Multifunctional Roles for Serum Protein Fetuin-A in Inhibition of Human Vascular Smooth Muscle Cell Calcification
Joanne L. Reynolds*,
Jeremy N. Skepper,
Rosamund McNair*,
Takeshi Kasama,
Kunal Gupta*,
Peter L. Weissberg*,
Willi Jahnen-Dechent and
Catherine M. Shanahan*
* Division of Cardiovascular Medicine, Addenbrooke’s Hospital, Cambridge, United Kingdom; Multi-Imaging Centre, Department of Anatomy, Cambridge, United Kingdom; Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, United Kingdom, and The Institute of Physical and Chemical Research, Hatoyama, Saitama, Japan; and IZKF Biomat Aachen University Clinics, Aachen, Germany
Address correspondence to: Dr. Catherine M. Shanahan, Division of Cardiovascular Medicine, Level 6, ACCI, Box 110, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK. Phone: 44-1223-331504; Fax: 44-1223-331505; cs131{at}mole.bio.cam.ac.uk
Received for publication October 29, 2004.
Accepted for publication July 2, 2005.
Vascular calcification predicts an increased risk for cardiovascularevents/mortality in atherosclerosis, diabetes, and ESRD. Serumconcentrations of 2-Heremens-Schmid glycoprotein, commonly referredto as fetuin-A, are reduced in ESRD, a condition associatedwith an elevated circulating calcium x phosphate product. Micethat lack fetuin-A exhibit extensive soft tissue calcification,which is accelerated on a mineral-rich diet, suggesting thatfetuin-A acts to inhibit calcification systemically. Westernblot and immunohistochemistry demonstrated that serum-derivedfetuin-A co-localized with calcified human vascular smooth musclecells (VSMC) in vitro and in calcified arteries in vivo. Fetuin-Ainhibited in vitro VSMC calcification, induced by elevated concentrationsof extracellular mineral ions, in a concentration-dependentmanner. This was achieved in part through inhibition of apoptosisand caspase cleavage. Confocal microscopy and electron microscopy–immunogolddemonstrated that fetuin-A was internalized by VSMC and concentratedin intracellular vesicles. Subsequently, fetuin-A was secretedvia vesicle release from apoptotic and viable VSMC. Vesicleshave previously been identified as the nidus for mineral nucleation.The presence of fetuin-A in vesicles abrogated their abilityto nucleate basic calcium phosphate. In addition, fetuin-A enhancedphagocytosis of vesicles by VSMC. These observations provideevidence that the uptake of the serum protein fetuin-A by VSMCis a key event in the inhibition of vesicle-mediated VSMC calcification.Strategies aimed at maintaining normal circulating levels offetuin-A may prove beneficial in patients with ESRD.
Elevated concentrations of circulating calcium (Ca) and/or phosphate(P) ions, such as occurs in ESRD or in rare genetic causes ofhypercalcemia, may result in catastrophic calcification of thevasculature and other soft tissues (1–5). Until recently,vascular calcification in the context of a mineral imbalancewas considered to be an unregulated consequence of the depositionof insoluble basic calcium-phosphate (BCP; a mixture of octacalciumphosphate, dicalcium phosphate dihydrate, and apatite) mineralin the extracellular matrix occurring when concentrations ofCa and/or P ions in the local environment/circulation exceededthe solubility product for calcium phosphate.
Recent evidence suggests that pathologic vascular calcificationshares many similarities with physiologic bone mineralization(6). Cultured human vascular smooth muscle cells (VSMC) spontaneouslyexpress the osteoblast transcription factor Cbfa1 and in postconfluentculture form "osteoblast-like" nodules that calcify after approximately28 d (7,8). VSMC in vitro and in vivo also express a numberof bone-associated, mineralization-regulating proteins suchas alkaline phosphatase, bone sialoprotein, matrix Gla protein(MGP), osteopontin, and osteocalcin, which can regulate thecalcification process (9). In response to raised concentrationsof extracellular Ca and/or P ions, VSMC calcification is accelerated(10). Under these conditions, VSMC shed numerous membrane-boundvesicles. These vesicles are a mixture of apoptotic bodies (AB)released from dying VSMC and matrix vesicles (MV) released fromviable cells. Both have the capacity to nucleate BCP, and theiraccumulation in the VSMC matrix results in rapid and widespreadcalcification (10,11). Thus, in the context of a raised extracellularCa x P product, the evidence suggests that VSMC calcificationis a cell-mediated, regulated process and therefore may be modifiablein ESRD, in which it is associated with a poor prognosis (1,12).
