Front. Med. DOI 10.1007/s11684-014-0379-2

REVIEW

Circulating microRNAs in cardiovascular diseases: from biomarkers to therapeutic targets ✉)

Feng Wang, Chen Chen, Daowen Wang (

Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2014

Abstract microRNAs (miRNAs) are a class of conserved, short, non-coding RNAs that have important and potent capacities to regulate gene expression at the posttranscriptional level. In the past several years, the aberrant expressions of miRNAs in the cardiovascular system have been widely reported, and the crucial roles of some special miRNAs in heart development and pathophysiology of various cardiovascular diseases have been gradually recognized. Recently, it was discovered that miRNAs are presented in peripheral circulation abundantly and stably. This has raised the possibility of using circulating miRNAs as biomarkers for diseases. Furthermore, some studies demonstrated that circulating miRNAs may serve as novel extracellular communicators of cell-cell communication. These discoveries not only reveal the functions of circulating miRNAs in cardiovascular system but also inform the development of miRNAs therapeutic strategies. In this review, we discuss the potential roles of circulating miRNAs in a variety of cardiovascular diseases from biomarkers to therapeutic targets to clearly understand the roles of circulating miRNAs in cardiovascular system. Keywords

microRNA; cardiovascular disease; biomarkers; therapeutic target

Introduction microRNAs are a class of evolutionarily conserved, endogenous, non-coding RNAs with 21–25 nucleotides in length [1,2]. miRNAs are transcribed by RNA polymerase II and can be derived from either intergenic, polycistronic or intronic regions [3,4]. Primary miRNAs (pri-miRNAs) are hundreds to thousands nucleotides long [5], and are processed by RNase enzyme Drosha into a 70– 100 nucleotide hairpin structure precursors named precursor miRNA (pre-miRNA) in the nucleus. The premiRNA is transported into the cytoplasm after interaction with Ran-GTP and exportin-5, where it finishes a second step of processing by Dicer to generate a small doublestranded RNA structure (~22nt) [6–8]. This transient oligonucleotide duplex is incorporated into the RNA induced silencing complex (RISC), and then a strand is stripped away from the double-stranded duplex to produce a mature miRNA [9]. The mature miRNA molecules which are loaded into RISC, are capable of negatively regulating

Received December 24, 2013; accepted October 15, 2014 Correspondence: [email protected]

gene expression by perfect or imperfect complement to the 3′ untranslated region (3′ UTR) of target mRNAs (mRNAs) [10–12]. Currently, about 1000 human miRNAs are identified [13,14], and they are estimated to regulate as many as 30% of mRNA transcripts [15]. Diseases of the cardiovascular system, such as myocardial infarction, heart failure, hypertension, stroke and coronary artery disease, represent the predominant causes of human morbidity and mortality worldwide [16–18]. The altered expression profiles of genes, which are important for cardiac function, have been implicated in pathological process of the heart. Correct diagnosis and appropriate therapies for cardiovascular diseases are critical for improving the prognosis and survival rate [16,19]. Over the past five years, the implications of miRNAs in a variety of physiological pathological processes of the cardiovascular system have been widely recognized [20,21]. Gain- and loss-of-function studies in mice and rats have revealed that some special miRNAs play important roles in the pathogenesis of various cardiovascular diseases and become an intriguing target for therapeutic intervention [22,23]. Recently, the theme on investigating the role of circulating miRNAs in cardiovascular diseases has become a rapidly evolving field. The

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stable presence of miRNAs in blood has raised the possibility of using circulating miRNAs as biomarkers or extracellular communicators for diagnosis or treatments of diseases [24–27]. In the present review, we summarize recent data about the roles of circulating miRNAs in cardiovascular system, and assess the novel opportunities for diagnosis and therapies of cardiovascular diseases.

Release and stabilization of circulating miRNAs In 2008, circulating miRNAs were discovered by Lawrie et al. As a seminal paper, the authors demonstrated that the elevated level of circulating miR-21 was associated with relapse-free survival in patients with diffuse large B cell lymphoma (DLBCL); thus, miR-21 may have potential as non-invasive diagnostic biomarker for DLBCL disease [28]. Since then, in another similar study, miR-15b, miR16 and miR-24 were found to present in blood in remarkably stable forms and may serve as biomarkers for certain cancer [29]. Later, some other studies revealed that plasma miRNAs can resist the degradation from endogenous RNase activity [30,31]. Even suffering some harsh conditions, such as boiling, extreme pH, prolonger storage

Fig. 1 Possible interactions of miRNAs between the organs.

Circulating microRNAs and applications in cardiovascular disease

at room temperature and multiple freeze-thaw cycles, endogenous miRNAs can remain stable in blood [32]. In contrast, exogenous synthetic miRNAs were quickly degraded in plasma by RNase [32]. Altogether, these observations suggest that some special manners are responsible for preventing endogenous plasma miRNAs from degradation. Recently, several underlying mechanisms have been demonstrated in miRNAs releasing from cell and escaping from degradation, including packaging into microvesicles, loading in lipoprotein complexes and combining with some special proteins to form proteinmiRNA complexes [31,33]. In this section, we will review the current knowledge about cellular release and stability of circulating miRNAs (Fig.1). Plasma miRNAs in microvesicles (MVs) Recently, several studies clearly confirmed that circulating miRNAs are associated with MVs in circulation [34,35], and these vesicles are defined as exosomes, microparticles (MPs) and apoptotic bodies based on their size and the release manner from cells [33]. The studies described below have investigated the hypothesis that MVs may play crucial roles in the secretion and stability of circulating miRNAs.

