EN. Toward microRNA-based therapeutics forheart disease: the sense in antisense. Circ Res 2008;103:919–28.

51] Pan Z, Sun X, Shan H, Wang N, Wang J, Ren J, Feng S, Xie L, Lu C, Yuan Y,Zhang Y, Wang Y, Lu Y, Yang B. MicroRNA-101 inhibited postinfarct cardiacfibrosis and improved left ventricular compliance via the FBJ osteosarcomaoncogene/transforming growth factor-�1 pathway. Circulation 2012;126:840–50.

52] Eulalio A, Mano M, Ferro MD, Zentilin L, Sinagra G, Zacchigna S, Giacca M.Functional screening identifies miRNAs inducing cardiac regeneration. Nature2012;492:376–81.

53] Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors ofsmall RNAs in mammalian cells. Nat Methods 2007;4:721–6.


54] Broderick JA, Zamore PD. MicroRNA therapeutics. Gene Ther 2011;18:1104–10.55] Xiao J, Yang B, Lin H, Lu Y, Luo X, Wang Z. Novel approaches for gene-

specific interference via manipulating actions of microRNAs: examinationon the pacemaker channel genes HCN2 and HCN4. J Cell Physiol 2007;212:285–92.

dx.doi.org/10.1016/j.jjcc.2013.01.012

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