REVIEWS MicroRNAs in platelet function and cardiovascular disease David D. McManus and Jane E. Freedman Abstract | Cardiovascular disease—a leading cause of morbidity and mortality among adults—is strongly influenced by platelet function through acute thrombotic and atherogenic mechanisms. Pathways that regulate platelet activity and lead to coronary occlusion are central to the pathogenesis of acute coronary syndromes. Platelet activation contributes to other thrombotic disorders and cardiovascular diseases, including stroke. Anucleate platelets are now understood to contain transcripts that might relate to other physiological or pathophysiological conditions, be released into the circulation, participate in protein formation, and engage in horizontal RNA transfer to other vascular cells. These platelet transcripts include microRNAs (miRNAs), which are small noncoding RNAs involved in many molecular processes, most notably regulation of gene expression. In platelets, these noncoding RNAs seem to participate in vascular homeostasis, inflammation, and platelet function. In addition, levels of platelet miRNAs in the circulation are associated with the presence or extent of cardiovascular diseases, such as atrial fibrillation and peripheral vascular disease. Accumulating data suggest mechanistic roles for platelet-derived miRNAs in haemostasis, thrombosis, and unstable coronary syndromes. In addition, evidence suggests that platelet-derived miRNAs might have important roles as biomarkers of cardiovascular disease susceptibility, prognosis, or treatment. McManus, D. D. & Freedman, J. E. Nat. Rev. Cardiol. advance online publication 7 July 2015; doi:10.1038/nrcardio.2015.101
Introduction
Cardiovascular disease (CVD) continues to be the major cause of morbidity and mortality in North America and is a growing problem in many industrialized and developing countries worldwide.1–3 CVD accounts for over onethird of deaths in the USA annually,1–3 and is the cause of more deaths each year than cancer, respiratory disease, and accidents combined.2 Platelets have long been known to have important roles in thrombosis, arteriosclerosis, and angiogenesis,4,5 but in the past decade, anucleate platelets have been shown to contain transcripts and the necessary molecular machinery to conduct translation. Subsequently, particular small RNAs, including microRNAs (miRNAs), have been found to be present inside mature platelets, and mRNAs and miRNAs have been shown to regulate gene expression in platelets. In this Review, we outline the role of platelets in CVD, and discuss miRNAs and their roles in platelet development, function, and CVD.
Platelets and cardiovascular disease Cardiology Division, Department of Medicine, University of Massachusetts Medical School, AS7‑1051, 368 Plantation Street, Worcester, MA 01605‑4319, USA (D.D.M., J.E.F.). Correspondence to: J.E.F. jane.freedman@ umassmed.edu
Platelets have a central role in CVD, particularly in acute coronary syndrome and stroke, contributing to the development of acute thrombotic events.6 Superficial and deep intimal injuries disturb the intact endothelium and disrupt the production of antiplatelet agents, including nitric oxide and prostacyclin. With exposure of collagen to the circulation, platelets adhere to the subendothelium, both directly and via von Willebrand factor.7 Platelets become Competing interests The authors declare no competing interests.
activated via soluble receptor-mediated stimulants, such as thrombin, ADP, and thromboxane A2, thereby enhancing the thrombogenicity of activated platelets. Platelet aggregation occurs when the glycoprotein IIb/IIIa receptor binds adhesive proteins. Platelets can release many cytokines and signalling molecules that can lead to a cascade of further platelet activation, thrombosis, and vasoconstriction. Activated platelets form a growing coronary thrombus with concomitant propagation of fibrin.8 Although these processes are in place to provide haemostasis in the context of vascular injury and allow for wound healing, they can induce vessel occlusion when they occur in the setting of coronary, peripheral arterial, or cerebrovascular plaque rupture, thereby contributing to an acute coronary syndrome or stroke. The specific mechanisms by which platelet function is regulated, particularly in the setting of vessel occlusion, have been reassessed in the past decade, with improved understanding of novel pathways that might alter thrombosis.
Platelets and transcripts
Appreciation of novel pathways regulating thrombosis has been growing, including the observation that platelets contain a variety of transcripts. Platelets are anucleate, and their cytoplasmic RNA has long been considered to be residual transcripts of their forming cell, the megakaryocyte. During platelet biogenesis, release of platelets of uncertain heterogeneity occurs, and these cells circulate for 7–10 days in humans, after which they are cleared in the spleen.4,5 Although lacking nuclei, platelets contain
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REVIEWS Key points ■■ Platelet function has an important role in mediating particular common forms of cardiovascular disease, such as acute coronary syndromes and stroke ■■ Although they lack nuclei, platelets contain RNAs, including small noncoding RNAs such as microRNAs, and the necessary machinery to perform translation ■■ Data suggest that microRNAs can influence platelet functions, including thrombosis, atherosclerosis, and angiogenesis ■■ Platelet RNA can also be transferred to other vascular cells, but further information about the role of microRNA transfer is needed
all the necessary components to perform translation in a signal-dependent manner,9 including functioning ribosomes.10–12 Investigators in several studies have used microarray and RNA sequencing to define the transcripts contained in platelets. Current estimates of the number of transcripts in platelets range from 1,500 to 9,500 in healthy human platelets,13,14 with notable differences in the number and types of transcripts in human compared with murine platelets—human platelets have more varied transcripts including those associated with surface receptors.11,12,15 The range in the number of reported transcripts might be related to platelet age, size, or method of measurement. Variable association between platelet transcripts and prote omics has been reported.16,17 Importantly, human platelets contain several forms of small RNAs, with miRNAs being the most abundant and comprising 80% of all small RNAs in platelets.18 These miRNA–mRNAs can be coexpressed.19
miRNAs
Small RNAs, including miRNAs, are important components of an evolutionarily conserved system of RNA-based gene regulation in eukaryotes.20 Small RNAs are involved in many molecular processes, including regulation of gene expression.21 Animal genomes contain an abundance of small genes that produce regulatory RNAs of ~22 nucleotides in length.22 These miRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread, suggesting that they might participate in a wide range of critical genetic regulatory pathways across species. miRNAs interfere with the expression of mRNA encoding factors that control developmental timing, stem-cell maintenance, and other physiological processes in animals.21 Research into small noncoding RNAs has fundamentally transformed our understanding of gene regulatory networks, especially at the post-transcriptional level.23,24 miRNAs are initially transcribed as long primary miRNAs (pri-miRNAs), which are processed by the RNase III enzyme Drosha (also known as ribonuclease 3) to generate stem–loop precursor miRNAs (pre-miRNAs) of ~70 nucleotides in length.21 These precursors are exported into the cytoplasm by exportin‑5 and, sub sequently, the cytoplasmic endoribonuclease Dicer cleaves pre-miRNA to release mature miRNA.22 Binding of miRNA to an mRNA with Argonaute proteins inhibits protein translation either directly or by promoting mRNA destruction. miRNAs regulate temporal transitions in gene expression associated with cell-fate progression and differentiation throughout animal development.22 By regulating cell-fate choices and transitions between pluripotency and differentiation, miRNAs help to orchestrate developmental
events in growing animals and have a role in tissue homeo stasis with relevance to disease states.24 Core areas of uncertainty remain regarding miRNA–mRNA target identification and miRNA target effects. However, new methods for validating miRNA–mRNA–Argonaute‑2 interactions in cells have been developed, including measuring gene expression after modulating miRNA expression, degradome sequencing, biotin-linked chroma tography, HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation), and CLASH (a variant of the HITS-CLIP method).25
