Fetuin-A Regulation of Calcified Matrix Metabolism
Abstract
The final step of biomineralization is a chemical precipitation reaction that occurs spontaneously in supersaturated or metastable salt solutions. Genetic programs direct precursor cells into a mineralization-competent state in physiological bone formation (osteogenesis) and in pathological mineralization (ectopic mineralization or calcification). 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-A protein binds small clusters of calcium and phosphate. This interaction results in the formation of prenucleation cluster-laden fetuin-A monomers, calciprotein monomers, and considerably larger aggregates of protein and mineral calciprotein particles. Both monomeric and aggregate forms of fetuin-A mineral accrue acidic plasma protein including albumin, thus stabilizing supersaturated and metastable mineral ion solutions as colloids. Hence, fetuin-A is a mineral carrier protein and a systemic inhibitor of pathological mineralization complementing local inhibitors that act in a cell-restricted or tissue-restricted fashion. Fetuin-A deficiency is associated with soft tissue calcification in mice and humans.
Fetuins are vertebrate plasma proteins. Fetuin-A/α2-Heremans Schmid (HS) glycoprotein homologues occur in reptiles, fish, birds, marsupials, and mammals. Bovine fetuin (derived from the Latin word fetus) was first described in 1944 by Pedersen1 as the most abundant globular plasma protein in fetal calf serum. The human species homologue was independently identified by Heremans2 and Schmidt and Bürgi.3 It was later named α2-HS-glycoprotein by Schultze4 in honor of two of the original codiscoverers. The name also indicates that α2-HS glycoprotein comigrates with the α-2-globulin fraction of serum proteins in cellulose acetate electrophoresis.
This protein is confused in the literature with α-2-Z-glycoprotein/Zn α-2 glycoprotein (Z for zinc-binding) or α-fetoprotein, two unrelated entities. Fetuin-A is member of a family of four structurally related plasma proteins containing cystatin-like protein domains. Cystatins can inhibit cysteine peptidases of the papain, calpain, cathepsin, and caspase families and play key roles in a wide array of physiological processes as well as in disease. The cystatin family harbors type 1 (mainly intracellular proteins), type 2 (mainly extracellular proteins), and type 3 cystatins (plasma proteins). Figure 1 shows cartoons of the type 3 family members fetuin-A/α2-HS glycoprotein, fetuin-B, histidine-rich glycoprotein, and kininogen. Cystatin domain 1 in fetuin-A is strongly negatively charged with a high affinity for calcium-rich minerals.5
Figure 1. Schematic structure of type 3 cystatins. Domain organization with indications of disulfide bridges, cystatin domains, and the His/Gly and His/Pro domains in kininogen (KNG) and histidine-rich glycoprotein (HRG), respectively. Molecular models of cystatin domains showing negative charge in red and positive charge in blue. The amino-terminal cystatin domain 1 in fetuin-A is particularly rich in acidic residues that mediate mineral binding to calcium-rich phases of calcium phosphates.
