Multifunctional Roles for Serum Fetuin-A in Inhibition of 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 system- ically. 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: 2920–2930, 2005. doi: 10.1681/ASN.2004100895

levated concentrations of circulating calcium (Ca) vivo also express a number of bone-associated, mineralization- and/or phosphate (P) ions, such as occurs in ESRD or regulating such as alkaline phosphatase, bone sialo- E in rare genetic causes of hypercalcemia, may result in protein, matrix Gla protein (MGP), osteopontin, and osteocal- catastrophic calcification of the vasculature and other soft tis- cin, which can regulate the calcification process (9). In response sues (1–5). Until recently, vascular calcification in the context of to raised concentrations of extracellular Ca and/or P ions, a mineral imbalance was considered to be an unregulated con- VSMC calcification is accelerated (10). Under these conditions, sequence of the deposition of insoluble basic calcium-phos- VSMC shed numerous membrane-bound vesicles. These vesi- phate (BCP; a mixture of octacalcium phosphate, dicalcium cles are a mixture of apoptotic bodies (AB) released from dying phosphate dihydrate, and apatite) mineral in the extracellular VSMC and matrix vesicles (MV) released from viable cells. Both matrix occurring when concentrations of Ca and/or P ions in have the capacity to nucleate BCP, and their accumulation in the local environment/circulation exceeded the solubility prod- the VSMC matrix results in rapid and widespread calcification uct for calcium phosphate. (10,11). Thus, in the context of a raised extracellular Ca ϫ P Recent evidence suggests that pathologic vascular calcifica- product, the evidence suggests that VSMC calcification is a tion shares many similarities with physiologic bone mineraliza- cell-mediated, regulated process and therefore may be modifi- tion (6). Cultured human vascular smooth muscle cells (VSMC) able in ESRD, in which it is associated with a poor prognosis spontaneously express the osteoblast transcription factor Cbfa1 (1,12). and in postconfluent culture form “osteoblast-like” nodules A number of naturally occurring, endogenous inhibitors of that calcify after approximately 28 d (7,8). VSMC in vitro and in vascular calcification have been identified, including MGP and pyrophosphate, both produced by medial VSMC (13,14). How- ever, emerging evidence suggests that there may also be circu- Received October 29, 2004. Accepted July 2, 2005. lating inhibitors of calcification. Using an in vitro model of Published online ahead of print. Publication date available at www.jasn.org. VSMC calcification induced by elevated levels of extracellular Address correspondence to: Dr. Catherine M. Shanahan, Division of Cardiovas- Ca and P ions, we observed that human serum prevented cular Medicine, Level 6, ACCI, Box 110, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK. Phone: 44-1223-331504; Fax: 44-1223-331505; E-mail: VSMC calcification by inhibiting apoptosis and by reducing the [email protected] calcification potential of shed membrane vesicles (10). Fetuin-A

Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1610-2920 J Am Soc Nephrol 16: 2920–2930, 2005 Fetuin-A Inhibits Vascular Smooth Muscle Cell Calcification 2921 was identified as a serum component that co-purified with precipitates were harvested by centrifugation, and 45Ca incorporation membrane vesicles and therefore potentially could be associ- in the pellet was measured as above (10). ated with their reduced calcification potential (10). Importantly, Experiments were performed using bovine fetuin-A (Sigma, St. there is clinical evidence to suggest that fetuin-A may be pro- Louis, MO) and verified with human AHSG/fetuin-A (Calbiochem, San Diego, CA). All experiments were performed in triplicate and tective in patients with ESRD. Its circulating levels are signifi- independently on at least five different VSMC isolates. cantly 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 Reverse Transcription–PCR Detection of Fetuin-A Human fetuin-A mRNA expression was investigated in a panel of to inhibit calcium phosphate