A number of naturally occurring, endogenous inhibitors of vascularcalcification have been identified, including MGP and pyrophosphate,both produced by medial VSMC (13,14). However, emerging evidencesuggests that there may also be circulating inhibitors of calcification.Using an in vitro model of VSMC calcification induced by elevatedlevels of extracellular Ca and P ions, we observed that humanserum prevented VSMC calcification by inhibiting apoptosis andby reducing the calcification potential of shed membrane vesicles(10). Fetuin-A was identified as a serum component that co-purifiedwith membrane vesicles and therefore potentially could be associatedwith their reduced calcification potential (10). Importantly,there is clinical evidence to suggest that fetuin-A may be protectivein patients with ESRD. Its circulating levels are significantlyreduced in patients with ESRD and calciphylaxis, a rapidly progressive,often fatal form of vascular calcification, with fetuin-A–deficientserum having impaired ex vivo capacity to inhibit calcium phosphateprecipitation (15,16). Fetuin-A deficiency also correlates withincreased cardiovascular mortality as well as atherosclerosisand coronary and valvular calcification in patients with ESRD(17–19).
Fetuin-A is a circulating plasma glycoprotein, produced abundantlyduring fetal development by multiple tissues, whereas in theadult, it is produced predominantly by the liver (20). It isa member of the cystatin superfamily of cysteine protease inhibitors.Ablation of the mouse fetuin-A gene in a strain of calcification-pronemice results in progressive, fatal calcification of soft tissues,including kidney, testis, skin, heart, and vasculature (16,21).These mice exhibit compromised serum inhibition of BCP formation,suggesting that fetuin-A may be important in preventing calcificationin the context of elevated concentrations of mineral ions. Invitro experiments have indicated that fetuin-A can inhibit mineralizationof primary rat calvaria cells by preventing BCP precipitationand modulating apatite formation during mineralization (16,22).
In this study, we show that fetuin-A can regulate several ofthe key cellular events that lead to VSMC calcification, includingapoptosis, vesicle calcification, and phagocytosis, providingnovel mechanistic insights into how a relative lack of fetuin-Amay contribute to vascular calcification. These studies pointto an important role for fetuin-A in inhibiting calcificationin ESRD, particularly at sites of tissue damage.
Cell Culture
VSMC that were derived from medial explants of human aortictissue (n = 15) were cultured in M199 with 20% FCS and usedbetween passages 3 and 10 (23). The apoptosis-sensitive cellline HASMC 66 SV40, Saos2 cells, and 293 kidney epithelial cellswere cultured as above in 10% FCS (24). For nodular cultures,VSMC were maintained in postconfluent conditions for 30 d untilnodules calcified. Calcification was visualized by alizarinred staining as described previously (23).
VSMC Calcification Assays
Calcification of VSMC in response to extracellular mineral ionswas performed as described previously using serum-free (SF)media designated control (1.8 mM Ca/1.0 mM P) and test; Cai(5.4 mM Ca), Pi (2.0 mM P), or CaPi (2.7 mM Ca/2.0 to 3.0 mMP), each containing 0.5% BSA and 45Ca (approximately 50,000cpm/ml) (10). Cells were transferred to SF control media for24 h before the addition of SF test media. Calcification couldbe monitored in live cultures by visualization of vesicle/mineraldeposition using phase contrast microscopy. After 24 h to 10d of treatment, the medium was removed and calcification wasvisualized by alizarin red staining and quantified by measuring45Ca incorporation. Briefly, VSMC were decalcified in 0.1 MHCL, neutralized with 0.1 M NaOH/0.1% SDS, and scraped, and45Ca incorporation was measured by liquid scintillation counting.In cell-free experiments, BCP precipitates were harvested bycentrifugation, and 45Ca incorporation in the pellet was measuredas above (10).
Experiments were performed using bovine fetuin-A (Sigma, St.Louis, MO) and verified with human AHSG/fetuin-A (Calbiochem,San Diego, CA). All experiments were performed in triplicateand independently on at least five different VSMC isolates.
Reverse Transcription–PCR Detection of Fetuin-A
Human fetuin-A mRNA expression was investigated in a panel ofcDNA samples (n = 40) that were composed of normal and atheroscleroticaortic samples (as described previously) and in cultured humanVSMC (9). Liver cDNA was used as a positive control. Fetuin-Aprimers were as follows: Forward, CCTGCTCCTTTGTCTTGC; reverse,CGGACTGGAGGAACCAC. PCR reactions were performed within the linearrange as standard for 30 cycles. -Microglobulin was used asa control for cDNA equality (9).
