**5. Circulating miRNAs as biomarkers and extracellular communicators**

miRNAs are present in blood (plasma, platelets, erythrocytes, nucleated blood cells) with high stability. miRNAs can circulate in extracellular vesicles [111], in a protein complex (Ago2), or in a lipoprotein complex (HDL) [38], which prevents their degradation. Depending on the size and type, extracellular vesicles are broadly classified as ectosomes (also called shedding microvesicles), exosomes, matrix vesicles (MVs), and apoptotic bodies. Ectosomes are large extracellular vesicles 50-1000 nm in diameter; exosomes are small membranous vesicles of endocytic origin, 40-100 nm in diameter; MVs are 30-300 nm in diameter, are produced by

bone Dkk-1 (miR-335-5p, miR-29a) increase with age [99, 100] — a risk for cardiovascular calcification. miR-335-5p directly targets and represses Wnt inhibitor Dkk-1, thereby enhanc‐ ing Wnt signaling and promoting osteoblast differentiation [101]. To date, no publications exist regarding the role of miR-335-5p in the cardiovascular system. Yet, the age-dependent increase of miR-335 in rat renal tissue inhibited the expression and function of the enzymes implicated in oxidative stress defense [99]. Likewise, miR-29a potentiates osteoblastogenesis by modu‐ lating Wnt signaling. Canonical Wnt signaling induces miR-29a expression, which negatively targets regulators of Wnt signaling, including Dkk-1, sFRP2, Kremen, and osteonectin [102, 103]. miR-29 increased age-dependently in mouse aortic tissue and associated with reduced extracellular matrix components, such as collagen and elastin [100]. Elastolysis accelerates arterial and aortic valve calcification [40]. Furthermore, MMP-2, another target of miR-29 [104], was shown to promote arterial calcification in CRD [105] and valvular calcification [106].

The contribution of osteoclasts to cardiovascular calcification is still controversial [59]. The observation of osteoclast-like cells in calcified atherosclerotic lesions suggested this bonerelated cell is active in the vessel wall. The evidence was strengthened recently by Sun et al., who demonstrated the functional role of SMC-derived Runx2 promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells — suggesting that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process [74]. The receptor activator of the nuclear factor-kappa B (NF-kappa B) ligand (RANKL)/osteoprotegerin (OPG) system controls proper osteoclasto‐ genesis, and actes as a biomarker for CAD [107, 108]. *In silico* analysis revealed RANKL as a target of miR-126 [109], which is decreased in the plasma of CAD [14] and T2D [33] patients. miR-146a, highly expressed in atherosclerotic arteries [16], inhibits osteoclastogenesis [46]. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was significantly reduced by miR-146a in a dose-dependent manner [46]. Furthermore, miR-155, which is decreased in plasma of CRD [37] and CAD [14] patients, was shown to inhibit osteoclast

Taken together, osteogenic processes in both bone and the cardiovascular system are tightly controlled by miRNAs (Figure 2). Further studies are needed to elucidate whether interplay of miRNAs could explain the bone-vascular axis "calcification paradox," or whether they act

**5. Circulating miRNAs as biomarkers and extracellular communicators**

miRNAs are present in blood (plasma, platelets, erythrocytes, nucleated blood cells) with high stability. miRNAs can circulate in extracellular vesicles [111], in a protein complex (Ago2), or in a lipoprotein complex (HDL) [38], which prevents their degradation. Depending on the size and type, extracellular vesicles are broadly classified as ectosomes (also called shedding microvesicles), exosomes, matrix vesicles (MVs), and apoptotic bodies. Ectosomes are large extracellular vesicles 50-1000 nm in diameter; exosomes are small membranous vesicles of endocytic origin, 40-100 nm in diameter; MVs are 30-300 nm in diameter, are produced by

function [110].

132 Calcific Aortic Valve Disease

independently in the calcifying vessel and bone.

**Figure 2. Potential and established miRNAs contributing to osteogenic regulation of vascular calcification.** Bold, established miRNAs in vascular calcification; underlined, dysregulated in cardiovascular disease (circulating or tis‐ sue); gray, predicted miRNA binding sites.

blebbing of the plasma membrane, and can calcify; and apoptotic bodies, 50-5000 nm in diameter, are released from fragmented apoptotic cells.

