Cardiovascular disease is a global health problem. Of all 60 million cases of deaths from all causes worldwide in 2005, an estimated 18 million were due to cardiovascular diseases, three times more than caused by infectious diseases including HIV/AIDS, tuberculosis, and malaria combined (World Health Organization, 2013). Current clinical diagnostics fail to identify early changes of adverse cardiac or vascular remodeling, forcing clinicians to wait for these cardiovascular disorders to become clinically evident before initiating intervention. Additionally, treatment efficacy cannot be reliably assessed in individual patients, in part as many interventions are merely treating symptoms (e.g., diuretics). Ideally, one would not assess intervention success based on survival or hospitalization, but build in intermediate end-points that can reliably assess therapeutic benefit. Thus, for the cardiovascular field, there is a need to identify intermediate diagnostic measures that monitor subtle biological changes in the heart or vasculature that directly reflect and predict adverse changes before they become clinically apparent.

To achieve the goal of early diagnosis and treatment, microRNAs (miRNAs) could play an unexpected role. MiRNAs are a group of non-coding regulatory RNAs of about 22 nucleotides that control gene expression at the post-transcriptional level (Bartel, 2004) and act as crucial regulators of most physiological and pathological processes. Indeed, dysregulation of intracellular miRNA expression has been linked to many clinically relevant cardiovascular conditions (Small and Olson, 2011; Da Costa Martins and De Windt, 2012; Gladka et al., 2012; van Empel et al., 2012). Unexpectedly, the recent discovery of circulating miRNAs has opened the possibility to study this class of biologically active agents as modes of inter-cellular information flow as well as biomarkers of disease. Here, we present an overview of the different carriers associated with extracellular miRNAs that render them stable in biological fluids, present the current level of understanding of their role in cell-to-cell communication and give an overview about the clinical utility of extracellular miRNAs as putative biomarkers for cardiovascular disease entities.

The first accounts of extracellular miRNA biomarkers were described in serum of lymphoma patients (Lawrie et al., 2008) and in plasma and serum of prostate cancer patients (Mitchell et al., 2008). Subsequently, it became evident that miRNAs can be exported from cells, and found in most extracellular biological fluids including plasma, serum, saliva, urine, tears, and breast milk (Chim et al., 2008; Weber et al., 2010; Boon and Vickers, 2013). Extracellular miRNAs are unexpectedly stable, and must be shielded from degradation, as naked RNA is readily targeted by exonucleases that are abundantly present in various extracellular fluids (Kamm and Smith, 1972). Indeed, miRNAs are packaged in microparticles (exosomes, microvesicles, and apoptotic bodies; Valadi et al., 2007; Hunter et al., 2008; Zernecke et al., 2009) or by their association with RNA-binding proteins including Argonaute 2 (Ago2; Arroyo et al., 2011) or lipoprotein complexes such as high-density lipoprotein (HDL; Kamm and Smith, 1972; Vickers et al., 2011; Figure 1).

The term exosomes was used for the first time in 1981 to describe exfoliated membrane vesicles (Trams et al., 1981). Exosomes are small (40–120 nm) extracellular microvesicles arising from multivesicular bodies (MVBs) and released by exocytosis of these MVBs (Heijnen et al., 1999). They are produced by a variety of cells including epithelial cells (Zhou et al., 2011), hematopoietic cells (Laulagnier et al., 2004), endothelial cells (Halkein et al., 2013), and tumor cells (Mitchell et al., 2008). Exosomes have also been identified in most circulating body fluids such as plasma, urine, milk, saliva, and sperm (Thery et al., 2006). The interest of exosome biology was increased following the demonstration that exosomes can serve as carriers for miRNAs (Valadi et al., 2007; Gallo et al., 2012). Selection processes must take place of miRNA uploading into exosomes, as some miRNAs can be either more or less expressed in donor cells or in the secreted exosomes (Valadi et al., 2007; Pigati et al., 2010), suggesting the existence of cellular mechanisms that actively concentrate specific miRNA species in exosomes (Valadi et al., 2007; Pigati et al., 2010).

Microvesicles or shedding microvesicles (SMVs) are another form of small, defined vesicles (Pant et al., 2012) that are shed from the plasma membrane by a wide variety of cells (Heijnen et al., 1999). They are larger (0.1–1 μm) than exosomes (Heijnen et al., 1999) and their mechanism of production is also different. While exosomes are produced by exocytic fusion of MVBs, microvesicles are produced by budding of vesicles from the plasma membrane (Mathivanan et al., 2010). The presence of miRNAs in microvesicles were described for the first time in 2008 (Hunter et al., 2008).