Immunohistochemical Detection of Fetuin-A
Human aortic (n = 3) and carotid endarterectomy specimens (n= 5) were formalin fixed and embedded in paraffin, and 6-µmsections were cut. Human fetuin-A was detected with a rabbitpolyclonal antibody (Behring, Marburg, Germany), and co-stainingfor -smooth muscle actin was performed using a mAb (Dako, Denmark;1:200) and counterstained blue using the Vector alkaline phosphatasesubstrate Kit III (SK-5300). Von Kossa staining was performedas standard. Negative controls included substitution of thetest antibody with PBS or with an irrelevant antibody.
For confocal microscopy, VSMC were cultured on 19-mm glass coverslipsand fixed in 3% formaldehyde/PBS. Coverslips were washed andblocked in 3% ovalbumin, and fetuin-A was detected using a rabbitpolyclonal antibody against human or bovine fetuin-A with AlexaFluor 488 anti-rabbit IgG secondary antibody before mountingin DAPI-containing medium (21).
VSMC were prepared for electron microscopy (EM) as describedpreviously (10). Immunogold was performed using a bovine fetuin-Aantibody (diluted 1:100) and a 10-nm gold-conjugated anti-rabbitIgG secondary antibody. PBS was substituted for primary antibodyas a control.
Protein Gels and Western Analysis
Nodular human VSMC that were maintained in growth medium for30 d were trypsinized, and monolayer and nodular cells wereseparated using a 70-µm cell sieve. Protein lysates wereprepared in RIPA buffer or SDS-PAGE denaturing sample buffer,and protein quantification was performed using the BioRad assay(Hercules, CA). Five micrograms of protein was electrophoresedthrough 10% acrylamide gels, and selected bands were subjectedto nine-residue N-terminal sequencing and identified using BLAST(http://www.ncbi.nlm.nih.gov/BLAST/).
Western blots were performed as standard using Immobilon-P membranewith horseradish peroxidase activity visualized using ECL (Amersham,UK). Antibodies used for detection were caspase 3, p17 subunit(Pharminogen, San Diego, CA); caspase 8, p18 subunit (UpstateBiotechnology, Lake Placid, NY); and caspase 9, p20 subunit(Pharminogen).
Time-Lapse Videomicroscopy and Immunofluorescent TUNEL Staining of Cells
Apoptosis was induced in the human coronary plaque cell lineHASMC 66 by serum starvation and in primary VSMC cultures byaddition of CaPi media. Video time-lapse microscopy was performedover 48 h, and apoptosis was recorded (24).
Fluorescence transferase-mediated dUTP nick-end labeling (TUNEL)assays were performed at time points between 6 and 24 h as describedpreviously using VSMC that were treated with test media ±5 µM fetuin (10,11). Coverslips were mounted in DAPI-containingmedium, 10 random images were captured digitally (Olympus, Tokyo,Japan) from each coverslip, and at least 100 nuclei countedper frame.
Apoptotic Body and Matrix Vesicle Isolation
AB and MV populations were isolated from the media of VSMC culturesby differential centrifugation, and the calcification potentialof isolated vesicle populations was measured as described previously(10). The calcifying reaction mixture described by Kirsch etal. (25) was used; it contains 45Ca (50,000 cpm) and 4 to 15µg of VSMC-derived MV or AB. Samples were incubated at37°C for 24 h, and assays were performed in triplicate (10,11).
Energy-Dispersive X-Ray Analysis and Electron Diffraction
Vesicle fractions that were isolated by differential centrifugation(MV and AB) were resuspended at a concentration of 10 µg/µland adsorbed onto glow-discharged, carbon-coated Formvar filmgrids for energy-dispersive X-ray analysis as described previously(10). For electron diffraction, thin sections (150 nm thick)of calcified VSMC cultures were examined by transmission electronmicroscopy (TEM) at 300 kv in a Philips CM30 in bright fieldmade at a magnification of x21,000. Calcified spicules wereidentified both within and without vesicles, and selected areadiffraction patterns were collected using a camera length of900 mm and compared with those from a calibration standard ofhydroxyapatite.
Apoptotic Body Binding Assay
Phagocytosis of AB by VSMC was assessed as described previously(11,26). AB from serum-starved HASMC66 cells were mixed withHoechst dye and either 5 to 10 µM BSA (protein control)or 5 to 10 µM bovine fetuin-A for 15 min before seedingonto VSMC at 1 x 106 AB/well. After 2 h, cells were washed vigorouslyand fixed in 4% formaldehyde, and random images were capturedusing an Olympus TV-1X digital camera with the number of boundAB counted for >30 VSMC in random areas of eight separatewells.
