Human Vascular Smooth Muscle Cells Undergo Vesicle-Mediated Calcification in Response to Changes in Extracellular Calcium and Phosphate Concentrations: A Potential Mechanism for Accelerated Vascular Calcification in ESRD
Joanne L. Reynolds*,
Alexis J. Joannides*,
Jeremy N. Skepper,
Rosamund McNair*,
Leon J. Schurgers,
Diane Proudfoot*,
Willi Jahnen-Dechent,
Peter L. Weissberg* and
Catherine M. Shanahan*
*Department of Medicine, Addenbrooke’s Hospital, Cambridge, United Kingdom; Multi-Imaging Centre, Department of Anatomy, Cambridge, United Kingdom; Department of Biochemistry, University of Maastricht, Maastricht, The Netherlands; and IZKF Biomat Aachen University Clinics, Aachen, Germany
Correspondence to Dr. Catherine M. Shanahan, Division of Cardiovascular Medicine, ACCI, Level 6, Box 110, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK. Phone/Fax: 44-1223-331504/5; E-mail: cs131{at}mole.bio.cam.ac.uk
Patients with ESRD have a high circulating calcium (Ca) x phosphate(P) product and develop extensive vascular calcification thatmay contribute to their high cardiovascular morbidity. However,the cellular mechanisms underlying vascular calcification inthis context are poorly understood. In an in vitro model, elevatedCa or P induced human vascular smooth muscle cell (VSMC) calcificationindependently and synergistically, a process that was potentlyinhibited by serum. Calcification was initiated by release fromliving VSMC of membrane-bound matrix vesicles (MV) and alsoby apoptotic bodies from dying cells. Vesicles released by VSMCafter prolonged exposure to Ca and P contained preformed basiccalcium phosphate and calcified extensively. However, vesiclesreleased in the presence of serum did not contain basic calciumphosphate, co-purified with the mineralization inhibitor fetuin-Aand calcified minimally. Importantly, MV released under normalphysiologic conditions did not calcify, and VSMC were also ableto inhibit the spontaneous precipitation of Ca and P in solution.The potent mineralization inhibitor matrix Gla protein was foundto be present in MV, and pretreatment of VSMC with warfarinmarkedly enhanced vesicle calcification. These data suggestthat in the context of raised Ca and P, vascular calcificationis a modifiable, cell-mediated process regulated by vesiclerelease. These vesicles contain mineralization inhibitors derivedfrom VSMC and serum, and perturbation of the production or functionof these inhibitors would lead to accelerated vascular calcification.
Patients with ESRD develop extensive medial calcification, orMonckeberg’s sclerosis, that causes increased arterialstiffness and contributes to the high cardiovascular mortality(1,2). Calciphylaxis is an increasingly common and life-threateningform of calcification characterized by destructive calcificationin the media of subcutaneous arterioles, leading to occlusionand subsequent widespread tissue necrosis (2,3). The precisepathophysiology of vascular calcification in ESRD is unknown,but risk factors include age, hypertension, time on dialysis,and, most significant, abnormalities in calcium (Ca) and phosphate(P) metabolism (4,5). Normal serum concentrations of Ca andinorganic P ions are metastable with respect to basic calciumphosphate (BCP; a mixture of octacalcium phosphate, dicalciumphosphate dihydrate, and apatite) precipitation but can supportgrowth of nascent crystals. In ESRD, systemic Ca and inorganicP concentrations typically exceed 2.4 and 2.0 mM, respectively(4). Consequently, calcification in ESRD has traditionally beenascribed to supersaturation and subsequent precipitation ofmineral ions. This has led to therapeutic measures to reducethe Ca/P product aimed mostly at reduction of P.
Recent studies have shown that vascular calcification is a regulatedprocess similar to bone formation (6,7). VSMC in the normalartery wall constitutively express potent inhibitors of calcification,such as matrix Gla protein (MGP), whose absence results in spontaneousmedial calcification (8). In atherosclerotic calcification anddiabetic Monckeberg’s sclerosis, expression of these endogenousinhibitors is reduced and VSMC express markers of both osteoblastand chondrocyte differentiation (7,9). Moreover, human VSMCin culture spontaneously convert to an osteo/chondrocytic phenotypeand form calcified nodules (7,9). Calcification is initiatedin nodules by release of apoptotic bodies (AB) and matrix vesicle(MV)-like structures from VSMC that act as a nidus for BCP nucleation(10). AB are calcium-enriched membrane-bound vesicles 0.3 to1.0 µm in diameter, released from apoptotic VSMC (10).MV are smaller (30 to 300 nm) and are released from the plasmamembrane of viable cells (11). VSMC-derived MV have been associatedwith calcification in vivo, but their composition and functionare poorly understood (12).