miRNAs as biomarkers Some miRNAs are expressed in a cell-specific manner. miRNAs are detectable in plasma, where they can be contained in microparticles or transported by lipoproteins.26–28 They are also found in circulating blood cells, and release of miRNAs into the circulation has been linked to cellular damage, such as cardiomyocyte injury, or active processes, including secretion. miRNAs seem to be stable in the circulation, and quantifiable with a high degree of accuracy. Even at low abundance, miRNAs are comprised of nucleic acids with sequences that can often be easily amplified, making them ideal biomarkers of disease.29 Moreover, and in contrast to many other cellular disease mediators, drugs can target miRNAs. Given that miRNAs promote homeostatic mechanisms and can transiently modulate disease pathways, miRNA-based therapeutics would theoretically enable modulation of the cardiac stress response in a unique and powerful manner.30,31 miRNAs in cardiovascular disease miRNAs have important roles in cardiac development, including stem-cell differentiation and proliferation of cardiomyocytes in the embryo; however, they are also important in susceptibility to CVD (Table 1).26 miRNAs in cardiac tissue show dynamic changes in several forms of heart disease, suggesting that they have important roles in regulating an acute injury response. For example, animal and human studies have clearly demonstrated that cardiac miRNAs have critical roles in regulating important pathophysiological processes underlying heart diseases, including electrical remodelling (miR‑1, miR‑21, miR‑133, and miR‑328), cardiac fibrosis (miR‑21 and miR‑29), hypertrophic structural remodel ling (miR‑208), and cardiac ischaemia–reperfusion injury (miR‑150 and miR‑320).26,32–37 Circulating miRNAs in whole blood and plasma have also been associated with various acute and chronic forms of CVD, including atrial fibrillation, coronary artery disease, myocardial infarction, heart failure, vascular disease, and cardiac death.31,34,36–41 miRNAs can be released or secreted by cardiomyocytes, endothelial cells, and fibroblasts, and the release of unique miRNA into the circulation in the context of myocardial injury suggests process specificity.39 However, most of these studies must be seen as preliminary, owing to either small numbers of miRNAs measured or limited numbers of patients included. Transcoronary miRNA gradients exist during an acute cardiac injury such as myocardial infarction or after
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REVIEWS Table 1 | Roles of microRNAs expressed in platelets and associated with CVD microRNA
Target genes and functional effects
Associated phenotypes
miR‑30c
Represses CTGF expression; matrix remodelling
Ventricular fibrosis, heart failure, STEMI/non-STEMI82
miR‑126
Regulates CXCL12, PIK3R2, and SPRED1; angiogenesis and vascular integrity
Regulates myocardial angiogenesis, platelet levels altered by aspirin administration63
miR‑150
Regulates EGR2, MYB, and P2RX7; H2O2-induced cardiac cell death, platelet biogenesis
Atherosclerosis, cardiac hypertrophy, heart failure, myocardial infarction, myocardial ischaemia–reperfusion injury, and platelet activation79
miR‑197
Regulates SERTAD4; functional effects unknown
Plasma level reduced in patients with myocardial infarction77 and associated with metabolic syndrome83
miR‑223
Regulates SLC2A4; insulin resistance
Associated with myocyte hypertrophy, ventricular remodelling, myocardial infarction, and platelet activation84
miR‑328
Regulates CACNA1C and CACNB1; encode cardiac L‑type calcium channels
Atrial fibrillation, electrical remodelling35
miR‑342
Regulates AKT1; glucose metabolism, apoptosis, cell proliferation
Inflammatory stimulation of macrophages63
Abbreviations: CVD, cardiovascular disease; STEMI, ST-segment elevation myocardial infarction.
septal ablation, and whole-blood levels of miRNAs (such as miR‑133a and miR‑499) can rise and fall hours after an acute coronary event, reinforcing their utility as biomarkers of disease.39,42 Whether the release of miRNAs into the circulation in the aforementioned CVD states is a passive or active process is unknown, but data suggest that miRNAs can be delivered to recipient cells and regulate protein expression.43 The distribution and function of miRNAs across circulating cell types and in plasma, and the effect of circulating miRNAs on other organ systems, such as the kidneys, are also presently unknown. However, evidence has linked cardiac remodelling in the context of heart failure to crosstalk between cardiomyocytes and fibroblasts via miRNA-enriched exosomes.44
miRNAs and megakaryopoiesis Megakaryocytes are derived from common myeloid progenitors, with platelets formed in turn from megakaryocytes. Most platelet-derived miRNAs are presumed to have been formed in the megakaryocyte (Figure 1). Of note, each mRNA has multiple miRNAs that bind and, given that each miRNA binds multiple mRNA targets, many miRNAs will regulate pathways in a ‘rheostat-like’ manner. miR‑155 is a common small RNA found in haemato poietic stem cells and implicated in platelet development. Specifically, upregulation of miR‑155 inhibits haemato poietic stem-cell differentiation into megakaryocytes, as shown by the transplantation of haematopoietic stem cells overexpressing miR‑155 into mice whose bone marrow had been depleted, which resulted in reduced numbers of megakaryocytes.45 Overexpression of miR‑150, by contrast, shifts megakaryocyte–erythroid precursors towards megakaryocyte differentiation both in vitro and in vivo, and is upregulated by thrombopoeitin.46 Megakaryopoiesis is also modified by miR‑146a, although effects differ according to experimental conditions.47,48 Additionally, several investigators have shown that patients with myeloproliferative neoplasms have upregulation or downregulation of miR‑10a, miR‑26b, miR‑148a, and miR‑490‑5p, further supporting the hypothesis that miRNAs have an important role in normal megakaryocyte development.49,50
miRNAs in platelets Platelets are known to have miRNAs as well as Dicer and Argonaute protein complexes, thereby enabling the processing of pre‑RNAs to miRNAs and control of reporter transcripts. On the basis of transcriptomic profiling, the vast majority of common small RNAs in the platelet are miRNAs.51 Profiling from healthy human platelets has characterized 492 platelet miRNAs and 40 novel miRNAs, with the most abundant being members of the let‑7 family.51 From analyses of platelet transcripts derived from parent megakaryocytes and comparisons with daughter platelet transcript profiles, specific RNAs might be sorted into platelets.47 Nonetheless, the regulation of this process and its link to circulating inflammatory, atherogenic, or thrombotic signalling remain unknown.52 Platelet miRNAs have intraindividual stability, particularly for the most abundant transcripts, suggesting that the platelet miRnome might be more stable than the miRnomes of other circulating cells or blood pools, such as plasma.49 Notably, almost half of miRNAs in microparticles—the main fraction of miRNAs in plasma—are platelet-derived.53 Effects on platelet function Transcripts are present in platelets in variable quantities and are associated with specific human phenotypes and disease states,54,55 but whether the platelet miRNA signature reflects or influences platelet function remains unknown. Such an association is certainly suggested by the distinct platelet miRNA profiles linked with age and sex 56 as well as diseases. For instance, atherosclerosis is known to be prothrombotic.57–61 In support of an association, platelet reactivity (as defined by platelet aggregation in response to epinephrine) has been demonstrated to be correlated with specific mRNA and miRNA patterns in platelets.19 The regulation of miRNAs is associated with changes in relevant gene and protein expression with functional consequences. Specifically, the presence of a miRNA network within platelets seems to allow the platelet to regulate expression of the P2Y12 receptor and thereby affect its own capacity to aggregate.62 Other studies
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REVIEWS Bone marrow
Endothelium
Circulation
Thrombin dsRNA RNA Pol III
pre-miRNA Drosha/ Pasha
pri-miRNA 5ʹ
Platelet
3ʹ
5ʹ
3ʹ
pre-miRNA Dicer miRNA
Immune/ inflammatory activation
Megakaryocyte
Leukocyte/neutrophil
Figure 1 | Platelet microRNA biogenesis and function. Intergenic miRNAs are thought to be transcribed megakaryocytes Nature in Reviews | Cardiology by RNA Pol III, generating a pri-miRNA molecule, which is processed into a pre-miRNA in the nucleus. Pre-miRNAs are exported to the cytoplasm and packaged by an unknown mechanism into platelets, which are released into the circulation. In the platelet, the pre-miRNA can be cleaved by the endoribonuclease Dicer to generate a miRNA duplex. Mature miRNA can bind to target mRNA and negatively regulate its expression, or be transferred to other vascular cells and translationally represses mRNA. Given that each mRNA has multiple miRNAs that can bind, and that each miRNA binds multiple mRNA targets, some miRNAs have been described as regulating pathways in a ‘rheostat-like’ manner. Abbreviations: dsRNA, double-stranded RNA; miRNA, microRNA; pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; RNA Pol III, RNA polymerase III.