Feng Wang et al.

Exosomes Exosomes are small (30–100 nm in size) membrane and secreted MVs that derive from the endosome and are released from cells after fusion of multivesicular bodies with the plasma membrane [36,37]. In an early study, more than 120 miRNAs had been indentified in exosomes from donor cells [34]. Intriguingly, the authors found that the levels of some special miRNAs were higher in exosomes than in their parent cells. Furthermore, another study found that some cellular miRNAs were selectively released into the extracellular space while others were kept in cells [34,38]. All these results imply that miRNAs can be selectively packaged in exosomes. In a recent study, Kosaka et al. revealed that miRNAs were secreted through a ceramide-dependent mechanism. Sphingomyelinase 2 (nSMase2), the rate-limiting enzyme of ceramide biosynthesis, is a crucial regulator for controlling the exosomal secretion of miRNA [39]. Consistently, blockading the activity of nSMase2 by either a chemical inhibitor (GW4869) or small interfering RNAs significantly reduced the secretion of miRNAs, whereas cellular expression of miRNAs remained unchanged. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. Furthermore, Hergenreider et al. confirmed that miR-143/145 cluster was also secreted via the exosomal pathway [40]. Tetraspanin-enriched microdomains (TEMs) are emerging as entities physically and functionally distinct from lipid rafts [41]. In 2010, it was reported that cell surface tetraspanin Tspan8 contributed to molecular pathways of exosome-induced endothelial cell activation [42]. By using exosomes derived from a cell line that expresses CD9, CD81, CD151, α3 and α4 and by transfecting with Tspan8, chimeric Tspan8 (replacement of the N-terminal region by that of CD9), or Tspan8 plus β4, it was demonstrated that exosomal tetraspanin-complexes involved in selective target binding [43]. It is conceivable that in the future expression modulation of exosomal integrins and tetraspanins may allow the targeting of therapeutic exosomes mediated nucleic acids, such as miRNAs, during systemic application [44]. MPs MPs are also membranous vesicles that originate from the direct budding of the cell plasma membrane [45]. MPs, with the diameter ranging from 100 nm to 1.0 μm, are larger than exosomes. An increasing body of evidences indicates that MPs, as secreted vesicles in peripheral circulation, play pivotal roles in intercellular information transmission [46,47]. By transferring chemokines, adhesion molecules, receptors, and bioactive lipids [48], MPs can influence the biological functions of receptor cells [46,49]. Previous studies have reported that certain miRNAs were secreted in the manner of MPs by a variety

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of cell types, including vascular cells, platelets and inflammatory cells [50,51], and this secretion was activated by various stimuli [52]. Importantly, different stimuli may alter the content of miRNAs in MPs, which suggesting that MPs-related secretion is an actively regulated process as well as in exosomes [53,54]. Recently, increased numbers of MPs have been reported related with cardiovascular diseases [55,56]. Philipp et al. showed that MPs are major transport vehicles in circulation for large numbers of specific miRNAs, which have been associated with cardiovascular diseases [57]. Apoptotic bodies Apoptotic bodies are the largest lipid-encapsulated vesicles (0.5–2.0 μm in size), which are released at the early stages of cell apoptosis [58,59]. Apoptotic bodies are generated in response to apoptotic stimuli [60]. Similar to MPs, apoptotic bodies contain cytoplasmic contents which derived from the parent cells, and nucleic acids which have been proved to present in apoptotic bodies previously [61]. However, the presence of miRNA in apoptotic bodies has not been confirmed until a few years ago [62]. In a landmark study, Zernecke et al. showed that miR-126 abundantly present in apoptotic bodies originate from endothelial cell (ECs), and can induce vascular protection via CXCL-12 in neighboring cells. Interestingly, although it had been proved that miR-126 was enriched in apoptotic bodies derived from ECs, the abundant presence of miR126 was not found in shear-flow induced exosomes from ECs [62]. These imply that the loading of miRNA into apoptotic bodies is as specific and selective as into exosomes and MPs. However, this view requires further investigations. Plasma miRNAs in high-density lipoproteins (HDLs) HDLs, with the average size of 9 to 13 nm, are known as the most important lipid carrier for returning excess cellular cholesterol to the liver for excretion. Previous studies demonstrated that lipoproteins could carry nucleic acids and were used as gene delivery vehicles [63,64]. Recently, Vickers et al. first presented evidences that HDLs could transport endogenous miRNAs and deliver them to recipient cells with functional targeting capabilities [65]. miR-223 is one of the most abundant miRNAs in both human and mouse HDLs, and HDLs-mediated delivery of miR-223 not only significantly but also directly reduced the mRNA levels of RhoB and EFNA1 in cocultured hepatocytes (60% decrease and 76% decrease, respectively). Intriguingly, the export of cellular miRNAs to HDLs was negatively regulated by neutral sphingomyelinase 2 via ceramide pathway, while the delivery of miRNAs by HDLs to recipient cells was dependent on scavenger receptor class B type I. Moreover, the human