Vascular calcification in ESRD may also be actively regulated.Bone matrix proteins are deposited in medial artery calcificationsof ESRD patients, suggesting osteogenic conversion of residentVSMC, while in vitro studies have shown increased calcificationand osteogenic changes in VSMC in the presence of elevated P(13,14). More recently, it was suggested that the circulatingserum protein fetuin-A, which is reduced in patients with ESRD,may play a role in regulating calcification. However, its mechanismof action is unclear (15).
In this study, we set out to determine the effects of increasedextracellular Ca and P concentrations on in vitro calcificationof human VSMC. Using this model, we demonstrate that VSMC calcificationis a vesicle-mediated phenomenon and identify potent local andcirculating inhibitors. Furthermore, we show that, in the presenceof raised P, a small increase in Ca concentration substantiallyincreases calcification.
In Vitro Model of VSMC Calcification: Media and Treatments
Explants of human aortic tissue (obtained with appropriate ethicalapproval) from organ donors aged 2 to 68 yr (n = 10) were established,and VSMC were cultured in M199 medium supplemented with 20%FCS and antibiotics (16). VSMC were grown to 80% confluenceand washed in Earle’s Balanced Salt Solution (EBSS) beforetreatment with control (1.8 mM Ca/1.0 mM P) or test media; highcalcium (Cai media; 2.0 to 2.8, 3.6, or 5.4 mM Ca), high phosphate(Pi media; 1.5 to 5.0 mM P), or both (CaPi media; 2.0 to 2.8mM Ca/2.0 to 3.0 mM P). The standard CaPi media used was 2.7mM Ca/2.0 mM P. CaCl2 and NaH2PO4 were used to supplement ioniccalcium and phosphate in the medium. Media either were serum-free(SF) and supplemented with 0.5% BSA or contained 20% FCS or1 to 10% human serum (HS). Experiments using the above controland test media were also performed in cell-free conditions.
For apoptosis experiments, 0.00025% DMSO (vehicle) or 100 µMof the caspase inhibitor ZVAD.fmk were added at the time oftest media. VSMC were pretreated for 3 d with warfarin (10 µM)before addition of test media.
Calcification Assays
Calcification was visualized 24 h to 10 d after treatment withtest media by alizarin red staining as described previously(10). Calcification was quantified by including 45Ca (50,000cpm/ml) in test and control media (17). Briefly, VSMC were decalcifiedin 0.1 M HCL, neutralized with 0.1 M NaOH/0.1% SDS, scrapedand 45Ca incorporation was measured by liquid scintillationcounting. In cell-free experiments, BCP precipitates were harvestedby centrifugation and 45Ca incorporation in the pellet measuredas above. Cell viability was measured by trypan blue (0.5%)staining as standard.
Time-Lapse Videomicroscopy and Detection of Apoptosis
Time-lapse videomicroscopy was performed at 37°C over 24h, and images were recorded as described previously (18). Apoptosiswas measured by direct counting of morphologically apoptoticcells and verified using fluorescence transferase-mediated dUTPnick-end labeling (TUNEL) staining visualized by a rhodamine-labeledanti-digoxigenin antibody (10). Nuclei were detected with DAPI,and >1000 nuclei were quantified.
Preparation of VSMC Vesicles and 45Ca Uptake
VSMC vesicles were harvested using a modified MV isolation protocol(19,20). Confluent VSMC were washed twice with EBSS and transferredto control or test media that contained 0.1% BSA. After 4 to24 h, the medium was decanted and spun at 2500 rpm in a SorvallRF7 centrifuge to remove AB. MV were then harvested from thesupernatant by centrifugation at 35,000 rpm (100,000 x g) for30 min at 4°C in a Beckman Ultracentrifuge. The MV and ABpellets were resuspended, and protein content was determinedusing a Biorad Kit (21,22). An increase in protein was indicativeof an increase in the number of vesicles isolated as confirmedby electron microscopy (EM) and FACS analysis (not shown). Invitro vesicle calcification assays were performed in triplicateover 24 h using a standard calcifying buffer and 4 to 15 µgof VSMC-derived MV or AB as described previously (10).