Statistical Analyses
Data were analyzed using t test or for multiple comparisonsANOVA with post hoc Scheffe test.
Fetuin-A Associates with Calcified VSMC In Vitro and In Vivo
Inhibitors of VSMC calcification are often deposited at sitesof mineralization (27). To identify proteins involved in VSMCcalcification, we used SDS-PAGE to compare protein profilesof cell lysates that were derived from noncalcified monolayerand calcified, nodular, human VSMC. A major band at approximately54 kD, in calcified VSMC, was identified by microsequencingas bovine fetuin-A, a protein derived from FCS (Figure 1A).Fetuin-A mRNA could not be detected by reverse transcription–PCRin cultured human VSMC, indicating that fetuin-A was not synthesizedby VSMC (Figure 1B).
Figure 1. Fetuin-A associates with calcified vascular smooth muscle cells (VSMC) in vitro. (A) Coomassie-stained gel comparing protein lysates from noncalcified monolayer VSMC with calcified nodular VMSC. The strong band (arrow) in calcified VSMC was identified as bovine fetuin-A by microsequencing. (B) Reverse transcription–PCR showing that VSMC do not express fetuin-A mRNA, confirming the derivation of fetuin-A protein from the serum.
Immunohistochemistry was performed on normal and calcified humanarterial specimens. Fetuin-A was barely detectable in the mediaor intima of uncalcified normal arteries (Figure 2A). However,fetuin-A staining was present in calcified medial and intimalareas of arteries (Figure 2, B and C). VSMC within calcifiedregions were strongly positive for fetuin-A, and much of thestaining appeared intracellular (Figure 2a), whereas in thecalcified acellular matrix, fetuin-A associated with microcalcifications(Figure 2b). Fetuin-A mRNA could not be detected by reversetranscription–PCR in cDNA samples that were derived fromnormal and calcified human arteries (n = 40), indicating thatdeposited fetuin-A was not synthesized locally but derived fromthe serum (Supplemental Figure 1, available online).
Figure 2. Fetuin-A associates with calcified VSMC in vivo. Immunohistochemistry for fetuin-A in normal and calcified human arteries. Fetuin-A (brown stain) was absent or present very weakly in a diffuse matrix pattern in the normal vessel wall. (A) Normal aorta and boxed region is enlarged. In calcified medial and intimal regions, fetuin-A was deposited in association with VSMC and the matrix. (B) Boxed regions (a and b) are enlarged to show intracellular distribution of fetuin-A in medial VSMC (a) and heavy fetuin-A deposition in association with microcalcifications (b). VSMC were identified by -smooth muscle actin immunostaining and are blue in A, B, a, and b. The distribution of intimal and medial calcification was identified by von Kossa stain (black in C).
Fetuin-A Inhibits VSMC Calcification Induced by Extracellular Mineral Ions In Vitro
Fetuin-A inhibited VSMC calcification, induced by CaPi media,in a concentration-dependent manner, with potent inhibitionoccurring at physiologic concentrations (10 µM; Figure 3).Similar inhibition of VSMC calcification by fetuin-A wasalso observed in Cai and Pi media (Supplemental Figure 2, availableonline). Fetuin-A was also able to inhibit calcification ofboth Saos2 osteoblasts and 293 kidney epithelial cells in responseto Cai and CaPi media (Supplemental Figure 3, available online).
Figure 3. Fetuin-A inhibits mineralization of VSMC in a concentration-dependent manner. (A) VSMC were treated with CaPi medium (2.7 mM Ca/2.0 mM P) in the presence or absence of fetuin-A, and alizarin red staining was used to visualize calcification after approximately 24 h. Calcification was inhibited in the presence of serum (positive control) and fetuin-A in a concentration-dependent manner. Similar results were obtained using Cai medium (data not shown). (B) Incorporation of 45Ca into VSMC that were treated with CaPi medium (2.7 mM Ca/2.0 mM P) was inhibited in a concentration-dependent manner by fetuin-A. Mean ± SD, n = 3, **P < 0.05.
Cell-Mediated Inhibition of VSMC Calcification by Fetuin-A
Fetuin-A is a binder of BCP and an inhibitor of spontaneousprecipitation of Ca and P in solution. To determine whetherthe ability of fetuin-A to inhibit VSMC calcification was dueto its capacity to inhibit BCP precipitation alone, we addedit to CaPi medium in cell-free conditions. This showed thatfetuin-A, as expected, could inhibit the spontaneous precipitationof BCP in solution. However, in the presence of VSMC, the inhibitorycapacity of fetuin-A was significantly increased, suggestingthat it also acted via cell-mediated mechanisms (Figure 4A).
