**7. Conclusion and perspectives**

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,

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

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

Osteoporosis frequently associates with cardiovascular calcification, and the severity of aortic calcification associates positively with bone loss [2, 129, 130]. The "calcification paradox" could be explained by the shared molecular pathways in bone remodeling and cardiovascular calcification [3]. How these two processes associate with each other and whether osteoporosis leads to cardiovascular calcification - or whether both disorders just share common risk factors - is unclear. In this section, we link cardiovascular calcification and bone loss and show

Studies of miRNA in patients with bone disease are lacking. A recent clinical study first reported miRNA as a potential biomarker for postmenopausal osteoporosis. Wang *et al*. demonstrated an association of miR-133a levels in circulating monocytes - osteoclast precur‐ sors - with postmenopausal osteoporosis [131]. Women with low bone mineral density showed higher circulating miR-133a levels [131], but the number of patients per group was small (n=10). Circulating miR-133a levels were also higher in patients with CAD [14]. Unfortunately, the study investigating bone mineral density in patients with osteoporosis did not mention characteristics of the cardiovascular patient population. miR-133a belongs to the Runx2-

which are then transported to SMCs to control their phenotype [118].

of their content into the cytosol of target cells [122].

134 Calcific Aortic Valve Disease

prevent calcification in the cardiovascular system.

**6. miRNAs in the "calcification paradox"**

commonalities in the systems' miRNA pathways/patterns.

targeting miRNA cluster [77].

*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‐ vascular calcification.

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 molecular pathogenesis of osteoporosis and vascular/valvular calcification.