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HDLs-miRNA profile of familial hypercholesterolemia patients was significantly different from that of control subjects [65]. Collectively, these observations indicate that HDLs participates involve in the transport and delivery of miRNAs. Plasma miRNAs in protein-miRNA complexes Besides MVs and HDLs, miRNAs may also be exported by protein complexes. Actually, accumulating evidences revealed that the majority of circulating miRNAs were cofractionated with RNA binding proteins rather than encapsulated within lipid-containing membranous vesicles [66]. The results from a recent study by Arroyo et al. showed that non membrane-bound miRNAs may count up ~90% of the total circulating miRNAs [66]. Although little is known regarding the origin of protein-bound miRNAs, they were detected both in cell culture supernatants and plasma samples [66,67]. Moreover, the release of proteinbound miRNAs may be simply due to the passive release from death or apoptotic cells; then remain in extracellular space due to the high stability of the protein-miRNA complex. Argonaute 2 Argonaute 2 (Ago2), which acts as a crucial intracellular effector protein of miRNA-mediated silencing, has been observed in the circulation associated with plasma miRNAs [66]. Using differential centrifugation and sizeexclusion chromatography approaches, Arroyo et al. systematically characterized circulating miRNA complexes in human plasma and serum. Surprisingly, they found that the majority of circulating miRNAs were bound with protein complexes, and unbounded miRNAs were still sensitive to protease treatment of plasma. These results indicated that protein complexes can prevent circulating miRNAs from degradation by plasma RNases. Further analysis found that many extracellular miRNAs were eluted with Ago2 by size-exclusion chromatography. Finally, these results identified Ago2 as an important and independent miRNA-bound protein complex in blood. Furthermore, another study by Turchinovich et al. showed that various miRNA-Ago2 protein complexes were identified in plasma and cell culture media [67,68]. However, it has been proposed that miRNA-Ago2 protein complexes are released into the circulation as a consequence of cell lysis/necrosis and may not participate in intercellular communication. Undoubtedly, this hypothesis should be carefully tested.

Circulating microRNAs and applications in cardiovascular disease

exactly identified 12 RNA binding proteins in the conditioned medium of human fibroblasts after 2 h of serum starvation. Besides several ribosomal proteins, the authors found a significant amount of nucleophosmin 1 (NPM1), a known nucleolar RNA binding protein, as well as nucleolin (a known NPM1-interacting protein) in the medium [69]. Indeed, NPM1 is located primarily in the nucleolus, where it is implicated in the nuclear export of the ribosome. Subsequent experiments showed that incubation of synthetic miR-122 with NPM1 protein can fully protect this miRNA from RNaseA digestion. It has been suggested that NPM1 may be involved in shuttling RNAs from the nucleus to the cytosol, and the NPM1 protein also was found in the extracellular space outside the cell. This shuttling mechanism implied a possible role of NPM1 protein in miRNA export. Taken together, it is interesting to speculate that NPM1 is involved in the processes for miRNA export and stability. However, whether miRNAs-NPM1 protein complex presents in blood is still not elucidated. In short, the stable existences of miRNAs in peripheral circulation were dependant on the combination between miRNAs and MVs/HDLs/binding protein. However, the underlying mechanisms, which account for the selective release of circulating miRNAs, were remained to be elucidated.

Circulating miRNAs as biomarkers for cardiovascular diseases The first discovery of circulating miRNAs, and the association between circulating miRNAs and certain cancer diseases was in 2008 [28]. Since then, circulating miRNAs have attracted more and more interests from the scientific community for their potential applications as novel clinical biomarkers of cardiovascular diseases [70,71]. In the past five years, based on their characters of tissue specificity, rapid release kinetics and stability in plasma, circulating miRNAs have been proposed as novel and potential biomarkers for diagnosing and prognosing various cardiovascular diseases, such as acute myocardial infarction (AMI) [72,73], heart failure (HF) [74], coronary artery disease (CAD) [75], hypertension [76], viral myocarditis (VM) [77], etc. In this section, we will summarize the current clinical and experimental studies, and discuss the role of circulating miRNAs as diagnostic or prognostic biomarkers of cardiovascular diseases (Table 1). AMI

Nucleophosmin Notably, Ago2 is not the only miRNA binding protein in cell culture supernatant. By mass spectrometry, Wang et al.

AMI, because of high morbidity and mortality, is the worst disease of cardiovascular diseases. A timely and correct diagnosis is essential for controlling the development of

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Table 1 Key miRNAs in cardiovascular diseases Diseases

miRNA

Patterns

Models

References

AMI

miR-208

Increased

ISO rat CAL rat Human patients

[76] [68] [68, 73]

miR-499

Increased

Human patients AMI mice

[66, 69] [73]

miR-1

Increased

CAL rat Human patients

[77] [78]

miR-133

Increased

Human patients

[66, 79, 81]

HF

miR-423-5p

Increased

Human patients

[70]

CAD

miR-126,-17,-92a,-155, -145 Decreased miR-133a, -208a Increased

Human patients Human patients

[71] [71, 81]

miR-135a

Increased

Human patients

[90]

miR-147

Decreased

Human patients

[90]

Hypertension

hcmv-miR-UL112

Increased

Human patients

[72]

VM

miR-208b, -499

Increased

Human patients

[73]

AMI: acute myocardial infarction; ISO: isoproterenol; CAL: coronary artery ligation; HF: heart failure; CAD: coronary artery disease; VM: viral myocarditis.