EM and Energy Dispersive X-Ray Analysis
VSMC for energy dispersive x-ray (EDX) analysis were grown onnucleopore filters and transferred to standard CaPi media for24 h. Samples were quench-frozen in melting propane, cooledin liquid nitrogen, then freeze-substituted against pure tetrahydrofuranfor 72 h at –85°C and embedded in Lowicryl HM20. Drysections (300 nm) were mounted on 100 mesh nickel grids ontoa Formvar film and coated with carbon before viewing in a PhilipsXL30 FEG-SEM with an Oxford Instruments GEM detector. Areasof calcification were identified using a solid-state, backscatteredelectron detector, and x-ray spectra were collected using anOxford Instruments INCA EDX system. Consecutive 50-nm-thicksections were stained with uranyl acetate and lead citrate andviewed in a Philips CM100. Sections were decalcified by floatingon 0.5% EDTA for 5 min before contrast staining to reveal anymasked structures.
Vesicle fractions isolated by differential centrifugation (MVand AB) were resuspended at a concentration of 10 µg/µland adsorbed onto glow-discharged, carbon-coated Formvar filmgrids. They were negatively stained with neutral potassium phosphotungstate,and vesicle diameters were measured with a Phillips CM100 withan on-line Kinetic Imaging analysis system. EDX analysis wascarried out on vesicle fractions adsorbed to grids, rinsed indeionized water, and air dried without staining.
Western Blot Analysis
Western blots for MGP and fetuin-A used 5 µg of vesicleprotein. Polyacrylamide gels (12.5% [wt/vol]) were run underdenaturing conditions in 8 M urea for MGP. After blotting, membraneswere incubated at 4°C overnight with biotin-conjugated a3-15MGP IgG or a rabbit polyclonal antibody raised against bovinefetuin (23,24). Visualization was via streptavidin–horseradishperoxidase (Amersham Pharmacia, Uppsala, Sweden) and developmentwith ECL Western blotting detection reagents (Amersham Pharmacia).Recombinant MGP was used as a positive control (24).
Statistical Analyses
Data were analyzed using t test or for multiple comparisonsANOVA with post hoc Scheffe test.
Ca and P Act Synergistically to Induce VSMC Calcification
VSMC were treated with increasing concentrations of Ca or Por both in the absence of serum. VSMC accumulated 45Ca in responseto elevated Ca or P. Ca was a more potent inducer of calcificationthan P at equivalent fold increases above normal physiologiclevels (Figure 1A, top). Treatment of VSMC with Ca and P togetherhad a synergistic effect on calcification in that a small elevationin extracellular Ca markedly enhanced the effects of elevatedphosphate (Figure 1A, bottom). In CaPi medium, extensive calcificationof the VSMC matrix was usually observed within 24 to 72 h andappeared as a granular precipitate on alizarin red staining(Figure 1C, 2).
Figure 1. Calcium (Ca) and phosphate (P) induce vascular smooth muscle cell (VSMC) calcification in the presence and absence of serum. Graphs show 45Ca accumulation in a VSMC isolate induced by Ca or P or both to calcify in serum-free (SF; 0.5% BSA) conditions for 24 h (A) or in the presence of serum (5% human serum [HS]) for 10 d (B). Note the synergistic effect on 45Ca accumulation of CaPi media (2.8 mM Ca/2.0 mM P) compared with Ca and P alone. Significant increases in calcification were observed even at lower concentrations of Ca in the presence of P (A, inset). In serum, calcification was induced in the same dose-dependent manner as in SF conditions but at a slower rate, and Ca accumulation was significantly less (note y-axis range). Results are normalized and expressed as fold increase in 45Ca accumulation compared with controls. The mean ± SD of a representative experiment performed in triplicate is shown. Similar results were obtained in five independent experiments performed on different VSMC isolates. **P < 0.05 versus controls analyzed by ANOVA. (C) Alizarin red staining demonstrating the punctate pattern of mineral accumulation occurring after treatment of VSMC with CaPi media (2.7 mM Ca/2.0 mM P) in the presence of serum after 10 d (right) and the granular pattern induced in the absence of serum after 24 h (middle). No calcification was observed in VSMC that were treated with control media (left).
