Calcium Regulates Key Components of Vascular Smooth Muscle Cell–Derived Matrix Vesicles to Enhance MineralizationNovelty and Significance
Rationale: Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)–derived MVs.
Objectives: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress.
Methods and Results: Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites.
Conclusions: In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.
Vascular calcification is the deposition of apatite mineral in the medial or intimal layers of the vessel wall and is a clinically significant pathology in atherosclerosis, diabetes, chronic kidney disease, and aging. Once established, vascular calcification is progressive, particularly in association with raised levels of extracellular mineral ions such as calcium and phosphate.1 Recent nuclear magnetic resonance studies have shown that the structural organization of the molecular components of vascular mineralizations are identical to those in bone.2,3 This implies mechanistic similarities during the earliest phases of initiation of mineral nucleation in both tissues.
During developmental osteogenesis/chondrogenesis, specialized membrane-bound bodies called matrix vesicles (MVs), which originate from the plasma membrane of chondrocytes and osteoblasts, serve as nucleation sites for hydroxyapatite.4 In cartilage, MV production occurs throughout the growth plate, but MVs are “mineralization competent” only in the hypertrophic zone.4 This transition is induced by an intracellular calcium signal that initiates changes in gene transcription and the subsequent release of MVs that are able to nucleate mineral to form hydroxyapatite nanocrystals.5 Mineralization-competent MVs are enriched with the calcium-binding annexins (Anx) A2, A5, and A6 and surface alkaline phosphatase (ALP), which releases phosphate by degrading pyrophosphate, a potent inhibitor of hydroxyapatite crystal growth; however, the exact mechanisms whereby MVs concentrate calcium and phosphate to enable mineral nucleation have not been resolved completely. A number of additional factors have been implicated, including sodium-dependent phosphate transporters and phospholipid components of the vesicle membrane such as phosphatidylserine.5–8
Ultrastructural studies have identified hydroxyapatite-containing membrane vesicles in the vessel wall, which suggests that these structures may also provide the first nidus for vascular calcification.9,10 Vascular smooth muscle cells (VSMCs) play a key role in initiating and regulating vascular calcification, and at sites of calcification, they undergo an osteocytic/chondrocytic phenotypic change and upregulate expression of mineralization-regulating proteins that are normally confined to bone and cartilage.11 Concomitant with this phenotypic transition, in vitro VSMCs also spontaneously release membrane-bound vesicles.12 Under normal conditions, these VSMC-derived vesicles do not calcify, because they are loaded with mineralization inhibitors such as matrix Gla protein (MGP) and fetuin-A, which act to block mineral nucleation.12 However, our previous studies have shown that treatment of VSMCs with elevated levels of extracellular calcium can stimulate the production of calcifying vesicles that contain preformed apatite, a hallmark of mineralization-competent MVs.12,13 Importantly, elevated extracellular phosphate could not induce the same effect on MV mineralization, which suggests that calcium uptake, raised intracellular calcium, or both may trigger VSMC MV release and mineralization. Indeed, elevated extracellular or intracellular calcium has been widely reported in pathologies associated with increased vascular calcification.10,14,15 Thus, the local environment of MV biogenesis is likely to affect their ability to calcify, and this notion is supported by studies showing that vesicles isolated from the normal vessel wall are inefficient at accumulating calcium compared with those from calcified or atherosclerotic regions.9
To identify the mechanisms of elevated calcium-induced MV mineralization, we used proteomics and molecular approaches to characterize the properties of VSMC-derived MVs (VSMC-MVs) released under basal noncalcifying conditions and in response to raised extracellular mineral ions. We show that key events in VSMC-MV calcification are calcium-induced loss of inhibitors and membrane association of AnxA6 and phosphatidylserine to form a complex that can nucleate hydroxyapatite. Alteration of the properties and biogenesis of VSMC-derived vesicles may be a useful strategy for limiting vascular calcification.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Extracellular Mineral Ion-Induced VSMC Calcification
Explanted cultures of human aortic VSMCs were established as described previously12 and used between passages 4 and 12. VSMCs were treated with calcification media supplemented with calcium (5.4 mmol/L) or calcium/phosphate (2.7 mmol/L calcium, 2.5 mmol/L phosphate) as indicated and 45Ca (≈50 000 cpm/mL) as described previously12 and exposed for between 16 hours and 5 days in the presence of 0.5% BSA. One to 5 μmol/L BAPTA-AM (1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid) was added at the time of calcium addition. Calcification was quantified by liquid scintillation counting for 45Ca and visualized by Alizarin Red staining.