AMI, initiating the appropriate therapy and reducing the mortality rate and improving prognosis, which prompted continuous searches for novel biomarkers with high sensitivity and specificity [78]. AMI is characterized by the sudden occurrence of cardiac cell death, which results in the release of cell contents such as the established biomarker cardiac troponin. Therefore, a number of initial studies supported the hypothesis that certain cardiacspecific miRNAs would be also released into blood during AMI. miR-208 miR-208, a cardiac-specific microRNA encoded by an intron of the α-MHC gene, had been reported involved in cardiomyocyte hypertrophy, fibrosis and expression of βMHC gene in response to stress [79]. An elegant study by Ji et al. discovered that plasma concentration of miR-208 was increased in isoproterenol-induced myocardial-injury rat, whereas the plasma level of miR-208 was not increased in renal injury model [80]. Plasma miR-208 concentration was significantly increased during the first several hours after myocardial-injury by isoproterenol, and it showed a significant positive correlation with the plasma level of cardiac troponin I. Another animal experiment showed that the peak of elevated miR-208 level appeared at 3 h after the onset of AMI in rat model of coronary artery ligation [72]. In humans, several studies identified the exclusive expression pattern of cardiac miR-208a by microarray screening and confirmed the highly elevated level of circulating miR-208 in blood sample of AMI patients [72]. Moreover, the receiver-operator-characteristics (ROC) curves analysis revealed that plasma miR-208 with a high AUC exhibited a strong discrimination between AMI patients and healthy controls [77]. These results indicated

that plasma miR-208 is a high sensitivity, specificity and new biomarker for AMI diagnosis. miR-499 Adachi et al. assessed the levels of plasma miR-499 in patients with AMI, unstable angina pectoris or congestive heart failure, respectively [73]. The results demonstrated that the levels of plasma miR-499 were remarkably increased in AMI patients, whereas plasma miR-499 concentrations in control and CHF groups were below the detective limitation. Interestingly, plasma miR-499 concentrations were rapidly increased in the acute phase (within 48 h of the last onset of chest pain) of AMI patients, and became undetectable before hospital discharge. In addition, Alessandra et al. determined the time course expressions of plasma miR-499 in patients with STsegment elevation myocardial infarction from another cohort [70]. Totally, 9 blood samples were collected from each AMI patients. The first sample was obtained 156  72 min after the onset of AMI symptoms (T0) and additional samples were obtained 3, 9, 15, 21, 33, 45, and 69 h after T0, respectively. Circulating miR-499 exhibited a slower time course and peak time after cTnI, which achieved a 299.1  106.4 fold peak at 9 h after T0 and returned close to its control level at the end of the 3day time course. A similar dynamic trend of plasma miR499 was observed in mice with AMI. ROC curve of circulating miR-499 showed a strong separation between AMI patients and healthy volunteers [77]. miR-1 The potential diagnostic value of circulating miR-1 has been profoundly studied in a rat AMI model induced by

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coronary ligation. The level of serum miR-1 was quickly increased after AMI with a peak at 6 h (over 200-fold) and returned to basal level at 3 days post-AMI [81]. Interestingly, serum miR-1 concentration positively correlated with myocardial infarct size in rats. Moreover, the elevated serum miR-1 induced by ischemia/reperfusion (I/ R) injury was reduced in an ischemic preconditioning model. In addition, several clinical studies were carried out for further investigating the possibility of miR-1 as a biomarker of AMI [70]. Consistent with animal experiments, an apparent elevation of plasma miR-1 was found in AMI patients, and plasma level of miR-1 has been showed to be well correlated with the QRS complex [82]. miR-133 Several studies showed that miR-133 levels were increased in both experimental AMI animal models and AMI patients [70]. In an ingenious study, Rosa et al. hold the hypothesis that cellular miRNAs would be released into the coronary circulation during myocardial injury. To assess the plasma levels of some certain miRNAs in blood from peripheral circulation and coronary circulation, blood samples were simultaneously obtained from the aorta and the coronary venous sinus in patients with acute coronary syndromes, with stable coronary artery disease, and without coronary artery disease. The results showed a significant increase in levels of circulating miR-133a and miR-499 after flowing through the coronary circulation in patients with acute coronary syndrome, suggesting that miR-133a and miR-499 were released from the heart into the coronary circulation during myocardial injury [83]. Consistent with this, another study showed that extracellular miR-133a in exosomes was released from injured myocardium after Ca2+ stimulation, and miR-133a was taken up by recipient cells where it exerted its effects [84]. In our previous study, we confirmed the dynamic levels of circulating miR-133a at the early phase of AMI, and showed a significant correlation between plasma miR-133a concentrations and severities of the coronary artery stenosis [85]. In summary, four miRNAs (miR-208a, miR-499, miR-1, and miR-133) were found elevated in plasma of both AMI patients and AMI animal models [72,86]. However, the use of these four circulating miRNAs as novel and specific biomarkers for AMI in clinical still remains controversial. On the one hand, unlike miR-208, the other 3 miRNAs (miR-499, miR-1, and miR-133) are highly expressed in heart as well as in skeletal muscle, indicating that elevated plasma levels of these three miRNAs might be induced by skeletal muscle injury [72,87,88]. Wang et al. demonstrated that miR-1, miR-133a, and miR-499 could also be released from wounded skeletal muscle during surgery procedure of thoracotomy and thus lack absolute cardio-

