Fetuin-A Regulation of Calcified Matrix...

This article is part of a thematic series on Pathobiology of Calcific Vasculopathy and Valvulopathy, which includedthe following articles:

Thematic series on the pathobiology of vascular calcification: An introduction [Circ Res. 2011;108:1378–1380]

Molecular imaging insights into early inflammatory stages of arterial and aortic valve calcification [Circ Res. 2011;108:1381–1391]

Calcific aortic valve stenosis: Methods, models, and mechanisms [Circ Res. 2011;108:1392–1412]

The roles of lipid oxidation products and receptor activator of nuclear factor-kappa B signaling in atherosclerotic calcification [CircRes. 2011;108:1482–1493]

Fetuin-A regulation of calcified matrix metabolism

Matricine cues and substrate compliance in the pathobiology of calcific valvular disease

Osteogenic BMP-Wnt signaling in valvular and vascular sclerosis

Calcium-phosphate homeostasis in the arterial calcification of CKD

Molecular genetics of calcific vasculopathy

Dwight A. Towler, Guest Editor

Fetuin-A Regulation of Calcified Matrix MetabolismWilli Jahnen-Dechent, Alexander Heiss, Cora Schafer, Markus Ketteler

Abstract: The final step of biomineralization is a chemical precipitation reaction that occurs spontaneously insupersaturated or metastable salt solutions. Genetic programs direct precursor cells into a mineralization-competent statein physiological bone formation (osteogenesis) and in pathological mineralization (ectopic mineralization or calcifica-tion). Therefore, all tissues not meant to mineralize must be actively protected against chance precipitation of mineral.Fetuin-A is a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization. Monomeric fetuin-Aprotein binds small clusters of calcium and phosphate. This interaction results in the formation of prenucleationcluster-laden fetuin-A monomers, calciprotein monomers, and considerably larger aggregates of protein and mineralcalciprotein particles. Both monomeric and aggregate forms of fetuin-A mineral accrue acidic plasma protein includingalbumin, thus stabilizing supersaturated and metastable mineral ion solutions as colloids. Hence, fetuin-A is a mineralcarrier protein and a systemic inhibitor of pathological mineralization complementing local inhibitors that act in acell-restricted or tissue-restricted fashion. Fetuin-A deficiency is associated with soft tissue calcification in mice andhumans. (Circ Res. 2011;108:1494-1509.)

Key Words: calcification � mineral metabolism � plasma proteins

Fetuins are vertebrate plasma proteins. Fetuin-A/�2-Heremans Schmid (HS) glycoprotein homologues occur

in reptiles, fish, birds, marsupials, and mammals. Bovinefetuin (derived from the Latin word fetus) was first describedin 1944 by Pedersen1 as the most abundant globular plasmaprotein in fetal calf serum. The human species homologuewas independently identified by Heremans2 and Schmidt and

Burgi.3 It was later named �2-HS-glycoprotein by Schultze4

in honor of two of the original codiscoverers. The name alsoindicates that �2-HS glycoprotein comigrates with the �-2-globulin fraction of serum proteins in cellulose acetateelectrophoresis.

This protein is confused in the literature with �-2-Z-gly-coprotein/Zn �-2 glycoprotein (Z for zinc-binding) or

Original received March 1, 2011; revision received April 18, 2011; accepted May 5, 2011. In April 2011, the average time from submission to firstdecision for all original research papers submitted to Circulation Research was 15 days.

From the RWTH Aachen University Clinics (W.J.D., A.H., C.S.), Department of Biomedical Engineering, Biointerface Lab, Aachen, Germany;Klinikum Coburg GmbH (M.K.), Division of Nephrology, Coburg, Germany.

Correspondence to Dr Willi Jahnen-Dechent, RWTH Aachen University Clinics, Pauwelsstrasse 30, 52074 Aachen, Germany. [email protected]

© 2011 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.234260

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�-fetoprotein, two unrelated entities. Fetuin-A is member ofa family of four structurally related plasma proteins contain-ing cystatin-like protein domains. Cystatins can inhibit cys-teine peptidases of the papain, calpain, cathepsin, and caspasefamilies and play key roles in a wide array of physiologicalprocesses as well as in disease. The cystatin family harborstype 1 (mainly intracellular proteins), type 2 (mainly extra-cellular proteins), and type 3 cystatins (plasma proteins).Figure 1 shows cartoons of the type 3 family membersfetuin-A/�2-HS glycoprotein, fetuin-B, histidine-rich glyco-protein, and kininogen. Cystatin domain 1 in fetuin-A isstrongly negatively charged with a high affinity for calcium-rich minerals.5

Fetuin-A BiosynthesisFetuins are highly expressed liver-derived plasma proteinsbearing posttranslational modifications proteolytic process-ing,6,7 complex glycosylation,8–10 phosphorylation (Ser andThr),6,11–15 and sulfation.16 Human fetuin-A/�2-HS glycopro-tein is processed from a single chain precursor to the maturecirculating two-chain form.6 Human fetuin-A is susceptible tofurther proteolytic cleavage in septicemia,7 and bovinefetuin-A is processed by matrix metalloproteinases.15 Figure2 illustrates secondary modifications and allelic variantsidentified in human fetuin-A/�2-HS glycoprotein. Thesemodifications may regulate protein expression levels, stabil-ity, and biological activity. Phosphorylation is indispensiblefor fetuin-A interaction with the insulin receptor,17,18 whereasphosphorylation seems not to be required for mineral inter-action.19 Desialylation will result in immediate hepatic clear-ing through the asialoglycoprotein receptor.20–22

Binding Properties of Fetuin-AThe type 3 members of the cystatin superfamily are glyco-proteins produced mainly in the liver, which circulate inplasma at high concentrations. Fetal calf serum contains morefetuin-A than albumin. Apart from the vasculature, fetuins arepresent throughout the extracellular spaces and the extracel-lular matrix. Given the high expression levels and the manypossible ways of molecular interaction with multiple ligands,it is fair to assume that fetuins primarily exercise carrier andscavenger functions like albumin. This poses an importantcomplication for ex vivo experimentation, because crudefetuin preparations contain impurities. Like pure albuminpreparations, pure fetuin preparations are difficult to make.High-abundance plasma protein preparations are notoriously

Non-standard Abbreviations and Acronyms

�2-HS-glycoprotein �2-Heremans Schmid glycoprotein/fetuin-A

AB Apoptotic body, membrane enclosed frag-ments of cells undergoing apoptosis

AFP Alpha-fetoprotein

AHSG Genetic symbol for �2-HS-glycoproteinANK Ankylosing spondylitis gene, mutation of this

gene causes spondyloarthritis

BCP Basic calcium phosphate (octacalcium phosphateor hydroxyapatite) as opposed to acidic calcium-like phosphate calcium pyrophosphate dihydrate

BMP Bone morphogenetic protein

BMPR-I BMP receptor-I

CKD Chronic kidney disease

CNP Calcifying nanoparticles

CPM Calciprotein monomer

CPP Calciprotein particle

CUA Calcific uremic arteriolopathy/calciphylaxis

DLA Dynamic light scattering

ENPP1 Extracellular nucleotide pyrophosphatase/phosphotransferase-1, a pyrophosphate-cleavingenzyme