Preparation of Vesicles and Assays
Apoptotic bodies and MVs were isolated by differential centrifugation as described previously, and MV calcification potential was measured by 45Ca incorporation as described previously.12,16 ALP activity was measured with a p-nitrophenyl phosphate substrate (Sigma, St Louis, MO). Protein content was measured with a Bio-Rad kit (Bio-Rad Laboratories, Hercules, CA). Matrix metalloproteinases (MMPs) were measured with zymography or fluorescence assay kits according to the manufacturer's instructions.
Protein Analysis of VSMC-MVs by Mass Spectrometry
MVs were resuspended in sample buffer, proteins were separated on a 4% to 15% gradient SDS-PAGE gel (Bio-Rad) and stained with Colloidal Blue (Invitrogen, Carlsbad, CA), and protein bands were excised. Excised proteins were identified with liquid chromatography tandem mass spectrometry, and the data were analyzed with the Mascot program (Matrix Science Inc, Boston, MA).
Two-dimensional difference gel electrophoresis was performed as described previously17 with modifications. Briefly, MV proteins (100 μg) were labeled with Cy3 DIGE Fluor minimal dye (GE Healthcare, Little Chalfont, United Kingdom) and separated by 2-dimensional difference gel electrophoresis, and proteins were silver stained with a Plus One silver stain kit with modification (Amersham, Uppsala, Sweden). Proteins were excised and identified by liquid chromatography tandem mass spectrometry. Detailed protocols for 2-dimensional gel electrophoresis and silver staining are provided in the online-only Data Supplement and online at http://www.vascular-proteomics.com.
Antibodies used were AnxA2 (BD Transduction Laboratories, San Diego, CA), AnxA5 (Abcam, Cambridge, United Kingdom), AnxA6 (BD Bioscience, San Diego, CA), vinculin (Sigma), MGP (a monoclonal antibody raised against aa 3-15), phosphatidylserine antibody (Abcam, Cambridge, United Kingdom), antibody for the extracellular domain of integrin β1 (Santa Cruz Biotechnology, Santa Cruz, CA), Alexa Fluor 488 goat anti-mouse antibody for immunofluorescence and flow cytometry (Invitrogen, Eugene, OR), and horseradish peroxidase–conjugated secondary antibody for immunohistochemistry (GE Healthcare, Little Chalfont, United Kingdom).
VSMC surface labeling was performed with a Pierce cell-surface protein isolation kit (Pierce, Rockford, IL) according to the manufacturer's protocol.
Small Interfering RNA Transfection
Small interfering RNA oligonucleotides targeting annexins predesigned by Qiagen (Valencia, CA) were transfected into VSMCs with HiPerFect (Qiagen) according to the manufacturer's protocol. VSMCs were transfected for 48 hours before addition of calcification medium that contained 1 μmol/L fetuin for a further 72 hours before analysis.
A total of 1×105 VSMCs were incubated with primary antibody or the corresponding isotype-matched IgG control for 30 minutes at 4°C. After they were washed, VSMCs were incubated with secondary antibody for 30 minutes at 4°C, washed again, and analyzed with a BD FACScalibur (BD Bioscience). MVs were coupled to 4-μm surfactant-free aldehyde/sulfate latex beads (Invitrogen) as described previously.18 As a control, 20 μL of beads was incubated with antibody and analyzed as above.
Biochemical Analysis of MVs
An aliquot of VSMC-MVs (5 μg) was treated with EGTA 10 mmol/L for 15 minutes at room temperature. MV membranes and extracted proteins were separated by ultracentrifugation at 100 000g for 30 minutes, and the pelleted membrane fraction was resuspended. Samples were then treated with or without EGTA 10 mmol/L or CaCl2 2 mmol/L and disrupted by freeze/thaw 4 times. Membrane and luminal extracted proteins were isolated by centrifugation as before, and the resultant pellet was resuspended. The fractions were analyzed by 10% (wt/vol) SDS-PAGE and Western blot.
Data were analyzed by 1-way ANOVA with Bonferroni post hoc test or t test as indicated with PRISM software (GraphPad, San Diego, CA). Data show the mean±SD. P<0.05 was considered statistically significant.
