European Heart Journal Supplements (2016) 18 (Supplement E), E31–E36 The Heart of the Matter doi:10.1093/eurheartj/suw012
microRNAs in ischaemic cardiovascular diseases Simona Greco, Germana Zaccagnini, Christine Voellenkle, and Fabio Martelli* Molecular Cardiology Laboratory, IRCCS Policlinico San Donato, via Morandi 30, 20097 San Donato Milanese, Milan, Italy
KEYWORDS microRNA; Cardiovascular diseases; Ischaemia; Hypoxia
microRNAs (miRNAs) are non-coding RNA molecules that modulate the stability and/or the translational efficiency of specific messenger RNAs. They have been shown to play a regulatory role in most biological processes and their expression is disrupted in many cardiovascular diseases. This review describes studies performed at Policlinico San Donato-IRCCS in cell cultures, animal models, and patients, showing a penetrant role of miRNAs in cell response to hypoxia and in ischaemic cardiovascular diseases. These experiments indicate miRNA as an emerging class of therapeutic targets.
Introduction microRNAs (miRNAs) are 20–25 nucleotide-long RNAs that regulate gene expression mainly suppressing the stability and/or the translation of a specific set of target mRNAs (Figure 1). They are synthesized in the nucleus and, after their maturation and export to the cytoplasm, are loaded on the effector complex called ‘RNA-induced silencing complex’ (RISC).1 microRNAs bind to their targets via partial complementary pairing, and while the targeting rules are still object of intense research, a 5′ region of the miRNA, called ‘seed sequence’, seems to play a particularly important role.2,3 Each miRNA can target multiple mRNAs, and since each mRNA can be targeted by more than one miRNA, miRNAs constitute an intricate and pervasive network regulating most aspects of biology, including the cardiovascular functions.4,5 Indeed, miRNAs regulate crucial aspects of angiogenesis, endothelial and myocyte growth, cardiogenesis, contractility, and cardiac rhythm. Accordingly, the expression of specific miRNAs is altered in many cardiovascular diseases and genetically engineered mice showed that miRNA deregulation is necessary and sufficient for many heart and blood vessel physiopathological conditions.4–8 Another element that is placing miRNAs at the centre stage is their perspective use as prognostic/diagnostic biomarkers. Indeed, miRNAs are present in the peripheral blood, in the haematic cellular components, but also outside the cells, both in plasma and in serum.9
* Corresponding author. Tel: +39 02 52 774 533, Fax: +39 02 52 774 666, Email: [email protected]
This review is aimed at describing a series studies performed in a variety of experimental and clinical settings at Policlinico San Donato-IRCCS. We show a wide role of miRNAs in ischaemic and post-ischaemic cardiovascular diseases, placing our observations in the context of the current literature.
microRNAs and ischaemic cardiovascular diseases Hypoxia response Hypoxia is a pivotal pathogenetic factor of ischaemic cardiovascular diseases.10 To investigate miRNA regulation in endothelial cell cultures exposed to 1% oxygen, miRNA expression profile was determined using RNA-Sequencing, a highthroughput technique allowing the quantitative measurement of known and novel miRNA species.11 This analysis allowed the identification of more than 400 annotated miRNA species with a wide range of expression. Indeed, miR-21 and miR-126 alone totalled almost half of all miRNAs present in endothelial cells. A custom pipeline of analysis, combining bioinformatics and molecular biology tools, allowed the discovery and characterization of 18 new miRNA species. Interestingly, the expression of two of these novel miRNAs was significantly down-modulated by hypoxia, while miR-210 was significantly induced. Indeed, miR-210 is generally recognized as the prototypical hypoxamiR,7,12 being robustly induced by hypoxia-inducible factor l a (HIF1A), both in vitro and in vivo. 7,13–15
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: [email protected]
E32
S. Greco et al.
Figure 1 microRNA biogenesis and function. microRNAs originate from the pri-miRNA, which is cleaved in the nucleus by the complex Drosha/DGCR8 to generate a 70–100 nucleotide-long hairpin-shaped precursor named pre-miRNA. The pre-miRNA is shuttled to the cytoplasm by Exportin 5, through nucleoporin (NPC) and further processed by the ribonuclease III Dicer, forming the mature 22-nt miRNA duplex. One strand from the miRNA duplex, the mature miRNA, is then incorporated into the effector complex, RNA-induced silencing complex, which recognizes specific target mRNAs through imperfect base-pairing, and down-regulates their expression by mRNA exonucleolitic degradation and/or inhibition of translation.
The identification of miRNA targets is instrumental for understanding their role.2,3 Several valid bioinformatics tools have been developed to this aim. However, all of them are burdened by a high number of false positives and negatives.8 Thus, to identify miR-210 targets, we took an experimental approach, using a combination of proteomics, transcriptomics, immunopurification of the RISC–miRNA complex, and molecular biology. This approach allowed us to identify 33 novel targets, both canonical7,16 and non-canonical, i.e. not requiring a seed region for target binding.17 The analysis of the targets identified by us and other authors18 suggested a role of miR-210 in cell migration and angiogenesis.7,16 Indeed, miR-210 promoted the formation of capillary-like structures in endothelial cells plated on Matrigel, as much as endothelial cell migration towards a vascular endothelial growth factor gradient.13 Both events required Ephrin-A3 (EFNA3) direct targeting by miR-21013 (Figure 2). miR-210 stimulates angiogenesis also through other mechanisms, both direct
and indirect. Indeed, increased angiogenic factor signalling has been observed in miR-210-expressing cells.19–21 Accordingly, increased tissue perfusion and capillary density have been found in mouse ischaemic muscles upon injection of umbilical cord blood CD34+ cells expressing miR-210.22 Other targets, such as ISCU1/2, COX10, NDUFA4, and FECH, indicated miR-210 involvement in mitochondrial metabolism. Indeed, miR-210 contributes to the oxidative phosphorylation decline observed in hypoxia and its repression triggers mitochondrial dysfunction and oxidative stress.23
Peripheral artery diseases Peripheral artery disease is commonly caused by an impairment of blood supply in the arteries of the leg, and is mostly caused by atherosclerosis and often results in critical limb ischaemia.24
microRNAs in ischaemic cardiovascular diseases
Figure 2 miR-210 and the hypoxia/ischaemia response. Cell culture in hypoxic conditions or acute tissue isachemia can both trigger the activation of HIF-1a, which in turn activates the expression of miR-210. miR-210 attenuates mitochondrial metabolism and reactive oxygen species (ROS) generation by the electron transport chain, thus preventing cell apoptosis and necrosis. Moreover, Ephrin-3 (EFNA3) is a direct target of miR-210, and its inhibition is necessary for miR-210 to stimulate capillary-like tubes formation (tubulogenesis) and angiogenesis.