EPIC European Prospective Investigation into Cancer andNutrition study involving 27 548 subjects

FABP Fatty acid binding protein

FMC Fetuin mineral complex

HMGB1 High-mobility group protein B1, a proinflam-matory nuclear protein

HRG Histidine-rich glycoprotein, a fetuin-related protein

KNG Kininogen, a fetuin-related protein

LPS Lipopolysaccharide, a strong inflammatoryagent from bacterial cell walls

MGP Matrix GLA protein

MO Monocyte

MIA Malnutrition-inflammation-atherosclerosissyndrome

MMP Matrix metalloproteinase

MP Macrophage

MV Matrix vesicles, mineral-laden vesicles be-lieved to mediate bone mineralization

NCP Noncollagenous proteins (from bone)

NLRP3 NOD-like receptor 3

NMP Nucleotide monophosphate

NTP Nucleotide triphosphate

OPN Osteopontin

PD Peritoneal dialysis

Pit-1 Sodium/phosphate cotransporter

PP Pyrophosphate a potent crystal growth inhibi-tor and anticalcification agent

PS Phosphatidylserine, membrane phospholipidexposed on the cell surface during apoptosis

Runx2/Cbfa-1 Runt-related transcription factor 2/core bindingfactor 1, an osteoblastic transcription factor

SANS Small-angle neutron scattering, an analyticaltechnique to determine molecular structure

SAXS Small angle x-ray scattering, an analytical tech-nique to study crystal growth at high resolution

Non-standard Abbreviations and Acronyms

Smad 6 Similar to mothers against decapentaplegic 6,an intracellular signaling protein

SM-MHC Smooth muscle myosin heavy chain

TGF-� Transforming growth factor beta

TNAP Tissue nonspecific alkaline phosphatase,cleaves pyrophosphate PP

TNF Tumor necrosis factor

VSMC Vascular smooth muscle cell

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contaminated with lower-abundance biological moleculesthat copurify either because of natural association or becauseof mere coincidence. Various growth factors or growth-promoting substances associate with crude fetuin prepara-tions and form the basis of the “enigmatic growth promotingproperties” of fetuin in cell culture.23

Whether associations of fetuin-A with certain ligands arephysiologically relevant is a matter of controversy and isalmost impossible to decide unless genetic models are devel-oped to test such interactions in vivo. We remind readers toheed the old wisdom of Racker24 that also became part of the“10 commandments” of Kornberg,25 “Do not waste cleanthinking on dirty enzymes.” Most commercial preparations offetuin-A on the market today do not go much beyond thequality of “fetuin” from 1944,1 which is better viewed as a“protein concept” like “globulin” or “albumin” rather thana clean product. We distinguish between fetuin and fetuin-Awhenever possible. Also, before the year 2000 when fetuin-Bwas cloned in silico26 and the year 2003 when it was finallyshown to be expressed as a plasma protein,10 both fetuin-Aand fetuin-B were collectively addressed as “fetuin,” andmost fetuin sold today is not screened for the presence of

fetuin-B. Studies of human �2-HS-glycoprotein/fetuin(�2-HS glycoprotein) will always signify fetuin-A; publica-tions studying “fetuin” protein are probably dealing withfetuin-A as well, but it is impossible to exclude that fetuin-Bwas also studied because of the physicochemical similitude ofboth proteins.

Fetuin-A binding is prodigious and reaches from smallmolecules to entire organisms. Plasmodium sporozoites usefetuin-A binding to “hitch-hike” their way into liver cells,27

suggesting that fetuin-A function is indispensable and despitenegative selection pressure has been maintained long enoughfor this docking mechanism to evolve. Soon after its discov-ery, fetuin has been shown to inhibit trypsin.28 Both positiveand negative fetuin interactions with proteases are docu-mented, including matrix metalloproteinase-9,29 matrixmetalloproteinase-3,30 meprin metalloproteinases,31 the cys-teine proteases m-calpain,32 cathepsin L in rat bone,33 andinhibition of recombinant human cathepsin V. Proteinaseinteractions are thought to be involved in the regulation oftumorigenesis and tumor progression.34,35

Fetuin markedly accelerates incorporation of exogenousfatty acids into cellular triglycerides.36–39 Thus, fetuin may

644

HRG 19 Cystatin 1 Cystatin 2 Pro-rich His / Pro-rich 525

KNG (LK) 19 Cystatin 1 Cystatin 2 Cystatin 3 434

KNG (HK) 19 Cystatin 1 Cystatin 2 Cystatin 3 His-rich HK light chain

Bradykinin

0 100 200 300 400 500 600 aa

FETUB 16 Cystatin 1 Cystatin 2 Domain 3 382

AHSGFETUA 19 Cystatin 1 Cystatin 2 Domain 3 367

Figure 1. Schematic structure of type 3 cystatins. Domain organization with indications of disulfide bridges, cystatin domains, and theHis/Gly and His/Pro domains in kininogen (KNG) and histidine-rich glycoprotein (HRG), respectively. Molecular models of cystatindomains showing negative charge in red and positive charge in blue. The amino-terminal cystatin domain 1 in fetuin-A is particularlyrich in acidic residues that mediate mineral binding to calcium-rich phases of calcium phosphates.

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share a function with fatty acid binding proteins, a family ofabundantly expressed 14-kDa to 15-kDa proteins. Like fetuin,fatty acid binding proteins reversibly bind hydrophobic li-gands, including saturated and unsaturated long-chain fattyacids, and other lipids with high affinity.40

Because of their rich complex glycosylation pattern, fe-tuins serve as model substances for lectin and glycoproteinresearch. Lectin binding should always be seriously consid-ered when fetuin binding to cells and to the extracellularmatrix is studied. On a practical note, the strong binding ofpertussis toxin to terminal sialic acid residues in fetuin formthe basis of a Food and Drug Administration-approvedpertussis toxin test.41,42 Fetuin-A sequestration of lectinsproved a major complication in experimental cytotoxic ther-apy using cancer cell–specific antibodies coupled to theRicinus communis agglutinin ricin.43 The immunotoxins wererapidly cleared by the asialoglycoprotein receptor and causedliver toxicity.44

In a search for natural transforming growth factor-beta(TGF-�) receptor antagonists, a sequence homology wasfound between TGF-� receptor type II and fetuin-A.45 TheTGF-� receptor II homology 1 domain from fetuin boundpreferentially to bone morphogenetic protein (BMP)-2. Full-length fetuin-A bound directly to TGF-�1 and TGF-�2 andwith greater affinity to the TGF-�–related BMP-2, BMP-4,and BMP-6. Finally, and likely impinging on fetuin’s role inmineralization biology, fetuin or neutralizing anti-TGF-�antibodies blocked osteogenesis and deposition of calcium-containing matrix in mineralizing cell cultures.46 An alteredbone phenotype in fetuin-A–deficient mice (Ahsg�/�) wasexplained accordingly in terms of failure to block TGF-�–dependent signaling in osteoblastic cells.47 Tumorigenesisexperiments using Ahsg�/� mice further supported the hy-pothesis that fetuin-A is an antagonist of TGF-� in vivo, inthat it inhibited intestinal tumor progression.48

