Elevated concentrations of circulating calcium (Ca)

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1 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 Vascular calcification predicts an increased risk for cardiovascular events/mortality in atherosclerosis, diabetes, and ESRD. Serum concentrations of 2 -Heremens-Schmid glycoprotein, commonly referred to as fetuin-a, are reduced in ESRD, a condition associated with an elevated circulating calcium 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. J Am Soc Nephrol 16: , doi: /ASN Elevated concentrations of circulating calcium (Ca) and/or phosphate (P) ions, such as occurs in ESRD or in rare genetic causes of hypercalcemia, may result in catastrophic calcification of the vasculature and other soft tissues (1 5). Until recently, vascular calcification in the context of a mineral imbalance was considered to be an unregulated consequence of the deposition of insoluble basic calcium-phosphate (BCP; a mixture of octacalcium phosphate, dicalcium phosphate dihydrate, and apatite) mineral in the extracellular matrix occurring when concentrations of Ca and/or P ions in the local environment/circulation exceeded the solubility product for calcium phosphate. Recent evidence suggests that pathologic vascular calcification shares many similarities with physiologic bone mineralization (6). Cultured human vascular smooth muscle cells (VSMC) spontaneously express the osteoblast transcription factor Cbfa1 and in postconfluent culture form osteoblast-like nodules that calcify after approximately 28 d (7,8). VSMC in vitro and in Received October 29, Accepted July 2, Published online ahead of print. Publication date available at 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: ; Fax: ; cs131@mole.bio.cam.ac.uk vivo also express a number of bone-associated, mineralizationregulating proteins such as alkaline phosphatase, bone sialoprotein, matrix Gla protein (MGP), osteopontin, and osteocalcin, which can regulate the calcification process (9). In response to raised concentrations of extracellular Ca and/or P ions, VSMC calcification is accelerated (10). Under these conditions, VSMC shed numerous membrane-bound vesicles. These vesicles are a mixture of apoptotic bodies (AB) released from dying VSMC and matrix vesicles (MV) released from viable cells. Both have the capacity to nucleate BCP, and their accumulation in the VSMC matrix results in rapid and widespread calcification (10,11). Thus, in the context of a raised extracellular Ca P product, the evidence suggests that VSMC calcification is a cell-mediated, regulated process and therefore may be modifiable in ESRD, in which it is associated with a poor prognosis (1,12). A number of naturally occurring, endogenous inhibitors of vascular calcification have been identified, including MGP and pyrophosphate, both produced by medial VSMC (13,14). However, emerging evidence suggests that there may also be circulating inhibitors of calcification. Using an in vitro model of VSMC calcification induced by elevated levels of extracellular Ca and P ions, we observed that human serum prevented VSMC calcification by inhibiting apoptosis and by reducing the calcification potential of shed membrane vesicles (10). Fetuin-A Copyright 2005 by the American Society of Nephrology ISSN: /

2 J Am Soc Nephrol 16: , 2005 Fetuin-A Inhibits Vascular Smooth Muscle Cell Calcification 2921 was identified as a serum component that co-purified with membrane vesicles and therefore potentially could be associated with their reduced calcification potential (10). Importantly, there is clinical evidence to suggest that fetuin-a may be protective in patients with ESRD. Its circulating levels are significantly reduced in patients with ESRD and calciphylaxis, a rapidly progressive, often fatal form of vascular calcification, with fetuin-a deficient serum having impaired ex vivo capacity to inhibit calcium phosphate precipitation (15,16). Fetuin-A deficiency also correlates with increased cardiovascular mortality as well as atherosclerosis and coronary and valvular calcification in patients with ESRD (17 19). Fetuin-A is a circulating plasma glycoprotein, produced abundantly during fetal development by multiple tissues, whereas in the adult, it is produced predominantly by the liver (20). It is a member of the cystatin superfamily of cysteine protease inhibitors. Ablation of the mouse fetuin-a gene in a strain of calcification-prone mice 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 calcification