Vesicles Mediate Calcification In Vivo
Transmission electron microscopy was used to examine MV deposition in normal and calcified arteries in different disease contexts. In young healthy arteries, numerous MVs 50 to 500 nm in size were observed in the extracellular matrix (ECM) of the vessel media; however, there was no evidence of crystalline mineral within these structures (Figure 1A). In minimally calcified arteries from patients with atherosclerosis and chronic kidney disease, mineralization was observed in a subset of MVs that clustered in association with elastin and collagen fibrils; however, mineralization did not extend into these ECM components (Figures 1B–D). Only in more heavily calcified vessels obtained from diabetic patients was mineralization observed to extend into the ECM and involve collagen; however, in these vessels, mineralization was also apparent on membrane surfaces, including the inner and outer membranes of MVs in close proximity to collagen and on the VSMC plasma membrane (Figure 1E). These observations suggest that mineral is nucleated on membranes and progresses to the ECM after initiation in MVs, a subset of which become mineralization competent in diseased arteries.
Elevated Extracellular Calcium Induces Alterations in Cytosolic Ca2+ Levels to Promote MV-Mediated Mineralization of VSMCs
Our previous studies showed that mineralization-competent MVs were released by VSMCs only in response to elevated extracellular calcium,5,12 and we found that blocking calcium uptake by VSMCs also inhibited VSMC calcification (Online Figure I). Calcium entry should lead to alterations in cytosolic calcium, and treatment of VSMCs with the intracellular calcium chelator BAPTA-AM in the presence of calcifying media decreased VSMC calcification in a dose-dependent manner (Figures 2A and 2B). Moreover, using a quantitative in vitro calcification assay, we found that MVs isolated from BAPTA-AM–treated VSMCs had a significant reduction in their capacity to mineralize (Figure 2C), which suggests extracellular calcium uptake and cytosolic calcium alterations are necessary for the production of mineralization-competent MVs.
Calcium Does Not Increase ALP in VSMC-MVs but Induces Loss of the Inhibitor MGP and Activation of MMP2
Next, we assessed the similarities between VSMCs and chondrocyte-derived MVs with respect to key regulators of mineralization. ALP is enriched in chondrocyte MVs4; however, in comparison, VSMCs show extremely low levels of activity of this enzyme. Although there was some enrichment of ALP in VMSC-MVs, ALP activity was decreased after treatment with extracellular calcium (Figure 3B). Treatment with BAPTA-AM failed to induce changes in ALP activity (data not shown), which suggests it is not a key calcium-responsive mediator of MV calcification in VSMCs.
MGP is a key inhibitor of MV calcification in both chondrocytes and VSMCs.19 Western blots showed that MVs isolated from VSMCs treated with calcifying media had a dramatic reduction in MGP content 48 hours after treatment (Figure 3B). To test the factors that contributed to this loss, we treated VSMCs with elevated phosphate or calcium alone. Treatment of VSMCs with phosphate had no effect on MGP content of MVs (data not shown). Treatment with elevated calcium induced an initial increase in MGP loading at 24 hours, but after 48 hours, MGP was significantly reduced in mineralization-competent MVs. This pattern of MGP loss was also mirrored in whole-cell lysates (Online Figure II).
MVs associated with ECM components, and previous studies have shown that elastin degradation promotes calcification.20 Zymography and fluorimetric assays showed that VSMC-MVs were enriched in MMP2 but contained no MMP9, whereas the majority of cathepsin S activity was present in apoptotic bodies (Online Figure III). Calcium treatment maximally activated MMP2, as indicated by no increased activation after treatment with APMA (4-aminophenylmercuric acetate), and calcium-dependent MMP2 activation could be blocked by treatment of VSMCs with BAPTA-AM (Figures 3C and 3D; Online Figure III). Treatment of VSMCs with the MMP inhibitor GM6001 dose dependently decreased calcification in response to calcium/phosphate after 16 hours (Figure 3E).
