Bonucci reported the appearance of “roundish bodies” in initiation of the calcification process in guinea pig and rat tibial–femoral epiphyseal plates in 1967 (1). One year later, Anderson used electron microscopy on tissue sections to demonstrate vesicular structures in the mouse cartilage epiphysis (2). Subsequent studies have suggested that mineralization depends on secretion of matrix vesicles (MVs), with diameter of 30–400 nm (3, 4), from chondrocytes, osteoblasts, odontoblasts, tenocytes, and cementoblasts (5). MVs released from specific sites on cell membranes [apical microvilli (3)] exhibit similar cytosolic and plasma membrane profiles apparent in their cell of origin (6). Compared to their parent cells, MVs carry augmented levels of acidic lipids such as phosphatidylserine (PS) and sphingomyelin, but diminished levels of neutral phospholipids of phosphatidylcholine and lysophospholipids (5). Chondrocytes residing in the epiphyseal plate experience hypoxic conditions due to presence of collagen fibrils and proteoglycans, which restrict oxygen and nutrients delivery (7). Development of blood microvessels into this zone creates oxidative stress caused by the sudden elevation in nutrients, oxygen, calcium ions (Ca²⁺), and phosphate ions (Pi) (8). This process leads to enrichment of mitochondria with Ca²⁺, which results in secretion of Ca²⁺-loaded vesicles into the cytosol. It has been proposed that the formation of complexes between PS and Ca²⁺ either with Pi or annexins in these vesicles, diminishes the Ca²⁺ level within the cytosol, expedites actin depolymerization, and consequently leads MVs to pinch-off from the cell and release into the extracellular environment (8, 9). Of note, apoptotic bodies originating from apoptotic cell membrane rearrangement during terminal stage of mineralization in epiphyseal plate differs from active formation and release of MVs (10). Released MVs interact with glycosaminoglycans and initiate extracellular mineral deposition (11).

Chondrocyte differentiating factors such as thyroxine (T3), bone morphogenetic protein 6, retinoic acid, and Indian hedgehog may give rise to MV generation by inducing changes in cell phenotype (12). Osteogenic cell types abundantly express annexin I, II, IV, V, VI, and VII. Annexins function as voltage-gated channels or Ca²⁺-binding agents, mediate inflammation responses, and regulate structural properties of both cells and MVs membranes (4, 11, 13). The most abundant proteins in MVs are annexins II, V, and VI, which can accelerate the calcification process by either providing required Ca²⁺ for mineralization or partaking in PS–Ca²⁺-annexin complexes (14). Additionally, other membrane proteins such as calbindin D9k can provide Ca²⁺ for MVs (15).

Pyrophosphate (PPi), which originates from nucleotide pyrophosphatase phosphodiesterase (NPP1) hydrolysis of nucleoside triphosphates, inhibits mineralization. Progressive ankylosis (ANK) carries PPi into extracellular milieu. Tissue non-specific alkaline phosphatase (TNAP, on the outer leaflet of MV membranes) hydrolyzes PPi into free phosphate (Pi) and provides free Pi for complexing with Ca²⁺ and mineral formation (5, 7, 16). Type III Na/Pi transporters (PiT-1) on the MV membrane facilitate Pi internalization. Bone morphogenetic protein 2 and parathyroid hormone upregulate expression of these transporters (7). Additionally, MVs contain phosphatases and membrane phosphohydrolases, such as TNAP, AMPase, ATPase, nucleoside triphosphate pyrophosphohydrolase (NTPPase, NPP1, and PC-1), and PHOSPHO-1 that elevate the intravesicular Pi concentration within the MVs (5, 17). This highly ions concentrated environment, adjacent to the MV membrane, where Ca²⁺ and Pi meet, provokes calcium phosphate precipitation followed by an increase in pH and mineral stabilization. Membrane–mineral associations are mediated by PS located on the luminal side of the MV lipid bilayer. PS is an anionic phospholipid with tendency to bind Ca²⁺. The complexes of PS–Ca²⁺–Pi may serve as initial nuclei for hydroxyapatite precipitation (7, 13, 18). Studies have also suggested that the mineralization process can begin intracellularly with contribution of pre-nucleation clusters within endosomes, which initiate mineralization in order to maintain Ca²⁺ and Pi in homeostatic concentration and fulfill energy trade-off (5).