Figure 4. Fetuin-A inhibits mineralization via a cell-mediated process. (A) Fetuin-A (5 µM) inhibited spontaneous basic calcium-phosphate (BCP) precipitation in CaPi medium (2.7 mM Ca/3.0 mM P) by approximately 50 to 60% in the absence of cells. In the presence of VSMC, the inhibitory effect of fetuin-A on calcification was increased to >90%. Mean ± SD, n = 3. (B) Fetuin-A inhibited VSMC calcification when added at the same time as CaPi test medium (2.7 mM Ca/2.0 mM P; 0 h). However, it had no inhibitory capacity when added 16 h after CaPi test medium and after calcification had been initiated (observed by phase contrast microscopy). Mean ± SD, n = 3, **P < 0.05.
The capacity for fetuin-A to act via cell-mediated mechanismswas suggested further when it was added to VSMC 16 h after theaddition of CaPi medium (i.e., after the onset of calcificationobserved by phase contrast microscopy). When added at this stage,fetuin-A had no effect on 45Ca incorporation, suggesting thatit could not inhibit precipitation/crystal growth once mineralnucleation had occurred, confirming previous in vitro studies(Figure 4B) (22).
Fetuin-A Inhibits VSMC Apoptosis
We showed previously, using the caspase inhibitor ZVAD.fmk,that apoptosis contributes to VSMC calcification induced byCaPi in SF conditions. However, CaPi did not induce apoptosisin the presence of serum, resulting in reduced calcification(10). Therefore, we tested whether fetuin-A was the componentin serum acting to inhibit VSMC apoptosis. ImmunofluorescentTUNEL labeling showed that apoptosis induced in response toCaPi medium was significantly reduced by 5 µM fetuin-A(Figure 5, A and B). To determine whether the antiapoptoticeffects of fetuin-A were context and cell specific, we testedits effect on HASMC66 apoptosis induced by serum starvation.Fetuin-A reduced apoptotic events by approximately half overa 48-h time course, and this was associated with reduced cleavageof caspases 3, 8, and 9 (Figure 5, C and D).
Figure 5. Fetuin-A inhibits VSMC apoptosis. (A) Immunofluorescent transferase-mediated dUTP nick-end labeling (TUNEL) of VSMC in CaPi medium (2.7 mM Ca/2.0 mM P). Nuclei were stained with DAPI (blue) to confirm TUNEL-positive cells as apoptotic by nuclear morphology. In the presence of fetuin-A, VSMC apoptosis was significantly inhibited. These results are shown graphically in B. Mean ± SD, n = 10. (C) Time-lapse video microscopy over 48 h was used to measure apoptosis in serum-starved HASMC66 SV40 cells in the presence of 2 µM BSA or fetuin-A. The cumulative percentage of apoptotic events is shown in 12-h increments. Fetuin-A significantly inhibited apoptosis at all time points. Mean ± SEM, n = 3, *P < 0.05. (D) Fetuin-A inhibited cleavage of caspases 3, 8, and 9 in serum-starved HASMC66 SV40 as shown by Western blot. In the presence of FCS, caspase cleavage is minimal but is induced in response to serum starvation and correlates with apoptosis. In the presence of 2 µM fetuin-A, caspase cleavage is minimal and similar to that observed in FCS, consistent with the inhibition of apoptosis.
Fetuin-A Localizes to Apoptotic Cells and Vesicles
Immunofluorescence showed that fetuin-A was not present in VSMCthat were maintained in SF control medium for >48 h (Figure 6A).VSMC that were cultured in the presence of serum or treatedwith fetuin-A showed fetuin-A distributed throughout the cytoplasmin a punctate pattern, suggestive of its localization in vesicle-likestructures (Figure 6B). In addition, confocal microscopy demonstratedintense staining throughout the cytoplasm of apoptotic cellsand within AB and other smaller MV released from the cells (Figure 6C).
Figure 6. Fetuin-A is intracellular in VSMC and localizes to vesicles. (A) VSMC that were cultured in serum-free (SF) media for 48 h contained little fetuin-A immunoreactivity. (B) VSMC that were cultured in the presence of serum or treated with 2.0 µM fetuin-A contained fetuin-A localized in discrete cytoplasmic vesicular structures (green stain). (C) Confocal microscopy confirmed the vesicular localization of fetuin-A and also showed that in apoptotic cells, fetuin-A was localized throughout the cytoplasm (arrow in C) and was concentrated in extracellular vesicles (arrowheads). Nuclei are stained with Hoechst. (Inset) Fetuin-A–loaded vesicles. (D) Western blots for fetuin-A in isolated apoptotic bodies (AB) and matrix vesicles (MV) confirmed its presence in these extracellular vesicles. Vesicles that were isolated from VSMC that were cultured in the presence of media that contained additional extracellular calcium (CaPi medium shown) contained approximately twice as much fetuin-A than vesicles from VSMC in control media. By densitometry, 1 versus 2.14 arbitrary units, control compared with test CaPi media (normalized to loading control -smooth muscle actin; data not shown). Multiple bands probably represent posttranslationally modified fetuin-A (e.g., sialylated).
