Exosomes, the message transporters in vascular calcification

Abstract Vascular calcification (VC) is caused by hydroxyapatite deposition in the intimal and medial layers of the vascular wall, leading to severe cardiovascular events in patients with hypertension, chronic kidney disease and diabetes mellitus. VC occurrences involve complicated mechanism networks, such as matrix vesicles or exosomes production, osteogenic differentiation, reduced cell viability, aging and so on. However, with present therapeutic methods targeting at VC ineffectively, novel targets for VC treatment are demanded. Exosomes are proven to participate in VC and function as initializers for mineral deposition. Secreted exosomes loaded with microRNAs are also demonstrated to modulate VC procession in recipient vascular smooth muscle cells. In this review, we targeted at the roles of exosomes during VC, especially at their effects on transporting biological information among cells. Moreover, we will discuss the potential mechanisms of exosomes in VC.


| INTRODUCTION
Vascular calcification (VC) is attributed to calcium and phosphate (Pi) metabolic dysfunction, osteogenic differentiation, inflammation and so on, leading to major adverse cardiovascular events (MACEs), especially in patients with chronic kidney disease (CKD). [1][2][3] Furthermore, VC occurs in the intimal and medial layers of vessel wall, which is linked to atherosclerotic plaque burden and consequent rupture. 4 In some clinical trials, moderate or severe calcification contributes to more MACE occurrences in patients treated with revascularization therapy, compared with non/mild calcification. 5 As VC increases MACE occurrences, many treatments are designed to counteract with VC, such as statins, Pi binders and so on. However, with more concentrations driving into this field, more shortages of such treatments are presented in the front. A meta-analysis revealed that statins failed to ameliorate coronary artery calcification procession despite reducing LDL-c level. 6 Moreover, Pi binders also enhance VC, which are mediated by calcium contained in such binders. 7,8 Pi binders are also demonstrated to limit bioavailability of vitamin K2, which further inhibits the activity of mineral deposition factor matrix Gla protein (MGP) to enhance VC occurrence. 9 MGP is an inhibitory factor for VC and inactivated MGP results in exacerbating VC. 9 Due to the limitation of present treatments in VC, novel targets and therapies for VC are demanded. 10,11 Importantly, exosomes have been demonstrated to be involved in VC recently. 11,12 Exosomes have up-regulated secretion from vascular smooth muscle cells (VSMCs) in vivo after pro-calcifying stimulation and become "calcifying" exosomes to induce VC. 11 Calcium binds with Pi to form hydroxyapatite nodes on the inner and outside of "calcifying" exosomes membranes, which further initializes mineral deposition. 11 Although these studies did reveal that exosomes participated in the calcification procession through promoting mineral deposition sites formation, they did not discuss exosomes functioning as mediators for RNAs transportation, which is vital for exosome function. 13 Exosomes are secreted by diverse cells to mediate cell-to-cell communications. 14 However, how exosomes regulating VC is only preliminarily explored recently. It is found that exosomes with diverse origins mainly mediate microRNAs (miRs) transporting to VSMCs in coronary artery calcification. 15 A bioinformatics analysis revealed that cultured in osteogenic medium, mesenchymal stem cells secreted exosomes with alterations of miRs, comparing with normal culturing. 16 Such alterations were suggested to accelerate calcification in other mesenchymal stem cells to modulate osteogenic phenotype transition. 16 Thus, it implies that besides heterogeneous mineral deposition inside vessel wall, 11 exosomes can also promote VC by transporting messages among cells.
In this review, we will summarize the roles of exosomes in VC and analyse the potential mechanisms associated with exosomes in VC.