A final vesicular form where miRNAs reside are apoptotic bodies or apoptotic blebs, byproducts of apoptotic cells. Apoptotic or dying cells release membrane vesicles into the extracellular environment via bleeding of the plasma membrane (Mathivanan et al., 2010). These are larger particles (1–5 μm) with heterogeneous shape (Gyorgy et al., 2011). In atherosclerotic vascular disease, endothelial cells can produce apoptotic bodies enriched with miR-126. These endothelial cell-derived apoptotic bodies trigger, via miR-126, the production of CXC chemokine CXCL12 in the recipient vascular cells which limits atherosclerosis and confers plaque stability (Zernecke et al., 2009).

Apart from packaging miRNAs in cell-derived vesicles, a significant fraction of extracellular miRNAs is associated with RNA binding proteins, including nucleophosmin (NPM1), that provide protection from degradation (Wang et al., 2010b). It was also demonstrated that many extracellular miRNAs are bound to proteins of the Argonaute family, primarily Ago2, although additional members such as Ago1, Ago3, and Ago4 might be also associated with miRNAs (Arroyo et al., 2011; Turchinovich et al., 2011). These latter studies are at odds with the findings by Gallo et al. (2012). This discrepancy may arise from the different protocols used for microvesicle- and RNA-isolation and subsequent data normalization, emphasizing the need for further protocol standardization (Turchinovich et al., 2012). Finally, it was recently shown that extracellular miRNAs can be transported by HDL (Vickers et al., 2011; Norata et al., 2013). Whereas vesicle carriers are composed of a bilayer of phospholipids, lipoproteins have a single layer of lipids (Boon and Vickers, 2013).

Interestingly, extracellular miRNAs also present a newly discovered potential of intercellular communication. It is now established that transfer of genetic information in the form of RNA exists (Valadi et al., 2007) and that this form of transfer between cells is of functional relevance by exerting gene silencing in the recipient cells (Kosaka et al., 2010; Mittelbrunn et al., 2011; Halkein et al., 2013; Figure 1). While the biological mechanisms driving the secretion of miRNAs are still under debate (Kosaka et al., 2010), this newly discovered manner of genetic exchange between cells opens a new aspect of how adjacent cells within an organ may communicate and how a miRNA can affect a cell type or a tissue where it is not produced. Since the first discovery of the extracellular miRNAs as intercellular communicators, this field of research is still growing. Increasing evidence suggests that this form of communication occurs in various physiological processes such as the regulation of the immunity (Mittelbrunn et al., 2011) or cellular migration (Zhang et al., 2010), but also participates in pathological situations including tumor development (Yang et al., 2011).

For cardiovascular diseases, only three examples of intercellular miRNA communication have been demonstrated. The first study presented evidence that endothelial cell-derived apoptotic bodies are generated during atherosclerosis and lead to the induction of the expression of CXCL12 in recipient endothelial cells. These endothelial cell-derived apoptotic bodies also induce the recruitment of progenitor cells in mice with atherosclerosis and reduce the extent of plaque formation. It was finally demonstrated that the atheroprotective effects of endothelial apoptotic bodies are mediated by miR-126 (Zernecke et al., 2009). Additionally, shear stress as well as the shear-responsive transcription factor Kruppel-like factor 2 (KLF2) induces the expression of the cluster miR-143/145 in endothelial cells and also its enrichment in extracellular vesicles produced by the treated-endothelial cells. It was demonstrated that these endothelial-derived miR-143/145-containing vesicles are transferred to smooth muscle cells and induce an atheroprotective phenotype in recipient cells. MiR-143/145 from endothelial cells repress target genes in recipient smooth muscle cells such as ELK1 and KFL4 implicated in smooth muscle cell fate and plasticity (Hergenreider et al., 2012).

More recently, it was demonstrated that the anti-angiogenic fragment 16K prolactin (PRL) positively regulates the expression of miR-146a in endothelial cells where it affects mainly the cell survival and proliferation by down-regulating NRAS gene expression. Even more, the treatment of endothelial cells with 16K PRL also increases miR-146a level in the exosomes secreted by the donor endothelial cells. There is an uptake of the endothelial cell-derived exosomes by cardiomyocytes and transferred miR-146a reduces the metabolism of the recipient cells. This model was proposed to play a central role in the development of peripartum cardiomyopathy since blocking miR-146a activity attenuated the disease in mice (Halkein et al., 2013).

The use of exosomes as therapeutic vehicles should now also be considered. In the field of cardiovascular diseases, a first study has presented the potential of cardiomyocyte progenitor cells-derived exosomes to stimulate endothelial cell migration in the treatment of myocardial infarction (MI) (Vrijsen et al., 2010). More recently, in vivo delivery of cardiac progenitor-derived exosomes has been shown to inhibit cardiomyocyte apoptosis in a mouse acute ischemia/reperfusion model (Chen et al., 2013). In the context of therapeutics, the first report is now also available demonstrating that cells can be engineering to express specific ligands at the surface of the exosomes and load these carriers with therapeutic siRNA species (Alvarez-Erviti et al., 2011). Additional efforts for a better understanding of the mechanisms of extracellular miRNA secretion and the targeting of recipient cells by microvesicles are expected in the future.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.