Fetuin-A Biosynthesis
Fetuins are highly expressed liver-derived plasma proteins bearing posttranslational modifications proteolytic processing,6,7 complex glycosylation,8–10 phosphorylation (Ser and Thr),6,11–15 and sulfation.16 Human fetuin-A/α2-HS glycoprotein is processed from a single chain precursor to the mature circulating two-chain form.6 Human fetuin-A is susceptible to further proteolytic cleavage in septicemia,7 and bovine fetuin-A is processed by matrix metalloproteinases.15Figure 2 illustrates secondary modifications and allelic variants identified in human fetuin-A/α2-HS glycoprotein. These modifications may regulate protein expression levels, stability, and biological activity. Phosphorylation is indispensible for fetuin-A interaction with the insulin receptor,17,18 whereas phosphorylation seems not to be required for mineral interaction.19 Desialylation will result in immediate hepatic clearing through the asialoglycoprotein receptor.20–22
Figure 2. Cartoon of human fetuin-A/α2-HS glycoprotein showing cystatin-like domains 1 and 2 and a third unrelated domain in green, yellow, and blue shading, respectively. The cotranslational and posttranslational modifications disulfide bridges (C-C in yellow) Ser-phosphorylation sites (S in red), protease-sensitive sites (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, and Ser/Thr O-glycosylation sites depicted as blue symbols, respectively. Data taken from.(6,7,13,96
Binding Properties of Fetuin-A
The type 3 members of the cystatin superfamily are glycoproteins produced mainly in the liver, which circulate in plasma at high concentrations. Fetal calf serum contains more fetuin-A than albumin. Apart from the vasculature, fetuins are present throughout the extracellular spaces and the extracellular matrix. Given the high expression levels and the many possible ways of molecular interaction with multiple ligands, it is fair to assume that fetuins primarily exercise carrier and scavenger functions like albumin. This poses an important complication for ex vivo experimentation, because crude fetuin preparations contain impurities. Like pure albumin preparations, pure fetuin preparations are difficult to make. High-abundance plasma protein preparations are notoriously contaminated with lower-abundance biological molecules that copurify either because of natural association or because of mere coincidence. Various growth factors or growth-promoting substances associate with crude fetuin preparations and form the basis of the “enigmatic growth promoting properties” of fetuin in cell culture.23
Whether associations of fetuin-A with certain ligands are physiologically relevant is a matter of controversy and is almost impossible to decide unless genetic models are developed to test such interactions in vivo. We remind readers to heed the old wisdom of Racker24 that also became part of the “10 commandments” of Kornberg,25 “Do not waste clean thinking on dirty enzymes.” Most commercial preparations of fetuin-A on the market today do not go much beyond the quality of “fetuin” from 1944,1 which is better viewed as a “protein concept” like “globulin” or “albumin” rather than a clean product. We distinguish between fetuin and fetuin-A whenever possible. Also, before the year 2000 when fetuin-B was cloned in silico26 and the year 2003 when it was finally shown to be expressed as a plasma protein,10 both fetuin-A and fetuin-B were collectively addressed as “fetuin,” and most fetuin sold today is not screened for the presence of fetuin-B. Studies of human α2-HS-glycoprotein/fetuin (α2-HS glycoprotein) will always signify fetuin-A; publications studying “fetuin” protein are probably dealing with fetuin-A as well, but it is impossible to exclude that fetuin-B was also studied because of the physicochemical similitude of both proteins.
Fetuin-A binding is prodigious and reaches from small molecules to entire organisms. Plasmodium sporozoites use fetuin-A binding to “hitch-hike” their way into liver cells,27 suggesting that fetuin-A function is indispensable and despite negative selection pressure has been maintained long enough for this docking mechanism to evolve. Soon after its discovery, fetuin has been shown to inhibit trypsin.28 Both positive and negative fetuin interactions with proteases are documented, including matrix metalloproteinase-9,29 matrix metalloproteinase-3,30 meprin metalloproteinases,31 the cysteine proteases m-calpain,32 cathepsin L in rat bone,33 and inhibition of recombinant human cathepsin V. Proteinase interactions are thought to be involved in the regulation of tumorigenesis and tumor progression.34,35