| Biological characters of exosomes
Widely found in body fluid, exosomes represent a group of extracellular vesicles (EVs) with intracellular contents, such as proteins and RNAs, which are transported among cells to mediate cell-to-cell communications under certain situations. 17 Exosomes originate from multivesicular bodies (MVBs) and are loaded with intracellular components upon biogenesis. 17 It is reported that after shear stress stimulation, EVs secreted from endothelial cells enriched with miR-143/145 and control the phenotype of VSMCs. 18 Such miRs transportation via exosomes regulates de-differentiation of VSMCs, which initializes phenotype transition during VC. 13,18 Recent study also revealed that increasing exosomes secreted by VSMCs promote VC via mineral deposition. 12 Attributing to EVs congestion in vascular wall, the calcification spheres contribute to heterogeneity of microcalcification formation via mineral deposition. 19,20 In the process of mineral deposition, comparisons based on previous researches demonstrated that tiny differences existed between exosomes and matrix vesicles (MVs) in size, morphology and lipid/protein contents, indicating that exosomes share characteristics with MVs during calcification procession. 21 Such vesicles secreted by VSMCs expressing exosome biomarker CD63 are regarded as exosomes. 11 Moreover, as Clotilde Thery et al suggested, exosomes represent the mixed population of small EVs which transport information among cells as the primary function. 17 Recently, exosomes derived from calcified VSMCs were proven to enhance calcification in the recipient VSMCs via activating mitogen-activated protein kinase. 22 Exosomes are also suggested to deliver intracellular contents such as proteins and RNAs, functioning as message transporters to promote VC. 23 Emerging evidences revealed that exosomal miRs were significant in diagnosis, prognosis or even therapeutic target selection in patients with cancer and heart failure. 24,25 Selective enrichments of miRs in exosomes were due to the alterations in parental or donor cells, from which exosomes are secreted or originated. 26 Of note, exosomes take part in cellular behaviour changes, such as phenotype transition and inflammatory reactions via transporting miRs to interfere with several signalling pathways. 27,28 Uptake of exosomes by osteoblasts is accelerated by increased receptors expressed on the cell surface, with transporting miRs from osteoclasts under osteoclastogenesis stimulation. 29 All of secretion, congestion and uptake processions of exosomes modulate VC from different aspects. Thus, exosomes participate in the procession of VC via partially promoting mineral deposition sites formation and transporting miRs as information among cells ( Figure 1).

| Initializing mineral deposition
Resembling to bone formation, mineral deposition is the characteristic feature of VC and MVs are regarded as the major players of calcification procession. 30 Elevated calcium combines with Pi to form mineral deposition sites which determines the outcome of calcification. 31 It is reported that MVs derived from macrophages enhanced ectopic mineralization after culturing in the high calcium/Pi medium. 32 During VC, MVs are proven to participate in mineral nucleation sites formation, and decreased MVs secretion results in amelioration of VC. 33,34 It is proven that exosomes, as MVs, obtain ZHANG ET AL. | 4025 mineral compounds to maintain the intracellular mineral metabolism homeostasis, which further aggravates the mineral deposition sites formation. 11,12 It is reported that exosomes secretion pathway is activated during VC and modulations on such procession exert as novel targets for VC prevention. 11 Specifically, elevated Pi and calcium and cytokines, including tumour necrosis factor a (TNF-a) and platelet-derived growth factor-BB (PDGF-BB) enhance exosomes secretion via elevating sphingomyelin phosphodiesterase 3 (SMPD3, also known as neutral sphingomyelinase 2, nSMase2) expression. 11 Pro-calcifying stimulation increases the expression of SMPD3/nSMase2 of VSMCs and leads to enhanced calcification. 2 SMPD3/nSMase2 converts sphingomyelin to ceramide which induces the conjunction of clathrincoated microdomains and further promote exosomes secretion. 35 As phenotype transiton of VC involving cytoskeleton remodelling, such intracellular alterations would promote exosomes secretion via ceramide. 2,36,37 Moreover, the "calcifying" exosomes secreted during VC are characterized with low MGP contents and high level of hydroxyapatite, which initialize mineral deposition as microcalcifiction. 11 It is known that Gla-rich proteins, including MGP, inhibit the nucleation sites formation on the surface of exosomes via binding with externalized phosphatidylserine (PS). 12 Such inhibitions of mineral-binding abilities further block calcium deposition in an exosomes-dependent manner and ameliorate calcification procession. 11,12 Besides mineral contents inside exosomes, externalized PS combines with calciumbinding protein (such as Annexin A2, A5, A6), which forms hydroxyapatite deposition inner and outside exosomes. 38 Moreover, Annexins are loaded into exosomes before releasing. 39 Extracellular Pi concentration is further enhanced by phosphatases on MVs surface via converting pyrophosphate to provide ectogenic Pi. Choline kinase mutant also enhances some phosphatase activities as compensatory mechanism to accelerate Pi production. 40 Thus, such evidences indicated that exosomes participated in VC via forming calcium deposition sites, which are attributed to exosomes contents and calcium-binding abilities.