Proteomic Characterization of Mineralization-Competent MVs Reveals a Role for Annexins
To identify additional mediators of calcification, we analyzed the composition of VSMC-MVs using protein mass spectrometry (Online Figures IV and V). Two approaches were used: (1) 1-dimensional gel electrophoresis to isolate MV components concentrated in response to calcium, and (2) CyDye tagging and 2-dimensional gel comparison to identify membrane components of MVs. The 79 identified proteins were broadly classified into 10 major groups (Online Tables I and II): 12 proteins potentially involved in mineralization processes, including calcium channels and ECM components; 24 proteins possibly related to MV biogenesis, including cytoskeletal proteins and proteins involved in intracellular vesicle trafficking; 15 different stress-related proteins, mainly involved in oxidative stress and protein folding; and 5 serum proteins. Comparative proteomic bioinformatics analysis identified 38 proteins that had been identified previously in chondrocyte MVs7,8 (Table). Of particular note was the identification of annexins, which have been shown to specifically accumulate in chondrocyte-derived mineralization-competent MVs in a calcium-dependent manner.5
AnxA6 Is a Biomarker of Vascular Calcification and Is Required for MV Mineralization
Western blots showed that only AnxA2, A5, and A6 were selectively enriched in MVs compared with VSMC lysates and apoptotic bodies, which implies they may play a specific role in MVs and not in other membrane-derived vesicles (Figure 4A). Treatment of VSMCs with calcifying media induced selective enrichment of AnxA6, whereas levels of AnxA2 and A5 remained relatively unchanged or were even reduced (Figure 4B).
The enrichment of AnxA6 in calcifying MVs was supported by immunohistochemistry of calcified human arteries (Figure 4C), which showed a significant deposition of AnxA6 at sites of calcification. In normal noncalcified arteries, AnxA5 and A6 were undetectable, whereas AnxA2 was detectable in VSMCs. In calcified arteries, AnxA2 staining remained unchanged, whereas some patchy AnxA5 staining was evident in association with VSMCs. AnxA6 staining was markedly upregulated, colocalized consistently with von Kossa–positive areas, and was deposited within the ECM surrounding calcified VSMCs. To evaluate the significance of annexins in VSMC mineralization, we depleted AnxA2, A5, and A6 using specific small interfering RNAs and found that only AnxA6 knockdown reduced calcification, which suggests a crucial role for AnxA6 in vascular mineralization (Figures 4D–F).
AnxA6 Translocates to the Plasma Membrane in Response to Cytosolic Calcium Elevation
Annexins are normally cytosolic, but in response to a rise in extracellular calcium, they can translocate to the plasma membrane from which MVs are shed.4,5 Immunofluorescence staining of VSMCs confirmed that AnxA6 was cytosolic under normal conditions (Figure 5A). In response to calcium, AnxA6 fluorescence became stronger at the plasma membrane and was enriched on the membrane of small vesicles, apparently budding from the plasma membrane surface. In contrast, localization and levels of AnxA2 and A5 were unchanged in response to calcium (Online Figure VI). Translocation of AnxA6 to the plasma membrane in response to calcium and inhibition of this translocation with BAPTA-AM were confirmed by biotin cross-linking and flow cytometry (Figures 5B–D), which supports the notion that AnxA6 enrichment at the plasma membrane is calcium dependent.
Annexin A6 Localizes to the Membrane of MVs in Response to Calcium
We next examined the localization of AnxA6 within MVs using flow cytometry. Using the cell-surface marker integrin-β1, we established that the orientation of the MV membrane was similar to the plasma membrane (Figure 6A). Annexins were also detected on the outer surface of the MV membrane; however, only AnxA6 was increased on the surface in response to calcium treatment (Figure 6A). To examine Anx6 localization further, EGTA was used to extract calcium-dependent binding of AnxA6 from the outer surface of MVs. This treatment released AnxA6 from the surface of calcifying MVs, whereas a significantly lower amount of AnxA6 was on the outer surface of control vesicles (Figure 6B). Next, to elute AnxA6 from the inner surface of MVs, we disrupted the membrane using a freeze/thaw cycle and again extracted with EGTA. We observed a significant extraction of AnxA6 from the inner membrane of MVs. In contrast, AnxA2 was only extracted by EGTA from the inner membrane (Figure 6B). As a further control, the intraluminal localization of AnxA2 in MV was confirmed with 2-dimensional difference gel electrophoresis proteomics (Figure 6C; Online Figure II).