Fetuin markedly accelerates incorporation of exogenous fatty acids into cellular triglycerides.36–39 Thus, fetuin may share a function with fatty acid binding proteins, a family of abundantly expressed 14-kDa to 15-kDa proteins. Like fetuin, fatty acid binding proteins reversibly bind hydrophobic ligands, including saturated and unsaturated long-chain fatty acids, and other lipids with high affinity.40
Because of their rich complex glycosylation pattern, fetuins serve as model substances for lectin and glycoprotein research. Lectin binding should always be seriously considered when fetuin binding to cells and to the extracellular matrix is studied. On a practical note, the strong binding of pertussis toxin to terminal sialic acid residues in fetuin form the basis of a Food and Drug Administration-approved pertussis toxin test.41,42 Fetuin-A sequestration of lectins proved a major complication in experimental cytotoxic therapy using cancer cell–specific antibodies coupled to the Ricinus communis agglutinin ricin.43 The immunotoxins were rapidly cleared by the asialoglycoprotein receptor and caused liver toxicity.44
In a search for natural transforming growth factor-beta (TGF-β) receptor antagonists, a sequence homology was found between TGF-β receptor type II and fetuin-A.45 The TGF-β receptor II homology 1 domain from fetuin bound preferentially to bone morphogenetic protein (BMP)-2. Full-length fetuin-A bound directly to TGF-β1 and TGF-β2 and with greater affinity to the TGF-β–related BMP-2, BMP-4, and BMP-6. Finally, and likely impinging on fetuin's role in mineralization biology, fetuin or neutralizing anti-TGF-β antibodies blocked osteogenesis and deposition of calcium-containing matrix in mineralizing cell cultures.46 An altered bone phenotype in fetuin-A–deficient mice (Ahsg−/−) was explained accordingly in terms of failure to block TGF-β–dependent signaling in osteoblastic cells.47 Tumorigenesis experiments using Ahsg−/− mice further supported the hypothesis that fetuin-A is an antagonist of TGF-β in vivo, in that it inhibited intestinal tumor progression.48
Antiinflammatory Role of Fetuin-A
Fetuin-A, one of the most abundant fetal plasma proteins, was found to be essential for the inhibition of the proinflammatory cytokine tumor necrosis factor production by spermine and its synthetic analogues.49,50 Accordingly, the strong fetal expression of fetuin and spermine have been associated with the tolerance of the fetus, “nature's transplant,” by mothers.51 The strong antiinflammatory effects of fetuin were verified in vivo using several models of inflammation, including lipopolysaccharide-induced miscarriage in rats,52 carrageenan injection,53 cerebral ischemic injury in rodents,54 and cecal ligation and puncture in mice.55 In all cases fetuin-A was associated with reduced inflammatory response and increased survival, and administering additional fetuin generally improved outcome.
Thus fetuin-A generally may be regarded as antiinflammatory.56 The antiinflammatory property of serum α2-HS glycoprotein/human fetuin-A was further supported by the demonstration that fetuin-A is a potent and specific crystal-bound inhibitor of neutrophil stimulation by hydroxyapatite crystals.57 Calcium phosphate crystals induce proinflammatory cytokine secretion through the NLRP3 inflammasome in monocytes/macrophages,58–60 cell death in human vascular smooth muscle cells,61 and cell activation in chondrocytes.62–64 Antiapoptotic activity of fetuin-A has been observed in smooth muscle cells65 and dampening of the cell-specific responses would generally be expected to alleviate the detrimental consequences of local inflammation, cell death, and cartilage degradation. The proven protective function of fetuin-A in many animal models of inflammation,52–55 the inhibition of proinflammatory compounds,50,51,66–68 and the inhibition of crystal-induced neutrophil activation57 collectively suggest that fetuin-A may generally protect during pathological mineralization as well.69
Fetuin-A in Metabolic Syndrome