| Transporting miRs to modulate VC
Cell-to-cell communication is a key mechanism for VC occurrence. 41 Recent findings showed that exosomes played important roles in transporting information among cells. 41 As plenty of works had focused on the roles of exosomes in mineral deposition during VC, limited insights into VC do not clearly explain the exact procession of exosomes as information transporters. 11 Exosomes mediate information transportation among cells, which are reported to depend on heparin sulphate proteoglycans (HSPG) for the internalization in cancer cells. 42 However, HSPG protects VSMCs from various toxic substances and circulating inflammatory cells to prevent VC. 43 Reduced HSPG expression in the extracellular matrix (ECM) exposes HSPG on cell surface, which further mediates bone morphogenetic protein 2 (BMP2) internalization to enhance osteogenic phenotype transition in myoblast cells. 44 Inhibition of HSPG expression on the cell surface leads to decreased efficiency of exosomes uptake. 29,45 Functioning as carriers to transport cargos among cells, exosomes trigger some reactions in recipient cells. Exosomes cargos contain RNAs (including mRNAs and miRs), cytokines, lipids and so on. 2 Exosomes released from mineralizing pre-osteoblast MC3T3-b1 cells promote osteogenic differentiation in ST2 cells, which is mediated by the complicated networks formed by exosomal miRs. 46 Other research also revealed that miRs expression in MVs during VC, suggesting that exosomes might transport vital information during VC. 47 Despite enhancing the exosomes secretion in VC, elevated SMPD3/nSMase2 expression also modulates miRs sorting into exosomes, and quantitative analysis revealed that inhibition of SMPD3/ nSMase2 led to significantly decreased expression of several miRs in exosomes. 48 Alteration of miRs inside exosomes regulates osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells. 16 Furthermore, these alterations of miRs inside exosomes could augment osteogenic phenotype transition via elevating runt-related transcription factor 2 (Runx2) expressions and activating several signalling pathways such as Wnt/b-catenin. 46 Osteogenic phenotype transition represents as a crucial characteristic of VC, with switching from contractile phenotype to osteoblast-like cells. 49 And such procession is mirrored by expression of osteogenic transcription factors such as Runx2 and loss of contractile phenotype such as a-smooth muscle actin (a-SMA). 50

| Autophagy
Autophagy is aimed to digest intracellular proteins and organelles when cells encounter with emergent situations, such as stress responses. 66 A series of studies have focused on the relationship between autophagy and VC, and it seems that autophagy ameliorates such procession through AMP-activated protein kinase (AMPK) activation under Pi-induced situation. 67 Autophagy involves autophagosomes formation mediated by LC3, Beclin1 and autophagic flux activated by autophagosomes fusing with lysosomes. 68 Up-regulating LC3 and Beclin1 expression blocks calcium deposition in high Pi stimulation, which indicates that autophagy might have the inhibitory role in VC. 67 It is also reported that 7ketecholesterol, a VC inducer, promotes VC through lysosomes dysfunction which blocks the fusion of autophagosomes and lysosomes. 69 Recently, autophagy is believed to be enhanced by high Pi stimulation and suppresses MVs secretion which further forms mineral nucleation sites and consequently ameliorates calcification in VSMCs. 34 It is well documented that autophagy accelerates MVBs degradation and decreases exosomes secretion, which is mediated by autophagosome-lysosome fusion. 21 Briefly, MVBs move to neighbourhood of the cell membrane and then dock to the membrane for exosomes releasing, which is regulated by Rab GTPases. 70 One of such GTPase, Rab11, then induces exosomes secretion by promoting MVBs docking and fusing to cytomembrane in a calciumdependent manner. 71 Rab11 also enhances autophagosomes fusing with MVBs to form amphisomes under interferon-c treatment, which promotes Annexins loading into exosomes. 39 Moreover, autophagy seems to interfere with miRs loading into exosomes during VC. It is reported that heterogeneous ribonuclear protein A2/B1 (hnRNPA2/B1) plays a vital role in promoting miRs loading into exosomes. 72 It is believed that small ubiquitin-like modifier (SUMOylation) of hnRNPA2/B1 promotes miRs loading into exosomes. 72 In addition, Ubc9, the E2-conjugating enzymes mediating SUMOylation, is degraded in autophagy procession. 73 Thus, autophagic flux partially decreases exosomes secretion and miRs loading into exosomes, which might interfere with mineral deposition and osteogenic phenotype transition. However, more researches need to distinguish the exact function of autophagy in VC.