have also linked specific mRNAs and miRNAs to platelet reactivity and activation.19 The platelet miRNA profile has also been suggested to be responsive to antiplatelet therapy, including aspirin.63 Specific mechanisms include a role for vesicle-associated membrane protein 8 (VAMP8, also known as endobrevin) with specific contribution from miRNA‑96 in the regulation of VAMP8 expression.64 In addition to their role in thrombosis, platelets might have an important role in maintaining vascular health (such as angiogenesis) and disease,65 including potentially contributing to the development of atherosclerotic plaque. This theory is supported by the observation that platelets adhere to the subendothelium and release substances that alter subendothelial cellular proliferation. Platelets also participate in cell-to-cell transfer of both mRNAs and miRNAs, providing a potential role for miRNAs as intercellular regulators of vascular homeostasis (Figure 1).66 Transferred RNA seems to be functional, given that posttransfer microarray gene expression analysis of recipient cells showed an increase in platelet-specific RNA and, by specifically transferring green fluorescent protein, external RNA was demonstrated to be functional in the recipient cells.66 Horizontal cell transfer of transcripts from platelets to other vascular cells is thought to occur via direct transfer and by release of microvesicles and exosomes.27,28,66–69 Several studies have been focused on the capacity of platelets to transfer miRNA to vascular cells. In an experimental model, transfer of miRNAs associated with myocardial infarction from platelets into endothelial cells occurred via platelet-derived microparticles in a
time-dependent and activation-dependent manner.59 This microparticle-mediated transfer was also demonstrated in another study, which showed that miR‑223 (a miRNA that is highly expressed in platelets) is shed after platelet activation by thrombin.70 In this study, uptake of miR‑223 by endothelial cells was demonstrated in vitro.70 The capacity of this transferred transcript to alter recipient-cell function was initially demonstrated with mRNA,66 and later for miRNA.59,70 Transfer of miRNA from platelets to endothelial cells might have functional consequences, given that after transfer of miRNA, mRNA targets are downregulated, particularly after platelet activation.59,70 Data showing that exogenous platelet miR‑223 decreases insulin-like growth factor I receptor and promotes endothelial cell apoptosis support the concept that platelet-released miR‑223 promotes glycation and vascular endothelial cell apoptosis.71 Roles in cardiovascular disease We and other groups have characterized the platelet miRNA profile associated with myocardial infarction and shown that it is distinct from that of other cell types and plasma (Figure 2).38,59,72 Moreover, platelet miRNA signatures differ between patients with ST-segment elevation myocardial infarction (STEMI), typically a more thrombosis-dependent form of myocardial infarction, and those with non-STEMI, suggesting that platelet levels of important miRNAs might influence platelet thrombogenicity and type of infarction.72 In further experiments to examine the role that platelet miRNAs
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REVIEWS
Plasma miR-624-5p miR-483-5p miR-30a-5p miR-218-5p
miR-30e-5p miR-30d-5p
miR-15b-5p miR-25-3p miR-27a-3p miR-27b-3p miR-29c-3p miR-103a-3p miR-145-5p miR-185-5p miR-186-5p miR-221-3p miR-374a-5p miR-554-3p
Platelets miR-127-3p miR-302c-3p miR-31-5p miR-423-5p miR-19b-3p miR-125a-5p RNU44 miR-342-3p miR-151a-5p miR-505-5p
Leukocytes miR-574-3p miR-146-3p miR-590a-5p miR-29c-3p miR-16-1-3p miR-212b-3p miR-140-3p miR-532-3p miR-16-5p miR-93-3p
Figure 2 | Common miRNAs associated with ST-segmentNature elevation myocardial Reviews | Cardiology infarction in plasma, platelets, and leukocytes.72 Modified from Ward, J. A. et al. Circulating cell and plasma microRNA profiles differ between non-ST-segment and ST-segment-elevation myocardial infarction. Fam. Med. Med. Sci. Res. 2 (2), 108 (2013), which is distributed under the terms of the Creative Commons Attribution License.