The majority of miRNAs are independent of vesicles [111] and co-purify with the Ago2 complex [112, 113]. But in CAD patients, most plasma miRNAs associate with extracellular vesicles, and only a small amount are found in extracellular vesicle-free plasma [114]. A celltype-specific miRNA release and different export systems are implicated, as the miRNA release pattern within vesicles is different from that associated with Ago2 complexes [112]. Thus, cells can select miRNA and pre-miRNA for controlled cellular release [115, 116]. miRNA profiles of extracellular vesicles are different from their maternal cell profiles, indicating an active mechanism of selective miRNA packing from cells into vesicles [114]. We have limited knowledge about miRNA secretion. Blockade of sphingomyelinase inhibits exosome genera‐ tion and miRNA secretion, and intercellular miRNA transfer implicates a ceramide-dependent mechanism [117, 118]. Ago2–miRNA complexes may be passively produced by dead cells, released by live cells, or actively transported though cell-membrane–associated channels or receptors [119].

Extracellular vesicles use miRNA to mediate intercellular communication over long distances or on a local tissue level [120]. Endothelial apoptotic bodies can convey miR-126 to athero‐ sclerotic lesions, which demonstrate uniquely paracrine-signaling function for miRNA during atherosclerosis [33, 121]. miRNA-containing vesicles can regulate intercellular communication between ECs and SMCs by selective packing of miR-143/145 in endothelial-derived vesicles, which are then transported to SMCs to control their phenotype [118].

Additionally, miR-2861 contributes to osteoporosis in mice and humans by targeting histone deacethylase 5, and thereby increasing Runx2 [132]. No studies of miR-2861 in the cardiovas‐ cular system have been reported. Patients with rheumatoid arthritis also suffer from vascular calcification in different vessel beds, in addition to osteoporosis; the pathogenesis includes pro-inflammatory cytokines and site-specific inflammation (reviewed in detail elsewhere [133]). miR-146a, a negative regulator of inflammation and osteoclastogenesis, also associates with rheumatoid arthritis [134]. Similar to patients with CAD, in patients with rheumatoid

Role of MicroRNAs in Cardiovascular Calcification

http://dx.doi.org/10.5772/55326

135

*In vitro* and *in vivo* studies have established miRNAs as biomarkers focusing on different aspects and providing circulating miRNA signatures for different diseases. But these circu‐ lating miRNAs may not have biological functions within the cell while circulating — instead, they act as intercellular communicators, and this communication may be disturbed by calcified vesicles. More studies are needed to fully exploit this potentially novel mechanism of cardio‐

Moreover, miRNA biology is very complex. Multiple miRNAs can target the same gene (e.g., Runx2–miRNA cluster), and one miRNA may have several targets. Only a small amount of these fine-tuned targets may alter biological responses and phenotypes. Understanding the role of miRNA in vascular calcification may be helpful in considering the paradoxical clinical observations of the concurrence of cardiovascular calcification and osteoporosis. Despite its global clinical burden, no medical therapies are available to treat cardiovascular calcification. Targeting of miRNA represents a novel therapeutic opportunity for treating calcification disorders. As vascular calcification and bone remodeling share common mechanisms, we have to understand in greater detail the functions of miRNAs and their association with the

1 Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Medicine, Brigham

2 Center for Excellence in Vascular Biology, Cardiovascular Medicine, Brigham and Wom‐

molecular pathogenesis of osteoporosis and vascular/valvular calcification.

and Elena Aikawa1,2

\*Address all correspondence to: eaikawa@partners.org

en's Hospital, Harvard Medical School, Boston, MA, USA

and Women's Hospital, Harvard Medical School, Boston, MA, USA

arthritis, miR-146a is up-regulated in PBMCs [25].

**7. Conclusion and perspectives**

vascular calcification.

**Author details**

Claudia Goettsch1

How miRNAs are taken up by target cells and remain biologically active is still unknown. We know little about the mechanisms of vesicle-mediated cargo transfer. In physiological condi‐ tions, extracellular vesicles may bind to the membrane proteins of the surface of target cells through receptor–ligand interaction, resulting in intracellular stimulation of genetic pathways. They can also fuse with cell–target membranes and release genetic content in a nonselective manner. Furthermore, vesicles can bind to surface receptors on target cells with endocytotic internalization by recipient cells, followed by fusion with the membranes, leading to a release of their content into the cytosol of target cells [122].

A key event in the initiation and promotion of VIC and SMC calcification is the release of extracellular vesicles [81, 123]. Treatment of SMCs with elevated calcium levels promotes the production of calcifying vesicles (MVs), and the loss of fetuin-A, an inhibitor of mineral nucleation [124]. These vesicles act as early nucleation sites for calcification. The phosphati‐ dylserine-membrane complex from SMC-derived and macrophage-derived MVs redistributes and nucleates hydroxyapatite [125-127]. In addition, hydroxyapatite nanocrystals shed from vesicles may further promote mineralization via direct effects on SMC phenotype [128].

Insight into the underlying mechanism of selective packing of miRNAs into extracellular vesicles and selective uptake into the target cell will help increase understanding of the role of miRNA-containing vesicles in physiological intercellular communication, which may prevent calcification in the cardiovascular system.