We also found that a high proportion of AnxA2 and A6 was not extractable by EGTA, which suggests a tight calcium-independent incorporation within the membrane bilayer (Figure 6B). This localization has been associated with AnxA5 calcium-channel activity in chondrocyte MVs.21 However, treatment of VSMCs and VSMC-MVs with the annexin calcium-channel blocker K201 did not reduce calcification of VSMCs or isolated MV nor did treatment with antibodies specific for AnxA2 or A6,21 which suggests that annexins do not act as calcium channels in VSMC-MVs (Online Figure VII).
Calcium-Dependent Phosphatidylserine-Annexin Complexes Mediate MV Calcification
It is known that annexin binding to the plasma membrane is mediated by negatively charged phospholipids, in particular phosphatidylserine.22 Moreover, complexes of annexins with phosphatidylserine have been shown to possess strong nucleation activity and significantly enhance crystalline mineral formation in chondrocyte MVs.6,21 Therefore, an accumulation of AnxA6 on the outer surface of MVs may occur simultaneous with externalization of phosphatidylserine in response to high calcium, leading to the formation of nucleation sites. Indeed, treatment of VSMCs in calcifying conditions resulted in the upregulation of phosphatidylserine on the VSMC surface (Figure 7A). This redistribution was calcium dependent and inhibited by BAPTA-AM (Figure 7B). Flow cytometry confirmed that phosphatidylserine was also present on the MV surface and was upregulated in the presence of calcifying media, and this phosphatidylserine externalization was calcium dependent and again inhibited by BAPTA-AM (Figure 7B; Online Figure VIII).
Mineralization-competent MVs were visualized with transmission electron microscopy to examine the sites of crystalline hydroxyapatite accumulation. MVs ranging in size from 50 to 500 nm showed accumulation of crystalline mineral on the surface and within the lumen (Figure 7C), consistent with nucleation complexes of AnxA6 and phosphatidylserine being present on both the inner and outer membranes of MVs (Figure 7D).
Mineralization-Competent VSMC-MVs Share Similarities With Chondrocyte-Derived MVs
In the present study, we have shown that VSMC calcification is first initiated in extracellular MVs. In vitro studies have demonstrated that MV calcification is induced by calcium-dependent loss of MGP loading and the concomitant upregulation and redistribution of phosphatidylserine and Anx6 complexes that act to nucleate hydroxyapatite on the inner and outer vesicle membranes. Although VSMC-MVs share some similarities with chondrocyte MVs, the absence of ALP activity and the abundance of Anx6 suggest they do not share all the properties of “professional” MVs released by hypertrophic chondrocytes, which are produced under regulated physiological conditions and primed for mineralization.4 Rather, the shift to mineralization competency in VSMC-MVs is dependent on pathological changes in intracellular calcium homeostasis that disrupt inhibitor loading and cause membrane and annexin changes that favor hydroxyapatite nucleation. These mechanisms are more consistent with the dystrophic nature of human vascular calcification, which accumulates over a long time period and rarely manifests as true cartilage or bone.
Production of Mineralization-Competent Matrix Vesicles Is Regulated by Intracellular Calcium Homeostasis
In growth plate chondrocytes, retinoic acid is the physiological signal that induces an intracellular calcium rise that triggers the production of mineralization-competent MVs.5,23 The mechanisms involve upregulation of AnxA2, A5, and A6 and their incorporation into mineralizing MVs, in which AnxA5, the most abundant annexin, is key in mediating mineral nucleation.5,6,24 In the present study, we also demonstrated a crucial role for cytosolic calcium homeostasis in the regulation of VSMC mineralization. Chelation of intracellular calcium blocked AnxA6 shuttling in response to extracellular calcium and MV calcification; however, in contrast to the physiological signaling observed in chondrocytes, pathological extracellular calcium stress induced VSMCs MV mineralization. Previous studies have suggested that MV release by VSMCs is an adaptive response aimed at preventing intracellular calcium overload.25,26 The normal loading of MVs with inhibitors such as MGP and fetuin-A, as well as the ability of these inhibitors to suppress MV calcification, supports this idea. The specific loading of VSMC-MVs with AnxA6 is also consistent with this notion in light of recent data that suggest that AnxA6 is involved in the maintenance of intracellular calcium homeostasis and in limiting cellular damage due to calcium overload.