Antiinflammatory Role of Fetuin-AFetuin-A, one of the most abundant fetal plasma proteins, wasfound to be essential for the inhibition of the proinflammatorycytokine tumor necrosis factor production by spermine and itssynthetic analogues.49,50 Accordingly, the strong fetal expres-sion of fetuin and spermine have been associated with thetolerance of the fetus, “nature’s transplant,” by mothers.51

The strong antiinflammatory effects of fetuin were verified invivo using several models of inflammation, includinglipopolysaccharide-induced miscarriage in rats,52 carrageenaninjection,53 cerebral ischemic injury in rodents,54 and cecalligation and puncture in mice.55 In all cases fetuin-A wasassociated with reduced inflammatory response and increasedsurvival, and administering additional fetuin generally im-proved outcome.

Thus fetuin-A generally may be regarded as antiinflam-matory.56 The antiinflammatory property of serum �2-HSglycoprotein/human fetuin-A was further supported by thedemonstration that fetuin-A is a potent and specificcrystal-bound inhibitor of neutrophil stimulation by hy-droxyapatite crystals.57 Calcium phosphate crystals induceproinflammatory cytokine secretion through the NLRP3inflammasome in monocytes/macrophages,58 – 60 cell deathin human vascular smooth muscle cells,61 and cell activa-tion in chondrocytes.62– 64 Antiapoptotic activity offetuin-A has been observed in smooth muscle cells65 anddampening of the cell-specific responses would generallybe expected to alleviate the detrimental consequences oflocal inflammation, cell death, and cartilage degradation.The proven protective function of fetuin-A in many animalmodels of inflammation,52–55 the inhibition of proinflam-matory compounds,50,51,66 – 68 and the inhibition of crystal-induced neutrophil activation57 collectively suggest thatfetuin-A may generally protect during pathological miner-alization as well.69

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Figure 2. Cartoon of human fetuin-A/�2-HS glycoprotein showing cystatin-likedomains 1 and 2 and a third unrelateddomain in green, yellow, and blue shad-ing, respectively. The cotranslational andposttranslational modifications disulfidebridges (C-C in yellow) Ser-phosphoryla-tion sites (S in red), protease-sensitivesites (R-K, dibasic tryptic cleavage site;L-L, chymotryptic cleavage site; R-T,furin-sensitive cleavage site; all in green),allelic variants (T230/T238; M230/S238)depicted as orange asterisks, and Asn-linked complex N-glycosylation sites, andSer/Thr O-glycosylation sites depicted asblue symbols, respectively. Data takenfrom.6,7,13,96



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Fetuin-A in Metabolic SyndromeSeveral clinical studies proposed that high-serum fetuin-Alevels are associated with metabolic syndrome (MetS) andthat fetuin-A therefore may present a risk factor forMetS.70–73 The association of fetuin-A, insulin signaling,diabetes type 2, and MetS dates back to a publication in 1989stating that pp63, a liver-secreted phosphoprotein, inhibitedinsulin receptor and downstream substrate phosphorylationsignaling.17 The cDNA sequence of pp63 was identified as ratfetuin-A,74 and it was disputed whether the activity tested inthe original article was actually rat fetuin-A or a copurifiedprotein.75,76 We tested authentic human fetuin-A and foundminor, if any, inhibitory activity at the insulin receptor anddownstream substrate level.74 We analyzed phosphotyrosinemodification of cellular proteins in rat fibroblasts expressingthe human insulin receptor and could not detect any robustand reproducible inhibition of insulin signaling by human orbovine fetuin including phosphofetuin-A immunoaffinity pu-rified from HepG2 cells. Testing of fetuin-A in relevant cellsshowed no classical insulin receptor inhibition, but showedunexplained downregulation of insulin-stimulatedElk1-phosphorylation.77

In another series of experiments, commercial bovine fetuinand supernatants of baculovirus-infected insect cells express-ing human fetuin-A did inhibit insulin receptor signaling.Furthermore, fetuin-A was shown by immunoprecipitation todirectly interact with the insulin receptor.18,78 These research-ers also showed that fetuin-A–deficient mice maintained on amixed genetic background of C57BL/6N and 129Sv hadimproved insulin response, resistance to weight gain, protec-tion against obesity, and protection against insulin resistanceassociated with aging.79 We back-crossed these mice thatwere originally generated in our laboratory80 onto pureC57BL/6N as well as DBA/2 genetic background, and wecould not detect evidence of diabetes-associated symptoms inthe genetically more homogeneous mice. The genetic back-ground of mice and even substrains of mice differ withrespect to insulin sensitivity. A deletion variant of nicotin-amide nucleotide transhydrogenase that has spontaneouslyoccurred in C57BL/6J, but not in C57BL/6N strains, isthought to influence insulin signaling.81,82 Therefore, siblingmice from the same colony should be ideally used whencomparing the influence of single gene deletions on insulinsensitivity. Mice with homogeneous defined genetic back-ground were not yet available at the time of the first study inthe case of fetuin-A–deficient mice.83

Thus, it is unclear if fetuin-A is a cause or consequence ofhigh-caloric feeding in mice or whether the association ofhigh-serum fetuin-A and MetS is a bystander phenomenon.First, fetuin-A is a highly expressed constitutively secretedhepatic serum glycoprotein. Using nuclear run-on assays,LeCam et al84 determined that the rat fetuin-A/pp63 promoterstrength was approximately three-times to four-times that ofthe strong liver-specific albumin promoter. Second, fetuin-Ais traditionally regarded as one of the few negative acutephase proteins55,85,86 and strong associations between low-serum fetuin-A levels and inflammatory markers likeC-reactive protein have been published.87–89 Thus, downregu-lation rather than upregulation of fetuin-A during “fat inflam-

mation” in obesity-related insulin resistance would be ex-pected.90 Therefore, it seems counterintuitive that fetuin-Alevels would be increased in MetS. Recent work showed thatfetuin-A induced inflammatory cytokines,91 and that one ofthe major mediators of inflammatory cytokine action, NF�B,further upregulated hepatic fetuin-A synthesis.39 The conflict-ing data on fetuin-A regulation clearly need to be reconciledin terms of timing of events and causal relationships vs mereassociation. The association of fetuin-A serum levels withMetS and type 2 diabetes in humans rests on single measure-ments, and most studies did not correct for a major con-founder, total liver protein synthesis. Therefore, we suggestthat association studies should at least be normalized to serumalbumin as an indicator of total liver protein synthesis, unlessserum albumin should also be called a risk factor of MetS.