have in myocardial infarction,58 specific miRNAs have been shown to be downregulated in platelets at the site of thrombus formation in patients with STEMI compared with those with non-STEMI.59 In follow-up experiments to examine the same set of thrombi miRNAs that are downregulated in STEMI (miR‑22, miR‑185, miR‑320b, and miR‑423‑5p), investigators have demonstrated an increase in the levels of these miRNAs in the supernatant of thrombin-stimulated platelets.59 In investigations focusing on other forms of heart disease, circulating levels of miR‑328, a platelet-enriched miRNA, were associated with atrial fibrillation (AF) in the Framingham Heart Study.35 In this study, the association between circulating miR‑328 levels and AF was attenuated when medication use, including antiplatelet drugs, was included in multivariable adjusted models.35 This finding suggests—as has been observed in other studies into platelet-derived miRNAs (such as miR‑126 in coronary disease73)—that the association between AF and miR‑328 was related to use of antiplatelet medication. Given that microparticles are shed by activated platelets, these microparticles include a large number of plateletderived miRNAs, and because the miRNA profiles of serum and platelets are so similar, considerable interest has been focused on platelet-derived microparticles as
important factors in CVD.53,74–76 Of note, several biomarkers associated with the risk of myocardial infarction (miR‑126, miR‑150, miR‑197, and miR‑223) are highly expressed in platelets and platelet microparticles.63,77 In a study of the levels of platelet miRNAs in patients with heart failure, platelet miR‑150 levels were associated with the risk of AF.78 The level of miR‑150 has also been noted to be lower in the plasma and cardiac tissue of patients with AF.36 Given that miR‑150 downregulates Egr2 and P2rx7 (proapoptotic and proinflammatory genes, respectively) in mouse cardiomyocytes, cardiac miR‑150 might be cardioprotective.79 Parallels between cardiac and platelet levels of miR‑150 warrant further examination, especially because miR‑150 has been implicated in megakaryocytopoiesis and is suppressed after platelet inhibition.63,65 miRNAs known to regulate inflammation have also been isolated from microparticles derived from platelets, endothelial cells, and other circulating blood cells, and are associated with metabolic status80 and CVD, reinforcing the concept that platelet-derived miRNAs, whether intracellular or extracellular, have important roles as CVD biomarkers and potential therapeutic targets. In addition to their potential roles as mediators of CVD, platelet miRNAs might provide a novel means to measure response to antiplatelet therapies used in CVD, as suggested by a study showing that circulating levels of miR‑223, another platelet-enriched miRNA, indicate platelet reactivity in patients with myocardial infarction.81 These findings suggest that miR‑223 profiling might be employed as a means to assess responsiveness to antiplatelet agents used in patients with acute coronary syndromes.81 In another investigation into the effects of antiplatelet therapies on platelet miRNA levels, in vitro platelet activation resulted in transfer of miR‑126 from platelets to the plasma, whereas platelets exposed to aspirin did not perform this transfer.73 Administration of aspirin in vivo resulted in platelet inhibition and was associated with lower levels of platelet-derived circulating miRNAs, including miR‑126, than were seen in untreated patients.73
Conclusions
In summary, our knowledge of platelet function has improved substantially over the past decade, particularly in the setting of CVD. Studies have defined a role for transcripts in anucleate platelets, which contain a broad number of miRNAs that seem to have functional relevance. However, important questions remain unanswered, including the origin of the transcriptomic material found in platelets, the specific function of these transcripts in acute and chronic CVDs, how miRNA transfers into and out of platelets, and whether this is a specific or targeted process. An accumulating body of evidence suggests that quantitative differences in the relative abundance of miRNAs contribute to susceptibility to, and prognosis of, human diseases, including atherothrombotic disease. Also intriguing is the role that platelets have in the transfer and distribution of vascular miRNAs. Improved understanding of the importance of platelet miRNAs in the setting of CVD might assist in the diagnosis and treatment of prothrombotic vascular diseases.
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22. Ambros, V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113, 673–676 (2003). 23. Ambros, V. MicroRNAs: tiny regulators with great potential. Cell 107, 823–826 (2001). 24. Ambros, V. MicroRNAs and developmental timing. Curr. Opin. Genet. Dev. 21, 511–517 (2011). 25. Cloonan, N. Re-thinking miRNA-mRNA interactions: intertwining issues confound target discovery. Bioessays 37, 379–388 (2015). 26. Small, E. M. & Olson, E. N. Pervasive roles of microRNAs in cardiovascular biology. Nature 469, 336–342 (2011). 27. Wahlgren, J. et al. Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Res. 40, e130 (2012). 28. Heijnen, H. F., Schiel, A. E., Fijnheer, R., Geuze, H. J. & Sixma, J. J. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 94, 3791–3799 (1999). 29. Small, E. M., Frost, R. J. A. & Olson, E. N. MicroRNAs add a new dimension to cardiovascular disease. Circulation 121, 1022–1032 (2010). 30. van Rooij, E. et al. Control of stress-dependent cardiac growth and gene expression by a microRNA. Science 316, 575–579 (2007). 31. Hinkel, R., Ng, J. K. M. & Kupatt, C. Targeting microRNAs for cardiovascular therapeutics in coronary artery disease. Curr. Opin. Cardiol. 29, 586–594 (2014). 32. Dong, S. et al. MicroRNA expression signature and the role of microRNA‑21 in the early phase of acute myocardial infarction. J. Biol. Chem. 284, 29514–29525 (2009). 33. Wang, R., Li, N., Zhang, Y., Ran, Y. & Pu, J. Circulating microRNAs are promising novel biomarkers of acute myocardial infarction. Intern. Med. 50, 1789–1795 (2011). 34. McManus, D. D. & Ambros, V. Circulating MicroRNAs in cardiovascular disease. Circulation 124, 1908–1910 (2011). 35. McManus, D. D. et al. Relations between circulating microRNAs and atrial fibrillation: data from the Framingham Offspring Study. Heart Rhythm 11, 663–669 (2014). 36. McManus, D. D. et al. Plasma microRNAs are associated with atrial fibrillation and change after catheter ablation (the miRhythm study). Heart Rhythm 12, 3–10 (2015). 37. Fichtlscherer, S. et al. Circulating microRNAs in patients with coronary artery disease. Circ. Res. 107, 677–684 (2010). 38. Freedman, J. E. et al. The distribution of circulating microRNA and their relation to coronary disease. F1000Res. 1, 50 (2012). 39. De Rosa, S. et al. Transcoronary concentration gradients of circulating microRNAs. Circulation 124, 1936–1944 (2011). 40. Fichtlscherer, S., Zeiher, A. M. & Dimmeler, S. Circulating microRNAs: biomarkers or mediators of cardiovascular diseases? Arterioscler. Thromb. Vasc. Biol. 31, 2383–2390 (2011). 41. Oliveira-Carvalho, V., da Silva, M. M. F., Guimarães, G. V., Bacal, F. & Bocchi, E. A. MicroRNAs: new players in heart failure. Mol. Biol. Rep. 40, 2663–2670 (2013). 42. Liebetrau, C. et al. Release kinetics of circulating muscle-enriched microRNAs in patients undergoing transcoronary ablation of septal hypertrophy. J. Am. Coll. Cardiol. 62, 992–998 (2013). 43. Das, S. & Halushka, M. K. Extracellular vesicle microRNA transfer in cardiovascular disease. Cardiovasc. Pathol. 24, 199–206 (2015).