Studies have shown that the calcium-dependent translocation of AnxA6 to the plasma membrane occurs after an influx of extracellular calcium.27–29 At the plasma membrane, AnxA6 functions as a membrane microdomain organizer and can regulate the activity of ion channels involved in both calcium entry and efflux in a cell- and context-specific manner.30–33 For example, calcium-induced binding of AnxA6 to the plasma membrane stabilizes the actin cytoskeleton and efficiently prevents store-operated calcium entry in HEK293 cells, whereas AnxA6 knockout mice show accelerated calcium efflux during cardiomyocyte contraction.30,31 AnxA6 translocation to the plasma membrane also preceded the sealing off of “hot spots” of extracellular calcium entry into cells and the subsequent shedding of microparticles, which led to a reduction in intracellular calcium and cell recovery.29 The present data are consistent with the notion that under sustained conditions of calcium overload, AnxA6 shuttles to the plasma membrane, where it acts to regulate calcium homeostasis and vesicle release. We also showed that AnxA6 knockdown acted to reduce VSMC calcification. Potentially, the lack of AnxA6 on MV membranes may have reduced their capacity to nucleate hydroxyapatite. Alternatively, AnxA6 may have a specific role in calcium homeostasis in VSMCs. Thus, loss of AnxA6 could potentially impinge on multiple cellular processes, including calcium transport, proliferation, apoptosis, differentiation, or membrane dynamics, and therefore, further investigation of these mechanisms is required.22
Phosphatidylserine and Annexin Complexes Mediate Calcification
VSMC mineralization-competent MVs were also enriched with phosphatidylserine on the outer surface compared with nonmineralizing MVs, and externalization of phosphatidylserine was induced by raised intracellular calcium. Studies have shown that phosphatidylserine is also abundant on chondrocyte-derived MVs, where it forms hydroxyapatite nucleation complexes, predominantly with AnxA5.6,34,35 In chondrocytes, phosphatidylserine exposure is induced by raised intracellular calcium, which stimulates the activity of a scramblase, which causes phosphatidylserine exposure, and this mechanism may also be active in VSMCs.34 Although nonspecific calcium-dependent flip-flop of phospholipids has been reported in apoptotic cells, and “default” calcification of apoptotic bodies is also thought to be mediated by externalized phosphatidylserine, the majority of VSMCs that exposed surface phosphatidylserine were not apoptotic.36,37
Indeed, although apoptosis is a key event in VSMC calcification, the present data suggest that MVs differed significantly from apoptotic bodies with respect to size, annexin composition, and mineralization capacity, which suggests MVs have additional mechanisms to enable luminal mineralization.12,16 Phosphatidylserine-annexin complexes may be sufficient to rapidly and efficiently nucleate hydroxyapatite without the requirement for annexin calcium channel activity6; however, proteomics revealed that VSMC-MVs contain additional proteins with possible roles in calcium and phosphate uptake. The plasma membrane enzyme 5′-nucleotidase hydrolyzes nucleotides to phosphate and their corresponding nucleosides, which provides an additional phosphate source.8,38 The voltage-dependent, anion-selective channel protein 1 (VDAC1) has calcium channel activity,39 although 2 additional proteins implicated in the regulation of cation channel activities, SLP-2 (stomatin-like protein 2) and calmodulin, were also identified.40,41 Whether these proteins are functional remains to be tested. To date, only raised levels of extracellular calcium have been shown to induce the formation of mineralization-competent MVs by VSMCs. Thus, VSMC-MVs may acquire elements of the calcium-homeostasis machinery because of the calcium-dependent accumulation of these ion channels in AnxA6-rich membrane microdomains (rafts), which are subsequently shed.27,29 Importantly, this mechanism is consistent with vascular mineralization occurring at sites of cellular damage in the media and intima, where cell death can induce microenvironments high in extracellular calcium.10,26 However, it will be important to determine whether other calcifying stimuli such as hyperlipidemia, hyperglycemia, or inflammatory cytokines can induce MV release and mineralization, potentially by affecting intracellular calcium homeostasis.