in the context of elevated concentrations of mineral ions. In vitro experiments have indicated that fetuin-a can inhibit mineralization of primary rat calvaria cells by preventing BCP precipitation and modulating apatite formation during mineralization (16,22). In this study, we show that fetuin-a can regulate several of the key cellular events that lead to VSMC calcification, including apoptosis, vesicle calcification, and phagocytosis, providing novel mechanistic insights into how a relative lack of fetuin-a may contribute to vascular calcification. These studies point to an important role for fetuin-a in inhibiting calcification in ESRD, particularly at sites of tissue damage. Materials and Methods Cell Culture VSMC that were derived from medial explants of human aortic tissue (n 15) were cultured in M199 with 20% FCS and used between passages 3 and 10 (23). The apoptosis-sensitive cell line HASMC 66 SV40, Saos2 cells, and 293 kidney epithelial cells were cultured as above in 10% FCS (24). For nodular cultures, VSMC were maintained in postconfluent conditions for 30 d until nodules calcified. Calcification was visualized by alizarin red staining as described previously (23). VSMC Calcification Assays Calcification of VSMC in response to extracellular mineral ions was 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 mm P), each containing 0.5% BSA and 45 Ca (approximately 50,000 cpm/ml) (10). Cells were transferred to SF control media for 24 h before the addition of SF test media. Calcification could be monitored in live cultures by visualization of vesicle/ mineral deposition using phase contrast microscopy. After 24 h to 10 d of treatment, the medium was removed and calcification was visualized by alizarin red staining and quantified by measuring 45 Ca incorporation. Briefly, VSMC were decalcified in 0.1 M HCL, neutralized with 0.1 M NaOH/0.1% SDS, and scraped, and 45 Ca incorporation was measured by liquid scintillation counting. In cell-free experiments, BCP precipitates were harvested by centrifugation, and 45 Ca incorporation in the pellet was measured as 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 triplicate and 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 of cdna samples (n 40) that were composed of normal and atherosclerotic aortic samples (as described previously) and in cultured human VSMC (9). Liver cdna was used as a positive control. Fetuin-A primers were as follows: Forward, CCTGCTCCTTTGTCTTGC; reverse, CG- GACTGGAGGAACCAC. PCR reactions were performed within the linear range as standard for 30 cycles. -Microglobulin was used as a 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- m sections were cut. Human fetuin-a was detected with a rabbit polyclonal antibody (Behring, Marburg, Germany), and co-staining for -smooth muscle actin was performed using a mab (Dako, Denmark; 1:200) and counterstained blue using the Vector alkaline phosphatase substrate Kit III (SK-5300). Von Kossa staining was performed as standard. Negative controls included substitution of the test antibody with PBS or with an irrelevant antibody. For confocal microscopy, VSMC were cultured on 19-mm glass coverslips and fixed in 3% formaldehyde/pbs. Coverslips were washed and blocked in 3% ovalbumin, and fetuin-a was detected using a rabbit polyclonal antibody against human or bovine fetuin-a with Alexa Fluor 488 anti-rabbit IgG secondary antibody before mounting in DAPI-containing medium (21). VSMC were prepared for electron microscopy (EM) as described previously (10). Immunogold was performed using a bovine fetuin-a antibody (diluted 1:100) and a 10-nm gold-conjugated anti-rabbit IgG secondary antibody. PBS was substituted for primary antibody as a control. Protein Gels and Western Analysis Nodular human VSMC that were maintained in growth medium for 30 d were trypsinized, and monolayer and nodular cells were separated using a 70- m cell sieve. Protein lysates were prepared 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 electrophoresed through 10% acrylamide gels, and selected bands were subjected to nine-residue N-terminal sequencing and identified using BLAST ( Western blots were performed as standard using Immobilon-P membrane with 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 (Upstate Biotechnology, 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 line HASMC 66 by serum starvation and in primary VSMC cultures by addition of CaPi media. Video time-lapse microscopy was performed over 48 h, and apoptosis was recorded (24).