Several clinical studies proposed that high-serum fetuin-A levels are associated with metabolic syndrome (MetS) and that fetuin-A therefore may present a risk factor for MetS.70–73 The association of fetuin-A, insulin signaling, diabetes type 2, and MetS dates back to a publication in 1989 stating that pp63, a liver-secreted phosphoprotein, inhibited insulin receptor and downstream substrate phosphorylation signaling.17 The cDNA sequence of pp63 was identified as rat fetuin-A,74 and it was disputed whether the activity tested in the original article was actually rat fetuin-A or a copurified protein.75,76 We tested authentic human fetuin-A and found minor, if any, inhibitory activity at the insulin receptor and downstream substrate level.74 We analyzed phosphotyrosine modification of cellular proteins in rat fibroblasts expressing the human insulin receptor and could not detect any robust and reproducible inhibition of insulin signaling by human or bovine fetuin including phosphofetuin-A immunoaffinity purified from HepG2 cells. Testing of fetuin-A in relevant cells showed no classical insulin receptor inhibition, but showed unexplained downregulation of insulin-stimulated Elk1-phosphorylation.77
In another series of experiments, commercial bovine fetuin and supernatants of baculovirus-infected insect cells expressing human fetuin-A did inhibit insulin receptor signaling. Furthermore, fetuin-A was shown by immunoprecipitation to directly interact with the insulin receptor.18,78 These researchers also showed that fetuin-A–deficient mice maintained on a mixed genetic background of C57BL/6N and 129Sv had improved insulin response, resistance to weight gain, protection against obesity, and protection against insulin resistance associated with aging.79 We back-crossed these mice that were originally generated in our laboratory80 onto pure C57BL/6N as well as DBA/2 genetic background, and we could not detect evidence of diabetes-associated symptoms in the genetically more homogeneous mice. The genetic background of mice and even substrains of mice differ with respect to insulin sensitivity. A deletion variant of nicotinamide nucleotide transhydrogenase that has spontaneously occurred in C57BL/6J, but not in C57BL/6N strains, is thought to influence insulin signaling.81,82 Therefore, sibling mice from the same colony should be ideally used when comparing the influence of single gene deletions on insulin sensitivity. Mice with homogeneous defined genetic background were not yet available at the time of the first study in the case of fetuin-A–deficient mice.83
Thus, it is unclear if fetuin-A is a cause or consequence of high-caloric feeding in mice or whether the association of high-serum fetuin-A and MetS is a bystander phenomenon. First, fetuin-A is a highly expressed constitutively secreted hepatic serum glycoprotein. Using nuclear run-on assays, LeCam et al84 determined that the rat fetuin-A/pp63 promoter strength was approximately three-times to four-times that of the strong liver-specific albumin promoter. Second, fetuin-A is traditionally regarded as one of the few negative acute phase proteins55,85,86 and strong associations between low-serum fetuin-A levels and inflammatory markers like C-reactive protein have been published.87–89 Thus, downregulation rather than upregulation of fetuin-A during “fat inflammation” in obesity-related insulin resistance would be expected.90 Therefore, it seems counterintuitive that fetuin-A levels would be increased in MetS. Recent work showed that fetuin-A induced inflammatory cytokines,91 and that one of the major mediators of inflammatory cytokine action, NFκB, further upregulated hepatic fetuin-A synthesis.39 The conflicting data on fetuin-A regulation clearly need to be reconciled in terms of timing of events and causal relationships vs mere association. The association of fetuin-A serum levels with MetS and type 2 diabetes in humans rests on single measurements, and most studies did not correct for a major confounder, total liver protein synthesis. Therefore, we suggest that association studies should at least be normalized to serum albumin as an indicator of total liver protein synthesis, unless serum albumin should also be called a risk factor of MetS.
Role of Fetuin-A in Mineralization Biology
We started working on fetuin-A as part of an ongoing research project studying the structure and function of type 3 members of the cystatin superfamily.6,92–96 At that time, fetuin-A/α2-HS glycoprotein was a “structure in search of a function.” Intrigued by the description of rat fetuin as a natural inhibitor of insulin receptor kinase inhibitor,17 and by the report on the identity of fetuin of hemonectin, a protein present in rabbit bone marrow extracellular matrix and a suspected homing factor for myeologenic cells in the bone marrow,97 we studied fetuin-A binding to cell membranes and extracellular targets. Binding to mineralized bone matrix as described by Triffitt et al98–100 turned out to be the most robust binding phenomenon by far. The high affinity of fetuin-A for bone mineral mediates selective fetuin-A accumulation