Circulating microRNAs and applications in cardiovascular disease

specificity. On the other hand, the elevated levels of plasma miR-208 in AMI patients were not observed in other studies, the levels of circulating miR-208 in AMI patients from these studied were very low, even undetectable [70,75]. So some large and profound explorations are required in the future. Other miRNAs It has been reported that several other plasma miRNAs are associated with AMI, such as miR-21, miR-126, and miR146a [71,89]. In our previous study, we found that circulating miR-30a and miR-195 concentrations were remarkably increased in AMI patients, whereas plasma level of let-7b was decreased [90]. Furthermore, we found that the reduced plasma miR-519e-3p exhibited a powerful differentiation value for AMI patients from healthy volunteers, ischemic stroke patients, or patients with pulmonary embolism [91]. In a prospective study, Zampetaki et al. explored the correction between baseline levels of miRNAs and myocardial infarction incidents. In multivariable Cox regression analysis, 3 circulating miRNAs derived from platelets were consistently and significantly correlated with the incidents of myocardial infarction. Plasma miR-126 showed a positive correlation, whereas circulating miR-223 and miR-197 were negatively associated with disease risk [92]. These observations suggest that circulating miRNAs may be used as predictors for evaluation of cardiovascular events. HF Recently, the expression profiles of miRNAs have been systematically assessed in blood from subjects with HF. Based on the results from a miRNA array performing on plasma of 12 HF patients and 12 healthy controls, 16 plasma miRNAs were validated by qPCR in a second cohort, including 30 HF patients, 20 non-HF patients with dyspnea and 39 healthy subjects. The results revealed that miR-423-5p was specifically enriched in blood of HF cases, and it can intensively distinguish HF patients from healthy controls and non-HF patients with dyspnea. Further, ROC curve analysis showed that miR-423-5p, with an AUC of 0.91, may be a strong diagnostic predictor for heart failure [74]. Cardiac resynchronization therapy (CRT) was a wellestablished therapy for HF patients with LVEF ≤ 35% and QRS duration ≥ 120 ms on the ECG to prolong their survival. However, a considerable part of HF patients do not respond clinically to CRT and exhibit no evidence in reversing LV remodeling, and the potential mechanisms for this were still not clear. In recent years, circulating miRNAs were evaluated in 81 HF patients treated with CRT for I2 months. Plasma miRNAs were differentially

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expressed between 55 responders and 26 non-responders, the levels of circulating miR-26b-5p, miR-145-5p, miR92a-3p, miR-30e-5p, and miR-29a-3p were significantly increased in responders compared to non-responders. The fold increases of these circulating miRNAs were directly correlated with EF and inversely correlated with NTproBNP in HF patients by depth analysis. These results suggested that circulating miRNAs may be involved in the cardiac functional recovery of patients with HF. Besides above miRNAs, miR-126 and miR-499 are also potential candidates of HF biomarkers. The level of circulating miR-499 was significantly increased in acute HF [77], whereas plasma concentration of miR-126 was negatively correlated with age and NYHA class in HF patients [93]. However, since plasma miR-499 level was below the limit of detection for individuals with congestive HF and the decreased circulating miR-126 was also observed in CAD and diabetic patients in some other studies [73,75,94], further explorations are required to confirm whether these plasma miRNAs are capable of acting as independent and specific HF biomarkers. CAD CAD is still the leading cause of death worldwide. The occurrence and progression of CAD are determined by the combination of genetic determinants and environmental factors. Activation of endothelial cells, invasion of proinflammatory cells, accumulation of lipid, and proliferation and dedifferentiation of smooth muscle cells are involved in the development of atherosclerotic lesion. Using microarray screening and TaqMan quantitative RT-PCR assays, Fichtlscherer’s group recently investigated the regulation of miRNAs in patients with CAD. Circulating levels of endothelial-enriched miR-126, miR-17, miR-92a, inflammation-associated miR-155, and smooth muscleenriched miR-145 were significantly reduced in patients with CAD, whereas plasma levels of cardiac muscleenriched miR-133a and miR-208a tended to be higher in patients with CAD than in healthy controls [75]. Based on the results from previous studies about delivering miRNAs into atherosclerotic lesion by apoptotic bodies, the authors speculated that the reduction of circulating miRNAs detected in CAD patients might be caused by an uptake of circulating miRNAs into atherosclerotic lesions. In our recent study, 154 CAD patients and 92 non-CHD patients were consecutively recruited for assessing the diagnostic value of circulating miR-133a for CAD. A significant elevated level of plasma miR-133a was observed in CAD patients, and ROC analysis revealed that circulating miR133a had considerable diagnostic accuracy for CHD with an AUC of 0.918. Importantly, circulating miR-133a has been found to be a more appropriate biomarker of underlying coronary artery stenosis than cardiac troponin [85].