precipitation (15,16). Fetuin-A cDNA samples (n ϭ 40) that were composed of normal and atheroscle- deficiency also correlates with increased cardiovascular mor- rotic aortic samples (as described previously) and in cultured human tality as well as atherosclerosis and coronary and valvular VSMC (9). cDNA was used as a positive control. Fetuin-A prim- calcification in patients with ESRD (17–19). ers were as follows: Forward, CCTGCTCCTTTGTCTTGC; reverse, CG- Fetuin-A is a circulating plasma glycoprotein, produced GACTGGAGGAACCAC. PCR reactions were performed within the abundantly during fetal development by multiple tissues, linear range as standard for 30 cycles. ␤-Microglobulin was used as a whereas in the adult, it is produced predominantly by the liver control for cDNA equality (9). (20). It is a member of the cystatin superfamily of cysteine protease inhibitors. Ablation of the mouse fetuin-A in a Immunohistochemical Detection of Fetuin-A strain of calcification-prone mice results in progressive, fatal Human aortic (n ϭ 3) and carotid endarterectomy specimens (n ϭ 5) calcification of soft tissues, including , testis, skin, , were formalin fixed and embedded in paraffin, and 6-␮m sections were and vasculature (16,21). These mice exhibit compromised se- cut. Human fetuin-A was detected with a rabbit polyclonal antibody ␣ rum inhibition of BCP formation, suggesting that fetuin-A may (Behring, Marburg, Germany), and co-staining for -smooth muscle be important in preventing calcification in the context of ele- actin was performed using a mAb (Dako, Denmark; 1:200) and coun- terstained blue using the Vector alkaline phosphatase substrate Kit III vated concentrations of mineral ions. In vitro experiments have (SK-5300). Von Kossa staining was performed as standard. Negative indicated that fetuin-A can inhibit mineralization of primary controls included substitution of the test antibody with PBS or with an calvaria cells by preventing BCP precipitation and modulating irrelevant antibody. apatite formation during mineralization (16,22). For confocal microscopy, VSMC were cultured on 19-mm glass cov- In this study, we show that fetuin-A can regulate several of erslips and fixed in 3% formaldehyde/PBS. Coverslips were washed the key cellular events that lead to VSMC calcification, includ- and blocked in 3% ovalbumin, and fetuin-A was detected using a rabbit ing apoptosis, vesicle calcification, and phagocytosis, providing polyclonal antibody against human or bovine fetuin-A with Alexa novel mechanistic insights into how a relative lack of fetuin-A Fluor 488 anti-rabbit IgG secondary antibody before mounting in may contribute to vascular calcification. These studies point to DAPI-containing medium (21). an important role for fetuin-A in inhibiting calcification in VSMC were prepared for electron microscopy (EM) as described ESRD, particularly at sites of tissue damage. 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 Materials and Methods control. Cell Culture VSMC that were derived from medial explants of human aortic tissue Protein Gels and Western Analysis ϭ (n 15) were cultured in M199 with 20% FCS and used between Nodular human VSMC that were maintained in growth medium for passages 3 and 10 (23). The apoptosis-sensitive cell line HASMC 66 30 d were trypsinized, and monolayer and nodular cells were separated SV40, Saos2 cells, and 293 kidney epithelial cells were cultured as above using a 70-␮m cell sieve. Protein lysates were prepared in RIPA buffer in 10% FCS (24). For nodular cultures, VSMC were maintained in or SDS-PAGE denaturing sample buffer, and protein quantification was postconfluent conditions for 30 d until nodules calcified. Calcification performed using the BioRad assay (Hercules, CA). Five micrograms of was visualized by alizarin red staining as described previously (23). protein was electrophoresed through 10% acrylamide gels, and selected bands were subjected to nine-residue N-terminal sequencing and iden- VSMC Calcification Assays tified using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Calcification of VSMC in response to extracellular mineral ions was Western blots were performed as