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44. Bang, C. et al. Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. J. Clin. Invest. 124, 2136–2146 (2014). 45. Georgantas, R. W. et al. CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc. Natl Acad. Sci. USA 104, 2750–2755 (2007). 46. Barroga, C. F., Pham, H. & Kaushansky, K. Thrombopoietin regulates c‑Myb expression by modulating micro RNA 150 expression. Exp. Hematol. 36, 1585–1592 (2008). 47. Opalinska, J. B. et al. MicroRNA expression in maturing murine megakaryocytes. Blood 116, e128–e138 (2010). 48. Labbaye, C. et al. A three-step pathway comprising PLZF/miR‑146a/CXCR4 controls megakaryopoiesis. Nat. Cell Biol. 10, 788–801 (2008). 49. Stratz, C. et al. Micro-array profiling exhibits remarkable intra-individual stability of human platelet micro-RNA. Thromb. Haemost. 107, 634–641 (2012). 50. Xu, X. et al. Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms. Blood 120, 3575–3585 (2012). 51. Plé, H. et al. The repertoire and features of human platelet microRNAs. PLoS ONE 7, e50746 (2012). 52. Cecchetti, L. et al. Megakaryocytes differentially sort mRNAs for matrix metalloproteinases and their inhibitors into platelets: a mechanism for regulating synthetic events. Blood 118, 1903–1911 (2011). 53. Diehl, P. et al. Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovasc. Res. 93, 633–644 (2012). 54. Freedman, J. E. et al. Relation of platelet and leukocyte inflammatory transcripts to body mass index in the Framingham heart study. Circulation 122, 119–129 (2010). 55. Lood, C. et al. Platelet transcriptional profile and protein expression in patients with systemic lupus erythematosus: up-regulation of the type I interferon system is strongly associated with vascular disease. Blood 116, 1951–1957 (2010). 56. Simon, L. M. et al. Human platelet microRNAmRNA networks associated with age and gender revealed by integrated plateletomics. Blood 123, e37–e45 (2014). 57. Jain, S. et al. Expression of regulatory platelet microRNAs in patients with sickle cell disease. PLoS ONE 8, e60932 (2013). 58. Healy, A. M. et al. Platelet expression profiling and clinical validation of myeloid-related protein‑14 as a novel determinant of cardiovascular events. Circulation 113, 2278–2284 (2006). 59. Gidlöf, O. et al. Platelets activated during myocardial infarction release functional miRNA, which can be taken up by endothelial cells and regulate ICAM1 expression. Blood 121, 3908–3917 (2013). 60. Kahr, W. H. A. et al. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat. Genet. 43, 738–740 (2011). 61. Gnatenko, D. V. et al. Class prediction models of thrombocytosis using genetic biomarkers. Blood 115, 7–14 (2010). 62. Landry, P. et al. Existence of a microRNA pathway in anucleate platelets. Nat. Struct. Mol. Biol. 16, 961–966 (2009). 63. Willeit, P. et al. Circulating microRNAs as novel biomarkers for platelet activation. Circ. Res. 112, 595–600 (2013).
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REVIEWS 64. Kondkar, A. A. et al. VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA. J. Thromb. Haemost. 8, 369–378 (2010). 65. Gatsiou, A., Boeckel, J.‑N., Randriamboavonjy, V. & Stellos, K. MicroRNAs in platelet biogenesis and function: implications in vascular homeostasis and inflammation. Curr. Vasc. Pharmacol. 10, 524–531 (2012). 66. Risitano, A., Beaulieu, L. M., Vitseva, O. & Freedman, J. E. Platelets and platelet-like particles mediate intercellular RNA transfer. Blood 119, 6288–6295 (2012). 67. Aatonen, M., Grönholm, M. & Siljander, P. R.‑M. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin. Thromb. Hemost. 38, 102–113 (2012). 68. Montecalvo, A. et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood 119, 756–766 (2012). 69. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007). 70. Laffont, B. et al. Activated platelets can deliver mRNA regulatory Ago2-microRNA complexes to endothelial cells via microparticles. Blood 122, 253–261 (2013). 71. Pan, Y. et al. Platelet-secreted microRNA‑223 promotes endothelial cell apoptosis induced by advanced glycation end products via targeting the insulin-like growth factor 1 receptor. J. Immunol. 192, 437–446 (2014).
72. Ward, J. A. et al. Circulating cell and plasma microRNA profiles differ between non-STsegment and ST-segment-elevation myocardial infarction. Fam. Med. Med. Sci. Res. 2, 108 (2013). 73. de Boer, H. C. et al. Aspirin treatment hampers the use of plasma microRNA‑126 as a biomarker for the progression of vascular disease. Eur. Heart J. 34, 3451–3457 (2013). 74. Hunter, M. P. et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS ONE 3, e3694 (2008). 75. Italiano, J. E., Mairuhu, A. T. A. & Flaumenhaft, R. Clinical relevance of microparticles from platelets and megakaryocytes. Curr. Opin. Hematol. 17, 578–584 (2010). 76. Cheng, H. H. et al. Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS ONE 8, e64795 (2013). 77. Zampetaki, A. et al. Prospective study on circulating microRNAs and risk of myocardial infarction. J. Am. Coll. Cardiol. 60, 290–299 (2012). 78. Goren, Y. et al. Relation of reduced expression of miR‑150 in platelets to atrial fibrillation in patients with chronic systolic heart failure. Am. J. Cardiol. 113, 976–981 (2014). 79. Tang, Y. et al. MicroRNA‑150 protects the mouse heart from ischaemic injury by regulating cell death. Cardiovasc. Res. 106, 387–397 (2015). 80. Hulsmans, M. & Holvoet, P. MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease. Cardiovasc. Res. 100, 7–18 (2013).
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81. Zhang, Y.‑Y. et al. Decreased circulating microRNA‑223 level predicts high on-treatment platelet reactivity in patients with troponinnegative non-ST elevation acute coronary syndrome. J. Thromb. Thrombolysis 38, 65–72 (2014). 82. Duisters, R. F. et al. miR‑133 and miR‑30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling. Circ. Res. 104, 170–178 (2009). 83. Karolina, D. S. et al. Circulating miRNA profiles in patients with metabolic syndrome. J. Clin. Endocrinol. Metab. 97, E2271–E2276 (2012). 84. Wang, Y.‑S., Zhou, J., Hong, K., Cheng, X.‑S. & Li, Y.‑G. MicroRNA‑223 displays a protective role against cardiomyocyte hypertrophy by targeting cardiac troponin I-interacting kinase. Cell. Physiol. Biochem. 35, 1546–1556 (2015). Acknowledgements The authors are supported by grants UH2TR000921 and U01OD019771 (J.E.F.) from the NIH Common Fund, through the Office of Strategic Coordination/ Office of the NIH Director, and KL2RR031981 (D.D.M.) from the University of Massachusetts Medical School’s Center for Clinical and Translational Science Award. Author contributions Both authors researched data for the article, discussed its content, and wrote, reviewed, and edited the manuscript before submission.
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Introduction
Cardiovascular disease (CVD) continues to be the major cause of morbidity and mortality in North America and is a growing problem in many industrialized and developing countries worldwide.1–3 CVD accounts for over onethird of deaths in the USA annually,1–3 and is the cause of more deaths each year than cancer, respiratory disease, and accidents combined.2 Platelets have long been known to have important roles in thrombosis, arteriosclerosis, and angiogenesis,4,5 but in the past decade, anucleate platelets have been shown to contain transcripts and the necessary molecular machinery to conduct translation. Subsequently, particular small RNAs, including microRNAs (miRNAs), have been found to be present inside mature platelets, and mRNAs and miRNAs have been shown to regulate gene expression in platelets. In this Review, we outline the role of platelets in CVD, and discuss miRNAs and their roles in platelet development, function, and CVD.