| Inflammation
It has been known that inflammation promotes VC, which is modulated by inflammatory cytokines secreted from inflammatory cells, such as macrophages. 74 Expression of TNF-a and interleukin (IL) family members, such as IL-1b and IL-6, is increased and such cytokines play pivotal roles in the procession of VC. 75 These cytokines enhanced the expression of BMP2 and reduced MGP expression, further promoting VC procession in VSMCs. 76 It is also reported that exosomes collected from body fluid promote inflammation. 77 Previous report indicates that ceramide is elevated due to inflammatory stimulation and promotes VC. 78 Moreover, macrophages are involved in inflammatory reaction during VC. Derived from monocytes, macrophages are recruited and activated in the calcification area to initialize the mineral deposition, which further enhances the production of inflammatory cytokines. 79 It is reported that in metabolic disorders, exosomes derived from macrophages shuttle miR-155 among cells to modulate insulin sensitivities in insulin target recipient cells. 80 MiRs-223 is also proven to be transferred by microvesicles from macrophages, and such microvesicles include exosomes and other kinds of EVs. 81 Both miR-155 and miR-223 are also proven to modulate VC, 13,56 suggesting that besides promoting inflammatory cytokines secretion, macrophages participate in VC via an exosomal miRs-dependent manner.
In addition, transforming growth factor b (TGF-b) signalling pathway is proven to promote VC, which is related to inflammation. 82 High Pi induces activation of TGF-b/Smad2/3 in VSMCs and Smads modulate specific genes transcription, including SMPD3/nSMase2 which converts sphingomyelin to ceramide. 48,83,84 However, miR-29b is proven to inhibit TGF-b/Smad3 axis activation via targeting at Smad3 and alteration of exosomal miR-29b modulates such mRNAs expression in the recipient infected cells concerning HIV study. 85,86 All these results indicate that exosomes modulate inflammation via mediating miRs transportation during VC.  reported that miR-30 was down-regulated after calcification stimulation, which also targeted at RAGEs to modulate AGE/RAGE activity and further decreased oxidative stress level. 51,91 In fact, miR-30 could be packed into exosomes and transport information among endothelial cells and mesenchymal stem cells. 92 The expression of miR-210 is also proven to be decreased in VC, 52 and exosomal miR-210 also ameliorated ROS production in the recipient endothelial cells. 93 Such results indicated that exosomes could regulate the oxidative stress via modulating ROS production.

| Immune response
Immune response is composed of innate and adaptive immunity, which is recently regarded as a major player in the occurrence of cardiovascular disease. 94

Vascular calcification
The potential regulatory mechanisms of exosomes during vascular calcification (VC). Several mechanisms of VC occurrence are modulated by exosomes, including autophagy, inflammation, oxidative stress and immune response. Transporting different miRs among cells, exosomes modulate several signalling pathways and further interfere with VC and Treg/T-helper cell 17 functional disequilibrium is also vital in such procession. 95,96 Exosomes are proven to be involved in Treg cells modulation. Exosomes derived from Treg cells transport exosomal contents including miRs to the recipient conventional T cells or recipient cells in tumour tissue, further modulating immune response or intracellular translation procession. 97 Indeed, Treg cell is regarded as the suppressive effector in immune system by delivering miRs via exosomes. 98 It is reported that Treg cells transfer miR-155 to recipient conventional T cells. 99 Importantly, miR-155 is vital in VC procession, 13 and exosomal miR-155 derived from Treg cells might function as an additional source of miRs during VC. Thus, exosomes might be a novel interaction point between immune response and VC procession, and such interaction may depend on the miRs transportation.
3.5 | Other mechanism relating to exosomes during VC Besides the mechanisms described above, mechanical stretch is regarded as a potential novel player of VC. Exosomes may regulate VC procession through this mechanism. Mechanical environment is recently proven to participate in calcification procession. Balachandran et al 100 reported that cyclic mechanical stretch could promote aortic valve calcification via elevating Runx2 expression and ALP activity. Mechanical membrane stretch enhanced exosomes secretion in cardiomyocytes, and contents inside exosomes were altered due to the mechanical environment. 101 Moreover, it was reported that under shear stress stimulation, BMP4 expression was down-regulated in endothelial cells, which is vital for osteogenic transition during VC. 102 Also, shear stress promotes miR-143 loading into exosomes rather than other miRs in endothelial cells, indicating that mechanic environment has effect on selective miRs secretion via exosomes. 103 Thus, mechanical environment is vital in the procession of VC via alterations of miRs inside exosomes and exosomes secretion.

CONFLI CT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.