Serum Inhibits VSMC Calcification Induced by Ca and P
The above experiments were repeated in the presence of serum.Normal HS inhibited VSMC calcification in a concentration-dependentmanner (data not shown) with maximal inhibition occurring ata concentration of 5%. Equivalent levels of calcification tothose achieved in SF media were not achieved even after 10 din HS (Figure 1B). The calcification induced in the presenceof serum was unevenly distributed in a punctate or nodular patternthroughout the VSMC matrix (Figure 1C, 3).
VSMC Inhibit Spontaneous BCP Precipitation
To assess the possibility that the calcification observed athigher Ca x P concentrations was due to spontaneous precipitationof Ca and P, we repeated the above experiments in cell-freeconditions. In the absence of cells, precipitation of Ca andP in SF media was observable by phase-contrast microscopy within6 h at a Ca x P of between 7.8 and 8.4 mM, resulting in high45Ca incorporation measured after 24 h (Figure 2B). However,in the presence of VSMC, this precipitation did not occur, andin some isolates, little calcification/45Ca incorporation occurredover 24 h (Figure 2).
Figure 2. VSMC inhibit precipitation of Ca and P. 45Ca accumulation in a VSMC primary culture (cells) compared with 45Ca accumulation in cell-free conditions using media that contained either 2.0 mM P (A) or 3.0 mM P (B) and 1.8 to 2.8 mM Ca. VSMC (•) calcified minimally (monitored by alizarin red staining) and accumulated little 45Ca and there was no evidence for mineral precipitation in solution over a 24-h period. In the absence of cells, extensive mineral precipitation was observed in the media (), particularly at higher concentrations of Ca and P, resulting in 45Ca accumulation. Mean ± SD of a representative experiment (of n = 4 experiments using different VSMC isolates) performed in triplicate is shown. **P < 0.05 (ANOVA).
VSMC Vesicle Release Contributes to Calcification
Time-lapse videomicroscopy was used to observe the calcificationprocess induced by extracellular Ca and/or P in the absenceof serum. This showed that VSMC in culture continually releasedsmall vesicles from the plasma membrane (Figure 3). Vesiclerelease increased within 60 min of treatment with Ca and/orP, and apoptosis, characterized by membrane blebbing, was alsoobserved (18). Apoptosis and AB and MV release continued untilrapid mineralization of the matrix was observed. In this context,calcification appeared as alizarin red–positive smallgranules (data not shown) that encased trypan blue excludingVSMC (Figure 3A, box). When these cells were released from thecalcified matrix by trypsinization, they proliferated in cellculture, confirming their viability (data not shown).
Figure 3. VSMC calcification is mediated by vesicle release. (A) VSMC in CaPi medium release vesicles (arrows, left) and calcification appears as a granular deposit on the VSMC and their surrounding matrix (middle). Trypan blue staining of calcified VSMC demonstrates that cells that are present within the calcified matrix remain viable and do no take up the dye (arrows in enlargement of boxed area in A). (B) Electron microscopy (EM) analysis of calcified VSMC showing calcification within (arrows) and on the surface of vesicles (arrowheads in i). Some calcified intracellular vesicles were observed (arrow in ii). Removal of Ca from the sections (iii) reveals "ghosts" of small (arrowheads) and larger underlying vesicles with some residual crystalline calcification (arrows), suggesting that vesicles act as the nidus for calcification. Energy dispersive X-ray analysis of vesicle content (inset) indicates basic calcium phosphate (note high Ca and P peaks), a basic Ca- and P-containing apatitic mineral.
Time-lapse experiments in the presence of serum revealed spontaneousvesicle release that was also further stimulated in the presenceof increased Ca and P. However, apoptosis was not induced, andthere was little subsequent calcification.
EM sections and EDX analysis of calcified VSMC showed the presenceof microspicules of crystalline mineral, predominantly composedof Ca and P, on the matrix, within vesicular structures andon the membrane surface of vesicles (Figure 3B). Most vesicleswere extracellular, but occasional intracellular calcified vesicleswere observed. It was not possible to determine whether thesehad formed in situ or had been phagocytosed. Removal of calciumfrom sections with EDTA revealed numerous vesicles underlyingall areas of matrix calcification, suggesting that vesicleswere the source of mineral nucleation (Figure 3B, iii). Calcifiedvesicles ranged in size from 70 nm to 1.0 µm, consistentwith their derivation from both apoptotic and viable cells (Figure 3B).