standard using Immobilon-P mem- performed as described previously using serum-free (SF) media desig- brane with horseradish peroxidase activity visualized using ECL (Am- nated control (1.8 mM Ca/1.0 mM P) and test; Cai (5.4 mM Ca), Pi (2.0 ersham, UK). Antibodies used for detection were caspase 3, p17 subunit mM P), or CaPi (2.7 mM Ca/2.0 to 3.0 mM P), each containing 0.5% BSA (Pharminogen, San Diego, CA); caspase 8, p18 subunit (Upstate Bio- and 45Ca (approximately 50,000 cpm/ml) (10). Cells were transferred to technology, Lake Placid, NY); and caspase 9, p20 subunit (Pharmino- SF control media for 24 h before the addition of SF test media. Calcifi- gen). cation could be monitored in live cultures by visualization of vesicle/ mineral deposition using phase contrast microscopy. After 24 h to 10 d Time-Lapse Videomicroscopy and Immunofluorescent of treatment, the medium was removed and calcification was visual- TUNEL Staining of Cells ized by alizarin red staining and quantified by measuring 45Ca incor- Apoptosis was induced in the human coronary plaque cell line poration. Briefly, VSMC were decalcified in 0.1 M HCL, neutralized HASMC 66 by serum starvation and in primary VSMC cultures by with 0.1 M NaOH/0.1% SDS, and scraped, and 45Ca incorporation was addition of CaPi media. Video time-lapse microscopy was performed measured by liquid scintillation counting. In cell-free experiments, BCP over 48 h, and apoptosis was recorded (24). 2922 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2920–2930, 2005

Fluorescence transferase-mediated dUTP nick-end labeling (TUNEL) Results assays were performed at time points between 6 and 24 h as described Fetuin-A Associates with Calcified VSMC In Vitro previously using VSMC that were treated with test media Ϯ 5 ␮M and In Vivo fetuin (10,11). Coverslips were mounted in DAPI-containing medium, Inhibitors of VSMC calcification are often deposited at sites 10 random images were captured digitally (Olympus, Tokyo, Japan) of mineralization (27). To identify proteins involved in VSMC from each coverslip, and at least 100 nuclei counted per frame. calcification, we used SDS-PAGE to compare protein profiles of cell lysates that were derived from noncalcified monolayer and Apoptotic Body and Matrix Vesicle Isolation calcified, nodular, human VSMC. A major band at approxi- AB and MV populations were isolated from the media of VSMC mately 54 kD, in calcified VSMC, was identified by microse- cultures by differential centrifugation, and the calcification potential of quencing as bovine fetuin-A, a protein derived from FCS (Fig- isolated vesicle populations was measured as described previously (10). The calcifying reaction mixture described by Kirsch et al. (25) was ure 1A). Fetuin-A mRNA could not be detected by reverse used; it contains 45Ca (50,000 cpm) and 4 to 15 ␮g of VSMC-derived MV transcription–PCR in cultured human VSMC, indicating that or AB. Samples were incubated at 37°C for 24 h, and assays were fetuin-A was not synthesized by VSMC (Figure 1B). performed in triplicate (10,11). Immunohistochemistry was performed on normal and calci- fied human arterial specimens. Fetuin-A was barely detectable Energy-Dispersive X-Ray Analysis and Electron Diffraction in the media or intima of uncalcified normal arteries (Figure Vesicle fractions that were isolated by differential centrifugation (MV 2A). However, fetuin-A staining was present in calcified medial and AB) were resuspended at a concentration of 10 ␮g/␮l and ad- and intimal areas of arteries (Figure 2, B and C). VSMC within sorbed onto glow-discharged, carbon-coated Formvar film grids for calcified regions were strongly positive for fetuin-A, and much energy-dispersive X-ray analysis as described previously (10). For elec- of the staining appeared intracellular (Figure 2a), whereas in tron diffraction, thin sections (150 nm thick) of calcified VSMC cultures the calcified acellular matrix, fetuin-A associated with micro- were examined by transmission electron microscopy (TEM) at 300 kv in calcifications (Figure 2b). Fetuin-A mRNA could not be de- ϫ a Philips CM30 in bright field made at a magnification of 21,000. tected by