from plasma into bone.98,99,101 Fetuin-A in bone accounts for 25% of the noncollagenous proteins and thus is one of the two most highly abundant noncollagenous proteins.102 We showed that fetuin-A also has a particularly high affinity to nascent apatite mineral and is an inhibitor of de novo apatite formation from supersaturated mineral solutions.19 The inhibitory effect is mediated by acidic amino acids clustering in cystatin-like domain D1. This fundamental observation formed the base of two decades of fetuin-A mineralization research and has been successfully repeated by many independent laboratories. In full accord with this biochemical finding, genetic work using fetuin-A–deficient mice80,103 likewise suggested that fetuin-A is a mineral chaperone104 mediating the transport of mineral from the extracellular space and the general circulation.103,105,106
Fetuin-A–Mineral Complexes, Calciprotein Particles, Calcifying Nanoparticles: Fetuin–Mineral Complexes, Calciprotein Monomers, Calciprotein Particles
Fetuin-A binds calcium phosphate and calcium carbonate with high affinity, and it binds magnesium phosphate less well.19 Importantly, fetuin-A only inhibits the de novo formation of calcium phosphate and does not dissolve preformed mineral. Fetuin-A is an inhibitor of mineralization in solution and of cell-mediated mineralization in that it regulates the process of matrix mineralization in rat calvaria osteoblastic cells.19 On addition of β-glycerophosphate and ascorbate, these cells formed mineralized bone nodules in DMEM culture medium containing 10% fetal calf serum or bovine fetuin-A or human α2-HS glycoprotein. However, when the cells were maintained serum-free and serum albumin was the sole bulk protein present in the medium, the cells catastrophically calcified and died.19 Fetuin-A inhibition of lethal calcification was dose-dependent in that higher concentrations of fetuin more consistently inhibited calcification than lower concentrations. Fetuin-A similarly inhibited vascular smooth muscle cell calcification induced by elevated concentrations of extracellular mineral ions.65 This was achieved in part through inhibition of apoptosis and caspase cleavage. Fetuin-A was internalized by vascular smooth muscle cells and concentrated in intracellular vesicles. Subsequently, fetuin-A was secreted via vesicle release from apoptotic and viable vascular smooth muscle cells. The presence of fetuin-A in vesicles abrogated their ability to nucleate calcium phosphate precipitation. In addition, fetuin-A enhanced phagocytosis of vesicles by vascular smooth muscle cells. These observations showed that the uptake of fetuin-A by cells and the vesicular recycling of fetuin–mineral complexes reduced both apoptosis and calcification in live cells subjected to elevated extracellular concentrations of mineral ions.107 Therefore, the stabilization of mineral solutions mediated by fetuin-A in test tube assays19 was also effective inside living cells, thus constituting a strong defense mechanism against pathological mineralization.
Fetuin-A stabilizes supersaturated mineral solutions by forming soluble colloidal nanospheres.5 Similar nanoparticles were detected in the ascites fluid of a pertioneal dialysis patient experiencing calcifying sclerosing peritonitis.108,109Figure 3 shows electron micrographs of nanoscopic protein–mineral particles generated in the laboratory (Figure 3A, B) or taken from the patient's ascites fluid (Figure 3C). These colloids are variously termed calciprotein particles in analogy to lipoprotein 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 the amino-terminal cystatin-like domain D1 are required for the inhibitory activity of fetuin-A.5,19 Using a wide array of high-resolution spectroscopic techniques including small-angle neutron scattering, small-angle X-ray scattering, dynamic light scattering, transmission electron microscopy, and conventional photometry, it was determined that the formation of calciprotein particles is a multistep process.
Figure 3. Electron microscopic pictures of synthetic and patient-derived calciprotein particles (CPP). A, Primary calciprotein particles have a diameter of 30 nm to 150 nm and contain amorphous mineral.5 B, Subsequently, the primary CPP were subjected to a major structural rearrangement. The resulting secondary CPP consist of a core of crystalline basic calcium phosphate, which is covered by a layer of fetuin-A (A and B: 50 μmol/L bovine fetuin-A, 10 mmol/L CaCl2, 6 mmol/L Na2HPO4, pH 7.4).113 These particles are stable 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.