Our group investigated the regulation and role of miRNAs in a mouse model of acute hindlimb ischaemia, where the femoral artery is dissected, causing severe ischaemia in the distal part of the affected limb.25 After acute ischaemic injury, the affected muscles undergo distinct phases, consisting of necrosis/degeneration and inflammation in the first few days, regeneration/repair from 2 to 4 weeks, and fibrosis at 2 or 3 weeks post-injury.26 Using this model, our group determined that the expression levels of some miRNAs decreased early at 2 days after ischaemia (miR-l, miR-29c, and miR-135a) correlating with the necrosis/degeneration phase. Inflammatory miRNAs miR-222 and miR-223 were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. Other miRNAs (miR-31, miR-34c, miR-335, miR-449, miR-494, and miR-206) peaked at 14 days correlating with the regeneration phase. The isolation of damaged, nondamaged, and regenerating myofibres corroborated these results.27 Our group also investigated the regulation and role of miR-210 in the mouse model of acute hindlimb ischaemia25 (Figure 2). To this end, miR-210 was blocked by the systemic injection of chemically modified Locked Nucleic Acid complementary oligonucleotides (antimiR-210). As expected, after femoral artery dissection, miR-210 expression was induced in the ischaemic gastrocnemius muscles, while the administration of antimiR-210 caused a marked
E33
decrease in miR-210 and increase in its targets. Histological evaluation of acute tissue damage upon miR-210 inhibition showed an increase in apoptosis and necrosis as well as a decrease in capillary density and residual limb perfusion. Transcriptomic data after ischaemia pointed out the deregulation of mitochondrial function and redox balance in antimiR-210-treated mice. These data are in keeping with miR-210 role as repressor of oxidative phosphorylation observed in vitro.23 Accordingly, following miR-210 blocking, mitochondrial metabolism was decreased and oxidative stress induced, both in vitro 23,28 and in vivo.25 Furthermore, oxidative stress-resistant p66Shc-null mice29 displayed decreased tissue damage following ischaemia when miR-210 was blocked.25 This study demonstrates that miR-210 has cytoprotective effects in the skeletal muscle, regulating oxidative metabolism and oxidative stress, thus being a crucial element of the adaptive mechanisms to acute ischaemia. Interestingly, miR-210 also seems to play a role in ischaemic ulcers. To elucidate how ischaemia complicates wound closure, Biswas et al.30 used a mouse model of wound healing. In ischaemic wounds, Hif1-a and miR-210 were strongly induced. Accordingly, miR-210 target E2F3, a cell cycle positive regulator, was down-regulated, correlating with decreased proliferation of keratinocytes. Considering that angiogenesis is also a key component of the wound healing process, it remains to be determined how the pro-angiogenic function of miR-210 integrates with its antiproliferative function in keratinocytes. microRNAs also contribute to diabetes mellitus-induced impairment of reparative angiogenesis after limb ischaemia. Indeed, miR-503 expression was increased in ischaemic muscles and in endothelial cells of a mouse model of diabetes mellitus, upon femoral artery dissection.31 More importantly, miR-503 blocking corrected diabetes-induced impairment of post-ischaemic angiogenesis and blood flow recovery. This does not seem limited to experimental animal models, since miR-503 expression was significantly increased in skeletal muscles of diabetic patients affected by critical limb ischaemia, while the expression of miR-503 targets decreased, suggesting miR-503 as a possible therapeutic target in diabetic patients.
Myocardial infarction Myocardial ischaemic injury arises from severe impairment of the coronary blood supply, usually caused by thrombosis or other acute alterations of coronary atherosclerotic plaques. After myocardial ischaemia, miR-l, miR-15, miR21, miR-92a, miR-208, and miR-126 expression levels increased in animal models of myocardial infarction,32–37 and in patients, who died after MI.38 In the border zone of myocardial infarction, in mice, miR-29,39 miR-24,40 and miR-101a/b41 levels were decreased. As expected, given its prominent role as hypoxamir, also miR-210 has been shown to be up-regulated following myocardial infarction in humans.42 The therapeutic potential of miR-210 has been shown in a mouse model of myocardial infarction.19 Upon hypoxic stimulation of a cardiomyocyte cell line, miR-210 levels significantly increased, promoting the release of several angiogenic factors, inhibiting
E34
S. Greco et al.
caspase activity, and preventing cell apoptosis, in keeping with the data obtained in endothelial cells. In in vivo experiments, after mid-left anterior descending artery ligation, miR-210 overexpression using a minicircle vector attenuated the decrease in left ventricle fractional shortening. Histological evaluation of explanted hearts demonstrated the reduction in both infarction size and apoptosis, as well as increased capillary density for the miR-210treated group, corroborating the imaging data.19 Thus, miR-210 can improve heart function by up-regulating angiogenesis and inhibiting apoptosis, suggesting that miR-210 delivery through non-viral minicircle has a therapeutic potential for treatment of ischaemic heart disease.
Heart failure Acute myocardial infarction triggers several structural changes of both the infarcted and non-infarcted regions of the ventricle, causing ventricle remodelling, chamber dilatation, and eventually, heart failure. This remodelling can importantly affect the function of the ventricle and the prognosis for survival.43 Several studies described miRNAs deregulation in heart failure. These data derive from studies in cell culture models of hypertrophy, in animal models of heart pathologic remodelling, as well as in humans.44 However, these human studies relied mostly on end-stage patients, thus lacking important information on early-stage determinants of the disease. To fill this knowledge gap, our group analysed the miRNA expression profiles of left ventricle biopsies from heart failure patients affected by non-end-stage dilated ischaemic cardiomyopathy.45 These biopsies were harvested in the remote nonischaemic area of the ventricle of patients undergoing ventricular restoration procedure by the surgical team of Lorenzo Menicanti at Policlinico San Donato.46,47 We found that 17 miRNAs were modulated in heart failure patients when compared with control subjects. In particular, miR-216a, an endothelial senescence-associated miRNA,48 was strongly increased in failing hearts and negatively correlated with left ventricle ejection fraction (Figure 3). Diabetes mellitus is a very serious co-morbidity factor in heart failure, being associated with increased morbidity and mortality.49 Comparing diabetic and nondiabetic heart failure patients, we found that six miRNAs were differently expressed: miR-34b, miR-34c, miR-199b, miR-210, miR-650, and miR-223. Interestingly, miR-199b and miR-210 were modulated by hypoxia and high glucose in both mouse neonatal cardiomyocytes and human endothelial cells. Accordingly, the whole hypoxia-inducible factor pathway was activated, indicating that diabetes mellitus is triggering a pseudo-hypoxic state also in the non-infarcted, vital myocardium.45
microRNAs as biomarkers In addition to their involvement in pathogenesis mechanisms, miRNAs are also attractive biomarkers. Several studies in different disciplines suggest that classifications based on miRNA profiling are more specific and sensitive compared with methods based on mRNA profiling.50,51
Figure 3 miR-216a in the senescence/autophagy network. miR-216a expression levels are induced in heart failure and are also increased in aged/ senescent cells and atherosclerotic plaques. The autophagy-related genes, ATG5 and BECN, are repressed by miR-216a, thus preventing autophagy.