reverse transcription–PCR in cDNA samples that Calcified spicules were identified both within and without vesicles, and were derived from normal and calcified human arteries (n ϭ selected area diffraction patterns were collected using a camera length 40), indicating that deposited fetuin-A was not synthesized of 900 mm and compared with those from a calibration standard of hydroxyapatite. locally but derived from the serum (Supplemental Figure 1, available online). Apoptotic Body Binding Assay Phagocytosis of AB by VSMC was assessed as described previously Fetuin-A Inhibits VSMC Calcification Induced by (11,26). AB from serum-starved HASMC66 cells were mixed with Extracellular Mineral Ions In Vitro Hoechst dye and either 5 to 10 ␮M BSA (protein control) or 5 to 10 ␮M Fetuin-A inhibited VSMC calcification, induced by CaPi bovine fetuin-A for 15 min before seeding onto VSMC at 1 ϫ 106 AB/well. After 2 h, cells were washed vigorously and fixed in 4% media, in a concentration-dependent manner, with potent ␮ formaldehyde, and random images were captured using an Olympus inhibition occurring at physiologic concentrations (10 M; Fig- TV-1X digital camera with the number of bound AB counted for Ͼ30 ure 3). Similar inhibition of VSMC calcification by fetuin-A was VSMC in random areas of eight separate wells. also observed in Cai and Pi media (Supplemental Figure 2, available online). Fetuin-A was also able to inhibit calcification Statistical Analyses of both Saos2 osteoblasts and 293 kidney epithelial cells in Data were analyzed using t test or for multiple comparisons ANOVA response to Cai and CaPi media (Supplemental Figure 3, avail- with post hoc Scheffe test. able 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. J Am Soc Nephrol 16: 2920–2930, 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 by CaPi in SF conditions. However, CaPi did not induce apo- Fetuin-A is a binder of BCP and an inhibitor of spontaneous ptosis in the presence of serum, resulting in reduced calcifica- precipitation of Ca and P in solution. To determine whether the tion (10). Therefore, we tested whether fetuin-A was the com- ability of fetuin-A to inhibit VSMC calcification was due to its ponent in serum acting to inhibit VSMC apoptosis. capacity to inhibit BCP precipitation alone, we added it to CaPi Immunofluorescent TUNEL labeling showed that apoptosis medium in cell-free conditions. This showed that fetuin-A, as induced in response to CaPi medium was significantly reduced expected, could inhibit the spontaneous precipitation of BCP in by 5 ␮M fetuin-A (Figure 5, A and B). To determine whether the solution. However, in the presence of VSMC, the inhibitory antiapoptotic effects of fetuin-A were context and cell specific, capacity of fetuin-A was significantly increased, suggesting we tested its effect on HASMC66 apoptosis induced by serum that it also acted via cell-mediated mechanisms (Figure 4A). starvation. Fetuin-A reduced apoptotic events by approxi- The capacity for fetuin-A to act via cell-mediated mechanisms mately half over a 48-h time course, and this was associated was suggested further when it was added to VSMC 16 h after with reduced cleavage of caspases 3, 8, and 9 (Figure 5, C the addition of CaPi medium (i.e., after the onset of calcification and D). observed by phase contrast microscopy). When added at this stage, fetuin-A had no effect on 45Ca incorporation, suggesting that it could not inhibit precipitation/crystal growth once min- Fetuin-A Localizes to Apoptotic Cells and Vesicles eral nucleation had occurred, confirming previous in vitro stud- Immunofluorescence showed that fetuin-A was not present ies (Figure 4B) (22). in VSMC that were maintained in SF control medium for Ͼ48 h (Figure 6A). VSMC that were cultured in the presence of Fetuin-A Inhibits VSMC Apoptosis serum or treated with fetuin-A showed fetuin-A distributed We showed previously, using the caspase inhibitor ZVAD- throughout the cytoplasm in a punctate pattern, suggestive of .fmk, that apoptosis contributes to VSMC calcification induced its localization in vesicle-like structures (Figure 6B). In addition, 2924 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2920–2930, 2005