Calcium Induces Loss of MGP, a Potent Inhibitor of MV Mineralization
Another crucial factor that led to increased production of mineralization-competent MVs by VSMCs in response to calcium was the reduction in MGP protein loading. In chondrocytes, MGP is present in nonmineralizing MVs but is absent in mineralization-competent MVs.42,43 Similarly, in the normal vessel wall, VSMC-MVs were noncalcified, which suggests they contained inhibitors; however, this inhibition is apparently lost in the diseased vessel wall.10 Downregulation of MGP loading into MVs could be due to exhaustion of the protein because of prolonged calcium overload or impairment of intracellular sorting mechanisms. Importantly, accumulation of the uncarboxylated form of MGP occurs at sites of calcification, and MGP posttranslational modifications occur in the endoplasmic reticulum, an organelle likely to be impaired by calcium overload.10
Origin and Alternate Functions for VSMC-MVs
The observation that VSMCs release MVs under noncalcifying conditions suggests that these particles may serve a functional role in the cell beyond pathological calcification. Proteomics analysis revealed a number of ECM components, as well as integrins, which suggests MVs may be involved in the formation of ECM and may also be targeted for integrin-dependent ECM binding. MVs also exhibited MMP2 activity, and freeze/thaw analysis (data not shown) indicated that this activity was most likely on the MV surface. MVs were found in tight association with collagen and elastin fibers, and under normal conditions, MMP2 activity may be required during matrix biogenesis. However, elastin degradation also promotes calcification, and the activation of MV MMP2 by calcium suggests this may lead to enhanced degradation, as well as the production of elastin peptides, both of which are key inducers of calcification.20,44 Signaling molecules, chaperones, and proinflammatory factors such as cyclophilin A were also detected, which suggests that MVs may act as intercellular signaling modules, similar to the role described for exosomes in other cell types.45 Although the presence of cell-surface markers on MVs and actin and actin regulatory proteins (moesin, cofilin, WD repeat-containing protein 1, F-actin–capping protein, and tropomyosin) is consistent with a plasma membrane origin, other proteins involved in vesicle biogenesis were also identified, which may point to an endosomal origin for some vesicles.46 Clearly, further studies are now required to characterize these VSMC-derived organelles more fully under normal and calcifying conditions.
Sources of Funding
This work was supported by a British Heart Foundation program grant to Catherine M. Shanahan (RG/05/001).
We acknowledge Dr Slawomir Pikula from the Nencki Institute of Experimental Biology for protocols and helpful discussion. We also acknowledge Dr Xiaoke Yin for excellent technical support.
In March 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days.
Non-standard Abbreviations and Acronyms
- alkaline phosphatase
- annexin A2
- annexin A5
- annexin A6
- 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester
- extracellular matrix
- matrix vesicle
- matrix Gla protein
- voltage-dependent anion-selective channel protein 1
- Received December 8, 2010.
- Revision received April 28, 2011.
- Accepted May 2, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known
Vascular smooth muscle cells (VSMCs) are known to be key participants in the initiation of vascular calcification in both the intima and media of the vessel wall.
Dysregulated mineral metabolism leading to increases in calcium and phosphate levels promotes VSMC calcification.
Matrix vesicles released by VSMCs are found at sites of calcification, but the mechanisms whereby these structures promote calcification in response to changes in calcium and phosphate levels are poorly understood.
What New Information Does This Article Contribute?
Calcification is initiated primarily in matrix vesicles rather than directly on the extracellular matrix.
Dysregulation of intracellular calcium plays an important role in initiating matrix vesicle calcification.
Elevated levels of extracellular calcium initiate matrix vesicle calcification by interfering with inhibitor loading and by promoting the formation of hydroxyapatite nucleation complexes.
Vascular calcification is a detrimental process associated with increased cardiovascular mortality. In association with dysregulated mineral metabolism, it is particularly prevalent in chronic kidney disease. VSMCs mediate the calcification process in response to elevated levels of extracellular calcium and phosphate. Although the mechanisms by which elevated phosphate induces calcification are well studied, the role of elevated calcium is less well understood. In the present study, we show that elevated calcium promotes calcification of matrix vesicles, small membrane-bound structures that are released by VSMCs that form the first nidus for calcification in the vessel wall. Elevated calcium triggers VSMC calcification by altering intracellular calcium levels. This calcium overload induces the production of specialized matrix vesicles that are enriched with annexins and membrane lipids. These vesicles are able to initiate calcification because they lack important calcification inhibitors. Importantly, maintenance of normal intracellular calcium levels prevents these events. Clinically, this suggests that the maintenance of plasma calcium levels in the normal range and reductions in bouts of transient hypercalcemia are important for the prevention of vascular calcification, particularly in patients with chronic kidney disease.