Unlike oncology, biopsies are only rarely indicated for cardiovascular diseases. Thus, the attention of the researchers has turned to peripheral blood that is accessible with a minimally invasive procedure. For instance, our group measured the miRNAs expressed by peripheral blood mononuclear cells (PBMCs) of heart failure patients affected by ischaemic and non-ischaemic dilated cardiomyopathy.52 We identified three miRNAs, miR-107, miR-139, and miR-142–5p, displaying decreased levels in both cardiomyopathies. Other miRNAs were deregulated in only one of the heart failure patient groups. Interestingly, many of these miRNAs are also deregulated in human and/or mouse failing hearts.44 As previously mentioned, some miRNAs are released from the cells of origin and can be measured in bodily fluids.50,51 Indeed, extracellular miRNAs are remarkably stable even outside the cell, where they are protected from degradation by their association with microvescicles or exosomes, high-density lipoproteins, or protein complexes, such as the RISC. Interestingly, disease-specific miRNA signatures can be identified in many physiopathological conditions and plasma or serum miRNAs have been proposed as biomarkers for diagnosis and prognosis of a variety of cardiovascular diseases.9 In particular, several promising miRNAs candidates have emerged so far for myocardial infarction, including miR-1, miR-133a, miR-133b, miR-208a, and miR-499.5,9,53 These miRNAs are all highly enriched in cardiomyocytes and their increase is most likely the result of massive cardiomyocyte necrosis and release into the bloodstream. Particularly interesting is the fact that miR-1, miR-133a, and miR-208a increased continuously during the first 4 h after MI, and before cardiac troponin T could be detected.54,55 Among these, miR-208 is noteworthy because it is cardiacspecific, and because it is absent in healthy subjects and patients without acute myocardial infarction.54,56 However, several conflicting results are present in the literature,
microRNAs in ischaemic cardiovascular diseases
most likely for the small size of most studies and because of the lack of standardized detection and normalization techniques. In this respect, new PCR techniques, such as digital PCR,57 will likely have a positive impact. Additionally, it should be kept in mind that the detection of protein humoral markers is very rapid and only requires standard laboratory equipment, while the detection of nucleic acids is still comparatively slow, requiring molecular biology equipment and expertise.
Concluding remarks Although the clinical value of miRNAs is only starting to be appreciated, it is clear that miRNAs seem very attractive therapeutic targets, since they can be easily synthesized, chemically modified for increased stability, and delivered via functionalized biomaterials. microRNAs could be used as therapeutic tools when increasing the levels of a specific miRNA is desirable. Conversely, in certain situations, the aim could be neutralizing pathological miRNAs, for instance, using complementary oligonucleotides. While no human experimentation targeting miRNAs in cardiovascular diseases has been reported so far, a phase I clinical trial for cancer therapy is ongoing (ClinicalTrials.gov Identifier: NCT01829971) and a phase IIa clinical trial for hepatitis C therapy (ClinicalTrials.gov Identifier: NCT01200420) has been completed successfully,58 showing the viability of miRNAs as therapeutic targets. Many hurdles must be overcome to achieve clinically feasible strategies for miRNA-based therapies in heart and vascular diseases, including the optimization of delivery tools and of the therapeutic molecule chemistry. Even more importantly however, given the intrinsically pleiotropic functions of miRNAs, we must first understand in detail the actions of the target miRNAs in each physio-pathological context, before progressing to clinical application.
Funding This work was supported by Ministero della Salute, Fondazione Cariplo (grant #2013-0887), Telethon-Italy (grant #GGP14092), and AFM Telethon (grant #18477). Conflict of interest: none declared.
References 1. Hammond SM. An overview of microRNAs. Adv Drug Deliv Rev 2015;87: 3–14. 2. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215–233. 3. Thomson DW, Bracken CP, Goodall GJ. Experimental strategies for microRNA target identification. Nucleic Acids Res 2011;39: 6845–6853. 4. Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature 2011;469:336–342. 5. Condorelli G, Latronico MV, Cavarretta E. microRNAs in cardiovascular diseases: current knowledge and the road ahead. J Am Coll Cardiol 2014;63:2177–2187.
E35 6. Greco S, Gorospe M, Martelli F. Noncoding RNA in age-related cardiovascular diseases. J Mol Cell Cardiol 2015;83:142–155. 7. Greco S, Gaetano C, Martelli F. HypoxamiR regulation and function in ischemic cardiovascular diseases. Antioxid Redox Signal 2014;21: 1202–1219. 8. Fasanaro P, Greco S, Ivan M, Capogrossi MC, Martelli F. microRNA: emerging therapeutic targets in acute ischemic diseases. Pharmacol Ther 2010;125:92–104. 9. Di Stefano V, Zaccagnini G, Capogrossi MC, Martelli F. microRNAs as peripheral blood biomarkers of cardiovascular disease. Vascul Pharmacol 2011;55:111–118. 10. Semenza GL. Hypoxia-inducible factor 1 and cardiovascular disease. Annu Rev Physiol 2014;76:39–56. 11. Voellenkle C, Rooij J, Guffanti A, Brini E, Fasanaro P, Isaia E, Croft L, David M, Capogrossi MC, Moles A, Felsani A, Martelli F. Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs. RNA 2012;18:472–484. 12. Devlin C, Greco S, Martelli F, Ivan M. miR-210: more than a silent player in hypoxia. IUBMB Life 2011;63:94–100. 13. Fasanaro P, D’Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, Capogrossi MC, Martelli F. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 2008;283:15878–15883. 14. Kulshreshtha R, Ferracin M, Negrini M, Calin GA, Davuluri RV, Ivan M. Regulation of microRNA expression: the hypoxic component. Cell Cycle 2007;6:1426–1431. 15. Kulshreshtha R, Ferracin M, Wojcik SE, Garzon R, Alder H, Agosto-Perez FJ, Davuluri R, Liu CG, Croce CM, Negrini M, Calin GA, Ivan M. A microRNA signature of hypoxia. Mol Cell Biol 2007;27: 1859–1867. 16. Fasanaro P, Greco S, Lorenzi M, Pescatori M, Brioschi M, Kulshreshtha R, Banfi C, Stubbs A, Calin GA, Ivan M. An integrated approach for experimental target identification of hypoxia-induced miR-210. J Biol Chem 2009;284:35134–35143. 17. Fasanaro P, Romani S, Voellenkle C, Maimone B, Capogrossi MC, Martelli F. ROD1 is a seedless target gene of hypoxia-induced miR-210. PLoS One 2012;7:e44651. 18. Bertero T, Robbe-Sermesant K, Le Brigand K, Ponzio G, Pottier N, Rezzonico R, Mazure NM, Barbry P, Mari B. MicroRNA target identification: lessons from hypoxamiRs. Antioxid Redox Signal 2014;21: 1249–1268. 19. Hu S, Huang M, Li Z, Jia F, Ghosh Z, Lijkwan MA, Fasanaro P, Sun N, Wang X, Martelli F, Robbins RC, Wu JC. MicroRNA-210 as a novel therapy for treatment of ischemic heart disease. Circulation 2010; 122(11 Suppl):S124–S131. 20. Liu W, Wen Y, Bi P, Lai X, Liu XS, Liu X, Kuang S. Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation. Development 2012;139:2857–2865. 21. Liu SC, Chuang SM, Hsu CJ, Tsai CH, Wang SW, Tang CH. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression. Cell Death Dis 2014;5:e1485. 22. Alaiti MA, Ishikawa M, Masuda H, Simon DI, Jain MK, Asahara T, Costa MA. Up-regulation of miR-210 by vascular endothelial growth factor in ex vivo expanded CD34+ cells enhances cell-mediated angiogenesis. J Cell Mol Med 2012;16:2413–2421. 23. Cottrill KA, Chan SY, Loscalzo J. Hypoxamirs and mitochondrial metabolism. Antioxid Redox Signal 2014;21:1189–1201. 24. Peach G, Griffin M, Jones KG, Thompson MM, Hinchliffe RJ. Diagnosis and management of peripheral arterial disease. BMJ 2012;345:e5208. 25. Zaccagnini G, Maimone B, Di Stefano V, Fasanaro P, Greco S, Perfetti A, Capogrossi MC, Gaetano C, Martelli F. Hypoxia-induced miR-210 modulates tissue response to acute peripheral ischemia. Antioxid Redox Signal 2014;21:1177–1188. 26. Huard J, Li Y, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 2002;84-A:822–832. 27. Greco S, De Simone M, Colussi C, Zaccagnini G, Fasanaro P, Pescatori M, Cardani R, Perbellini R, Isaia E, Sale P, Meola G, Capogrossi MC, Gaetano C, Martelli F. Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia. FASEB J 2009;23:3335–3346. 28. Cicchillitti L, Di Stefano V, Isaia E, Crimaldi L, Fasanaro P, Ambrosino V, Antonini A, Capogrossi MC, Gaetano C, Piaggio G, Martelli F.