Matrix vesicles are equipped with matrix metalloproteases (MMPs) that degrade and remodel the ECM (3, 19). Particularly, MMP-2, MMP-3, MMP-9, and MMP-13 are located in MV membranes and play a key role in matrix remodeling and propagation of mineralization (5, 19, 20). In ECM, collagen fibrils and proteoglycans provide charged regions, which are favorable sites for accumulation of the calcium phosphate nanoparticle clusters that form in MVs. The amorphous apatite resulting from this accumulation transforms into structured and crystalline mineral following ECM associations (5). MVs interact with ECM proteins via their integrin receptors and surface motifs, such as CD9, CD63, and Hsp70 (5) (Figure 1). In addition to their remodeling potential and ECM binding, MVs in growth plate ECM can affect the proliferation and fate of resident cells, due to the activation of parathyroid hormones and gene-related peptide through their loaded proteins (5). This paracrine signaling property is similar to other subtypes of extracellular vesicles (EVs) ubiquitous to many cells and tissues.

It is worth mentioning that skepticism exists on the existence and role of MVs in bone growth and formation. Studies have suggested that calcification originates from cell death and debris, which nucleate mineral, and observations of MVs are attributed to sample preparation artifacts (21). In recent studies, however, isolation of MVs from chicken tibias using multiple centrifugations and application of microscopy techniques such as transmission electron microscopy on growth plate during bone formation have provided support for MVs as mediators of mineralization (22, 23). Furthermore, knockout of Stx4a, a regulatory factor for secretion of MVs, decreases number of MVs and leads to reduced bone mineral density (24). Recent interest in non-calcifying EVs (e.g., exosomes) and the related development of assay techniques may build on the observational studies of the past and help clarify the derivation and function of MVs in the mineralization of hard tissues.

Matrix vesicles represent one specific subtype of EVs. Generally, EVs function to maintain both intracellular and extracellular homeostasis. Two major pathways mediate EV release into the ECM: (i) via multivesicular bodies (MVB), containing several EVs wrapped by plasma membrane and (ii) through direct budding of a single EV from cellular plasma membrane (4). Depending on the release mechanism, EVs are generally referred to as exosomes and microparticles, respectively. Both EV types carry a subset of cargos representing their parental cell (11, 25). MVB trafficking and fusion/fission from the plasma membrane requires activity of Rab GTPases, specifically Rab27a, Rab27b, Rab35, and Rab11 (26, 27). EV structure consists of a metabolically active membrane with transmembrane proteins and an inner core, which typically carries RNAs, soluble proteins, and lipids (11, 25, 28). EVs often contain sets of small RNAs, such as miRNA, tRNA, mRNA, piRNA, snRNA, Y-RNA, and vault RNA, which can be protected from RNase degradation and encoded at target cells (27, 29). The difference in RNA ingredients of parental cells and EV cargo demonstrates the selective mechanism of RNA loading. EV RNA cargo can serve as biomarker that indicate the phenotypic state of parental cells, as well as messengers that can interact with other cells (27). EVs play a key role in cell–cell interaction and data trafficking in both normal and pathological conditions. For instance, miRNA-enriched EVs from endothelial cells can regulate gene expression and resultant phenotypic transitions in smooth muscle cells (SMCs) (30). Divergence from normal physiological conditions toward pathological ones induces release of dysfunctional EVs with pathologic cargo and may affect tissue homeostasis and cellular phenotypes (4). This section serves as a short primer on the complex mechanisms associated with EV release and cargo loading. These processes are reviewed in greater detail elsewhere (31–34). The current review focuses on EVs that play a direct role in depositing mineral in cardiovascular tissues. Current evidence, discussed in the following sections, suggests that these EVs share commonalities with both MVs and other EV populations, such as exosomes.