Western blotting confirmed that VSMC AB contained fetuin-A (Figure 6D).In addition, smaller MV, isolated by ultracentrifugation,contained fetuin-A. The amount of fetuin-A concentrated withinthese MV was increased when VSMC were cultured in Cai (not shown)or CaPi media (Figure 6D).
EM immunogold labeling of cultured VSMC that were grown in SFCai or CaPi medium demonstrated little fetuin-A in VSMC, extracellularvesicles, or the calcified matrix (Figure 7, A and B). Of note,vesicles that were released by cells in the absence of fetuin-Awere associated with both intravesicular and extravesicularcrystalline BCP. The presence of crystalline hydroxyapatitewas confirmed by selected area electron diffraction. Polycrystallinediffraction patterns with concentric rings similar to thoseof hydroxyapatite standards were collected from electron-densecrystals within and without vesicles (Figure 7B, inset). Incontrast, in the presence of 5 µM fetuin-A, immunogoldlabeling showed fetuin-A at the cell membrane, within the VSMCcytoplasm, and, most striking, within vesicles and/or AB (Figure 7,C through E). Calcification was minimal in treated cells,and vesicles were generally not associated with crystallineBCP. However, fetuin-A was deposited in rare regions of calcifiedmatrix in association with crystalline calcification (Figure 7F).
Figure 7. Electron microscopy immunogold localization of fetuin-A in VSMC. (A and B) VSMC that were cultured in CaPi media in the absence of fetuin-A calcified via a vesicle-mediated process. Minimal fetuin-A immunogold labeling was detected in association with vesicles. Crystalline BCP deposition could be observed both within vesicles (arrow in A) and on the surface of vesicles (arrow in A and B). The heterogeneity in the size of vesicles was consistent with their derivation from both AB and MV. Bar = 500 nm for A through C. (B, inset) Diffraction pattern from the hydroxyapatite standard (HA) consisting of a series of concentric rings. The adjacent diffraction pattern (MV) from calcified spicules in a typical calcified vesicle is not as strong as the standard but consistent with polycrystalline hydroxyapatite. (C through F) VSMC treated with fetuin-A in the presence of CaPi medium calcified minimally. Note absence of crystalline material in C. Dense immunogold decoration was observed within vesicles (inset in C and E) that were seen to bud from VSMC (C). Gold decoration was also observed within vesicular structures present within the cytoplasm of VSMC (D) and associated with areas of calcification of the extracellular matrix (ECM; arrow in F). These areas of calcification were associated with vesicle "ghosts" (arrowheads in F).
Fetuin-A Inhibits Calcification of Isolated Apoptotic Bodies and Matrix Vesicles
The above studies suggested that the presence of fetuin-A invesicles inhibited their capacity to nucleate BCP. Therefore,the calcification potential of isolated AB and MV that werederived from VSMC that were treated with CaPi medium in thepresence or absence of fetuin-A was tested in vitro. MV thatwere released from VSMC that were cultured in the presence offetuin-A did not calcify. In contrast, MV that were releasedfrom VSMC in the absence of fetuin-A calcified extensively (Figure 8A).Fetuin-A also inhibited to a lesser extent the calcificationof AB.
Figure 8. Fetuin-A inhibits calcification of MV and AB. (A) Isolated MV and AB were incubated in calcifying buffer for 24 h. VSMC that were cultured in CaPi medium released MV that contained preformed BCP that calcified extensively, as well as AB that also calcified. Calcification was significantly inhibited in both MV and AB when 5.0 µM fetuin-A was added to the CaPi medium. (B) Inhibition of MV and AB calcification was also observed when VSMC were pretreated with fetuin-A for 24 h and then washed before immediate addition of CaPi medium for an additional 24 h. Mean ± SD, n = 3, **P < 0.0001.
In the next experiment, VSMC were pretreated with fetuin-A for24 h and washed extensively before the immediate addition ofCaPi medium. MV that were derived from pretreated VSMC containedfetuin-A as confirmed by Western blot (data not shown). MV thatwere released from pretreated cells did not calcify, demonstratingthat the presence of fetuin-A in the CaPi medium was not requiredfor its inhibitory effects and indicating that uptake of fetuin-Aby VSMC and intracellular loading of vesicles with fetuin-A,before their release into the medium, is the likely mechanismof inhibition of MV calcification (Figure 8B).