E36
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
S. Greco et al. Hypoxia-inducible factor 1-alpha induces miR-210 in normoxic differentiating myoblasts. J Biol Chem 2012;287:44761–44771. Zaccagnini G, Martelli F, Fasanaro P, Magenta A, Gaetano C, Di Carlo A, Biglioli P, Giorgio M, Martin-Padura I, Pelicci PG, Capogrossi MC. p66ShcA modulates tissue response to hindlimb ischemia. Circulation 2004;109:2917–2923. Biswas S, Roy S, Banerjee J, Hussain SR, Khanna S, Meenakshisundaram G, Kuppusamy P, Friedman A, Sen CK. Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds. Proc Natl Acad Sci USA 2010;107:6976–6981. Caporali A, Meloni M, Vollenkle C, Bonci D, Sala-Newby GB, Addis R, Spinetti G, Losa S, Masson R, Baker AH, Agami R, le Sage C, Condorelli G, Madeddu P, Martelli F, Emanueli C. Deregulation of microRNA-503 contributes to diabetes mellitus-induced impairment of endothelial function and reparative angiogenesis after limb ischemia. Circulation 2011;123:282–291. 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, Dimmeler S. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science 2009;324:1710–1713. 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 Rooij E. Inhibition of miR-15 protects against cardiac ischemic injury. Circ Res 2012;110:71–81. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES, Zhang C. MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 2009;284: 29514–29525. van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stress-dependent cardiac growth and gene expression by a microRNA. Science 2007;316:575–579. Cardin S, Guasch E, Luo X, Naud P, Le Quang K, Shi Y, Tardif JC, Comtois P, Nattel S. Role for MicroRNA-21 in atrial profibrillatory fibrotic remodeling associated with experimental postinfarction heart failure. Circ Arrhythm Electrophysiol 2012;5:1027–1035. Shan ZX, Lin QX, Fu YH, Deng CY, Zhou ZL, Zhu JN, Liu XY, Zhang YY, Li Y, Lin SG, Yu XY. Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun 2009;381: 597–601. Bostjancic E, Zidar N, Stajner D, Glavac D. MicroRNA miR-1 is up-regulated in remote myocardium in patients with myocardial infarction. Folia Biol (Praha) 2010;56:27–31. van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN. Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci USA 2008;105:13027–13032. Qian L, Van Laake LW, Huang Y, Liu S, Wendland MF, Srivastava D. miR-24 inhibits apoptosis and represses Bim in mouse cardiomyocytes. J Exp Med 2011;208:549–560. 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 cardiac fibrosis and improved left ventricular compliance via the FBJ osteosarcoma oncogene/transforming growth factor-beta1 pathway. Circulation 2012;126:840–850. Bostjancic E, Zidar N, Glavac D. MicroRNA microarray expression profiling in human myocardial infarction. Dis Markers 2009;27:255–268.
43. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 1990;81:1161–1172. 44. Melman YF, Shah R, Das S. MicroRNAs in heart failure: is the picture becoming less miRky? Circ Heart Fail 2014;7:203–214. 45. Greco S, Fasanaro P, Castelvecchio S, D’Alessandra Y, Arcelli D, Di Donato M, Malavazos A, Capogrossi MC, Menicanti L, Martelli F. MicroRNA dysregulation in diabetic ischemic heart failure patients. Diabetes 2012;61:1633–1641. 46. Castelvecchio S, Menicanti L, Donato MD. Surgical ventricular restoration to reverse left ventricular remodeling. Curr Cardiol Rev 2010;6: 15–23. 47. Di Donato M, Menicanti L, Ranucci M, Castelvecchio S, de Vincentiis C, Salvia J, Yussuf T. Effects of surgical ventricular reconstruction on diastolic function at midterm follow-up. J Thorac Cardiovasc Surg 2010; 140:285–291.e1. 48. Menghini R, Casagrande V, Marino A, Marchetti V, Cardellini M, Stoehr R, Rizza S, Martelli E, Greco S, Mauriello A, Ippoliti A, Martelli F, Lauro R, Federici M. MiR-216a: a link between endothelial dysfunction and autophagy. Cell Death Dis 2014;5:e1029. 49. Dei Cas A, Khan SS, Butler J, Mentz RJ, Bonow RO, Avogaro A, Tschoepe D, Doehner W, Greene SJ, Senni M, Gheorghiade M, Fonarow GC. Impact of diabetes on epidemiology, treatment, and outcomes of patients with heart failure. JACC Heart Fail 2015;3:136–145. 50. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008;105:10513–10518. 51. Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, Galas DJ, Wang K. The microRNA spectrum in 12 body fluids. Clin Chem 2010;56: 1733–1741. 52. Voellenkle C, van Rooij J, Cappuzzello C, Greco S, Arcelli D, Di Vito L, Melillo G, Rigolini R, Costa E, Crea F. MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients. Physiol Genomics 2010;42:420–426. 53. D’Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, Rubino M, Carena MC, Spazzafumo L, De Simone M, Micheli B, Biglioli P, Achilli F, Martelli F, Maggiolini S, Marenzi G, Pompilio G, Capogrossi MC. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J 2010;31:2765–2773. 54. Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y, Iwai N. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem 2009;55: 1944–1949. 55. Liebetrau C, Mollmann H, Dorr O, Szardien S, Troidl C, Willmer M, Voss S, Gaede L, Rixe J, Rolf A, Hamm C, Nef H. Release kinetics of circulating muscle-enriched microRNAs in patients undergoing transcoronary ablation of septal hypertrophy. J Am Coll Cardiol 2013;62:992–998. 56. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW, Jing Q. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010;31:659–666. 57. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 2013;10:1003–1005. 58. Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, van der Meer AJ, Patick AK, Chen A, Zhou Y, Persson R, King BD, Kauppinen S, Levin AA, Hodges MR. Treatment of HCV infection by targeting microRNA. N Engl J Med 2013;368:1685–1694.
microRNAs in ischaemic cardiovascular diseases Simona Greco, Germana Zaccagnini, Christine Voellenkle, and Fabio Martelli* Molecular Cardiology Laboratory, IRCCS Policlinico San Donato, via Morandi 30, 20097 San Donato Milanese, Milan, Italy
KEYWORDS microRNA; Cardiovascular diseases; Ischaemia; Hypoxia
microRNAs (miRNAs) are non-coding RNA molecules that modulate the stability and/or the translational efficiency of specific messenger RNAs. They have been shown to play a regulatory role in most biological processes and their expression is disrupted in many cardiovascular diseases. This review describes studies performed at Policlinico San Donato-IRCCS in cell cultures, animal models, and patients, showing a penetrant role of miRNAs in cell response to hypoxia and in ischaemic cardiovascular diseases. These experiments indicate miRNA as an emerging class of therapeutic targets.