Platelets and cardiovascular disease Cardiology Division, Department of Medicine, University of Massachusetts Medical School, AS7‑1051, 368 Plantation Street, Worcester, MA 01605‑4319, USA (D.D.M., J.E.F.). Correspondence to: J.E.F. jane.freedman@ umassmed.edu
Platelets have a central role in CVD, particularly in acute coronary syndrome and stroke, contributing to the development of acute thrombotic events.6 Superficial and deep intimal injuries disturb the intact endothelium and disrupt the production of antiplatelet agents, including nitric oxide and prostacyclin. With exposure of collagen to the circulation, platelets adhere to the subendothelium, both directly and via von Willebrand factor.7 Platelets become Competing interests The authors declare no competing interests.
activated via soluble receptor-mediated stimulants, such as thrombin, ADP, and thromboxane A2, thereby enhancing the thrombogenicity of activated platelets. Platelet aggregation occurs when the glycoprotein IIb/IIIa receptor binds adhesive proteins. Platelets can release many cytokines and signalling molecules that can lead to a cascade of further platelet activation, thrombosis, and vasoconstriction. Activated platelets form a growing coronary thrombus with concomitant propagation of fibrin.8 Although these processes are in place to provide haemostasis in the context of vascular injury and allow for wound healing, they can induce vessel occlusion when they occur in the setting of coronary, peripheral arterial, or cerebrovascular plaque rupture, thereby contributing to an acute coronary syndrome or stroke. The specific mechanisms by which platelet function is regulated, particularly in the setting of vessel occlusion, have been reassessed in the past decade, with improved understanding of novel pathways that might alter thrombosis.
Platelets and transcripts
Appreciation of novel pathways regulating thrombosis has been growing, including the observation that platelets contain a variety of transcripts. Platelets are anucleate, and their cytoplasmic RNA has long been considered to be residual transcripts of their forming cell, the megakaryocyte. During platelet biogenesis, release of platelets of uncertain heterogeneity occurs, and these cells circulate for 7–10 days in humans, after which they are cleared in the spleen.4,5 Although lacking nuclei, platelets contain
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REVIEWS Key points ■■ Platelet function has an important role in mediating particular common forms of cardiovascular disease, such as acute coronary syndromes and stroke ■■ Although they lack nuclei, platelets contain RNAs, including small noncoding RNAs such as microRNAs, and the necessary machinery to perform translation ■■ Data suggest that microRNAs can influence platelet functions, including thrombosis, atherosclerosis, and angiogenesis ■■ Platelet RNA can also be transferred to other vascular cells, but further information about the role of microRNA transfer is needed
all the necessary components to perform translation in a signal-dependent manner,9 including functioning ribosomes.10–12 Investigators in several studies have used microarray and RNA sequencing to define the transcripts contained in platelets. Current estimates of the number of transcripts in platelets range from 1,500 to 9,500 in healthy human platelets,13,14 with notable differences in the number and types of transcripts in human compared with murine platelets—human platelets have more varied transcripts including those associated with surface receptors.11,12,15 The range in the number of reported transcripts might be related to platelet age, size, or method of measurement. Variable association between platelet transcripts and prote omics has been reported.16,17 Importantly, human platelets contain several forms of small RNAs, with miRNAs being the most abundant and comprising 80% of all small RNAs in platelets.18 These miRNA–mRNAs can be coexpressed.19
miRNAs
Small RNAs, including miRNAs, are important components of an evolutionarily conserved system of RNA-based gene regulation in eukaryotes.20 Small RNAs are involved in many molecular processes, including regulation of gene expression.21 Animal genomes contain an abundance of small genes that produce regulatory RNAs of ~22 nucleotides in length.22 These miRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread, suggesting that they might participate in a wide range of critical genetic regulatory pathways across species. miRNAs interfere with the expression of mRNA encoding factors that control developmental timing, stem-cell maintenance, and other physiological processes in animals.21 Research into small noncoding RNAs has fundamentally transformed our understanding of gene regulatory networks, especially at the post-transcriptional level.23,24 miRNAs are initially transcribed as long primary miRNAs (pri-miRNAs), which are processed by the RNase III enzyme Drosha (also known as ribonuclease 3) to generate stem–loop precursor miRNAs (pre-miRNAs) of ~70 nucleotides in length.21 These precursors are exported into the cytoplasm by exportin‑5 and, sub sequently, the cytoplasmic endoribonuclease Dicer cleaves pre-miRNA to release mature miRNA.22 Binding of miRNA to an mRNA with Argonaute proteins inhibits protein translation either directly or by promoting mRNA destruction. miRNAs regulate temporal transitions in gene expression associated with cell-fate progression and differentiation throughout animal development.22 By regulating cell-fate choices and transitions between pluripotency and differentiation, miRNAs help to orchestrate developmental
events in growing animals and have a role in tissue homeo stasis with relevance to disease states.24 Core areas of uncertainty remain regarding miRNA–mRNA target identification and miRNA target effects. However, new methods for validating miRNA–mRNA–Argonaute‑2 interactions in cells have been developed, including measuring gene expression after modulating miRNA expression, degradome sequencing, biotin-linked chroma tography, HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation), and CLASH (a variant of the HITS-CLIP method).25
miRNAs as biomarkers Some miRNAs are expressed in a cell-specific manner. miRNAs are detectable in plasma, where they can be contained in microparticles or transported by lipoproteins.26–28 They are also found in circulating blood cells, and release of miRNAs into the circulation has been linked to cellular damage, such as cardiomyocyte injury, or active processes, including secretion. miRNAs seem to be stable in the circulation, and quantifiable with a high degree of accuracy. Even at low abundance, miRNAs are comprised of nucleic acids with sequences that can often be easily amplified, making them ideal biomarkers of disease.29 Moreover, and in contrast to many other cellular disease mediators, drugs can target miRNAs. Given that miRNAs promote homeostatic mechanisms and can transiently modulate disease pathways, miRNA-based therapeutics would theoretically enable modulation of the cardiac stress response in a unique and powerful manner.30,31 miRNAs in cardiovascular disease miRNAs have important roles in cardiac development, including stem-cell differentiation and proliferation of cardiomyocytes in the embryo; however, they are also important in susceptibility to CVD (Table 1).26 miRNAs in cardiac tissue show dynamic changes in several forms of heart disease, suggesting that they have important roles in regulating an acute injury response. For example, animal and human studies have clearly demonstrated that cardiac miRNAs have critical roles in regulating important pathophysiological processes underlying heart diseases, including electrical remodelling (miR‑1, miR‑21, miR‑133, and miR‑328), cardiac fibrosis (miR‑21 and miR‑29), hypertrophic structural remodel ling (miR‑208), and cardiac ischaemia–reperfusion injury (miR‑150 and miR‑320).26,32–37 Circulating miRNAs in whole blood and plasma have also been associated with various acute and chronic forms of CVD, including atrial fibrillation, coronary artery disease, myocardial infarction, heart failure, vascular disease, and cardiac death.31,34,36–41 miRNAs can be released or secreted by cardiomyocytes, endothelial cells, and fibroblasts, and the release of unique miRNA into the circulation in the context of myocardial injury suggests process specificity.39 However, most of these studies must be seen as preliminary, owing to either small numbers of miRNAs measured or limited numbers of patients included. Transcoronary miRNA gradients exist during an acute cardiac injury such as myocardial infarction or after
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REVIEWS Table 1 | Roles of microRNAs expressed in platelets and associated with CVD microRNA
Target genes and functional effects
Associated phenotypes
miR‑30c
Represses CTGF expression; matrix remodelling
Ventricular fibrosis, heart failure, STEMI/non-STEMI82
miR‑126
Regulates CXCL12, PIK3R2, and SPRED1; angiogenesis and vascular integrity
Regulates myocardial angiogenesis, platelet levels altered by aspirin administration63
miR‑150
Regulates EGR2, MYB, and P2RX7; H2O2-induced cardiac cell death, platelet biogenesis
Atherosclerosis, cardiac hypertrophy, heart failure, myocardial infarction, myocardial ischaemia–reperfusion injury, and platelet activation79
miR‑197
Regulates SERTAD4; functional effects unknown
Plasma level reduced in patients with myocardial infarction77 and associated with metabolic syndrome83
miR‑223
Regulates SLC2A4; insulin resistance
Associated with myocyte hypertrophy, ventricular remodelling, myocardial infarction, and platelet activation84
miR‑328
Regulates CACNA1C and CACNB1; encode cardiac L‑type calcium channels
Atrial fibrillation, electrical remodelling35
miR‑342
Regulates AKT1; glucose metabolism, apoptosis, cell proliferation
Inflammatory stimulation of macrophages63
Abbreviations: CVD, cardiovascular disease; STEMI, ST-segment elevation myocardial infarction.