Role of Fetuin-A in Mineralization BiologyWe started working on fetuin-A as part of an ongoingresearch project studying the structure and function of type 3members of the cystatin superfamily.6,92–96 At that time,fetuin-A/�2-HS glycoprotein was a “structure in search of afunction.” Intrigued by the description of rat fetuin as anatural inhibitor of insulin receptor kinase inhibitor,17 and bythe report on the identity of fetuin of hemonectin, a proteinpresent in rabbit bone marrow extracellular matrix and asuspected homing factor for myeologenic cells in the bonemarrow,97 we studied fetuin-A binding to cell membranes andextracellular targets. Binding to mineralized bone matrix asdescribed by Triffitt et al98–100 turned out to be the mostrobust binding phenomenon by far. The high affinity offetuin-A for bone mineral mediates selective fetuin-A accu-mulation from plasma into bone.98,99,101 Fetuin-A in boneaccounts for 25% of the noncollagenous proteins and thus isone of the two most highly abundant noncollagenous pro-teins.102 We showed that fetuin-A also has a particularly highaffinity to nascent apatite mineral and is an inhibitor of denovo apatite formation from supersaturated mineral solu-tions.19 The inhibitory effect is mediated by acidic aminoacids clustering in cystatin-like domain D1. This fundamentalobservation formed the base of two decades of fetuin-Amineralization research and has been successfully repeated bymany independent laboratories. In full accord with thisbiochemical finding, genetic work using fetuin-A–deficientmice80,103 likewise suggested that fetuin-A is a mineralchaperone104 mediating the transport of mineral from theextracellular space and the general circulation.103,105,106

Fetuin-A–Mineral Complexes, CalciproteinParticles, Calcifying Nanoparticles:Fetuin–Mineral Complexes, CalciproteinMonomers, Calciprotein ParticlesFetuin-A binds calcium phosphate and calcium carbonatewith high affinity, and it binds magnesium phosphate lesswell.19 Importantly, fetuin-A only inhibits the de novo for-mation of calcium phosphate and does not dissolve preformedmineral. Fetuin-A is an inhibitor of mineralization in solutionand of cell-mediated mineralization in that it regulates theprocess of matrix mineralization in rat calvaria osteoblasticcells.19 On addition of �-glycerophosphate and ascorbate,



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these cells formed mineralized bone nodules in DMEMculture medium containing 10% fetal calf serum or bovinefetuin-A or human �2-HS glycoprotein. However, when thecells were maintained serum-free and serum albumin was thesole bulk protein present in the medium, the cells catastroph-ically calcified and died.19 Fetuin-A inhibition of lethalcalcification was dose-dependent in that higher concentra-tions of fetuin more consistently inhibited calcification thanlower concentrations. Fetuin-A similarly inhibited vascularsmooth muscle cell calcification induced by elevated concen-trations of extracellular mineral ions.65 This was achieved inpart through inhibition of apoptosis and caspase cleavage.Fetuin-A was internalized by vascular smooth muscle cellsand concentrated in intracellular vesicles. Subsequently,fetuin-A was secreted via vesicle release from apoptotic andviable vascular smooth muscle cells. The presence offetuin-A in vesicles abrogated their ability to nucleate cal-cium phosphate precipitation. In addition, fetuin-A enhancedphagocytosis of vesicles by vascular smooth muscle cells.These observations showed that the uptake of fetuin-A bycells and the vesicular recycling of fetuin–mineral complexesreduced both apoptosis and calcification in live cells sub-jected to elevated extracellular concentrations of mineralions.107 Therefore, the stabilization of mineral solutionsmediated by fetuin-A in test tube assays19 was also effectiveinside living cells, thus constituting a strong defense mecha-nism against pathological mineralization.

Fetuin-A stabilizes supersaturated mineral solutions byforming soluble colloidal nanospheres.5 Similar nanoparticleswere detected in the ascites fluid of a pertioneal dialysispatient experiencing calcifying sclerosing peritonitis.108,109

Figure 3 shows electron micrographs of nanoscopic protein–mineral particles generated in the laboratory (Figure 3A, B) ortaken from the patient’s ascites fluid (Figure 3C). These colloidsare variously termed calciprotein particles in analogy to lipopro-tein particles5,19,109–113 or fetuin–mineral complexes114–118 or,most recently, calcifying nanoparticles119 or nanons.120,121

Aspartic acid and glutamic acid residues located in theamino-terminal cystatin-like domain D1 are required for theinhibitory activity of fetuin-A.5,19 Using a wide array ofhigh-resolution spectroscopic techniques including small-angle neutron scattering, small-angle X-ray scattering, dy-namic light scattering, transmission electron microscopy, andconventional photometry, it was determined that the forma-tion of calciprotein particles is a multistep process.

Figure 4 depicts schematically the sequence of calciproteinparticles/fetuin–mineral complexes formation, starting with

the spontaneous formation of prenucleation clusters frommetastable solutions, their sequestration to acidic asparticacid, and glutamic acid side chains in the cystatin-like domain1 of fetuin-A–forming mineral-laden fetuin-A monomer orcalciprotein monomer. Calciprotein monomer was discoveredwhen quantitative small-angle neutron scattering data analy-sis revealed that even at a fetuin-A concentration close to thestability limit of supersaturated salt solutions, only approxi-mately one-half of the mineral ions and only 5% of thefetuin-A were contained in the calciprotein particles. Theremaining supersaturated mineral ion fraction and 95% ofnoncalciprotein particles of fetuin-A were associated with amineral-laden fetuin-A monomer fraction that could be sep-arated from mature calciprotein particles by ultrafiltrationthrough a 300-KDa cut-off membrane. Small-angle neutronscattering analysis showed that the fetuin-A monomer in thisfraction was closely associated with coalesced subnanome-ter-size mineral ions clusters reminiscent of Ca9(PO4)6 Pos-ner clusters (Figure 4A).111

Fetuin-A binds to apatitic mineral surfaces, as shown inFigure 4C. Fetuin-A does not influence the formation ofmineral nuclei. However, fetuin-A prevents the growth andaggregation of nuclei to larger entities and ultimately mineralprecipitation.112 Thus, fetuin-A effectively shields spontane-ously formed mineral nuclei, leading to transiently stablecalciprotein particles.

A hierarchical role of fetuin-A was established in thatfetuin-A was critically required during the early steps ofcalciprotein particles formation and stabilization and thatother acidic plasma proteins including serum albumin furtherstabilized the initial colloids and could substitute for fetuin-Aat later stages of calciprotein particles formation.109,122 Theselater stages were studied in detail using small-angle neutronscattering. Figure 4D illustrates the aggregation of manycalciprotein monomers and other mineral nuclei into tran-siently stable primary calciprotein particles of initially �100nanometers in diameter. After a lag period, these particlesgrow into elongated and more crystalline particles of approx-imately twice the initial size, termed secondary or maturecalciprotein particles (Figure 4E). Thus, formation and mat-uration are two separate and successive processes followingthe principles of Ostwald ripening.5,110–112 Similar particlesmay be generated with other acidic macromolecules as well.However, fetuin-A is exceptionally potent regarding activityand specificity of inhibition. Increased fetuin-A concentrationleads to smaller particles. An increased temperature, mineralion concentration, and a reduced fetuin-A concentration,

Figure 3. Electron microscopic pictures of syn-thetic and patient-derived calciprotein particles(CPP). A, Primary calciprotein particles have adiameter of 30 nm to 150 nm and contain amor-phous mineral.5 B, Subsequently, the primary CPPwere subjected to a major structural rearrange-ment. The resulting secondary CPP consist of acore of crystalline basic calcium phosphate, whichis covered by a layer of fetuin-A (A and B:50 �mol/L bovine fetuin-A, 10 mmol/L CaCl2,6 mmol/L Na2HPO4, pH 7.4).113 These particles arestable for days at 37°C. C, Similar particles have

been detected in ascites of patients with sclerosing calcifying peritonitis.108,109 Bars represent 100 nm.