Introduction microRNAs (miRNAs) are 20–25 nucleotide-long RNAs that regulate gene expression mainly suppressing the stability and/or the translation of a specific set of target mRNAs (Figure 1). They are synthesized in the nucleus and, after their maturation and export to the cytoplasm, are loaded on the effector complex called ‘RNA-induced silencing complex’ (RISC).1 microRNAs bind to their targets via partial complementary pairing, and while the targeting rules are still object of intense research, a 5′ region of the miRNA, called ‘seed sequence’, seems to play a particularly important role.2,3 Each miRNA can target multiple mRNAs, and since each mRNA can be targeted by more than one miRNA, miRNAs constitute an intricate and pervasive network regulating most aspects of biology, including the cardiovascular functions.4,5 Indeed, miRNAs regulate crucial aspects of angiogenesis, endothelial and myocyte growth, cardiogenesis, contractility, and cardiac rhythm. Accordingly, the expression of specific miRNAs is altered in many cardiovascular diseases and genetically engineered mice showed that miRNA deregulation is necessary and sufficient for many heart and blood vessel physiopathological conditions.4–8 Another element that is placing miRNAs at the centre stage is their perspective use as prognostic/diagnostic biomarkers. Indeed, miRNAs are present in the peripheral blood, in the haematic cellular components, but also outside the cells, both in plasma and in serum.9
* Corresponding author. Tel: +39 02 52 774 533, Fax: +39 02 52 774 666, Email: [email protected]
This review is aimed at describing a series studies performed in a variety of experimental and clinical settings at Policlinico San Donato-IRCCS. We show a wide role of miRNAs in ischaemic and post-ischaemic cardiovascular diseases, placing our observations in the context of the current literature.
microRNAs and ischaemic cardiovascular diseases Hypoxia response Hypoxia is a pivotal pathogenetic factor of ischaemic cardiovascular diseases.10 To investigate miRNA regulation in endothelial cell cultures exposed to 1% oxygen, miRNA expression profile was determined using RNA-Sequencing, a highthroughput technique allowing the quantitative measurement of known and novel miRNA species.11 This analysis allowed the identification of more than 400 annotated miRNA species with a wide range of expression. Indeed, miR-21 and miR-126 alone totalled almost half of all miRNAs present in endothelial cells. A custom pipeline of analysis, combining bioinformatics and molecular biology tools, allowed the discovery and characterization of 18 new miRNA species. Interestingly, the expression of two of these novel miRNAs was significantly down-modulated by hypoxia, while miR-210 was significantly induced. Indeed, miR-210 is generally recognized as the prototypical hypoxamiR,7,12 being robustly induced by hypoxia-inducible factor l a (HIF1A), both in vitro and in vivo. 7,13–15
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: [email protected]
E32
S. Greco et al.
Figure 1 microRNA biogenesis and function. microRNAs originate from the pri-miRNA, which is cleaved in the nucleus by the complex Drosha/DGCR8 to generate a 70–100 nucleotide-long hairpin-shaped precursor named pre-miRNA. The pre-miRNA is shuttled to the cytoplasm by Exportin 5, through nucleoporin (NPC) and further processed by the ribonuclease III Dicer, forming the mature 22-nt miRNA duplex. One strand from the miRNA duplex, the mature miRNA, is then incorporated into the effector complex, RNA-induced silencing complex, which recognizes specific target mRNAs through imperfect base-pairing, and down-regulates their expression by mRNA exonucleolitic degradation and/or inhibition of translation.
The identification of miRNA targets is instrumental for understanding their role.2,3 Several valid bioinformatics tools have been developed to this aim. However, all of them are burdened by a high number of false positives and negatives.8 Thus, to identify miR-210 targets, we took an experimental approach, using a combination of proteomics, transcriptomics, immunopurification of the RISC–miRNA complex, and molecular biology. This approach allowed us to identify 33 novel targets, both canonical7,16 and non-canonical, i.e. not requiring a seed region for target binding.17 The analysis of the targets identified by us and other authors18 suggested a role of miR-210 in cell migration and angiogenesis.7,16 Indeed, miR-210 promoted the formation of capillary-like structures in endothelial cells plated on Matrigel, as much as endothelial cell migration towards a vascular endothelial growth factor gradient.13 Both events required Ephrin-A3 (EFNA3) direct targeting by miR-21013 (Figure 2). miR-210 stimulates angiogenesis also through other mechanisms, both direct
and indirect. Indeed, increased angiogenic factor signalling has been observed in miR-210-expressing cells.19–21 Accordingly, increased tissue perfusion and capillary density have been found in mouse ischaemic muscles upon injection of umbilical cord blood CD34+ cells expressing miR-210.22 Other targets, such as ISCU1/2, COX10, NDUFA4, and FECH, indicated miR-210 involvement in mitochondrial metabolism. Indeed, miR-210 contributes to the oxidative phosphorylation decline observed in hypoxia and its repression triggers mitochondrial dysfunction and oxidative stress.23
Peripheral artery diseases Peripheral artery disease is commonly caused by an impairment of blood supply in the arteries of the leg, and is mostly caused by atherosclerosis and often results in critical limb ischaemia.24
microRNAs in ischaemic cardiovascular diseases
Figure 2 miR-210 and the hypoxia/ischaemia response. Cell culture in hypoxic conditions or acute tissue isachemia can both trigger the activation of HIF-1a, which in turn activates the expression of miR-210. miR-210 attenuates mitochondrial metabolism and reactive oxygen species (ROS) generation by the electron transport chain, thus preventing cell apoptosis and necrosis. Moreover, Ephrin-3 (EFNA3) is a direct target of miR-210, and its inhibition is necessary for miR-210 to stimulate capillary-like tubes formation (tubulogenesis) and angiogenesis.