Figure 4 depicts schematically the sequence of calciprotein particles/fetuin–mineral complexes formation, starting with the spontaneous formation of prenucleation clusters from metastable solutions, their sequestration to acidic aspartic acid, and glutamic acid side chains in the cystatin-like domain 1 of fetuin-A–forming mineral-laden fetuin-A monomer or calciprotein monomer. Calciprotein monomer was discovered when quantitative small-angle neutron scattering data analysis revealed that even at a fetuin-A concentration close to the stability limit of supersaturated salt solutions, only approximately one-half of the mineral ions and only 5% of the fetuin-A were contained in the calciprotein particles. The remaining supersaturated mineral ion fraction and 95% of noncalciprotein particles of fetuin-A were associated with a mineral-laden fetuin-A monomer fraction that could be separated from mature calciprotein particles by ultrafiltration through a 300-KDa cut-off membrane. Small-angle neutron scattering analysis showed that the fetuin-A monomer in this fraction was closely associated with coalesced subnanometer-size mineral ions clusters reminiscent of Ca9(PO4)6 Posner clusters (Figure 4A).111
Figure 4. Formation of primary and secondary calciprotein particles (CPP) from fetuin-A monomer and amorphous mineral precursors. A, Hydroxyapatite precursor viewed along the [001] axis. The dotted lines confine the hexagonal unit cell. The circle represents a Ca9(PO4)6 Posner cluster as a building block of the apatite structure. B, A three-dimensional view of five Posner clusters juxtaposed to the acidic β-sheet of the computer-modeled aminoterminal fetuin-A domain D1. The domain surface is plotted semitransparent with negative charge indicated in red and positive charge indicated in blue. C, A computer-generated homology model structure illustrating the fetuin-A–mineral interface. The fetuin-A protein structure was modeled after published structures for cystatin-like domains 1 (green) and 2 (yellow).176 Domain 3 (blue) was modeled after the 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 precipitation.112 D, Primary CPP are spherical and rather unstructured agglomerates of mineral (clusters) and fetuin-A, whereas (E) secondary CPP consist of a crystalline mineral core covered by fetuin-A. In conclusion, fetuin-A effectively shields the mineral phase, leading to stable CPP that can be transported and cleared, as shown in Figure 5. The computer graphics were generated using the software packages VESTA,178 VMD,179 and APBS.180
Fetuin-A binds to apatitic mineral surfaces, as shown in Figure 4C. Fetuin-A does not influence the formation of mineral nuclei. However, fetuin-A prevents the growth and aggregation of nuclei to larger entities and ultimately mineral precipitation.112 Thus, fetuin-A effectively shields spontaneously formed mineral nuclei, leading to transiently stable calciprotein particles.
A hierarchical role of fetuin-A was established in that fetuin-A was critically required during the early steps of calciprotein particles formation and stabilization and that other acidic plasma proteins including serum albumin further stabilized the initial colloids and could substitute for fetuin-A at later stages of calciprotein particles formation.109,122 These later stages were studied in detail using small-angle neutron scattering. Figure 4D illustrates the aggregation of many calciprotein monomers and other mineral nuclei into transiently stable primary calciprotein particles of initially <100 nanometers in diameter. After a lag period, these particles grow into elongated and more crystalline particles of approximately twice the initial size, termed secondary or mature calciprotein particles (Figure 4E). Thus, formation and maturation are two separate and successive processes following the principles of Ostwald ripening.5,110–112 Similar particles may be generated with other acidic macromolecules as well. However, fetuin-A is exceptionally potent regarding activity and specificity of inhibition. Increased fetuin-A concentration leads to smaller particles. An increased temperature, mineral ion concentration, and a reduced fetuin-A concentration, respectively, all accelerate the particle ripening process, demonstrating that calciprotein particle maturation follows the Arrhenius law. Secondary calciprotein particles are stabilized by a compact outer fetuin-A monolayer against further growth (Figure 4E) for up to 30 hours at body temperature, which is ample time for clearing of calciprotein particles from circulation.
The formation and maturation of calciprotein particles can be followed by optical monitoring110 of mineralizing solutions. Supplemental Figure I (available online at http://www.circresaha.org) shows dynamic light scatter diagrams illustrating that the transformation from primary to secondary calciprotein particles is fetuin-A concentration-dependent. Higher fetuin-A concentrations cause prolonged stability of both calciprotein monomers and calciprotein particles, and thus a right-shift in the transformation curves. A clinical test could use the kinetic parameters of the precipitation reaction, thus measuring the overall calcification risk in biological fluids, eg, in the blood of dialysis patients.