Calcification contributes to pathological remodeling in different locations throughout the cardiovascular system, such as the arterial intima and media and the aortic valve (35, 36). Electron microscopy demonstrated the presence of needle-like hydroxyapatite nanocrystals in EVs extracted from atherosclerotic lesions of apolipoprotein E-deficient mice (37). EVs released into the atherosclerotic lesion have a Ca/P ratio of 0.66, indicating incomplete calcification [compared to the ratio in normal adult murine bones of 1.71 (38)]. High resolution microcomputed tomography imaging revealed microcalcification in the fibrous cap of atherosclerotic plaques composed of calcified EV aggregates (39, 40). These observations indicate a role for EVs in the formation and progression of cardiovascular calcification, but mechanistic studies demonstrating causality are difficult. One major challenge in EV research is to distinguish between different EV populations, such as calcifying EVs, MVs, exosomes, apoptotic bodies, and microparticles, due to their shared size and shape characteristics (41). Application of multiple and consecutive centrifugations followed by size-based filtration and sucrose gradient-based ultracentrifugation to isolate EVs of known density have been used to separate different cellular-derived particles and EVs (25, 42). EVs with various sizes and densities pellet based on the centrifugation speed, i.e., large, medium, and small EVs precipitate at low, moderate, and high speeds, respectively (25). However, these techniques are often unable to separate the various EV populations. Calcifying EVs secreted by SMCs cultured under osteogenic conditions exhibit increased mass density compared to other EVs, likely due to mineral formation. Therefore, calcifying EVs precipitate more quickly under the application of ultracentrifugation (~100,000g) (41), permitting enrichment of these EVs and subsequent proteomic profiling (25, 41). Once proteomic fingerprints are established, membrane proteins (tetraspanins), ER-related proteins, mitochondrial proteins, exosomal markers, endosomal markers, and ECM factors (such as surface molecules and integrins) specific to various EV populations help map and separate EVs into subsets based on their origins (25).

In certain pathological conditions (such as chronic kidney disease), extracellular Ca²⁺/Pi concentrations increase, thereby cells release EVs with high levels of these ions (43). This may be in contrast to inflammation-driven osteogenic differentiation (e.g., in atherosclerosis). Release of EVs from cardiovascular cells with osteogenic phenotypes promotes mineralization via generation of free phosphate from sources such as ATP and pyrophosphate (44). Further, dystrophic mineral deposition resulting from cell death may also contribute to a significant portion of cardiovascular calcification (45). The diverse contributors have led to confusion surrounding the mechanisms of cardiovascular calcification (46); however, numerous studies have demonstrated similarities between cardiovascular calcification and bone metabolism (47–51). Annexins present on calcifying EVs play a dual role of Ca²⁺ uptake and counteracting the calcification inhibitory activity of fetuin-A during ectopic mineralization (4, 11). High phosphate imbalance present in chronic kidney disease may accelerate calcification nucleation in EVs (46). In this condition, TNAP activity may accelerate the calcification process in both non-osteogenic and osteogenic EVs through removing PPi, a competitive inhibitor to Ca²⁺ associations with Pi (39). EVs with calcifying potential may also contain imbalanced and dysfunctional miRNAs, which induce the gene expression and protein synthesis of osteogenic markers, such as RUNX2, Smad1, osterix, TNAP, chaperones, and pro-inflammatory factors (4, 11). Unlike the physiological process of MV-mediated mineral deposition, however, calcification of EVs may inhibit their ability to reach their intended target cells, further promoting a loss in tissue homeostasis and pathological remodeling (52) (Figure 1).

Future works are needed to improve upon EV isolation techniques in order to reduce noise from non-calcifying EVs. Advancements in EV characterization tools may allow careful comparative studies to understand the similarities and differences between MVs and calcifying EVs liberated from the three reported cellular contributors to calcification in cardiovascular tissues: vascular SMCs, macrophages, and valvular interstitial cells (VICs). While these cell populations do not represent all cells involved in cardiovascular calcification, direct EV-related contributions to mineral nucleation from other cells remain unreported. Improvement of EV isolation and assay techniques may also allow for determination of the relative roles of the various cell populations in the mineralization process.

Calcifying EVs play an important role in the initiation and development of cardiovascular calcification. Though calcifying EVs in cardiovascular tissues appear to share commonalities with MVs, they seem to be derived from different origins within cells. The overlapping characteristics between EVs and MVs underscore the fact that research in cardiovascular calcification has been informed by pioneering research in bone physiology. However, the noted differences between cardiovascular calcifying EVs and bone MVs warrant further investigation. The type and quality of mineral that forms appear different in the two tissues. Further, the appearance of calcific mineral in cardiovascular tissues associates strongly with a decrease in bone mineral density—a phenomenon known as the calcification paradox (59). Studies into the differences between the fundamental building blocks of calcification—calcifying EVs in cardiovascular tissues and MVs in bone—may provide new insight into the observed divergence in mineral within these tissues and present therapeutic options that avoid unwanted off-target effects.

AN researched the topic and drafted the manuscript. JH helped draft the original manuscript and edited and revised the manuscript text. EA provided intellectual contributions to the original manuscript draft and substantially edited and revised the manuscript text.

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.