VSMC Vesicle Release is Increased by Ca and P
Differential centrifugation revealed two populations of vesicleswith mean diameters of 220 ± 24 and 127 ± 7 nm(Figure 4A), consistent with their being derived predominantlyfrom AB and MV, respectively. The time-lapse studies suggestedthat vesicle release was potentiated in the presence of increasedextracellular Ca and P. Therefore, vesicle release was quantifiedfrom equivalent numbers of VSMC treated with Ca and/or P for24 h. This demonstrated that vesicle release was stimulatedmore than twofold in the presence of extracellular Ca and Ca/Pions both in serum (not shown) and in SF conditions (Figure 4B).AB release was increased only in the absence of serum,consistent with the stimulation of apoptosis (Figure 4B).
Figure 4. Ca and P induce VSMC vesicle release. (A) EM analysis of isolated vesicle populations. Differential centrifugation isolated two vesicle populations. The smaller population was uniform in size (arrows) and most likely represents matrix vesicles (MV). The population of larger vesicles were of a size consistent with being derived from apoptotic bodies (AB; white arrows). However, there was some contamination with smaller vesicle structures (arrowhead), probably isolated by "sticking" to the larger vesicles during centrifugation. Graph shows mean population sizes ± SD (n = 30). Bar = 500 nm. (B) Slightly elevated extracellular Ca (2.7 mM Ca) induced increased MV release from VSMC but had no effect on AB release. CaPi media significantly increased MV release. AB release was also increased in CaPi medium but only in the absence of serum, consistent with the induction of apoptosis. Results are shown for SF conditions. Mean ± SD shown (n = 6 independent experiments for each treatment analyzed by t test).
Inhibition of Apoptosis Decreased Calcification
To test the role of apoptosis in VSMC calcification, we quantifiedapoptotic cells in test media in the absence of serum usingtime-lapse videomicroscopy and TUNEL. VSMC apoptosis was markedlyinduced, particularly when Ca and P were added together (Figure 5A).TUNEL staining confirmed that 4 h after the addition ofextracellular mineral ions, 20% of cells were apoptotic (Figure 5B).
Figure 5. Ca and P induce VSMC apoptosis. (A) Ca and P alone at high concentrations and together at lower concentrations significantly induced VSMC apoptosis in SF conditions as measured by time-lapse over 24 h. The lower level of apoptosis observed in 2.7 mM Ca/3.0 mM P was due to calcification occurring before the end of the 24-h time period; therefore, apoptosis could no longer be measured. Mean ± SD shown for n = 3 independent experiments for each treatment normalized to each control. **P < 0.05 (t test). (B) Apoptosis was further confirmed by transferase-mediated dUTP nick-end labeling (TUNEL) staining. Note the presence of TUNEL-labeled cells in standard CaPi media. (C) ZVAD.fmk significantly inhibited VSMC calcification in Cai and CaPi media in SF conditions at 24 h. Mean ± SD of three independent experiments for each treatment shown. **P < 0.05 (t test).
For measuring the contribution of apoptosis to calcification,VSMC in SF Cai or CaPi media were treated with ZVAD.fmk. ZVAD.fmkreduced incorporation of 45Ca into VSMC by 20 to 30% (Figure 5C).However, the pattern of calcification that occurred wasidentical to that observed in the absence of ZVAD.fmk and appearedas a granular alizarin red–positive extracellular deposit(data not shown).
Vesicle Calcification Is Increased by Ca and P
EM analysis of calcified VSMC suggested that vesicles were responsiblefor initiating calcification. We therefore measured the abilityof isolated VSMC-derived vesicles to accumulate calcium usingan in vitro assay. Vesicles that were released by VSMC in theabsence of serum after treatment with 2.7 mM Ca or 3.0 mM Pincorporated two- to threefold more 45Ca than vesicles thatwere released at physiologic concentrations of Ca and P. Vesiclesthat were released in CaPi medium (2.7 mM Ca/2 mM P) concentrated50- to 200-fold more calcium (Figure 6A). However, consistentwith our previous results, vesicles that were released in thepresence of serum and CaPi were suppressed in their abilityto calcify (Figure 6B). In experiments in which the same VSMCisolate was treated with CaPi medium in the presence or absenceof serum, SF-derived vesicles consistently exhibited a significantlygreater capacity to calcify than those isolated from serum-treatedVSMC (Figure 6C).