3 2922 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 Fluorescence transferase-mediated dutp nick-end labeling (TUNEL) assays were performed at time points between 6 and 24 h as described previously using VSMC that were treated with test media 5 M fetuin (10,11). Coverslips were mounted in DAPI-containing medium, 10 random images were captured digitally (Olympus, Tokyo, Japan) from each coverslip, and at least 100 nuclei counted per frame. Apoptotic Body and Matrix Vesicle Isolation AB and MV populations were isolated from the media of VSMC cultures by differential centrifugation, and the calcification potential of isolated vesicle populations was measured as described previously (10). The calcifying reaction mixture described by Kirsch et al. (25) was used; it contains 45 Ca (50,000 cpm) and 4 to 15 g of VSMC-derived MV or AB. Samples were incubated at 37 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 g/ l and adsorbed onto glow-discharged, carbon-coated Formvar film grids 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 electron microscopy (TEM) at 300 kv in a Philips CM30 in bright field made at a magnification of 21,000. Calcified spicules were identified both within and without vesicles, and selected area diffraction patterns were collected using a camera length of 900 mm and compared with those from a calibration standard of hydroxyapatite. Apoptotic Body Binding Assay Phagocytosis of AB by VSMC was assessed as described previously (11,26). AB from serum-starved HASMC66 cells were mixed with Hoechst dye and either 5 to 10 M BSA (protein control) or 5 to 10 M bovine fetuin-a for 15 min before seeding onto VSMC at AB/well. After 2 h, cells were washed vigorously and fixed in 4% formaldehyde, and random images were captured using an Olympus TV-1X digital camera with the number of bound AB counted for 30 VSMC in random areas of eight separate wells. Statistical Analyses Data were analyzed using t test or for multiple comparisons ANOVA with post hoc Scheffe test. Results Fetuin-A Associates with Calcified VSMC In Vitro and In Vivo Inhibitors of VSMC calcification are often deposited at sites of mineralization (27). To identify proteins involved in VSMC calcification, we used SDS-PAGE to compare protein profiles of cell lysates that were derived from noncalcified monolayer and calcified, nodular, human VSMC. A major band at approximately 54 kd, in calcified VSMC, was identified by microsequencing as bovine fetuin-a, a protein derived from FCS (Figure 1A). Fetuin-A mrna could not be detected by reverse transcription PCR in cultured human VSMC, indicating that fetuin-a was not synthesized by VSMC (Figure 1B). Immunohistochemistry was performed on normal and calcified human arterial specimens. Fetuin-A was barely detectable in the media or intima of uncalcified normal arteries (Figure 2A). However, fetuin-a staining was present in calcified medial and intimal areas of arteries (Figure 2, B and C). VSMC within calcified regions were strongly positive for fetuin-a, and much of the staining appeared intracellular (Figure 2a), whereas in the calcified acellular matrix, fetuin-a associated with microcalcifications (Figure 2b). Fetuin-A mrna could not be detected by reverse transcription PCR in cdna samples that were derived from normal and calcified human arteries (n 40), indicating that deposited fetuin-a was not synthesized locally but derived from the serum (Supplemental Figure 1, available online). 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 inhibition occurring at physiologic concentrations (10 M; Figure 3). Similar inhibition of VSMC calcification by fetuin-a was also observed in Cai and Pi media (Supplemental Figure 2, available online). Fetuin-A was also able to inhibit calcification of both Saos2 osteoblasts and 293 kidney epithelial cells in response to Cai and CaPi media (Supplemental Figure 3, available online). 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.