septal ablation, and whole-blood levels of miRNAs (such as miR‑133a and miR‑499) can rise and fall hours after an acute coronary event, reinforcing their utility as biomarkers of disease.39,42 Whether the release of miRNAs into the circulation in the aforementioned CVD states is a passive or active process is unknown, but data suggest that miRNAs can be delivered to recipient cells and regulate protein expression.43 The distribution and function of miRNAs across circulating cell types and in plasma, and the effect of circulating miRNAs on other organ systems, such as the kidneys, are also presently unknown. However, evidence has linked cardiac remodelling in the context of heart failure to crosstalk between cardiomyocytes and fibroblasts via miRNA-enriched exosomes.44
miRNAs and megakaryopoiesis Megakaryocytes are derived from common myeloid progenitors, with platelets formed in turn from megakaryocytes. Most platelet-derived miRNAs are presumed to have been formed in the megakaryocyte (Figure 1). Of note, each mRNA has multiple miRNAs that bind and, given that each miRNA binds multiple mRNA targets, many miRNAs will regulate pathways in a ‘rheostat-like’ manner. miR‑155 is a common small RNA found in haemato poietic stem cells and implicated in platelet development. Specifically, upregulation of miR‑155 inhibits haemato poietic stem-cell differentiation into megakaryocytes, as shown by the transplantation of haematopoietic stem cells overexpressing miR‑155 into mice whose bone marrow had been depleted, which resulted in reduced numbers of megakaryocytes.45 Overexpression of miR‑150, by contrast, shifts megakaryocyte–erythroid precursors towards megakaryocyte differentiation both in vitro and in vivo, and is upregulated by thrombopoeitin.46 Megakaryopoiesis is also modified by miR‑146a, although effects differ according to experimental conditions.47,48 Additionally, several investigators have shown that patients with myeloproliferative neoplasms have upregulation or downregulation of miR‑10a, miR‑26b, miR‑148a, and miR‑490‑5p, further supporting the hypothesis that miRNAs have an important role in normal megakaryocyte development.49,50
miRNAs in platelets Platelets are known to have miRNAs as well as Dicer and Argonaute protein complexes, thereby enabling the processing of pre‑RNAs to miRNAs and control of reporter transcripts. On the basis of transcriptomic profiling, the vast majority of common small RNAs in the platelet are miRNAs.51 Profiling from healthy human platelets has characterized 492 platelet miRNAs and 40 novel miRNAs, with the most abundant being members of the let‑7 family.51 From analyses of platelet transcripts derived from parent megakaryocytes and comparisons with daughter platelet transcript profiles, specific RNAs might be sorted into platelets.47 Nonetheless, the regulation of this process and its link to circulating inflammatory, atherogenic, or thrombotic signalling remain unknown.52 Platelet miRNAs have intraindividual stability, particularly for the most abundant transcripts, suggesting that the platelet miRnome might be more stable than the miRnomes of other circulating cells or blood pools, such as plasma.49 Notably, almost half of miRNAs in microparticles—the main fraction of miRNAs in plasma—are platelet-derived.53 Effects on platelet function Transcripts are present in platelets in variable quantities and are associated with specific human phenotypes and disease states,54,55 but whether the platelet miRNA signature reflects or influences platelet function remains unknown. Such an association is certainly suggested by the distinct platelet miRNA profiles linked with age and sex 56 as well as diseases. For instance, atherosclerosis is known to be prothrombotic.57–61 In support of an association, platelet reactivity (as defined by platelet aggregation in response to epinephrine) has been demonstrated to be correlated with specific mRNA and miRNA patterns in platelets.19 The regulation of miRNAs is associated with changes in relevant gene and protein expression with functional consequences. Specifically, the presence of a miRNA network within platelets seems to allow the platelet to regulate expression of the P2Y12 receptor and thereby affect its own capacity to aggregate.62 Other studies
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REVIEWS Bone marrow
Endothelium
Circulation
Thrombin dsRNA RNA Pol III
pre-miRNA Drosha/ Pasha
pri-miRNA 5ʹ
Platelet
3ʹ
5ʹ
3ʹ
pre-miRNA Dicer miRNA
Immune/ inflammatory activation
Megakaryocyte
Leukocyte/neutrophil
Figure 1 | Platelet microRNA biogenesis and function. Intergenic miRNAs are thought to be transcribed megakaryocytes Nature in Reviews | Cardiology by RNA Pol III, generating a pri-miRNA molecule, which is processed into a pre-miRNA in the nucleus. Pre-miRNAs are exported to the cytoplasm and packaged by an unknown mechanism into platelets, which are released into the circulation. In the platelet, the pre-miRNA can be cleaved by the endoribonuclease Dicer to generate a miRNA duplex. Mature miRNA can bind to target mRNA and negatively regulate its expression, or be transferred to other vascular cells and translationally represses mRNA. Given that each mRNA has multiple miRNAs that can bind, and that each miRNA binds multiple mRNA targets, some miRNAs have been described as regulating pathways in a ‘rheostat-like’ manner. Abbreviations: dsRNA, double-stranded RNA; miRNA, microRNA; pre-miRNA, precursor microRNA; pri-miRNA, primary microRNA; RNA Pol III, RNA polymerase III.