Energy-dispersive X-ray analysis of isolated MV and AB showedthat, in the absence of fetuin-A, MV contained preformed BCPevident as strong peaks for oxygen (O), P, and Ca in spectra(data not shown). The presence of BCP accounts for their increasedcalcification potential and is consistent with EM analysis above.BCP was not present in MV or AB that were isolated from fetuin-A–treatedVSMC (Table 1).
Table 1. EDX analysis of mineral content in isolated VSMC MV and ABa
Fetuin-A Promotes Binding of Apoptotic Bodies to VSMC
Phagocytosis of vesicles is an important mechanism for theirremoval with a reduction in phagocytosis of AB associated withincreased VSMC calcification (28). Using a quantitative AB bindingassay indicative of phagocytosis, we found that VSMC in thepresence of fetuin-A had a greater capacity to bind AB thanin its absence (Figure 9) (24,28).
Figure 9. Fetuin enhances phagocytosis of AB by VSMC. AB were Hoechst labeled, and binding to VSMC was quantified by counting. VSMC bound significantly more AB in the presence of fetuin-A (B) than in its absence (A). Representative individual VSMC delineated by broken lines. Quantification shown graphically in C. Mean ± SD, n = 8, **P < 0.05.
Multiple Roles for Fetuin-A in Inhibition of VSMC Calcification at Sites of Damage
Previous studies have shown that one of the earliest eventsin VSMC calcification, induced by high concentrations of extracellularCa and P, is the nucleation of BCP in vesicles that are releasedfrom both dying and viable VSMC (10). Vesicle release by VSMCis thought to be a protective mechanism used to remove excessintracellular Ca to prevent overload and subsequent apoptosis(29). In this study, we demonstrate that the circulating protein,fetuin-A, potentially plays multiple roles in protecting VSMCfrom the detrimental effects of Ca overload and subsequent calcification.First, by perturbing death-signaling pathways, it inhibits VSMCapoptosis. Second, it is taken up by VSMC and loaded into intracellularvesicles, where it prevents nucleation of BCP. Third, it enhancesbinding of AB to adjacent viable cells, thereby enhancing thepotential for AB clearance and limiting their capacity to bindand nucleate BCP in the extracellular matrix. These novel cellbiologic effects are in addition to fetuin-A’s role instabilizing Ca and P in serum and preventing its precipitation(30).
In vivo localization of fetuin-A in atherosclerotic and ESRDarteries has revealed that it is deposited at sites of calcification(19,31) and importantly, in this study, that it is intracellularin VSMC associated with calcification. These VSMC have lostmany of their contractile properties and may display osteo/chondrocyticcharacteristics, properties similar to VSMC in vitro (8,9,32).This observation suggests that inhibition of vascular calcificationby fetuin-A might be most relevant at or restricted to sitesof vessel wall damage, where VSMC have become phenotypicallymodulated in response to injury. Importantly, patients withESRD are subjected to multiple insults that additively contributeto vascular damage. They are often atherosclerotic and hypertensive,have a high Ca x P product, and circulating levels of toxins.In addition, they can be treated with high doses of vitaminD3 and the anticoagulant warfarin, an inhibitor of MGP function.Many of these factors have been shown to induce Ca overloadand/or vesicle release and calcification in animal models (10,33,34).Coupled with low serum fetuin-A levels, they are likely to contributeto the accelerated vascular calcification observed in this patientgroup (15).
Fetuin as an Antiapoptotic Molecule and Opsonin
A role for fetuin-A in inhibiting calcification at sites oftissue damage is supported further by its roles in inhibitingapoptosis and aiding phagocytosis. Fetuin-A inhibited VSMC apoptosisand reduced the cleavage of caspases 3, 8, and 9 into theiractive subunits. Caspases are cysteine proteases that on cleavagepromote the apoptotic cascade, making inhibition of caspaseactivity vital for cell survival (35). Fetuin-A, like othercystatins, has been reported to have antiproteolytic activityresiding in domains D1 and D2 (22,36). Thus, intracellular fetuin-Amay function as an inhibitor of caspase cleavage by direct interactionwith caspases, in a manner similar to its ability to inhibitMMP9 cleavage, but this remains to be tested experimentally(37).