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respectively, all accelerate the particle ripening process,demonstrating that calciprotein particle maturation followsthe Arrhenius law. Secondary calciprotein particles are sta-bilized by a compact outer fetuin-A monolayer against furthergrowth (Figure 4E) for up to 30 hours at body temperature,which is ample time for clearing of calciprotein particles fromcirculation.

The formation and maturation of calciprotein particles canbe followed by optical monitoring110 of mineralizing solu-tions. Supplemental Figure I (available online at http://www.circresaha.org) shows dynamic light scatter diagramsillustrating that the transformation from primary to secondarycalciprotein particles is fetuin-A concentration-dependent.Higher fetuin-A concentrations cause prolonged stability ofboth calciprotein monomers and calciprotein particles, andthus a right-shift in the transformation curves. A clinical testcould use the kinetic parameters of the precipitation reaction,thus measuring the overall calcification risk in biologicalfluids, eg, in the blood of dialysis patients.

Protein–mineral complexes are soluble precursors of phys-iological mineralization of bones and teeth as well. Recentresearch has shown that biomineralization starts with amor-phous mineral–protein complexes containing fetuin-A.123–126

Price et al127–129 have shown that fetuin-A is criticallyrequired to direct mineralization to the interior of syntheticmatrices that have size exclusion characteristics similar tothose of collagen. Fetuin-A does so by selectively inhibitingmineral growth outside of these matrices. This mineralizationby inhibitor exclusion is likely redundant, because fetuin-A–deficient mouse bone is mineralized perfectly well. The micenevertheless show a bone phenotype of stunted femur length,indicating premature growth plate mineralization and dis-turbed osteogenic signaling.47

Nanobacteria: A Red Herring From MarsNanobacteria initially described nanoscopic life forms de-tected by electron microscopy in rock sediments.130 Similarentities were discovered in meterorites from Mars131 and,finally, in cell culture.132 Nanobacteria attracted a lot ofscientific and economical attention as a causal agent of majordiseases, including vascular calcification, kidney stones, andcancer.132–136 In the year 2000, Cisar et al137 determined thatsimple mixtures of phospholipids and calcium phosphatecrystals closely resembled nanobacteria in that they showedlife-like growth and replication. Nucleic acid sequencespreviously thought to be diagnostic markers of nanobacteriawere in fact diagnostic of common laboratory contami-nants.137 Two groups of researchers finally solved the riddleof pathological mineralizing nanobacteria.120,138 A series ofexperiments on the origin of putative nanobacteria showedthat calcium phosphate together with proteins and furthernonmineral compounds formed nanoparticles that resemblednanobacteria in shape and behavior.139 Minerals containingnanoparticles had a high binding capacity for charged mole-cules, including ions, carbohydrates, lipids, and nucleic acids.Depending on the exact composition, mixtures of mineral andnonmineral compounds sustained either crystallization ofhydroxyapatite mineral or the formation of complex protein–mineral complexes.122,140 It was found that the main proteincomponent of the nanobacteria was fetuin-A.120 Besidesfetuin-A, serum albumin and apolipoproteins were also iden-tified;121 hence, the term nanobacteria was exchanged for themore apt term calcifying nanoparticles, virtually identicalwith the protein–mineral complexes, calciprotein particles, orfetuin–mineral complexes described before.

Calciprotein Particles Metabolism and ClearingFigure 5 illustrates the putative metabolism of calciproteinparticles, soluble protein–mineral complexes, which are now

Figure 4. Formation of primary and sec-ondary calciprotein particles (CPP) fromfetuin-A monomer and amorphous mineralprecursors. A, Hydroxyapatite precursorviewed along the [001] axis. The dottedlines confine the hexagonal unit cell. Thecircle represents a Ca9(PO4)6 Posner clus-ter as a building block of the apatite struc-ture. B, A three-dimensional view of fivePosner clusters juxtaposed to the acidic�-sheet of the computer-modeled ami-noterminal fetuin-A domain D1. Thedomain surface is plotted semitransparentwith negative charge indicated in red andpositive charge indicated in blue. C, Acomputer-generated homology modelstructure illustrating the fetuin-A–mineralinterface. The fetuin-A protein structurewas modeled after published structures forcystatin-like domains 1 (green) and 2 (yel-low).176 Domain 3 (blue) was modeled afterthe Escherichia coli protein malonyl-CoA:acyl carrier protein transacylase(1MLA).177 Fetuin-A does not influence the

formation of mineral nuclei. However, fetuin-A does prevent the rapid growth and aggregation of nuclei and, thus, mineral precipita-tion.112 D, Primary CPP are spherical and rather unstructured agglomerates of mineral (clusters) and fetuin-A, whereas (E) secondaryCPP consist of a crystalline mineral core covered by fetuin-A. In conclusion, fetuin-A effectively shields the mineral phase, leading tostable CPP that can be transported and cleared, as shown in Figure 5. The computer graphics were generated using the softwarepackages VESTA,178 VMD,179 and APBS.180



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regarded as a physiological byproduct of mineral metabolism.The scheme is partly hypothetical and requires experimentalverification. Soluble protein–mineral complexes includingfetuin-A have been detected in serum from etidronate over-dosed rats,118 in adenine-treated rats,141 in peritoneal dialysispatients with sclerosing calcifying peritonitis,108,109 and indialysis patients.142 The scheme is modeled after lipoproteinparticle metabolism, a well-known transport system for oth-erwise insoluble lipids like cholesterol esters.

The basic tenet of calciprotein particles metabolism holdsthat fetuin-A stabilizes mineral complexes and at the sametime mediates their transport and clearing. Fetuin-A should beregarded as an opsonizing serum protein with a high affinityfor mineral complexes and debris. The opsonizing propertiesof fetuin-A have been determined several decades ago.143–145

Fetuin-A affects microparticle phagocytosis by dendriticcells,146 phagocytosis of apoptotic cells by macrophages,147

and opsonizes phospholipid particles.148

Fetuin-A is, however, not generally sticky like the “big 12”plasma proteins, which adhere to most materials in blood

contact,149 and fetuin-A was also not listed among specificproteins interacting with Sepharose beads in a study that usedquantitative mass spectrometry to determine bead pro-teomes.150 Therefore, binding may be restricted to a narrowrange of cationic, lipidic, or mineral ligands, and a few moreinteracting TGF-�–related cytokines mentioned earlier in thistext. Given the high abundance of fetuin-A in plasma, anyclearing mechanism would have to rely on conformational orstructural changes in fetuin-A or on multivalent binding thatturns low-affinity binding into high-avidity binding.