Figure 6. Ca and P increase vesicle calcification, and serum inhibits the calcification potential of MV. (A) In vitro calcification of both MV and AB isolated from SF media was significantly increased (2.5-fold) in response to mild Cai, whereas mild Pi increased only MV calcification by approximately the same amount. Strikingly, vesicles that were isolated from cells that were treated with CaPi media had a >70-fold increase in MV calcification and 15-fold increase in AB calcification. Results show the mean ± SD of n = 5 independent experiments. All controls were normalized to 1 to allow comparisons between treatments. **P < 0.05 t test. (B) The calcification potential of MV that were isolated from cells that were treated with CaPi media in the presence of serum (5% HS) for 24 h was not significantly increased compared with untreated VSMC in normal medium (this compares to the 70-fold induction observed in SF conditions after 24 h). Mean ± SD of n = 4 independent experiments. (C) The in vitro calcification potential of MV that were derived from VSMC that were treated with CaPi media in the presence of serum is significantly reduced compared with MV that were isolated from SF CaPi media. Mean ± SD of n = 3 independent experiments shown.
To determine why vesicles that were released in CaPi mediumcalcified so readily, we analyzed them by EDX. This revealedthat MV that were released from VSMC in physiologic concentrationsof Ca and P, both in the presence and the absence of serum,contained no BCP (Table 1). In contrast, MV that were releasedin SF CaPi medium contained BCP, whereas those that were releasedin the presence of serum did not. AB did not contain BCP, suggestingthat there are differences in the properties of the two vesiclepopulations. However, when placed in the in vitro calcifyingbuffer for 24 h, both AB and MV that were released in SF CaPimedium were able to nucleate BCP (Table 1).
Table 1. EDX analysis of mineral content of VSMC-derived vesicle populationsa
MV Contain Serum- and Cell-Derived Inhibitors of Mineralization
To determine whether the reduced calcification potential ofvesicles isolated from VSMC in medium that contained serum wasdue to the presence of serum-derived calcification inhibitors,we performed Western blots for fetuin-A in isolated vesiclepopulations. Fetuin-A was present a low levels in vesicles thatwere derived from VSMC in SF conditions but was highly concentratedin vesicles that were isolated in the presence of serum (Figure 7A).Thus, the association of serum-derived proteins with MVmay inhibit their ability to nucleate BCP.
Figure 7. MV contain the calcification inhibitors fetuin-A and matrix Gla protein (MGP). (A) Western blot showing the presence of fetuin-A in VSMC-derived MV. Small amounts are seen in MV derived from SF VSMC, but the concentration is increased in MV derived from VSMC cultured in 5% HS. (B) Western blot analysis demonstrating that MGP protein is present in VSMC-derived MV. Recombinant MGP (recMGP) used as a control is fused to dihydrofolate reductase and therefore has a molecular weight of 30 kD. MGP from vesicles is detected as a 15-kD band, slightly larger than that predicted by the amino acid sequence, i.e., 12 kD, probably as a result of posttranslational modifications.
Our previous data showed that VSMC inhibit spontaneous BCP precipitation,and Western blot showed that the endogenous mineralization inhibitorMGP was also concentrated in VSMC-derived MV (Figure 7B). Wehypothesized that inhibition of MGP -carboxylation by warfarinwould potentiate VSMC calcification in response to increasedCa and P. Warfarin had no effect on MV release by VSMC (Figure 8B).However, it significantly increased calcification of VSMCin response to increased Ca and P at all concentrations tested(Figure 8A). MV that were released after warfarin treatmentexhibited a threefold increase in their basal ability to calcifyafter incubation in vitro in calcifying buffer (Figure 8C).
Figure 8. Warfarin accelerates VSMC calcification and increases the calcification potential of MV. (A) Pretreatment of VSMC with warfarin significantly increased 45Ca incorporation into VSMC cultures in response to extracellular mineral ions. Mean ± SD of an experiment performed in quadruplicate shown. *P < 0.05 and **P < 0.001 (ANOVA). (B) Treatment with warfarin did not increase MV release from VSMC. (C). MV released from warfarin-treated VSMC calcified in vitro three times more potently than control MV from untreated cells. Mean ± SD of n = 3 independent experiments shown (t test).