Our group investigated the regulation and role of miRNAs in a mouse model of acute hindlimb ischaemia, where the femoral artery is dissected, causing severe ischaemia in the distal part of the affected limb.25 After acute ischaemic injury, the affected muscles undergo distinct phases, consisting of necrosis/degeneration and inflammation in the first few days, regeneration/repair from 2 to 4 weeks, and fibrosis at 2 or 3 weeks post-injury.26 Using this model, our group determined that the expression levels of some miRNAs decreased early at 2 days after ischaemia (miR-l, miR-29c, and miR-135a) correlating with the necrosis/degeneration phase. Inflammatory miRNAs miR-222 and miR-223 were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. Other miRNAs (miR-31, miR-34c, miR-335, miR-449, miR-494, and miR-206) peaked at 14 days correlating with the regeneration phase. The isolation of damaged, nondamaged, and regenerating myofibres corroborated these results.27 Our group also investigated the regulation and role of miR-210 in the mouse model of acute hindlimb ischaemia25 (Figure 2). To this end, miR-210 was blocked by the systemic injection of chemically modified Locked Nucleic Acid complementary oligonucleotides (antimiR-210). As expected, after femoral artery dissection, miR-210 expression was induced in the ischaemic gastrocnemius muscles, while the administration of antimiR-210 caused a marked
E33
decrease in miR-210 and increase in its targets. Histological evaluation of acute tissue damage upon miR-210 inhibition showed an increase in apoptosis and necrosis as well as a decrease in capillary density and residual limb perfusion. Transcriptomic data after ischaemia pointed out the deregulation of mitochondrial function and redox balance in antimiR-210-treated mice. These data are in keeping with miR-210 role as repressor of oxidative phosphorylation observed in vitro.23 Accordingly, following miR-210 blocking, mitochondrial metabolism was decreased and oxidative stress induced, both in vitro 23,28 and in vivo.25 Furthermore, oxidative stress-resistant p66Shc-null mice29 displayed decreased tissue damage following ischaemia when miR-210 was blocked.25 This study demonstrates that miR-210 has cytoprotective effects in the skeletal muscle, regulating oxidative metabolism and oxidative stress, thus being a crucial element of the adaptive mechanisms to acute ischaemia. Interestingly, miR-210 also seems to play a role in ischaemic ulcers. To elucidate how ischaemia complicates wound closure, Biswas et al.30 used a mouse model of wound healing. In ischaemic wounds, Hif1-a and miR-210 were strongly induced. Accordingly, miR-210 target E2F3, a cell cycle positive regulator, was down-regulated, correlating with decreased proliferation of keratinocytes. Considering that angiogenesis is also a key component of the wound healing process, it remains to be determined how the pro-angiogenic function of miR-210 integrates with its antiproliferative function in keratinocytes. microRNAs also contribute to diabetes mellitus-induced impairment of reparative angiogenesis after limb ischaemia. Indeed, miR-503 expression was increased in ischaemic muscles and in endothelial cells of a mouse model of diabetes mellitus, upon femoral artery dissection.31 More importantly, miR-503 blocking corrected diabetes-induced impairment of post-ischaemic angiogenesis and blood flow recovery. This does not seem limited to experimental animal models, since miR-503 expression was significantly increased in skeletal muscles of diabetic patients affected by critical limb ischaemia, while the expression of miR-503 targets decreased, suggesting miR-503 as a possible therapeutic target in diabetic patients.
Myocardial infarction Myocardial ischaemic injury arises from severe impairment of the coronary blood supply, usually caused by thrombosis or other acute alterations of coronary atherosclerotic plaques. After myocardial ischaemia, miR-l, miR-15, miR21, miR-92a, miR-208, and miR-126 expression levels increased in animal models of myocardial infarction,32–37 and in patients, who died after MI.38 In the border zone of myocardial infarction, in mice, miR-29,39 miR-24,40 and miR-101a/b41 levels were decreased. As expected, given its prominent role as hypoxamir, also miR-210 has been shown to be up-regulated following myocardial infarction in humans.42 The therapeutic potential of miR-210 has been shown in a mouse model of myocardial infarction.19 Upon hypoxic stimulation of a cardiomyocyte cell line, miR-210 levels significantly increased, promoting the release of several angiogenic factors, inhibiting
E34
S. Greco et al.
caspase activity, and preventing cell apoptosis, in keeping with the data obtained in endothelial cells. In in vivo experiments, after mid-left anterior descending artery ligation, miR-210 overexpression using a minicircle vector attenuated the decrease in left ventricle fractional shortening. Histological evaluation of explanted hearts demonstrated the reduction in both infarction size and apoptosis, as well as increased capillary density for the miR-210treated group, corroborating the imaging data.19 Thus, miR-210 can improve heart function by up-regulating angiogenesis and inhibiting apoptosis, suggesting that miR-210 delivery through non-viral minicircle has a therapeutic potential for treatment of ischaemic heart disease.
Heart failure Acute myocardial infarction triggers several structural changes of both the infarcted and non-infarcted regions of the ventricle, causing ventricle remodelling, chamber dilatation, and eventually, heart failure. This remodelling can importantly affect the function of the ventricle and the prognosis for survival.43 Several studies described miRNAs deregulation in heart failure. These data derive from studies in cell culture models of hypertrophy, in animal models of heart pathologic remodelling, as well as in humans.44 However, these human studies relied mostly on end-stage patients, thus lacking important information on early-stage determinants of the disease. To fill this knowledge gap, our group analysed the miRNA expression profiles of left ventricle biopsies from heart failure patients affected by non-end-stage dilated ischaemic cardiomyopathy.45 These biopsies were harvested in the remote nonischaemic area of the ventricle of patients undergoing ventricular restoration procedure by the surgical team of Lorenzo Menicanti at Policlinico San Donato.46,47 We found that 17 miRNAs were modulated in heart failure patients when compared with control subjects. In particular, miR-216a, an endothelial senescence-associated miRNA,48 was strongly increased in failing hearts and negatively correlated with left ventricle ejection fraction (Figure 3). Diabetes mellitus is a very serious co-morbidity factor in heart failure, being associated with increased morbidity and mortality.49 Comparing diabetic and nondiabetic heart failure patients, we found that six miRNAs were differently expressed: miR-34b, miR-34c, miR-199b, miR-210, miR-650, and miR-223. Interestingly, miR-199b and miR-210 were modulated by hypoxia and high glucose in both mouse neonatal cardiomyocytes and human endothelial cells. Accordingly, the whole hypoxia-inducible factor pathway was activated, indicating that diabetes mellitus is triggering a pseudo-hypoxic state also in the non-infarcted, vital myocardium.45
microRNAs as biomarkers In addition to their involvement in pathogenesis mechanisms, miRNAs are also attractive biomarkers. Several studies in different disciplines suggest that classifications based on miRNA profiling are more specific and sensitive compared with methods based on mRNA profiling.50,51
Figure 3 miR-216a in the senescence/autophagy network. miR-216a expression levels are induced in heart failure and are also increased in aged/ senescent cells and atherosclerotic plaques. The autophagy-related genes, ATG5 and BECN, are repressed by miR-216a, thus preventing autophagy.