4 J Am Soc Nephrol 16: , 2005 Fetuin-A Inhibits Vascular Smooth Muscle Cell Calcification 2923 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). Cell-Mediated Inhibition of VSMC Calcification by Fetuin-A Fetuin-A is a binder of BCP and an inhibitor of spontaneous precipitation of Ca and P in solution. To determine whether the ability of fetuin-a to inhibit VSMC calcification was due to its capacity to inhibit BCP precipitation alone, we added it to CaPi medium in cell-free conditions. This showed that fetuin-a, as expected, could inhibit the spontaneous precipitation of BCP in solution. However, in the presence of VSMC, the inhibitory capacity of fetuin-a was significantly increased, suggesting that it also acted via cell-mediated mechanisms (Figure 4A). The capacity for fetuin-a to act via cell-mediated mechanisms was suggested further when it was added to VSMC 16 h after the addition of CaPi medium (i.e., after the onset of calcification observed by phase contrast microscopy). When added at this stage, fetuin-a had no effect on 45 Ca incorporation, suggesting that it could not inhibit precipitation/crystal growth once mineral nucleation 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 by CaPi in SF conditions. However, CaPi did not induce apoptosis in the presence of serum, resulting in reduced calcification (10). Therefore, we tested whether fetuin-a was the component in serum acting to inhibit VSMC apoptosis. Immunofluorescent TUNEL labeling showed that apoptosis induced in response to CaPi medium was significantly reduced by 5 M fetuin-a (Figure 5, A and B). To determine whether the antiapoptotic effects of fetuin-a were context and cell specific, we tested its effect on HASMC66 apoptosis induced by serum starvation. Fetuin-A reduced apoptotic events by approximately half over a 48-h time course, and this was associated with reduced cleavage of caspases 3, 8, and 9 (Figure 5, C and D). Fetuin-A Localizes to Apoptotic Cells and Vesicles Immunofluorescence showed that fetuin-a was not present in VSMC that were maintained in SF control medium for 48 h (Figure 6A). VSMC that were cultured in the presence of serum or treated with fetuin-a showed fetuin-a distributed throughout the cytoplasm in a punctate pattern, suggestive of its localization in vesicle-like structures (Figure 6B). In addition,

5 2924 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 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 45 Ca 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 confocal microscopy demonstrated intense staining throughout the cytoplasm of apoptotic cells and within AB and other smaller MV released from the cells (Figure 6C). 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 within these 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 SF Cai or CaPi medium demonstrated little fetuin-a in VSMC, extracellular vesicles, or the calcified matrix (Figure 7, A and B). Of note, vesicles that were released by cells in the absence of fetuin-a were associated with both intravesicular and extravesicular crystalline BCP. The presence of crystalline hydroxyapatite was confirmed by selected area electron diffraction. Polycrystalline diffraction patterns with concentric rings similar to those of hydroxyapatite standards were collected from electron-dense crystals within and without vesicles (Figure 7B, inset). In contrast, in the presence of 5 M fetuin-a, immunogold labeling showed fetuin-a at the cell membrane, Figure 4. Fetuin-A inhibits mineralization via a cell-mediated process. (A) Fetuin-A (5 M) 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 within the VSMC cytoplasm, 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 crystalline BCP. However, fetuin-a was deposited in rare regions of calcified matrix in association with crystalline calcification (Figure 7F). Fetuin-A Inhibits Calcification of Isolated Apoptotic Bodies and Matrix Vesicles The above studies suggested that the presence of fetuin-a in vesicles inhibited their capacity to nucleate BCP. Therefore, the calcification potential of isolated AB and MV that were derived from VSMC that were treated with CaPi medium in the presence or absence of fetuin-a was tested in vitro. MV that were released from VSMC that were cultured in the presence of fetuin-a did not calcify. In contrast, MV that were released from VSMC in the absence of fetuin-a calcified extensively

6 J Am Soc Nephrol 16: , 2005 Fetuin-A Inhibits Vascular Smooth Muscle Cell Calcification 2925 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 M 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 (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 M fetuin-a, caspase cleavage is minimal and similar to that observed in FCS, consistent with the inhibition of apoptosis. (Figure 8A). Fetuin-A also inhibited to a lesser extent the calcification of AB. In the next experiment, VSMC were pretreated with fetuin-a for 24 h and washed extensively before the immediate addition of CaPi medium. MV that were derived from pretreated VSMC contained fetuin-a as confirmed by Western blot (data not shown). MV that were released from pretreated cells did not calcify, demonstrating that the presence of fetuin-a in the CaPi medium was not required for its inhibitory effects and indicating that uptake of fetuin-a by VSMC and intracellular loading of vesicles with fetuin-a, before their release into the medium, is the likely mechanism of inhibition of MV calcification (Figure 8B). Energy-dispersive X-ray analysis of isolated MV and AB showed that, in the absence of fetuin-a, MV contained preformed BCP evident as strong peaks for oxygen (O), P, and Ca in spectra (data not shown). The presence of BCP accounts for their increased calcification potential and is consistent with EM analysis above. BCP was not present in MV or AB that were isolated from fetuin-a treated VSMC (Table 1).