Figure 3. Fetuin-A inhibits mineralization of VSMC in a concen- tration-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 calci- Figure 4. Fetuin-A inhibits mineralization via a cell-mediated ␮ fication after approximately 24 h. Calcification was inhibited in process. (A) Fetuin-A (5 M) inhibited spontaneous basic cal- the presence of serum (positive control) and fetuin-A in a cium-phosphate (BCP) precipitation in CaPi medium (2.7 mM concentration-dependent manner. Similar results were ob- Ca/3.0 mM P) by approximately 50 to 60% in the absence of tained using Cai medium (data not shown). (B) Incorporation of cells. In the presence of VSMC, the inhibitory effect of fetuin-A Ͼ Ϯ ϭ 45Ca into VSMC that were treated with CaPi medium (2.7 mM on calcification was increased to 90%. Mean SD, n 3. (B) Ca/2.0 mM P) was inhibited in a concentration-dependent Fetuin-A inhibited VSMC calcification when added at the same manner by fetuin-A. Mean Ϯ SD, n ϭ 3, **P Ͻ 0.05. time as CaPi test medium (2.7 mM Ca/2.0 mM P; 0 h). How- ever, 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 Ϯ SD, n ϭ 3, **P Ͻ 0.05. 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 fe- within the VSMC cytoplasm, and, most striking, within vesicles tuin-A (Figure 6D). In addition, smaller MV, isolated by ultra- and/or AB (Figure 7, C through E). Calcification was minimal centrifugation, contained fetuin-A. The amount of fetuin-A con- in treated cells, and vesicles were generally not associated with centrated within these MV was increased when VSMC were crystalline BCP. However, fetuin-A was deposited in rare re- cultured in Cai (not shown) or CaPi media (Figure 6D). gions of calcified matrix in association with crystalline calcifi- EM immunogold labeling of cultured VSMC that were cation (Figure 7F). grown in SF Cai or CaPi medium demonstrated little fetuin-A in VSMC, extracellular vesicles, or the calcified matrix (Figure Fetuin-A Inhibits Calcification of Isolated Apoptotic Bodies 7, A and B). Of note, vesicles that were released by cells in the and Matrix Vesicles absence of fetuin-A were associated with both intravesicular The above studies suggested that the presence of fetuin-A in and extravesicular crystalline BCP. The presence of crystalline vesicles inhibited their capacity to nucleate BCP. Therefore, the hydroxyapatite was confirmed by selected area electron diffrac- calcification potential of isolated AB and MV that were derived tion. Polycrystalline diffraction patterns with concentric rings from VSMC that were treated with CaPi medium in the pres- similar to those of hydroxyapatite standards were collected ence or absence of fetuin-A was tested in vitro. MV that were from electron-dense crystals within and without vesicles (Fig- released from VSMC that were cultured in the presence of ure 7B, inset). In contrast, in the presence of 5 ␮M fetuin-A, fetuin-A did not calcify. In contrast, MV that were released immunogold labeling showed fetuin-A at the cell membrane, from VSMC in the absence of fetuin-A calcified extensively J Am Soc Nephrol 16: 2920–2930, 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 Ϯ SD, n ϭ 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 Ϯ SEM, n ϭ 3, *P Ͻ 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 ␮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 cal- of vesicles with fetuin-A, before their release into the medium, cification of AB. is the likely mechanism of inhibition of MV calcification (Fig- In the next experiment, VSMC were pretreated with fetuin-A ure 8B). for 24 h and washed extensively before the immediate addition Energy-dispersive X-ray analysis of isolated MV and AB of CaPi medium. MV that were derived from pretreated VSMC showed that, in the absence of fetuin-A, MV contained pre- contained fetuin-A as confirmed by Western blot (data not formed BCP evident as strong peaks for oxygen (O), P, and Ca shown). MV that were released from pretreated cells did not in spectra (data not shown). The presence of BCP accounts for calcify, demonstrating that the presence of fetuin-A in the CaPi their increased calcification potential and is consistent with EM medium was not required for its inhibitory effects and indicat- analysis above. BCP was not present in MV or AB that were ing that uptake of fetuin-A by VSMC and intracellular loading isolated from fetuin-A–treated VSMC (Table 1). 