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In another study, Hoekstra et al. determined the miRNA signature of peripheral blood mononuclear cells (PBMCs) from CAD patients [95]. The expression of miR-135a in PBMCs achieved a marked 5-fold increase in CAD patients, while the expression of miR-147 was 4-fold decreased. microRNA/target gene/biological function linkage analysis suggested that the dynamic changes of PBMCs miRNAs in CAD patients were probably associated with intracellular cadherin/Wnt signaling. Furthermore, patients with unstable angina pectoris could be distinguished from stable patients based upon elevated levels of a cluster of three PBMCs miRNAs, suggesting that miR-134, miR-198, and miR-370 can be used to identify patients at risk for acute coronary syndromes. Hypertension Microarray screening of plasma miRNAs revealed a novel and unexpected link between human cytomegalovirus and essential hypertension [76]. Twenty-seven differentially expressed miRNAs were identified in plasma samples from hypertensive patients. A human cytomegalovirus (HCMV)-encoded miRNA, hcmv-miR-UL112, had the highest expression level (approximate 2.5-fold) in hypertensive patients. Subsequent results showed that virus titers of HCMV were also substantially higher in the hypertension patients than in control subjects. After adjustment for the confounding risk factors of hypertension, the authors found that the seropositivity and copy number of HCMV were independently and positively associated with an increased risk of essential hypertension. Importantly, a significant correlation was found between HCMV virus titers and hcmv-miR-UL112 levels in hypertensive patients. Interferon regulatory factor 1 (IRF-1), which can regulate blood pressure by inducing the expressions of angiotensin II type 2 receptor and nitric oxide synthase, is a direct target of hcmv-miR-UL112. Additionally, increasing evidences indicated that IRF-1 plays critical roles in immunological, inflammatory, and anti-infection responses. Taken together, it is not difficult to speculate that IRF-1 repression by hcmv-miR-UL112 could be unifying mechanisms of human cytomegalovirus for evading the immune response and increasing blood pressure in host. These findings may provide possible mechanisms of HCMV in the pathogenesis of essential hypertension and suggest potential therapeutic targets. Previous study demonstrated that human angiotensin II type-1 receptor (AT1R), which plays an important role in the regulation of blood pressure, was a target gene of miR155 [96]. However, a silent polymorphism ( + 1166 A/C single-nucleotide polymorphism) localized in the 3′-UTR of AT1R may affect the combination between miR-155 and AT1R mRNA. In a recent study, Giulio Ceolotto et al. found that the expression of miR-155 was significantly decreased in PBMC from hypertension patients with CC

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genotype in comparison to AA and AC genotype, whereas AT1R protein expression was significantly increased in the CC group compared to AA and AC groups. Therefore, the silent polymorphism ( + 1166 A/C) of AT1R may be a underlying mechanism of hypertension [97]. Hypertension was an important cause of stroke. In previous studies, circulating miRNAs had been proved to serve as potential non-invasive biomarkers for the early detection of hypertension-related stroke [26]. For example, circulating miR-145 level was significantly increased in patients with ischemic stroke compared to control [98]. In our recent study, we verified that miR-30a, miR-126 and let-7b may be useful biomarkers for ischemic stroke in humans [99]. VM Myocarditis is an inflammatory disease of the myocardium caused by viral infections, postinfectious immune reactions, or organ-specific autoimmune reactions. In a clinical experiment, Corsten’s group subsequently determined the levels of some special circulating miRNA in 14 patients with acute VM, 20 patients in the post-VM phase, and 20 healthy control subjects, respectively [77]. The plasma levels of cardiac-enriched miR-208b and miR-499 were significantly elevated at the acute phase of VM, and their levels were remarkably correlated with the severities of VM. However, leukocyte-associated miR-146a, miR146b, miR-155, and miR-223 were not increased following the significant leukocytosis in the acute VM group. Besides viral infections, the elevated levels of cardiacenriched miR-208b and miR-499 were also observed in patients with AMI or with HF. Therefore, it is readily plausible that plasma miR-208 and miR-499 could reflect myocardial damages caused by various kinds of pathogens in cardiovascular diseases. Collectively, all these findings support the notion that circulating miRNAs can be promising biomarkers for early detection of various cardiovascular diseases.

Circulating miRNAs as extracellular communicators in cardiovascular diseases The remarkable stability of miRNAs in the circulation raises the intriguing possibility that functional miRNAs packaged in microparticles, HDL or Ago2 were released into the peripheral circulation, and taken up by distant recipient cells to regulate their gene expression. Besides their potential roles as biomarkers, circulating miRNAs may have other potential biological functions. Currently, accumulating evidences suggest that circulating miRNAs play a previously unrecognized role in intercellular communication. In this section, we focus on discussing the available evidences regarding the potential functions of

Circulating microRNAs and applications in cardiovascular disease

circulating miRNAs in extracellular signal transmission (Fig.1). Communication by membrane-bound miRNAs Early studies of microvesicles, including exosomes and MPs, had demonstrated that these membrane-vesicles could indeed transport tRNA and protein contents to distant cells and influence the behaviors of the recipient cells. For instance, microvesicles derived from glioblastoma tumor cells transport genetic information and proteins to recipient cells in the tumor environment which promoted tumor growth [100]. In addition, embryonic stem cell-derived microvesicles are capable of reprogramming hematopoietic progenitors [35]. The results of these studies imply that miRNAs in vesicles, as well as other bioactive molecules in vesicles, may also have ability to modulate distant cell function. In 2009, it was first confirmed that miRNAs in membrane-vesicles may indeed have effects on cell-to-cell communication [62]. As previously described, originated from endothelial cells, miR-126 secreted in apoptotic bodies could directly regulate the expression of its target gene RGS16 in recipient cells, subsequently trigger an autoregulatory feedback loop to increase the production of chemokine receptor CXCL12 in target cells, and ultimately induce CXCL12-dependent vascular protection. Soon thereafter, Zhang et al. clearly demonstrated that blood cells and cultured monocytes cells (THP-1) selectively packaged immune-related miR-150 into microvesicles and actively secreted it into the peripheral circulation or the culture medium in response to diverse stimuli. The monocytic miR-150 in MVs was delivered into human HMEC-1 cells, and enhanced HMEC-1 cells migration by directly and effectively repressing the expression of c-Myb gene in HMEC-1 cells. Interestingly, MVs isolated from the plasma of patients with atherosclerosis were enriched with miR-150, and could enhance the migration of recipient HMEC-1 cells. Altogether, elevated miR-150 secreted in MVs may play an important role in regulating endothelial cell function during the pathological processes of atherosclerosis [53]. Moreover, Hergenreider et al. demonstrated that miR-143 and miR-145 regulated by atheroprotective flow and Krüppel-like factor 2, a shearresponsive transcription factor, were transferred by extracellular vesicles from endothelial cells to smooth muscle cell (SMC), and then reduced the expressions of their target genes to induce an atheroprotective SMC phenotype [40]. Interestingly, in 2013, it was demonstrated that 16-kDa Nterminal prolactin fragment (16K PRL) induced miR-146a expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in