Protein–mineral complexes are soluble precursors of physiological mineralization of bones and teeth as well. Recent research has shown that biomineralization starts with amorphous mineral–protein complexes containing fetuin-A.123–126 Price et al127–129 have shown that fetuin-A is critically required to direct mineralization to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen. Fetuin-A does so by selectively inhibiting mineral growth outside of these matrices. This mineralization by inhibitor exclusion is likely redundant, because fetuin-A–deficient mouse bone is mineralized perfectly well. The mice nevertheless show a bone phenotype of stunted femur length, indicating premature growth plate mineralization and disturbed osteogenic signaling.47
Nanobacteria: A Red Herring From Mars
Nanobacteria initially described nanoscopic life forms detected by electron microscopy in rock sediments.130 Similar entities were discovered in meterorites from Mars131 and, finally, in cell culture.132 Nanobacteria attracted a lot of scientific and economical attention as a causal agent of major diseases, including vascular calcification, kidney stones, and cancer.132–136 In the year 2000, Cisar et al137 determined that simple mixtures of phospholipids and calcium phosphate crystals closely resembled nanobacteria in that they showed life-like growth and replication. Nucleic acid sequences previously thought to be diagnostic markers of nanobacteria were in fact diagnostic of common laboratory contaminants.137 Two groups of researchers finally solved the riddle of pathological mineralizing nanobacteria.120,138 A series of experiments on the origin of putative nanobacteria showed that calcium phosphate together with proteins and further nonmineral compounds formed nanoparticles that resembled nanobacteria in shape and behavior.139 Minerals containing nanoparticles had a high binding capacity for charged molecules, including ions, carbohydrates, lipids, and nucleic acids. Depending on the exact composition, mixtures of mineral and nonmineral compounds sustained either crystallization of hydroxyapatite mineral or the formation of complex protein–mineral complexes.122,140 It was found that the main protein component of the nanobacteria was fetuin-A.120 Besides fetuin-A, serum albumin and apolipoproteins were also identified;121 hence, the term nanobacteria was exchanged for the more apt term calcifying nanoparticles, virtually identical with the protein–mineral complexes, calciprotein particles, or fetuin–mineral complexes described before.
Calciprotein Particles Metabolism and Clearing
Figure 5 illustrates the putative metabolism of calciprotein particles, soluble protein–mineral complexes, which are now regarded as a physiological byproduct of mineral metabolism. The scheme is partly hypothetical and requires experimental verification. Soluble protein–mineral complexes including fetuin-A have been detected in serum from etidronate overdosed rats,118 in adenine-treated rats,141 in peritoneal dialysis patients with sclerosing calcifying peritonitis,108,109 and in dialysis patients.142 The scheme is modeled after lipoprotein particle metabolism, a well-known transport system for otherwise insoluble lipids like cholesterol esters.
Figure 5. Hypothetical pathway of calciprotein particle (CPP) circulation and clearing by endothelial cells and tissues resident phagocytes. Fetuin-A stabilizes CPP in the circulation and mediates their efficient uptake. In healthy individuals, CPP may form spontaneously and be present in small numbers throughout all tissues, or may occur substantially where bone resorption by osteoclasts (OC) is releasing high levels of calcium and phosphate by acid-mediated mineral dissolution. CPP may be transported in the blood or retrieved locally after nearby osteoclastic resorption, and are used locally by osteoblasts (OB) during bone formation. When mineral homeostasis is severely disturbed, eg, in dialysis patients or in severely etidronate-overdosed rodents, bouts of hypercalcemia and hyperphosphatemia will result in the formation of high numbers of CPP and fetuin-A will be consumed in the process as part of the CPP complex. CPP in the interstitial spaces will be cleared by monocytes (MC) or macrophages (MP) or by any other endocytosing cell types. Overly abundant CPP may overwhelm the clearing capacity of the reticuloendothelial system phagocytes and may result in apoptosis 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 experimentally verified. It may also be of significance that many inhibitors of calcification activate monocytes/macrophages and stimulate phagocytosis; therefore, this mode of calcified remnant clearance may be of similar importance. Reproduced from Jahnen-Dechent W, Schäfer C, Ketteler M, et al. Mineral chaperones: a role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification. J Mol Med. 2008;86:379–389.