7 2926 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 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 M 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). Fetuin-A Promotes Binding of Apoptotic Bodies to VSMC Phagocytosis of vesicles is an important mechanism for their removal with a reduction in phagocytosis of AB associated with increased VSMC calcification (28). Using a quantitative AB binding assay indicative of phagocytosis, we found that VSMC in the presence of fetuin-a had a greater capacity to bind AB than in its absence (Figure 9) (24,28). Discussion Multiple Roles for Fetuin-A in Inhibition of VSMC Calcification at Sites of Damage Previous studies have shown that one of the earliest events in VSMC calcification, induced by high concentrations of extracellular Ca and P, is the nucleation of BCP in vesicles that are released from both dying and viable VSMC (10). Vesicle release 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). by VSMC is thought to be a protective mechanism used to remove excess intracellular 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 VSMC from the detrimental effects of Ca overload and subsequent calcification. First, by perturbing death-signaling pathways, it inhibits VSMC apoptosis. Second, it is taken up by VSMC and loaded into intracellular vesicles, where it pre-

8 J Am Soc Nephrol 16: , 2005 Fetuin-A Inhibits Vascular Smooth Muscle Cell Calcification 2927 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 M 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 Table 1. EDX analysis of mineral content in isolated VSMC MV and AB a Treatment MV AB Cai or CaPi Cai or CaPi Fetuin (5 m) Cai or CaPi b Cai or CaPi Fetuin (5 m) b a EDX, energy-dispersive X-ray analysis; VSMC, vascular smooth muscle cells; MV, matrix vesicles; AB, apoptotic bodies; BCP, basic calcium-phosphate. EDX analysis (data not shown) demonstrated that MV but not AB isolated from VSMC that were treated with Cai (5.4 mm Ca) or CaPi (2.7 mm Ca/2.0 mm P) media for 24 h contained preformed BCP. Fetuin-A inhibited the formation of BCP within VSMC vesicles. b After incubation in vitro in calcifying buffer (23) (see Figure 8), both MV and AB contained BCP by EDX. However, formation was inhibited when the VSMC had been treated with fetuin., BCP detected;, BCP not detected. vents nucleation of BCP. Third, it enhances binding of AB to adjacent viable cells, thereby enhancing the potential for AB clearance and limiting their capacity to bind and nucleate BCP in the extracellular matrix. These novel cell biologic effects are in addition to fetuin-a s role in stabilizing Ca and P in serum and preventing its precipitation (30). In vivo localization of fetuin-a in atherosclerotic and ESRD arteries has revealed that it is deposited at sites of calcification (19,31) and importantly, in this study, that it is intracellular in VSMC associated with calcification. These VSMC have lost many of their contractile properties and may display osteo/ chondrocytic characteristics, properties similar to VSMC in vitro (8,9,32). This observation suggests that inhibition of vascular calcification by fetuin-a might be most relevant at or restricted 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 to sites of vessel wall damage, where VSMC have become phenotypically modulated in response to injury. Importantly, patients with ESRD are subjected to multiple insults that additively contribute to vascular damage. They are often atherosclerotic and hypertensive, have a high Ca P product, and circulating levels of toxins. In addition, they can be treated with