We have demonstrated that concentrations of extracellular Caand P commonly found in serum of patients on dialysis induceVSMC calcification and that this effect is potentiated in theabsence of serum. Our findings suggest that in the presenceof increased P, even modest increases in Ca can substantiallyexacerbate calcification, which is induced by nucleation ofBCP in vesicles that are released from both viable and apoptoticVSMC. Our results suggest that under normal physiologic conditions,MV do not calcify as a result of the presence of mineralizationinhibitors derived from both cells and serum. However, in thepresence of raised extracellular concentrations of Ca and P,if serum proteins are limiting or the action of endogenous inhibitorsis compromised, then vascular damage is exacerbated, vesiclerelease is potentiated, and MV can nucleate BCP. Taken together,these studies suggest that "metastatic" calcification observedin patients with ESRD results from a reduced capacity of VSMCto inhibit mineralization via cell-mediated mechanisms.
Vesicle-Mediated VSMC Calcification
We have previously shown that apoptosis and AB release can initiateVSMC calcification in vitro, and abundant AB have been demonstratedin calcified atherosclerotic plaques (10,25). Indeed, the concentrationof Ca in atherosclerotic plaques may exceed 30 mM; therefore,the apoptosis observed in high Cai medium may model processesin atherosclerotic calcification (26). However, our presentdata clearly show that cell death is not the only mechanismresponsible for VSMC calcification, because we were able todistinguish AB from MV and demonstrate that both are capableof initiating calcification, albeit by possibly different mechanisms.In our experiments, VSMC apoptosis was rare in the presenceof serum, whereas MV were released by viable VSMC, particularlyin the presence of elevated levels of extracellular Ca and P.Vesicle release by VSMC has been described in vivo in a numberof conditions, including atherosclerosis, hypertension, andCa overload induced by vitamin D3 toxicity (27–29). Ithas been suggested that Ca-loaded vesicles are released fromVSMC to protect against the cytotoxic effects of intracellularCa overload (29). Our data showing that VSMC increased vesiclerelease in response to extracellular Ca and remained viablewithin the calcified matrix supports this notion but suggeststhat if phagocytosis were limited, then the extracellular accumulationof these vesicles, particularly in the context of elevated Caand P, would lead to calcification.
In chondrocytes, MV become "mineralization competent" duringhypertrophic differentiation, when they are exposed to retinoicacid. A transient rise in intracellular Ca triggers the relocationof annexins II, V, and VI from the cytoplasm to the plasma membrane,where they act as Ca channels (11,22). Chondrocyte MV also containsodium-dependent P channels and concentrate alkaline phosphatase,thereby providing an internal environment suitable for the nucleationof BCP (30). Our data suggest that in the presence of elevatedCa and P, VSMC can also release mineralization-competent MV;however, the mechanisms regulating this process in VSMC remainto be determined. There is evidence to suggest that VSMC-derivedMV have similarities with those derived from chondrocytes. Aprevious study showed that in the presence of elevated P, VSMCcalcification could be inhibited if Pit-1, a sodium-dependentphosphate transporter, were blocked, suggesting a vesicle-mediatedprocess (14).
We demonstrated that VSMC could inhibit spontaneous precipitationof BCP. There are a number of mechanisms that may underlie thisobservation. Many of the bone-associated proteins expressedby osteo/chondrocytic VSMC are regulators or inhibitors, ratherthan nucleators of mineralization. In addition, VSMC possesa G protein–coupled cation–sensing mechanism thatcan induce expression of MGP in response to increased extracellularCa (31). Results of this study suggest that MGP is producedand packaged, along with excess intracellular Ca, into vesiclesthat are released back into the extracellular environment. Theincrease in calcification seen after inhibiting the functionof MGP with warfarin treatment is consistent with this mechanism,with the presence of undercarboxylated MGP in MV increasingtheir calcification potential (32). Furthermore, calcificationprogressed much faster once BCP had been nucleated, and MV producedin CaPi conditions already contained BCP. Thus, acceleratedvascular matrix calcification will result if endogenous inhibitorsof calcification are either impaired or absent from MV.