Unlike oncology, biopsies are only rarely indicated for cardiovascular diseases. Thus, the attention of the researchers has turned to peripheral blood that is accessible with a minimally invasive procedure. For instance, our group measured the miRNAs expressed by peripheral blood mononuclear cells (PBMCs) of heart failure patients affected by ischaemic and non-ischaemic dilated cardiomyopathy.52 We identified three miRNAs, miR-107, miR-139, and miR-142–5p, displaying decreased levels in both cardiomyopathies. Other miRNAs were deregulated in only one of the heart failure patient groups. Interestingly, many of these miRNAs are also deregulated in human and/or mouse failing hearts.44 As previously mentioned, some miRNAs are released from the cells of origin and can be measured in bodily fluids.50,51 Indeed, extracellular miRNAs are remarkably stable even outside the cell, where they are protected from degradation by their association with microvescicles or exosomes, high-density lipoproteins, or protein complexes, such as the RISC. Interestingly, disease-specific miRNA signatures can be identified in many physiopathological conditions and plasma or serum miRNAs have been proposed as biomarkers for diagnosis and prognosis of a variety of cardiovascular diseases.9 In particular, several promising miRNAs candidates have emerged so far for myocardial infarction, including miR-1, miR-133a, miR-133b, miR-208a, and miR-499.5,9,53 These miRNAs are all highly enriched in cardiomyocytes and their increase is most likely the result of massive cardiomyocyte necrosis and release into the bloodstream. Particularly interesting is the fact that miR-1, miR-133a, and miR-208a increased continuously during the first 4 h after MI, and before cardiac troponin T could be detected.54,55 Among these, miR-208 is noteworthy because it is cardiacspecific, and because it is absent in healthy subjects and patients without acute myocardial infarction.54,56 However, several conflicting results are present in the literature,
microRNAs in ischaemic cardiovascular diseases
most likely for the small size of most studies and because of the lack of standardized detection and normalization techniques. In this respect, new PCR techniques, such as digital PCR,57 will likely have a positive impact. Additionally, it should be kept in mind that the detection of protein humoral markers is very rapid and only requires standard laboratory equipment, while the detection of nucleic acids is still comparatively slow, requiring molecular biology equipment and expertise.
Concluding remarks Although the clinical value of miRNAs is only starting to be appreciated, it is clear that miRNAs seem very attractive therapeutic targets, since they can be easily synthesized, chemically modified for increased stability, and delivered via functionalized biomaterials. microRNAs could be used as therapeutic tools when increasing the levels of a specific miRNA is desirable. Conversely, in certain situations, the aim could be neutralizing pathological miRNAs, for instance, using complementary oligonucleotides. While no human experimentation targeting miRNAs in cardiovascular diseases has been reported so far, a phase I clinical trial for cancer therapy is ongoing (ClinicalTrials.gov Identifier: NCT01829971) and a phase IIa clinical trial for hepatitis C therapy (ClinicalTrials.gov Identifier: NCT01200420) has been completed successfully,58 showing the viability of miRNAs as therapeutic targets. Many hurdles must be overcome to achieve clinically feasible strategies for miRNA-based therapies in heart and vascular diseases, including the optimization of delivery tools and of the therapeutic molecule chemistry. Even more importantly however, given the intrinsically pleiotropic functions of miRNAs, we must first understand in detail the actions of the target miRNAs in each physio-pathological context, before progressing to clinical application.
Funding This work was supported by Ministero della Salute, Fondazione Cariplo (grant #2013-0887), Telethon-Italy (grant #GGP14092), and AFM Telethon (grant #18477). Conflict of interest: none declared.
References 1. Hammond SM. An overview of microRNAs. Adv Drug Deliv Rev 2015;87: 3–14. 2. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136:215–233. 3. Thomson DW, Bracken CP, Goodall GJ. Experimental strategies for microRNA target identification. Nucleic Acids Res 2011;39: 6845–6853. 4. Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature 2011;469:336–342. 5. Condorelli G, Latronico MV, Cavarretta E. microRNAs in cardiovascular diseases: current knowledge and the road ahead. J Am Coll Cardiol 2014;63:2177–2187.
E35 6. Greco S, Gorospe M, Martelli F. Noncoding RNA in age-related cardiovascular diseases. J Mol Cell Cardiol 2015;83:142–155. 7. Greco S, Gaetano C, Martelli F. HypoxamiR regulation and function in ischemic cardiovascular diseases. Antioxid Redox Signal 2014;21: 1202–1219. 8. Fasanaro P, Greco S, Ivan M, Capogrossi MC, Martelli F. microRNA: emerging therapeutic targets in acute ischemic diseases. Pharmacol Ther 2010;125:92–104. 9. Di Stefano V, Zaccagnini G, Capogrossi MC, Martelli F. microRNAs as peripheral blood biomarkers of cardiovascular disease. Vascul Pharmacol 2011;55:111–118. 10. Semenza GL. Hypoxia-inducible factor 1 and cardiovascular disease. Annu Rev Physiol 2014;76:39–56. 11. Voellenkle C, Rooij J, Guffanti A, Brini E, Fasanaro P, Isaia E, Croft L, David M, Capogrossi MC, Moles A, Felsani A, Martelli F. Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs. RNA 2012;18:472–484. 12. Devlin C, Greco S, Martelli F, Ivan M. miR-210: more than a silent player in hypoxia. IUBMB Life 2011;63:94–100. 13. Fasanaro P, D’Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, Capogrossi MC, Martelli F. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 2008;283:15878–15883. 14. Kulshreshtha R, Ferracin M, Negrini M, Calin GA, Davuluri RV, Ivan M. Regulation of microRNA expression: the hypoxic component. Cell Cycle 2007;6:1426–1431. 15. Kulshreshtha R, Ferracin M, Wojcik SE, Garzon R, Alder H, Agosto-Perez FJ, Davuluri R, Liu CG, Croce CM, Negrini M, Calin GA, Ivan M. A microRNA signature of hypoxia. Mol Cell Biol 2007;27: 1859–1867. 16. Fasanaro P, Greco S, Lorenzi M, Pescatori M, Brioschi M, Kulshreshtha R, Banfi C, Stubbs A, Calin GA, Ivan M. An integrated approach for experimental target identification of hypoxia-induced miR-210. J Biol Chem 2009;284:35134–35143. 17. Fasanaro P, Romani S, Voellenkle C, Maimone B, Capogrossi MC, Martelli F. ROD1 is a seedless target gene of hypoxia-induced miR-210. PLoS One 2012;7:e44651. 18. Bertero T, Robbe-Sermesant K, Le Brigand K, Ponzio G, Pottier N, Rezzonico R, Mazure NM, Barbry P, Mari B. MicroRNA target identification: lessons from hypoxamiRs. Antioxid Redox Signal 2014;21: 1249–1268. 19. Hu S, Huang M, Li Z, Jia F, Ghosh Z, Lijkwan MA, Fasanaro P, Sun N, Wang X, Martelli F, Robbins RC, Wu JC. MicroRNA-210 as a novel therapy for treatment of ischemic heart disease. Circulation 2010; 122(11 Suppl):S124–S131. 20. Liu W, Wen Y, Bi P, Lai X, Liu XS, Liu X, Kuang S. Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation. Development 2012;139:2857–2865. 21. Liu SC, Chuang SM, Hsu CJ, Tsai CH, Wang SW, Tang CH. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression. Cell Death Dis 2014;5:e1485. 22. Alaiti MA, Ishikawa M, Masuda H, Simon DI, Jain MK, Asahara T, Costa MA. Up-regulation of miR-210 by vascular endothelial growth factor in ex vivo expanded CD34+ cells enhances cell-mediated angiogenesis. J Cell Mol Med 2012;16:2413–2421. 23. Cottrill KA, Chan SY, Loscalzo J. Hypoxamirs and mitochondrial metabolism. Antioxid Redox Signal 2014;21:1189–1201. 24. Peach G, Griffin M, Jones KG, Thompson MM, Hinchliffe RJ. Diagnosis and management of peripheral arterial disease. BMJ 2012;345:e5208. 25. Zaccagnini G, Maimone B, Di Stefano V, Fasanaro P, Greco S, Perfetti A, Capogrossi MC, Gaetano C, Martelli F. Hypoxia-induced miR-210 modulates tissue response to acute peripheral ischemia. Antioxid Redox Signal 2014;21:1177–1188. 26. Huard J, Li Y, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 2002;84-A:822–832. 27. Greco S, De Simone M, Colussi C, Zaccagnini G, Fasanaro P, Pescatori M, Cardani R, Perbellini R, Isaia E, Sale P, Meola G, Capogrossi MC, Gaetano C, Martelli F. Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia. FASEB J 2009;23:3335–3346. 28. Cicchillitti L, Di Stefano V, Isaia E, Crimaldi L, Fasanaro P, Ambrosino V, Antonini A, Capogrossi MC, Gaetano C, Piaggio G, Martelli F.