2926 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2920–2930, 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 cytoplas- mic vesicular structures (green stain). (C) Confocal microscopy Figure 7. Electron microscopy immunogold localization of fe- confirmed the vesicular localization of fetuin-A and also tuin-A in VSMC. (A and B) VSMC that were cultured in CaPi showed that in apoptotic cells, fetuin-A was localized through- media in the absence of fetuin-A calcified via a vesicle-mediated out the cytoplasm (arrow in C) and was concentrated in extra- process. Minimal fetuin-A immunogold labeling was detected cellular vesicles (arrowheads). Nuclei are stained with Hoechst. in association with vesicles. Crystalline BCP deposition could (Inset) Fetuin-A–loaded vesicles. (D) Western blots for fetuin-A be observed both within vesicles (arrow in A) and on the in isolated apoptotic bodies (AB) and matrix vesicles (MV) surface of vesicles (arrow in A and B). The heterogeneity in the confirmed its presence in these extracellular vesicles. Vesicles size of vesicles was consistent with their derivation from both that were isolated from VSMC that were cultured in the pres- AB and MV. Bar ϭ 500 nm for A through C. (B, inset) Diffrac- ence of media that contained additional extracellular calcium tion pattern from the hydroxyapatite standard (HA) consisting (CaPi medium shown) contained approximately twice as much of a series of concentric rings. The adjacent diffraction pattern fetuin-A than vesicles from VSMC in control media. By densi- (MV) from calcified spicules in a typical calcified vesicle is not tometry, 1 versus 2.14 arbitrary units, control compared with as strong as the standard but consistent with polycrystalline test CaPi media (normalized to loading control ␣-smooth mus- hydroxyapatite. (C through F) VSMC treated with fetuin-A in cle actin; data not shown). Multiple bands probably represent the presence of CaPi medium calcified minimally. Note absence posttranslationally modified fetuin-A (e.g., sialylated). 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 Fetuin-A Promotes Binding of Apoptotic Bodies to VSMC vesicular structures present within the cytoplasm of VSMC (D) Phagocytosis of vesicles is an important mechanism for their and associated with areas of calcification of the extracellular removal with a reduction in phagocytosis of AB associated with matrix (ECM; arrow in F). These areas of calcification were increased VSMC calcification (28). Using a quantitative AB associated with vesicle “ghosts” (arrowheads in F). 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). by VSMC is thought to be a protective mechanism used to Discussion remove excess intracellular Ca to prevent overload and subse- Multiple Roles for Fetuin-A in Inhibition of VSMC quent apoptosis (29). In this study, we demonstrate that the Calcification at Sites of Damage circulating protein, fetuin-A, potentially plays multiple roles in Previous studies have shown that one of the earliest events in protecting VSMC from the detrimental effects of Ca overload VSMC calcification, induced by high concentrations of extracel- and subsequent calcification. First, by perturbing death-signal- lular Ca and P, is the nucleation of BCP in vesicles that are ing pathways, it inhibits VSMC apoptosis. Second, it is taken up released from both dying and viable VSMC (10). Vesicle release by VSMC and loaded into intracellular vesicles, where it pre- J Am Soc Nephrol 16: 2920–2930, 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 Ϯ SD, n ϭ 3, **P Ͻ 0.0001.

Table 1. EDX analysis of mineral content in isolated VSMC MV and ABa

Treatment MV AB

Cai or CaPi ϩϪ Cai or CaPi ϩ Fetuin (5 ␮m) ϪϪ Cai or CaPib ϩϩ Cai or CaPi ϩ Fetuin (5 ␮m)b ϪϪ

aEDX, 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. bAfter 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. ϩ, BCP detected; Ϫ, BCP not detected.