Feng Wang et al.

metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a peripartum cardiomyopathy (PPCM)-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antagomiRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling. These results show that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as not only a biomarker but also therapeutic target for PPCM [101,102]. Recently, Wang et al. discovered that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells [103]. Besides cardiovascular diseases, some similar studies were carried out in other diseases, such as cancer and virus infection. Recently, Iguchi’s group has revealed that exosomal miR-16 acted as a translational inhibitor for suppressing its target gene in the recipient cells in nude mice [104]. In addition, exosomes-mediated circulating miRNAs have been found involving in the underlying mechanisms of Epstein-Barr virus (EBV) infection. Pegtel et al. demonstrated that EBV-encoded miRNAs secreted in exosomes by EBV-infected B cells could be released to peripheral circulation and taken up by neighboring monocyte-derived dendritic cells, where they could suppress the expressions of confirmed EBV target genes, such as immunoregulatory gene CXCL11/ITAC [105]. The results from Pegtel’s study raised an intriguing speculate that the significantly elevated level of circulating hcmvmiR-UL112 in patients with hypertension as previously described might also be transported into the recipient cells and exerted its effects as a silencer of gene expressions. Previous studies proposed that the internalization of microvesicles by distant cells was involved in the process of endocytosis or membrane fusion. Recently, Montecalvo et al. demonstrated that circulating miRNAs were taken into the cytosol after exosomes fused with the target dendritic cells, and repressed target mRNAs of acceptor cells [106]. These results indicate that the uptake of membrane-bound circulating miRNAs may associate with underlying mechanisms of internalization of vesicles. Communication by non-membrane-bound miRNAs Direct delivery of circulating miRNAs to recipient cells can also be mediated by HDL [65]. To test whether miRNAs can be delivered by HDL to recipient cells and sufficiently alter their target genes expressions in recipient cells, Vickers et al. incorporated native HDL with exogenous miR-223, and introduced the HDL-miR-375 complexes to cultured hepatocytes. The results showed that cellular delivery of HDL incorporated with miR-223 remarkably elevated miR-223 expression levels in hepa-

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tocytes, which resulted in a significant decrease of the levels of its targets mRNA (RhoB and EFNA1). Furthermore, HDL isolated from familial hypercholesterolemia and normal subjects were incubated with hepatocytes. Compared with normal HDL, familial hypercholesterolemia HDL-miRNA delivery increased intracellular miR-105 levels, and significantly repressed its target mRNA levels. Interestingly, sequential results revealed that the uptake of HDL-medicated miRNAs was dependent on scavenger receptor class B type I (SR-BI). The delivery of HDL-miR-223 (both endogenous and exogenous) complexes significantly increased the intracellular level of miR-223 in SR-BI-expressing cells compared with non-SR-BI-expressing cells, and knockdown of SRBI by siRNA resulted in a remarkable loss of HDL-miR223 delivery. As demonstrated by Arroyo’s team, the majority of miRNAs in circulation appeared to be bound with Ago2 protein [66], and this raises the possibility that functional miRNA-induced silencing complexes were released into circulation from host cells and might alter the expressions of their target genes in recipient cells. However, until now, no reports indicated that miRNAAgo2 complexes can be taken up by distant recipient cells. In summary, all these observations strongly denominated that circulating miRNAs either stored in MVs or incorporated with HDL might have biological functions to influence recipient cellular activities and play a very important role in cellular communication.

Prospects of circulating miRNAs As discussed above, miRNAs exhibit significant stability and detestability in circulation, and their expression profiles are often cell/tissue-specific. The differential expressions of circulating miRNAs have been associated with the processes of diverse cardiovascular disorders. All these biological characteristics of circulating miRNAs make them an attractive class of biomarkers with high sensitivity and specificity. Moreover, several studies indicated that certain plasma miRNAs were more sensitive and specific than well-established biomarkers and the combination of circulating miRNAs to traditional biological markers, such as troponin, could significantly increase the diagnostic accuracy of cardiovascular diseases [71,85]. Importantly, besides diagnosing of diseases, abnormal circulating miRNAs also have ability to identify subjects who are at risk of the development of acute manifestations of cardiovascular diseases [92]. Not yet clinically established, but recently discovered and investigated is the potential role of mRNA and miRNAs as biomarkers in cardiovascular diseases. However, before using circulating miRNAs as novel and meaningful diagnostic tools clinically, several restrictive problems need to be urgently addressed. First, numerous experimental procedures and