Uptake of fetuin-A by cultured human vascular smoothmuscle cells has been demonstrated,65,107,151 but the exactform of fetuin-A was not determined. Clustering of fetuin-Amolecules on the surface of secondary calciprotein particlesas demonstrated111 ideally fulfills the ligand clustering re-quired to increase binding strength. Our preliminary results offetuin-A monomer and fetuin-A containing calciprotein par-ticle clearing in vivo show that calciprotein particles arecleared vastly more efficiently than fetuin-A monomer. Nev-ertheless, specific receptors for calciprotein particle clearingdiscriminating against fetuin-A monomer remain to bedetermined.

Fetuin-A Knockout PhenotypeThe use of animal models has uncovered major inhibitors ofpathological calcification and their mode of action.152

Fetuin-A is one of the major systemic inhibitors of patholog-ical mineralization, but it is not the only one. This is vividlyillustrated by the history of fetuin-A–deficient mice (Ahsg�/�).While establishing that fetuin-A is a regulator of mineraliza-tion,19 we also generated Ahsg�/� mice to test this hypothesisin an animal model. For technical reasons, the first Ahsg�/�

mice had a mixed 129Sv � C57BL/6 genetic backgrounddesignated 129,B6Ahsgtm1mbl.80 Out of an initial colony ofapproximately 200 mice, only approximately 10 femaleex-breeders showed a spontaneous calcification phenotype.80

All other mice were apparently normal. Force-feeding supra-physiological doses of active vitamin D (calcitriol) and ahigh-mineral diet (phosphate-rich) resulted in nephrocalcino-sis and pulmonary and myocardial calcification of thesemice.103

Unsatisfied by this highly variable yet low-penetratingcalcification phenotype, we decided to combine the fetuin-Adeficiency with the calcification-prone DBA/2 genetic back-ground.153–155 For comparison, we also backcrossed the miceonto the widely used calcification-resistant genetic back-ground C57BL/6 and thus generated two more Ahsg�/�

mouse strains, D2-Ahsgtm1wja and B6-Ahsgtm1wja. The lattermice behaved like the mixed genetic background mice in thatthey scarcely had any spontaneous calcification. They did,however, have calcification when challenged with hemine-phrectomy and a high-mineral diet.105,106 In stark contrast,virtually all D2-Ahsgtm1wja mice show spontaneous massivesoft tissue calcification throughout their bodies.103 This strainis arguably the most calcifying mouse strain in existence inthat the myocardium of D2-Ahsg�/� mice contains almostone-tenth the calcium content of normal bone.156 Kidney,lung, skin, brown fat, pancreas, and reproductive organs areequally strongly calcified.

Figure 5. Hypothetical pathway of calciprotein particle (CPP)circulation and clearing by endothelial cells and tissues residentphagocytes. Fetuin-A stabilizes CPP in the circulation and medi-ates their efficient uptake. In healthy individuals, CPP may formspontaneously and be present in small numbers throughout alltissues, or may occur substantially where bone resorption byosteoclasts (OC) is releasing high levels of calcium and phos-phate by acid-mediated mineral dissolution. CPP may be trans-ported in the blood or retrieved locally after nearby osteoclasticresorption, and are used locally by osteoblasts (OB) during boneformation. When mineral homeostasis is severely disturbed, eg,in dialysis patients or in severely etidronate-overdosed rodents,bouts of hypercalcemia and hyperphosphatemia will result in theformation of high numbers of CPP and fetuin-A will be con-sumed in the process as part of the CPP complex. CPP in theinterstitial spaces will be cleared by monocytes (MC) or macro-phages (MP) or by any other endocytosing cell types. Overlyabundant CPP may overwhelm the clearing capacity of thereticuloendothelial system phagocytes and may result in apo-ptosis of endothelial cells (EC), MP, and smooth muscle cells(SMC), and in deposition of calcified apoptotic cell remnants.This pathway is hypothetical and needs to be experimentallyverified. It may also be of significance that many inhibitors ofcalcification activate monocytes/macrophages and stimulatephagocytosis; therefore, this mode of calcified remnant clear-ance may be of similar importance. Reproduced from Jahnen-Dechent W, Schafer C, Ketteler M, et al. Mineral chaperones: arole for fetuin-A and osteopontin in the inhibition and regressionof pathologic calcification. J Mol Med. 2008;86:379–389.



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Supplemental Figure II (available online at http://www.cir-cresaha.org) shows random examples of 11-month-old wild-type or Ahsg�/� mice maintained against the genetic back-ground C57BL/6 or DBA/2. Even from the low-resolutionradiology photographs, the uniformity of genetic penetranceand the extent of the calcification in D2-Ahsg�/� mice can bereadily appreciated. Kidney and skin calcification are easilyvisible. Details may be glanced at higher magnifications ofthe electronic pictures provided with this article. We point outthat the pictures were taken of live anesthetized mice.Therefore, myocardium, lung, and pancreas calcification areinvisible or blurred because of breathing motion of the chestregion. Surprisingly, the soft tissue calcification is not lethallike the arterial calcification of matrix GLA protein–deficientmice.157 Calcification does, however, greatly compromisereproduction in mice aged 6 months and older. The 1-yearsurvival rate of D2-Ahsg�/� mice in our specified pathogen-free colony is 60% to 70% of all born pups, compared to 90%to 95% in DBA/2 wild-type mice.

These results showed that fetuin-A is a systemic andsoluble inhibitor of pathological mineralization that isbacked-up by other genetic factors rendering mouse strainsprone to or resistant to dystrophic calcification. Integrativegenomics of the so-called Dyscalc locus led to the identifi-cation of Abcc6 as the major gene determining dystrophiccardiac calcification.158 Abcc6-deficient mice have soft tissuecalcifications develop, like D2-Ahsg�/� mice, albeit they areless extensive in both localization and extent. Interestingly,Abcc6 deficiency is associated with reduced plasma fetuin-Alevels and calcifications can be partially corrected by over-expressing fetuin-A,159 suggesting that fetuin-A acts down-stream or in concert with Abcc6. Despite heavy early-onsetand life-long progressing dystrophic calcification inAbcc6�/� and especially in D2- Ahsg�/� mice, true osteo-genesis involving clearly identifiable osteochondrocytic cellshas never been reported in these mouse models of calcifica-tion. This casts doubt on the now popular hypothesis thatpathological calcification is always a form of ectopic osteo-genesis. More likely, both genes seem to be involved insystemic mineral homeostasis and transport. Therefore, wehave termed fetuin-A a mineral chaperone mediating thesolubilization, transport, and elimination from circulation ofotherwise insoluble minerals, much like apolipoproteins helpin lipid transport and metabolism.104