Serum Contains Inhibitors of VSMC Calcification
Previous in vitro studies showing accelerated calcificationof bovine VSMC in uremic HS were interpreted as demonstratingthe presence of potentiators of calcification (33). However,an alternative interpretation is that uremic serum lacks calcificationinhibitors. We found that both VSMC calcification and isolatedvesicle calcification were inhibited by normal HS and that MVcontain the potent serum-derived calcification inhibitor 2-Hereman’sSchmid glycoprotein/fetuin-A. Fetuin-A is produced in the liver,binds Ca and P in extracellular fluids, and can occur withincells, and knockout mice develop extensive soft tissue calcification(15,34). Circulating levels of fetuin-A are substantially reducedin patients with ESRD and correlate inversely with cardiovascularmortality (3,15). Our study shows that fetuin-A was concentratedin MV derived from VSMC in normal serum. Thus, calcificationin ESRD may be due to the combined effect of an elevated Cax P in the context of reduced levels of serum inhibitory proteins,leading to increased apoptosis and vesicle calcification. Investigationof the effects of individual serum proteins on VSMC calcification/phenotypeare now required.
Clinical Implications
Our in vitro model suggests that, in the context of raised P,VSMC calcification is extremely sensitive to elevations in serumCa and that even transient increases in Ca would lead to releaseof vesicles that contain high concentrations of both Ca andP from stressed or dying VSMC. In the context of reduced levelsof circulating inhibitors such as fetuin-A, this would leadto matrix mineralization. Calcification would be further exacerbatedif the activity of local inhibitors, such as MGP, were reduced.Our data therefore provide a cell biologic explanation for theobserved effects of warfarin on the progression of calciphylaxisin some patients with ESRD (3,32,35). They may also explainsome of the observed effects of Ca channel blockers and drugsthat reduce serum Ca levels on calcification (36). Blockadeof L-type Ca channels prevents VSMC Ca overload, vesicle release,and calcification in animal models of hypertension and vitaminD3 toxicity (29). It also inhibits the progression of vascularcalcification in hypertensives and improves outcome in ESRD(37–39). Perhaps the most important observation is thepivotal role played by Ca in the context of raised P. However,it will be important to determine how raised Ca and P in thismodel system equates to a serum electrolyte imbalance in uremia.Thus, further experiments incorporating uremic serum into themodel are warranted but beyond the scope of the present study(33). Despite these limitations, our findings do suggest thatgreater attention should be paid to preventing elevation inserum Ca than has traditionally been the case and that strategiesaimed at enhancing endogenous and circulating inhibitors ofcalcification may have therapeutic benefits.
Acknowledgments
This work was supported by a British Heart Foundation programgrant to C.M.S. and P.L.W. (RG/200004). P.L.W. is a BHF Professorof Cardiovascular Medicine; C.M.S. is a BHF Basic Sciences Lecturer.
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Received for publication March 16, 2004.
Accepted for publication June 28, 2004.
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Top
Abstract
Introduction
0.3 to 1.0 µm in diameter, released from apoptotic VSMC (10). MV are smaller (30 to 300 nm) and are released from the plasma membrane of viable cells (11). VSMC-derived MV have been associated with calcification in vivo, but their composition and function are poorly understood (12). 
-carboxylation by warfarin would potentiate VSMC calcification in response to increased Ca and P. Warfarin had no effect on MV release by VSMC (Figure 8B). However, it significantly increased calcification of VSMC in response to increased Ca and P at all concentrations tested (Figure 8A). MV that were released after warfarin treatment exhibited a threefold increase in their basal ability to calcify after incubation in vitro in calcifying buffer (Figure 8C). 
2-Hereman’s Schmid glycoprotein/fetuin-A. Fetuin-A is produced in the liver, binds Ca and P in extracellular fluids, and can occur within cells, and knockout mice develop extensive soft tissue calcification (15,34). Circulating levels of fetuin-A are substantially reduced in patients with ESRD and correlate inversely with cardiovascular mortality (3,15). Our study shows that fetuin-A was concentrated in MV derived from VSMC in normal serum. Thus, calcification in ESRD may be due to the combined effect of an elevated Ca x P in the context of reduced levels of serum inhibitory proteins, leading to increased apoptosis and vesicle calcification. Investigation of the effects of individual serum proteins on VSMC calcification/phenotype are now required. 
), particularly at higher concentrations of Ca and P, resulting in 45Ca accumulation. Mean ± SD of a representative experiment (of n = 4 experiments using different VSMC isolates) performed in triplicate is shown. **P < 0.05 (ANOVA).