9 2928 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 high doses of vitamin D 3 and the anticoagulant warfarin, an inhibitor of MGP function. Many of these factors have been shown to induce Ca overload and/or vesicle release and calcification in animal models (10,33,34). Coupled with low serum fetuin-a levels, they are likely to contribute to the accelerated vascular calcification observed in this patient group (15). Fetuin as an Antiapoptotic Molecule and Opsonin A role for fetuin-a in inhibiting calcification at sites of tissue damage is supported further by its roles in inhibiting apoptosis and aiding phagocytosis. Fetuin-A inhibited VSMC apoptosis and reduced the cleavage of caspases 3, 8, and 9 into their active subunits. Caspases are cysteine proteases that on cleavage promote the apoptotic cascade, making inhibition of caspase activity vital for cell survival (35). Fetuin-A, like other cystatins, has been reported to have antiproteolytic activity residing in domains D1 and D2 (22,36). Thus, intracellular fetuin-a may function as an inhibitor of caspase cleavage by direct interaction with caspases, in a manner similar to its ability to inhibit MMP9 cleavage, but this remains to be tested experimentally (37). In cells that were undergoing apoptosis, fetuin-a was not confined to vesicles but distributed throughout the cell, perhaps to ensure its association with cell-derived fragments and vesicles. A similar localization has been described in colloid and parenchymal cells in the human fetal pituitary gland, where it was suggested that fetuin-a was tagging cells for elimination (38). In support of this, our data and other recent reports show that fetuin-a is important in phagocytosis (39). Fetuin-A has also been shown to inhibit the inflammatory response of macrophages after phagocytosis (40). Efficient phagocytosis may be particularly important in limiting accelerated atherosclerotic calcification in patients with ESRD, which is associated with inflammatory macrophages and 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 developmental and pathologic mineralization. In both processes, inhibitors of mineralization, for example MGP, are incorporated into released vesicles to inhibit or regulate the timing of mineralization (43,44). The mechanisms by which fetuin-a is internalized and localized to what are most probably intracellular endosomal vesicles in VSMC are unknown (45,46). Potentially, posttranslational modifications such as proteolytic processing and/or sialylation may facilitate its interaction with plasma membrane proteins or receptors (47). Recent evidence suggests that annexins might act as cell surface fetuin-a receptors, in a Ca-dependent manner (48). Annexins act as calcium channels in chondrocyte MV, and it will be important to analyze VSMCderived MV for annexin content and function (49). Fetuin-A may also have a role in the poorly understood process of intracellular Ca loading of VSMC MV, a notion supported by the observation that MV contained more fetuin-a protein in the presence of elevated extracellular calcium. Fetuin-A as a Regulator of Mineral Metabolism in ESRD Bone turnover and vascular and other soft tissue calcifications are physiologically linked processes in patients with ESRD (2). Our studies on the effects of fetuin-a on mineralization of osteoblast and kidney epithelial cells in vitro support a general role for fetuin-a in mineral metabolism. This is supported further by the observation that fetuin-a null mice also have a perturbation in bone mineralization and that fetuin-a is a major protein component of bone (16,20,50). A lack of fetuin-a in patients with ESRD could impinge on bone health by increasing osteoblast apoptosis in response to elevated Ca and P (50). Fetuin-A also has other functions that might impinge on both VSMC and bone biology. It can modulate TGF- signaling and regulate osteoblast phenotype; therefore, its potential role in regulating the osteoblastic phenotype of VSMC should be investigated (51). Finally, it will be important to determine whether the circulating fetuin/mgp/mineral complex described in rats that were treated with bisphosphonates is present under certain pathologic conditions in patients with ESRD (52). Acknowledgments This work was supported by grants from the British Heart Foundation to C.M.S. and P.L.W. 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