vents nucleation of BCP. Third, it enhances binding of AB to Figure 9. Fetuin enhances phagocytosis of AB by VSMC. AB adjacent viable cells, thereby enhancing the potential for AB were Hoechst labeled, and binding to VSMC was quantified by clearance and limiting their capacity to bind and nucleate BCP counting. VSMC bound significantly more AB in the presence in the extracellular matrix. These novel cell biologic effects are of fetuin-A (B) than in its absence (A). Representative individ- in addition to fetuin-A’s role in stabilizing Ca and P in serum ual VSMC delineated by broken lines. Quantification shown and preventing its precipitation (30). graphically in C. Mean Ϯ SD, n ϭ 8, **P Ͻ 0.05. 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 to sites of vessel wall damage, where VSMC have become VSMC associated with calcification. These VSMC have lost phenotypically modulated in response to injury. Importantly, many of their contractile properties and may display osteo/ patients with ESRD are subjected to multiple insults that addi- chondrocytic characteristics, properties similar to VSMC in vitro tively contribute to vascular damage. They are often atheroscle- (8,9,32). This observation suggests that inhibition of vascular rotic and hypertensive, have a high Ca ϫ P product, and calcification by fetuin-A might be most relevant at or restricted circulating levels of toxins. In addition, they can be treated with 2928 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2920–2930, 2005

high doses of vitamin D3 and the anticoagulant warfarin, an ESRD (2). Our studies on the effects of fetuin-A on mineraliza- inhibitor of MGP function. Many of these factors have been tion of osteoblast and kidney epithelial cells in vitro support a shown to induce Ca overload and/or vesicle release and calci- general role for fetuin-A in mineral metabolism. This is sup- fication in models (10,33,34). Coupled with low serum ported further by the observation that fetuin-A null mice also fetuin-A levels, they are likely to contribute to the accelerated have a perturbation in bone mineralization and that fetuin-A is vascular calcification observed in this patient group (15). a major protein component of bone (16,20,50). A lack of fe- tuin-A in patients with ESRD could impinge on bone health by Fetuin as an Antiapoptotic Molecule and Opsonin increasing osteoblast apoptosis in response to elevated Ca and A role for fetuin-A in inhibiting calcification at sites of tissue P (50). Fetuin-A also has other functions that might impinge on damage is supported further by its roles in inhibiting apoptosis both VSMC and bone biology. It can modulate TGF-␤ signaling and aiding phagocytosis. Fetuin-A inhibited VSMC apoptosis and and regulate osteoblast phenotype; therefore, its potential role reduced the cleavage of caspases 3, 8, and 9 into their active in regulating the osteoblastic phenotype of VSMC should be subunits. Caspases are cysteine proteases that on cleavage pro- investigated (51). Finally, it will be important to determine mote the apoptotic cascade, making inhibition of caspase activity whether the circulating fetuin/MGP/mineral complex de- vital for cell survival (35). Fetuin-A, like other cystatins, has been scribed in that were treated with bisphosphonates is reported to have antiproteolytic activity residing in domains D1 present under certain pathologic conditions in patients with and D2 (22,36). Thus, intracellular fetuin-A may function as an ESRD (52). 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). Acknowledgments This work was supported by grants from the British Heart Founda- In cells that were undergoing apoptosis, fetuin-A was not tion to C.M.S. and P.L.W. P.L.W. is a BHF Professor of Cardiovascular confined to vesicles but distributed throughout the cell, per- Medicine; C.M.S. is a BHF Basic Sciences Lecturer. haps to ensure its association with cell-derived fragments and Thanks to Nikki Figg for expert assistance with immunohistochem- vesicles. A similar localization has been described in colloid istry. and parenchymal cells in the human fetal pituitary gland, where it was suggested that fetuin-A was “tagging” cells for elimination (38). 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