Exosomes, the message transporters in vascular calcification.

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.

EXOSOMES PARTICIPATE 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).

Figure 1

The functions of exosomes during vascular calcification (VC) as initializers and transporters for microRNAs (miRs). Exosomes function as mineral nucleation sites extracellularly and transport miRs among cells targeting at mRNAs in the recipient vascular smooth muscle cells. Exosomes intake further promotes miRs transportation among cells, which is in a heparin sulphate proteoglycans (HSPG)‐dependent manner. Moreover, under pro‐calcific milieu, exosomes secretion is enhanced by sphingomyelin phosphodiesterase 3 (SMPD3)

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 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 α (TNF‐α) 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 clathrin‐coated 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 calcium‐binding 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/β‐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 α‐smooth muscle actin (α‐SMA).50 It is revealed that some miRs with elevated expression during VC promote osteogenesis via targeting at anti‐calcification proteins or contractile markers, whereas some other miRs with decreased expression suppress osteogenesis of VSMCs through targeting at osteogenic transcription factors13, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 (shown in Table 1). As described, some of such miRs, including miR‐133b,55 miR‐204,57 miR‐211,55 alter during VC and are also proven to be transported by exosomes to modulate the biological behaviour in various kinds of recipient cells.62, 63, 64 Such results indicated that exosomes could participate in VC through transporting miRs to influence phenotype transition. However, Ulbing et al65 reported that circulating miR‐223 was down‐regulated in CKD patients and decreased expression of miR‐223 was regarded as a risk factor for VC occurrence which might be packaged into exosomes. Moreover, miR‐223 expression is up‐regulated in VSMCs under elevated Pi stimulation.56 Such contradiction implies that besides message transporters, exosomes function more than what the present knowledge obtains and more attentions need to be paid in such field.

Table 1

Targets and expression changes of different miRs in VC procession

miR(s)	Target molecule	Pro‐calcific stimulation	Cell Source/Tissue	Function	Reference number	miRNA expression
miR‐29b	ACVR2A CTNNBIP	Pi‐induced	Rat VSMCs	Inhibition of osteoblast differentiation	55	↓
miR‐30b/c	Runx2	rhBMP2‐induced	Human coronary artery SMCs	Inhibition of osteoblast differentiation	51	↓
miR‐32	PTEN	β‐glycerophosphate‐induced	Mouse VMSCs	Promotion of osteoblast differentiation	52	↑
miR‐34b/c	SATB2	Aldosterone‐induced	Rat VSMCs	Suppression of osteogenesis transdifferentiation	53	↓
miR‐125b	Osterix	β‐glycerophosphate‐induced	Human coronary artery SMCs	Decreasing ALP expression and matrix mineralization	54	↓
miR‐133b	Runx2	Pi‐induced	Rat VSMCs	Inhibition of osteoblast differentiation	55	↓
miR‐143/145	KLF4/KLF5	Pi‐induced	HAVSMCs	Phenotype transition preservation	56	↓
miR‐155	AT1R	CKD(transgenic rat)	Rat VSMCs	Inhibitions to VC	13	↓
miR‐204	Runx2	β‐glycerophosphate‐induced	Mouse VSMCs	Inhibition of osteoblast differentiation	57	↓
miR‐211	Runx2	Pi‐induced	Rat VSMCs	Inhibition of osteoblast differentiation	55	↓
miR‐223	Mef2c/RhoB	Pi‐induced	Human VSMCs	Phenotype transition from contractile to synthesis and calcification induction	56	↑
miR‐712	NCKX4	Klotho homozygous mutant	Mouse VSMCs	Disrupt calcium transporters and promote calcium deposition	60	↑
miR‐714	PMCA1	Klotho homozygous mutant	Mouse VSMCs	Disrupt calcium transporters and promote calcium deposition	60	↑
miR‐762	NCX1	Klotho homozygous mutant	Mouse VSMCs	Disrupt calcium transporters and promote calcium deposition	60	↑
miR‐2861	HDAC5	β‐glycerophosphate‐induced	Mouse VMSCs	Promotion of osteoblast differentiation	61	↑
miR‐3960	HoxA2	β‐glycerophosphate‐induced	Mouse VMSCs	Increasing osteoblastogenesis	61	↑

ACVR2A, activin A receptor type II A; AT1R, angiotensin type 1 receptor; CTNNBIP, β‐catenin interacting protein; HDAC5, histone deacetylase 5; HoxA2, homeobox A2; Mef2C, myocyte enhancer factor 2C; NCKX4, sodium/calcium exchange member 1; NCX1, sodium/calcium exchange member 1; PMCA1, plasma membrane calcium pump isoform 1; PTEN, phosphate and tensin homologue; RhoB, ras homologue family member B; SATB2, special AT‐rich sequence‐binding protein 2.

POTENTIAL REGULATORY MECHANISMS OF EXOSOMES IN VC

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 7‐ketecholesterol, 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 calcium‐dependent manner.71 Rab11 also enhances autophagosomes fusing with MVBs to form amphisomes under interferon‐γ 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‐α and interleukin (IL) family members, such as IL‐1β 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 β (TGF‐β) signalling pathway is proven to promote VC, which is related to inflammation.82 High Pi induces activation of TGF‐β/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‐β/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.

Oxidative stress

Intracellular calcium overloading triggers disruption of superoxide metabolism, and further induces oxidative stress. Excessive production of reactive oxygen species (ROS) promotes VC via inducing osteogenic phenotype transition.87 Advanced glycation end‐products (AGEs) are the key factors for ROS production in diabetes mellitus (DM) patients, which activates the receptors to initialize oxidative stress procession. It was found that in DM, AGE up‐regulated ROS production, elevated alkaline phosphatase (ALP) activity and promoted VC via receptors for advanced glycation end‐products (RAGEs).88 In another study, Kay et al89 demonstrated that AGE/RAGE axis accelerated ROS production via nicotinamide adenine dinucleotide phosphate oxidase 1 (Nox1) to enhance oxidative stress in VSMCs and subsequently enhanced VC. It has been shown that exosomes are associated with oxidative stress. Patel et al90 previously found that exosomes from breast cancer cells promoted ROS production in the recipient primary mammary epithelial cells. It is also 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 Regulatory T (Treg) cells are of great significance in immune response, which might negatively regulate inflammatory reaction.94 In haemodialysis patients, coronary artery calcification score is negatively correlated with Treg cell frequencies 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.

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 al100 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.

CONCLUSION

Vascular calcification elevates the probabilities for patients to encounter with MACEs. In this review, we have discussed the roles of exosomes as message transporters in VC. Exosomes accelerate VC through mediating miRs transportation among cells to regulate autophagy, inflammation, oxidative stress, immune response and other possible mechanisms (Figure 2). Interfering exosomes secretion and miRs alterations inside might provide novel targets for treating VC.

Figure 2

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

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.