E36
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
S. Greco et al. Hypoxia-inducible factor 1-alpha induces miR-210 in normoxic differentiating myoblasts. J Biol Chem 2012;287:44761–44771. Zaccagnini G, Martelli F, Fasanaro P, Magenta A, Gaetano C, Di Carlo A, Biglioli P, Giorgio M, Martin-Padura I, Pelicci PG, Capogrossi MC. p66ShcA modulates tissue response to hindlimb ischemia. Circulation 2004;109:2917–2923. Biswas S, Roy S, Banerjee J, Hussain SR, Khanna S, Meenakshisundaram G, Kuppusamy P, Friedman A, Sen CK. Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds. Proc Natl Acad Sci USA 2010;107:6976–6981. Caporali A, Meloni M, Vollenkle C, Bonci D, Sala-Newby GB, Addis R, Spinetti G, Losa S, Masson R, Baker AH, Agami R, le Sage C, Condorelli G, Madeddu P, Martelli F, Emanueli C. Deregulation of microRNA-503 contributes to diabetes mellitus-induced impairment of endothelial function and reparative angiogenesis after limb ischemia. Circulation 2011;123:282–291. 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, Dimmeler S. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science 2009;324:1710–1713. 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 Rooij E. Inhibition of miR-15 protects against cardiac ischemic injury. Circ Res 2012;110:71–81. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES, Zhang C. MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 2009;284: 29514–29525. van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stress-dependent cardiac growth and gene expression by a microRNA. Science 2007;316:575–579. Cardin S, Guasch E, Luo X, Naud P, Le Quang K, Shi Y, Tardif JC, Comtois P, Nattel S. Role for MicroRNA-21 in atrial profibrillatory fibrotic remodeling associated with experimental postinfarction heart failure. Circ Arrhythm Electrophysiol 2012;5:1027–1035. Shan ZX, Lin QX, Fu YH, Deng CY, Zhou ZL, Zhu JN, Liu XY, Zhang YY, Li Y, Lin SG, Yu XY. Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun 2009;381: 597–601. Bostjancic E, Zidar N, Stajner D, Glavac D. MicroRNA miR-1 is up-regulated in remote myocardium in patients with myocardial infarction. Folia Biol (Praha) 2010;56:27–31. van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN. Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci USA 2008;105:13027–13032. Qian L, Van Laake LW, Huang Y, Liu S, Wendland MF, Srivastava D. miR-24 inhibits apoptosis and represses Bim in mouse cardiomyocytes. J Exp Med 2011;208:549–560. 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 cardiac fibrosis and improved left ventricular compliance via the FBJ osteosarcoma oncogene/transforming growth factor-beta1 pathway. Circulation 2012;126:840–850. Bostjancic E, Zidar N, Glavac D. MicroRNA microarray expression profiling in human myocardial infarction. Dis Markers 2009;27:255–268.
43. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation 1990;81:1161–1172. 44. Melman YF, Shah R, Das S. MicroRNAs in heart failure: is the picture becoming less miRky? Circ Heart Fail 2014;7:203–214. 45. Greco S, Fasanaro P, Castelvecchio S, D’Alessandra Y, Arcelli D, Di Donato M, Malavazos A, Capogrossi MC, Menicanti L, Martelli F. MicroRNA dysregulation in diabetic ischemic heart failure patients. Diabetes 2012;61:1633–1641. 46. Castelvecchio S, Menicanti L, Donato MD. Surgical ventricular restoration to reverse left ventricular remodeling. Curr Cardiol Rev 2010;6: 15–23. 47. Di Donato M, Menicanti L, Ranucci M, Castelvecchio S, de Vincentiis C, Salvia J, Yussuf T. Effects of surgical ventricular reconstruction on diastolic function at midterm follow-up. J Thorac Cardiovasc Surg 2010; 140:285–291.e1. 48. Menghini R, Casagrande V, Marino A, Marchetti V, Cardellini M, Stoehr R, Rizza S, Martelli E, Greco S, Mauriello A, Ippoliti A, Martelli F, Lauro R, Federici M. MiR-216a: a link between endothelial dysfunction and autophagy. Cell Death Dis 2014;5:e1029. 49. Dei Cas A, Khan SS, Butler J, Mentz RJ, Bonow RO, Avogaro A, Tschoepe D, Doehner W, Greene SJ, Senni M, Gheorghiade M, Fonarow GC. Impact of diabetes on epidemiology, treatment, and outcomes of patients with heart failure. JACC Heart Fail 2015;3:136–145. 50. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008;105:10513–10518. 51. Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, Galas DJ, Wang K. The microRNA spectrum in 12 body fluids. Clin Chem 2010;56: 1733–1741. 52. Voellenkle C, van Rooij J, Cappuzzello C, Greco S, Arcelli D, Di Vito L, Melillo G, Rigolini R, Costa E, Crea F. MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients. Physiol Genomics 2010;42:420–426. 53. D’Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, Rubino M, Carena MC, Spazzafumo L, De Simone M, Micheli B, Biglioli P, Achilli F, Martelli F, Maggiolini S, Marenzi G, Pompilio G, Capogrossi MC. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J 2010;31:2765–2773. 54. Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y, Iwai N. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem 2009;55: 1944–1949. 55. Liebetrau C, Mollmann H, Dorr O, Szardien S, Troidl C, Willmer M, Voss S, Gaede L, Rixe J, Rolf A, Hamm C, Nef H. Release kinetics of circulating muscle-enriched microRNAs in patients undergoing transcoronary ablation of septal hypertrophy. J Am Coll Cardiol 2013;62:992–998. 56. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW, Jing Q. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010;31:659–666. 57. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 2013;10:1003–1005. 58. Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, Patel K, van der Meer AJ, Patick AK, Chen A, Zhou Y, Persson R, King BD, Kauppinen S, Levin AA, Hodges MR. Treatment of HCV infection by targeting microRNA. N Engl J Med 2013;368:1685–1694.