have also linked specific mRNAs and miRNAs to platelet reactivity and activation.19 The platelet miRNA profile has also been suggested to be responsive to antiplatelet therapy, including aspirin.63 Specific mechanisms include a role for vesicle-associated membrane protein 8 (VAMP8, also known as endobrevin) with specific contribution from miRNA‑96 in the regulation of VAMP8 expression.64 In addition to their role in thrombosis, platelets might have an important role in maintaining vascular health (such as angiogenesis) and disease,65 including potentially contributing to the development of atherosclerotic plaque. This theory is supported by the observation that platelets adhere to the subendothelium and release substances that alter subendothelial cellular proliferation. Platelets also participate in cell-to-cell transfer of both mRNAs and miRNAs, providing a potential role for miRNAs as intercellular regulators of vascular homeostasis (Figure 1).66 Transferred RNA seems to be functional, given that posttransfer microarray gene expression analysis of recipient cells showed an increase in platelet-specific RNA and, by specifically transferring green fluorescent protein, external RNA was demonstrated to be functional in the recipient cells.66 Horizontal cell transfer of transcripts from platelets to other vascular cells is thought to occur via direct transfer and by release of microvesicles and exosomes.27,28,66–69 Several studies have been focused on the capacity of platelets to transfer miRNA to vascular cells. In an experimental model, transfer of miRNAs associated with myocardial infarction from platelets into endothelial cells occurred via platelet-derived microparticles in a
time-dependent and activation-dependent manner.59 This microparticle-mediated transfer was also demonstrated in another study, which showed that miR‑223 (a miRNA that is highly expressed in platelets) is shed after platelet activation by thrombin.70 In this study, uptake of miR‑223 by endothelial cells was demonstrated in vitro.70 The capacity of this transferred transcript to alter recipient-cell function was initially demonstrated with mRNA,66 and later for miRNA.59,70 Transfer of miRNA from platelets to endothelial cells might have functional consequences, given that after transfer of miRNA, mRNA targets are downregulated, particularly after platelet activation.59,70 Data showing that exogenous platelet miR‑223 decreases insulin-like growth factor I receptor and promotes endothelial cell apoptosis support the concept that platelet-released miR‑223 promotes glycation and vascular endothelial cell apoptosis.71 Roles in cardiovascular disease We and other groups have characterized the platelet miRNA profile associated with myocardial infarction and shown that it is distinct from that of other cell types and plasma (Figure 2).38,59,72 Moreover, platelet miRNA signatures differ between patients with ST-segment elevation myocardial infarction (STEMI), typically a more thrombosis-dependent form of myocardial infarction, and those with non-STEMI, suggesting that platelet levels of important miRNAs might influence platelet thrombogenicity and type of infarction.72 In further experiments to examine the role that platelet miRNAs
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REVIEWS
Plasma miR-624-5p miR-483-5p miR-30a-5p miR-218-5p
miR-30e-5p miR-30d-5p
miR-15b-5p miR-25-3p miR-27a-3p miR-27b-3p miR-29c-3p miR-103a-3p miR-145-5p miR-185-5p miR-186-5p miR-221-3p miR-374a-5p miR-554-3p
Platelets miR-127-3p miR-302c-3p miR-31-5p miR-423-5p miR-19b-3p miR-125a-5p RNU44 miR-342-3p miR-151a-5p miR-505-5p
Leukocytes miR-574-3p miR-146-3p miR-590a-5p miR-29c-3p miR-16-1-3p miR-212b-3p miR-140-3p miR-532-3p miR-16-5p miR-93-3p
Figure 2 | Common miRNAs associated with ST-segmentNature elevation myocardial Reviews | Cardiology infarction in plasma, platelets, and leukocytes.72 Modified from Ward, J. A. et al. Circulating cell and plasma microRNA profiles differ between non-ST-segment and ST-segment-elevation myocardial infarction. Fam. Med. Med. Sci. Res. 2 (2), 108 (2013), which is distributed under the terms of the Creative Commons Attribution License.
have in myocardial infarction,58 specific miRNAs have been shown to be downregulated in platelets at the site of thrombus formation in patients with STEMI compared with those with non-STEMI.59 In follow-up experiments to examine the same set of thrombi miRNAs that are downregulated in STEMI (miR‑22, miR‑185, miR‑320b, and miR‑423‑5p), investigators have demonstrated an increase in the levels of these miRNAs in the supernatant of thrombin-stimulated platelets.59 In investigations focusing on other forms of heart disease, circulating levels of miR‑328, a platelet-enriched miRNA, were associated with atrial fibrillation (AF) in the Framingham Heart Study.35 In this study, the association between circulating miR‑328 levels and AF was attenuated when medication use, including antiplatelet drugs, was included in multivariable adjusted models.35 This finding suggests—as has been observed in other studies into platelet-derived miRNAs (such as miR‑126 in coronary disease73)—that the association between AF and miR‑328 was related to use of antiplatelet medication. Given that microparticles are shed by activated platelets, these microparticles include a large number of plateletderived miRNAs, and because the miRNA profiles of serum and platelets are so similar, considerable interest has been focused on platelet-derived microparticles as
important factors in CVD.53,74–76 Of note, several biomarkers associated with the risk of myocardial infarction (miR‑126, miR‑150, miR‑197, and miR‑223) are highly expressed in platelets and platelet microparticles.63,77 In a study of the levels of platelet miRNAs in patients with heart failure, platelet miR‑150 levels were associated with the risk of AF.78 The level of miR‑150 has also been noted to be lower in the plasma and cardiac tissue of patients with AF.36 Given that miR‑150 downregulates Egr2 and P2rx7 (proapoptotic and proinflammatory genes, respectively) in mouse cardiomyocytes, cardiac miR‑150 might be cardioprotective.79 Parallels between cardiac and platelet levels of miR‑150 warrant further examination, especially because miR‑150 has been implicated in megakaryocytopoiesis and is suppressed after platelet inhibition.63,65 miRNAs known to regulate inflammation have also been isolated from microparticles derived from platelets, endothelial cells, and other circulating blood cells, and are associated with metabolic status80 and CVD, reinforcing the concept that platelet-derived miRNAs, whether intracellular or extracellular, have important roles as CVD biomarkers and potential therapeutic targets. In addition to their potential roles as mediators of CVD, platelet miRNAs might provide a novel means to measure response to antiplatelet therapies used in CVD, as suggested by a study showing that circulating levels of miR‑223, another platelet-enriched miRNA, indicate platelet reactivity in patients with myocardial infarction.81 These findings suggest that miR‑223 profiling might be employed as a means to assess responsiveness to antiplatelet agents used in patients with acute coronary syndromes.81 In another investigation into the effects of antiplatelet therapies on platelet miRNA levels, in vitro platelet activation resulted in transfer of miR‑126 from platelets to the plasma, whereas platelets exposed to aspirin did not perform this transfer.73 Administration of aspirin in vivo resulted in platelet inhibition and was associated with lower levels of platelet-derived circulating miRNAs, including miR‑126, than were seen in untreated patients.73
Conclusions
In summary, our knowledge of platelet function has improved substantially over the past decade, particularly in the setting of CVD. Studies have defined a role for transcripts in anucleate platelets, which contain a broad number of miRNAs that seem to have functional relevance. However, important questions remain unanswered, including the origin of the transcriptomic material found in platelets, the specific function of these transcripts in acute and chronic CVDs, how miRNA transfers into and out of platelets, and whether this is a specific or targeted process. An accumulating body of evidence suggests that quantitative differences in the relative abundance of miRNAs contribute to susceptibility to, and prognosis of, human diseases, including atherothrombotic disease. Also intriguing is the role that platelets have in the transfer and distribution of vascular miRNAs. Improved understanding of the importance of platelet miRNAs in the setting of CVD might assist in the diagnosis and treatment of prothrombotic vascular diseases.
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