Because fetuin-A is highly expressed in bone and accountsfor 25% of all noncollagen proteins of bone (noncollagenous

proteins),102 we examined bone growth and remodeling phe-notypes in mixed background 129,B6-Ahsg�/� mice.47 Theskeletal structure of these mice appeared normal at birth, butabnormalities were observed in adult 129,B6-Ahsg�/� mice.Maturation of growth plate chondrocytes was impaired, andfemurs lengthened more slowly between 3 and 18 months ofage. Previously, it had been found that fetuin-A is a solubleTGF-�/BMP-binding protein controlling cytokine access tomembrane signaling receptors.45,46,160 Hence, the altered bonephenotype was explained in terms of failure to block TGF-�–dependent signaling in osteoblastic cells. Mice lackingfetuin-A displayed growth plate defects, increased boneformation with age, and enhanced cytokine-dependentosteogenesis.47

In view of the mineralization regulation effected byfetuin-A, we revisited the bone phenotype of pure-bredB6-Ahsg�/� mice. These mice are unaffected by the second-ary hyperparathyroidism and osteoporosis typical of D2-Ahsg�/� mice.103 We essentially reproduced all major find-ings of the previous study performed in 129,B6-Ahsg�/�

mice.47 The mice displayed normal trabecular bone mass inthe spine but increased cortical thickness in the femur. Bonecomposition and mineral and collagen characteristics ofcortical bone were unaffected by the absence of fetuin-A. Thelong bones, especially femora, were severely stunted inB6-Ahsg�/� mice compared to wild-type littermates, result-ing in increased biomechanical stability. In addition, wedetermined increased mineral content in the growth plates ofB6-Ahsg�/� long bones, corroborating its physiological roleas an inhibitor of excessive mineralization in the growth platecartilage matrix, a site of vigorous physiological mineraliza-tion. We thus demonstrated that growth plate chondrocytesare prone to “pathological calcification” in the absence offetuin-A, and that active mineralization inhibition is a neces-sity for proper long bone growth, at least in mice.

The fact that three of the most potent anticalcificationcompounds, matrix GLA protein,157 pyrophosphate,161 andfetuin-A, all operate in and around growth plate chondrocytessuggests that this cell type is physiologically extremely proneto premature calcification. The pathomechanism of osteo-genic vascular calcification likewise critically requires chon-drogenic, but not necessarily osteoblastic, differentiation ofvascular cells.162–164 It will be interesting to revisit theinfluence of fetuin-A alone and in combination on chondro-cyte mineralization in a way similar to that previouslyperformed with primary osteoblasts19 and smooth muscle

Table 1. Fetuin-A Bioactivities With References

Fetuin-A Bioactivity Biochemical and Cell Culture Work Animal Study Clinical/Observational Study

Proteinase interaction 28–32 34, 35

Lipid binding 36–39

Lectin binding 41–43 44

TGF-� antagonism 45, 46 47, 48

Antiinflammatory activity 49, 50, 56, 57 52–55

Insulin receptor antagonism, role in MetS 17, 18, 78 79, 83 70–73

Calcification Inhibition 5, 19, 65, 109–121, 127–129 47, 80, 103, 105, 106, 118, 141, 156, 159 88, 89, 103, 108, 142, 169–171

MetS indicates metabolic syndrome; TGF, transforming growth factor.



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cells65 as models of physiological and pathological mineral-ization, respectively.

Clinical Epidemiology of Serum Fetuin-A Levelsand PolymorphismsApproaching the clinical situation in humans and based onthe outlined experiments, it is strongly suggested to generallyview serum fetuin-A levels in combination with serumalbumin as an indicator of nutritional state, as anothernegative acute phase protein, and as an indicator of overallhepatic protein synthesis, especially in dialysis patients.Patients in advanced stages of chronic kidney disease (CKD)clinically have the most serious cardiovascular and soft tissuecalcifications develop, more than any other population, and itis now well-understood that the individual magnitude ofcalcification significantly corresponds with impaired survivalin CKD.165–167 Furthermore, CKD patients tend to be in astate of malnutrition and microinflammation or macroinflam-mation, or both, in which downregulation of proteins such asfetuin-A may be expected.168

When the biochemical properties of fetuin-A became moreand more apparent, it was a straightforward approach toobserve the relationship between serum fetuin-A levels andoutcomes in large dialysis populations. In a cohort of �300hemodialysis patients, the lowest tertile of serum fetuin-Alevels was associated with significantly increased all-causeand cardiovascular mortality.89 Fetuin-A was also found to beinversely correlated with C-reactive protein levels, emphasiz-ing its nature as a negative acute phase reactant. These datawere subsequently confirmed, demonstrating mortality riskprediction by fetuin-A deficiency in nearly 300 incidentdialysis patients (including patients using peritoneal dialy-sis).169 In this study, a specific fetuin-A gene polymorphism(Thr256Ser) was shown to predict particularly low fetuin-Alevels and to be associated with an adverse prognosis com-pared to patients carrying alternative polymorphisms. Hy-poalbuminemia was strongly correlated with fetuin-A defi-ciency, suggesting the expected involvement in themalnutrition-inflammation-atherosclerosis syndrome in clin-ical fetuin-A deficiency. In a pure cohort of prevalentperitoneal dialysis patients, fetuin-A deficiency was alsoshown to be linked to features of the malnutrition-inflammation-atherosclerosis syndrome (low albumin, ele-vated C-reactive protein) and to cardiovascular events andmortality, respectively.88 Moreover, this study demonstratedan association between low fetuin-A levels and the magnitudeof valvular calcification, emphasizing the hypothesized linkbetween progressive calcification and impaired outcomes. Ina smaller cohort of hemodialyis patients, a significant asso-ciation between coronary calcification and fetuin-A defi-ciency was shown.170 Taken together, these observationsindicate that fetuin-A deficiency may be an importantinflammation-related link between cardiovascular calcifica-tion and mortality in patients using dialysis.

In patients with normal renal function as well as inpredialysis CKD patients, the available information on therelationship between fetuin-A levels, the degree of calcifica-tion, and mortality is less clear. Recently, we have shown inthe first prospective longitudinal study that low baseline

serum levels of fetuin-A are associated with the increase ofaortic valve calcification in 77 patients.171 However, in nearly1000 patients from the Heart and Soul study focusing onpatients at cardiovascular risk, mostly without renal dysfunc-tion, high fetuin-A levels were found to be strongly associ-ated with hyperlipidemia and features of the metabolicsyndrome, but not with hard outcome parameters.172 Inpatients with diabetes mellitus spanning CKD stages 1 to 4,the magnitude of coronary artery calcification correlated withincreased rather than with decreased fetuin-A levels.173 Thisfinding may be specific for a pure diabetic cohort in which adifferent pattern of calcium-regulatory systems may be im-plicated and in which downregulation of fetuin-A may bepartially compensated or masked by overnutrition. However,these data may imply that fetuin-A upregulation initially actsas a systemic defense mechanism (early warning system)trying to protect from or counteract against vascular calcifi-cations in their early stages. Such an interpretation is indi-rectly supported by immunohistochemical findings showingstrong fetuin-A deposition, but not synthesis, in areas ofvascular calcification65,170 and may be better-understood oncethe metabolism of fetuin-A mineral complexes (calciproteinmonomers, calciprotein particles/fetuin–mineral complexes)and concomitant fetuin-A serum depletion is known. A recentstudy suggested that serum measurements of fetuin-A have totake into account that mineral-bound fetuin-A may competewith free fetuin-A, and that centrifuge sedimentation of serumsamples may considerably influence certain assay readoutsbut not others.142

When the calcification burden increases beyond a certainpoint in chronic long-term CKD, compensatory systems suchas fetuin-A release may finally become exhausted and con-secutive fetuin-A deficiency may start a vicious cycle of evenmore progressive extraosseous calcification and fetuin-Aconsumption. If this hypothesis of initial adaptive fetuin-Aoverproduction and later exhaustion of the system is correct,then future research should attempt to identify factors influ-encing fetuin-A expression and secretion.