In cells that were undergoing apoptosis, fetuin-A was not confinedto vesicles but distributed throughout the cell, perhaps toensure its association with cell-derived fragments and vesicles.A similar localization has been described in colloid and parenchymalcells in the human fetal pituitary gland, where it was suggestedthat fetuin-A was "tagging" cells for elimination (38). In supportof this, our data and other recent reports show that fetuin-Ais important in phagocytosis (39). Fetuin-A has also been shownto inhibit the inflammatory response of macrophages after phagocytosis(40). Efficient phagocytosis may be particularly important inlimiting accelerated atherosclerotic calcification in patientswith ESRD, which is associated with inflammatory macrophagesand cell death, and where AB form the nidus for calcification(39,41,42).
Fetuin as an Inhibitor of Vesicle Mineralization
Cell death and vesicle release are common in both developmentaland pathologic mineralization. In both processes, inhibitorsof mineralization, for example MGP, are incorporated into releasedvesicles to inhibit or regulate the timing of mineralization(43,44). The mechanisms by which fetuin-A is internalized andlocalized to what are most probably intracellular endosomalvesicles in VSMC are unknown (45,46). Potentially, posttranslationalmodifications such as proteolytic processing and/or sialylationmay facilitate its interaction with plasma membrane proteinsor receptors (47). Recent evidence suggests that annexins mightact as cell surface fetuin-A receptors, in a Ca-dependent manner(48). Annexins act as calcium channels in chondrocyte MV, andit will be important to analyze VSMC-derived MV for annexincontent and function (49). Fetuin-A may also have a role inthe poorly understood process of intracellular Ca loading ofVSMC MV, a notion supported by the observation that MV containedmore fetuin-A protein in the presence of elevated extracellularcalcium.
Fetuin-A as a Regulator of Mineral Metabolism in ESRD
Bone turnover and vascular and other soft tissue calcificationsare physiologically linked processes in patients with ESRD (2).Our studies on the effects of fetuin-A on mineralization ofosteoblast and kidney epithelial cells in vitro support a generalrole for fetuin-A in mineral metabolism. This is supported furtherby the observation that fetuin-A null mice also have a perturbationin bone mineralization and that fetuin-A is a major proteincomponent of bone (16,20,50). A lack of fetuin-A in patientswith ESRD could impinge on bone health by increasing osteoblastapoptosis in response to elevated Ca and P (50). Fetuin-A alsohas other functions that might impinge on both VSMC and bonebiology. It can modulate TGF- signaling and regulate osteoblastphenotype; therefore, its potential role in regulating the osteoblasticphenotype of VSMC should be investigated (51). Finally, it willbe important to determine whether the circulating fetuin/MGP/mineralcomplex described in rats that were treated with bisphosphonatesis present under certain pathologic conditions in patients withESRD (52).
Acknowledgments
This work was supported by grants from the British Heart Foundationto C.M.S. and P.L.W. P.L.W. is a BHF Professor of CardiovascularMedicine; C.M.S. is a BHF Basic Sciences Lecturer.
Thanks to Nikki Figg for expert assistance with immunohistochemistry.
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Received for publication October 29, 2004.
Accepted for publication July 2, 2005.
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Top
Abstract
Introduction
2-Heremens-Schmid glycoprotein, commonly referred to as fetuin-A, are reduced in ESRD, a condition associated with an elevated circulating calcium x phosphate product. Mice that lack fetuin-A exhibit extensive soft tissue calcification, which is accelerated on a mineral-rich diet, suggesting that fetuin-A acts to inhibit calcification systemically. Western blot and immunohistochemistry demonstrated that serum-derived fetuin-A co-localized with calcified human vascular smooth muscle cells (VSMC) in vitro and in calcified arteries in vivo. Fetuin-A inhibited in vitro VSMC calcification, induced by elevated concentrations of extracellular mineral ions, in a concentration-dependent manner. This was achieved in part through inhibition of apoptosis and caspase cleavage. Confocal microscopy and electron microscopy–immunogold demonstrated that fetuin-A was internalized by VSMC and concentrated in intracellular vesicles. Subsequently, fetuin-A was secreted via vesicle release from apoptotic and viable VSMC. Vesicles have previously been identified as the nidus for mineral nucleation. The presence of fetuin-A in vesicles abrogated their ability to nucleate basic calcium phosphate. In addition, fetuin-A enhanced phagocytosis of vesicles by VSMC. These observations provide evidence that the uptake of the serum protein fetuin-A by VSMC is a key event in the inhibition of vesicle-mediated VSMC calcification. Strategies aimed at maintaining normal circulating levels of fetuin-A may prove beneficial in patients with ESRD. 
-Microglobulin was used as a control for cDNA equality (9). 