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techniques were applied to investigate the levels of circulating miRNAs in different studies, which make it difficult to compare the data of circulating miRNA obtained from different academic researches. So far, the methods for testing the absolute concentrations of plasma miRNAs have yet not been determined. A standardized and normalized method of miRNAs extraction and quantification is badly required. Secondly, most previous studies were based on small number of patients and did not have appropriate controls, which may be responsible for some inconsistent observations from different studies. Some further studies with large number of participants are expected in the future. Thirdly, appropriate reference small RNAs are also important for detecting circulating miRNAs. Plasma housekeeping gene U6 or other miRNAs were used as endogenous control for data normalization in previous studies. However, the changes of these RNAs plasma levels have been reported under some pathological conditions. A spiked-in normalization approach reported by Mitchell et al. suggested that synthetic exogenous miRNAs derived from C. elegans could be used as normalizers [29], but this still needs careful verification because of these synthetic miRNAs may be less stable than endogenous miRNAs in plasma with high level of RNase activity. Moreover, the choice of a matrix (serum, plasma, PBMC or whole blood) for the detection of miRNAs can have a direct impact on the expression profiles of these novel biomarkers [107]. Taken together, challenges remain in the process to prepare miRNAs as biomarkers for prognosis or therapy response for the clinical transfer. In recent years, genetic gain- and loss-of-function studies have revealed the prominent roles of miRNAs in various cardiovascular disorders, including HF [108,109], cardiac hypertrophy and fibrosis [108,110], AMI [111], and angiogenesis, etc. [112]. The ability to selectively manipulate the expressions and functions of miRNAs in cardiovascular system has provoked the idea that certain miRNAs might be potential therapeutic targets for cardiovascular diseases [22]. As discussed above, miRNAs in circulation may be involved in cell-to-cell communication by mediating the repression of critical mRNA targets in distant recipient cells, which may trigger the possibility that administration of miRNAs through the peripheral circulation might effectively achieve miRNA-based therapeutics. To date, various synthetic miRNA small molecule analogs and antisense miRNA oligonucleotides are designed and adapted to manipulate the expression of miRNA in vivo [23]. miRNAs mimics, a class of synthetic double-stranded miRNA analogs, are widely used to regulate gene expression by simulating the functions of miRNA-medicated gene silencing in vitro. However, in vivo, miRNA mimics have not yet been fully demonstrated. Recombinant adeno-associated virus, with low toxicity and antigenicity, may be a promising vector for long-term and effective enhancing miRNAs-medicated

Circulating microRNAs and applications in cardiovascular disease

gene therapy. Antisense miRNA chemistries, various highaffinity 2' sugar modified oligonucleotides, can inhibit miRNA expression and function through complementary base pairing with miRNAs. Recently, antisense miRNA chemistries are widely used for therapeutically regulating miRNA expression in vivo and the developments are far ahead of the miRNAs mimics. Miravirsen, a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide, is the first and successful miRNA therapeutic drug in human clinical trial for suppression of hepatitis C virus replication by sequestering liver-expressed miR-122 in a highly stable heteroduplex [113]. Recently, several studies have demonstrated antimiR chemistries, such as antagomir and locked nucleic acid, can effectively block the development and process of cardiovascular diseases by therapeutically manipulating miRNAs expression. miR208, encoded by an intron of the human and mouse βMHC gene, has been involved in regulating cardiac remodeling and β-MHC expression in response to increased heart afterload by thoracic aortic banding [79]. A subsequent study by Montgomery et al. showed that therapeutic inhibition of miR-208a by subcutaneous delivery of an LNA-modified oligonucleotide inhibitor (antimiR-208a) can profoundly suppresses cardiac remodeling and myosin-7 expression while improve cardiac function and survival in hypertension-induced heart failure rat model [114]. Most notably, delivery of anti-miR-208a via several routes of administration, including intravenous, intraperitoneal, and subcutaneous, showed no significant differences in anti-miR-208a detection and all of them exhibited a robust and long-term inhibition of miR208a in vivo. Importantly, another study demonstrated that anti-miR-208a displayed an unexpected resistance to obesity in mice [115]. In addition, the dysregulation of miR-15 family, including 6 closely related miRNAs, have been indicated in response to myocardial infarction, which might result in death of cardiomyocytes and loss of pump function [111]. Hullinger et al. determined that therapeutic delivery of LNA-modified anti-miR-15 chemistries significantly reduced infarct size, repressed cardiac remodeling, and enhanced cardiac function in response to AMI [116]. Most importantly, to date, the anti-miR-208 and anti-miR-15 family chemistries have been delivered into the pre-clinical applications for treatments of heart failure and post-myocardial infarction remodeling, respectively. In conclusion, the potential functions of circulating miRNAs are unexpectedly strong, which may provoke great expectations of using them for the diagnosis, prognosis and therapeutic of cardiovascular diseases.

Acknowledgements This work was supported by grant from the National Natural Science Foundation of China (No. 31200594), Research Fund for the

Feng Wang et al.

11

Doctoral Program of Higher Education of China (No. 20120142120056) and Project from Hubei Province (JX6A02).

Abbreviations and acronyms Ago2

Argonaute 2

AMI

acute myocardial infarction

CAD

coronary artery disease

CRT

cardiac resynchronization therapy

EBVs

Epstein-Barr virus

ECs

endothelial cells

HCMV

human cytomegalovirus

HDLs

high-density lipoproteins

HF

heart failure

IRF-1

interferon regulatory factor 1

miRNAs

microRNAs

MPs

microparticles

mRNAs

mRNAs

MVs

microvesicles

NPM1

nucleophosmin 1

nSMase2

sphingomyelinase 2

PBMCs

peripheral blood mononuclear cells

pre-miRNA

precursor miRNA

pri-miRNAs

primary miRNAs

RISC

RNA induced silencing complex

ROC

receiver operator characteristics

SMC

smooth muscle cell

SR-BI

scavenger receptor class B type I

TEMs

tetraspanin-enriched microdomains

VMs

viral myocarditis

Compliance with ethics guidelines Feng Wang, Chen Chen, and Daowen Wang declare that they have no conflict of interest. This manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee.

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