Fetuin-A Consumption and Downregulation inCalciphylaxis/Calcific Uremic ArteriolopathyPerhaps the most intriguing experimental example offetuin-A consumption was published by Price et al,118 whodemonstrated transient increases of total serum calcium up to10 mmol/L by high-dose vitamin D treatment in rats withina few hours and a return to normocalcemia within 1 day. Aconcomitant loss of half the serum fetuin-A that formed ahigh-molecular-weight fetuin–mineral complex was ob-served, and it was probably cleared by the reticuloendothelialsystem or deposited in the bone or in extraskeletal sites.115 Apotential clinical example of fetuin-A consumption and ex-haust may be calciphylaxis (calcific uremic arteriolopathy), arare but potentially life-threatening syndrome characterizedby progressive and painful skin ulcerations associated withmedia calcification of medium and small cutaneous arterialvessels.174,175 Calciphylaxis primarily affects patients usingdialysis or after renal transplantation. We reported fetuin-Adeficiency with very low serum levels in a case series ofcalciphylaxis patients.103 To demonstrate the functional cal-



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cification inhibitory capacity of fetuin-A deficiency, we usedsera from calcific uremic arteriolopathy patients in an ex vivo[45]CaCl2 radioisotope assay, which enables quantification ofserum-induced inhibition of a calcium phosphate precipita-tion. These fetuin-A–deficient calcific uremic arteriolopathysera were significantly less effective at inhibiting calciumphosphate crystal formation than sera from healthy subjectswith appropriate fetuin-A concentrations. This lack of effi-cacy could be reversed by the addition of purified fetuin-A tothe calciphylaxis sera in quantities restoring normal serumlevels. In this context, it remained unclear whether calcificuremic arteriolopathy was initially triggered by a lack offetuin-A in the circulation, whether the system was alreadyexhausted by a major attempt to counteract this fulminantcalcification process, or whether levels were low secondary toa calciphylaxis-induced systemic inflammatory reaction.

ConclusionsAlthough we have focused solely on fetuin-A, we do notadvertise fetuin-A as the “holy grail” of mineralizationresearch. We are fully aware that fetuin-A plays its roletoward the very end of pathological mineralization, whenmost of the damage is done and mineralization is imminentthat would not occur in the presence of potent mineralizationinhibitors like pyrophosphate and matrix GLA protein, and inthe absence of local inflammation, cell death, destruction ofmatrix, and so on. Figure 6 concatenates major principles ofpathological mineralization known to date and puts fetuin-Ain perspective. Fetuin-A serves as a mineral chaperone, acarrier protein facilitating transport and clearing of poten-tially proinflammatory and procalcific cargo (waste). Themechanistic and functional analogy between lipoproteins

Figure 6. Calcification-related genes “atwork” in vascular smooth muscle cells(VSMC) undergoing metaplasia. After“transdifferentiation” into mineralizingVSMC, the cells elaborate markers of theosteogenic/chondrogenic lineage. Thesodium/phosphate cotransporter Pit-1mediates phosphate transport into the cell.Elevated phosphate levels in the cyto-plasm upregulate expression of Runx2/Cbfa-1, an osteogenic transcription factor.In addition, hyperphosphatemia enhancesproduction of apoptotic bodies and matrixvesicles that nucleate vascular mineraldeposition. The transforming growth factor(TGF)-�/TGF-like cytokine BMP-7 main-tains the contractile phenotype (via Smad6/similiar to mothers against decapen-taplegic 6 and Smad 7 signaling) andBMP-2 and TGF-�1 enhance the osteo-genic phenotype. Extracellular calcium istransported into matrix vesicles by Ca2�channel-forming annexins II, V, and VI.Calcium enhances the phosphate-dependent osteogenic differentiation byupregulation of Pit-1 expression. Pyro-phosphate (PP) acts as an inhibitor ofbasic calcium phosphate crystal growth.The concentration of PP is controlledby nucleotide pyrophosphatase/phosphotransferase-1 (ENPP1), whichgenerates PP, the PP transporter ANK,and tissue-nonspecific alkaline phospha-tase (TNAP), which cleaves PP. When cellsfail to properly handle a high mineral loadbecause of elevated extracellular calciumphosphate, especially in the absence ofextracellular fetuin-A, they will succumb toapoptosis.65 Apoptotic bodies (AB) con-taining high amounts of mineral-like matrixvesicles readily mineralize and form apotent nidus for further extracellular matrixcalcification. Unlike matrix vesicle-mediated mineralization, AB-mediatedmineralization does not require alkalinephosphatase and annexins. In addition,phosphatidylserine (PS) is localized onopposite sides of the plasma membrane ofmatrix vesicles (inside) and AB (outside).PS is externalized to the outer membrane

leaflet during apoptosis. Fetuin-A prevents extracellular and intravesicular basic calcium phosphate growth in matrix vesicles and thusreduces calcium-induced apoptosis in VSMC. BMPR-I indicates BMP receptor-I; MGP, matrix GLA protein; NTP, nucleotide triphos-phate; NMP, nucleotide monophosphate; OPN, osteopontin; SM-MHC, smooth muscle myosin heavy chain; TNF-�, tumor necrosisfactor-�. Illustration credit: Cosmocyte/Ben Smith.



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and fetuin-A– based calciprotein particles is obvious. Nomatter what the preferred mechanism of atheroscleroticlesion calcification is, deranged mineral homeostasis, dys-lipidemia, compromised scavenging and debris clearing,inflammation, apoptosis, matrix mineralization, and osteo-genesis are all known pundits (“partners in crime”), and itseems like fetuin-A counters many of them and thus is ahighly pleomorphic protein and is truly a systemic regu-lator of mineralization.

Sources of FundingThis work was funded by the Deutsche Forschungsgemeinschaft(project JA562/10) to W. Jahnen-Dechent.

DisclosuresNone.

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Willi Jahnen-Dechent, Alexander Heiss, Cora Schäfer and Markus KettelerFetuin-A Regulation of Calcified Matrix Metabolism

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