The basic tenet of calciprotein particles metabolism holds that fetuin-A stabilizes mineral complexes and at the same time mediates their transport and clearing. Fetuin-A should be regarded as an opsonizing serum protein with a high affinity for mineral complexes and debris. The opsonizing properties of fetuin-A have been determined several decades ago.143–145 Fetuin-A affects microparticle phagocytosis by dendritic cells,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 specific proteins interacting with Sepharose beads in a study that used quantitative mass spectrometry to determine bead proteomes.150 Therefore, binding may be restricted to a narrow range of cationic, lipidic, or mineral ligands, and a few more interacting TGF-β–related cytokines mentioned earlier in this text. Given the high abundance of fetuin-A in plasma, any clearing mechanism would have to rely on conformational or structural changes in fetuin-A or on multivalent binding that turns low-affinity binding into high-avidity binding.
Uptake of fetuin-A by cultured human vascular smooth muscle cells has been demonstrated,65,107,151 but the exact form of fetuin-A was not determined. Clustering of fetuin-A molecules on the surface of secondary calciprotein particles as demonstrated111 ideally fulfills the ligand clustering required to increase binding strength. Our preliminary results of fetuin-A monomer and fetuin-A containing calciprotein particle clearing in vivo show that calciprotein particles are cleared vastly more efficiently than fetuin-A monomer. Nevertheless, specific receptors for calciprotein particle clearing discriminating against fetuin-A monomer remain to be determined.
Fetuin-A Knockout Phenotype
The use of animal models has uncovered major inhibitors of pathological calcification and their mode of action.152 Fetuin-A is one of the major systemic inhibitors of pathological mineralization, but it is not the only one. This is vividly illustrated by the history of fetuin-A–deficient mice (Ahsg−/−). While establishing that fetuin-A is a regulator of mineralization,19 we also generated Ahsg−/− mice to test this hypothesis in an animal model. For technical reasons, the first Ahsg−/− mice had a mixed 129Sv × C57BL/6 genetic background designated 129,B6Ahsgtm1mbl.80 Out of an initial colony of approximately 200 mice, only approximately 10 female ex-breeders showed a spontaneous calcification phenotype.80 All other mice were apparently normal. Force-feeding supraphysiological doses of active vitamin D (calcitriol) and a high-mineral diet (phosphate-rich) resulted in nephrocalcinosis and pulmonary and myocardial calcification of these mice.103
Unsatisfied by this highly variable yet low-penetrating calcification phenotype, we decided to combine the fetuin-A deficiency with the calcification-prone DBA/2 genetic background.153–155 For comparison, we also backcrossed the mice onto the widely used calcification-resistant genetic background C57BL/6 and thus generated two more Ahsg−/− mouse strains, D2-Ahsgtm1wja and B6-Ahsgtm1wja. The latter mice behaved like the mixed genetic background mice in that they scarcely had any spontaneous calcification. They did, however, have calcification when challenged with heminephrectomy and a high-mineral diet.105,106 In stark contrast, virtually all D2-Ahsgtm1wja mice show spontaneous massive soft tissue calcification throughout their bodies.103 This strain is arguably the most calcifying mouse strain in existence in that the myocardium of D2-Ahsg−/− mice contains almost one-tenth the calcium content of normal bone.156 Kidney, lung, skin, brown fat, pancreas, and reproductive organs are equally strongly calcified.
| 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-β 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 |
Supplemental Figure II (available online at http://www.circresaha.org) shows random examples of 11-month-old wild-type or Ahsg−/− mice maintained against the genetic background C57BL/6 or DBA/2. Even from the low-resolution radiology photographs, the uniformity of genetic penetrance and the extent of the calcification in D2-Ahsg−/− mice can be readily appreciated. Kidney and skin calcification are easily visible. Details may be glanced at higher magnifications of the electronic pictures provided with this article. We point out that the pictures were taken of live anesthetized mice. Therefore, myocardium, lung, and pancreas calcification are invisible or blurred because of breathing motion of the chest region. Surprisingly, the soft tissue calcification is not lethal like the arterial calcification of matrix GLA protein–deficient mice.157 Calcification does, however, greatly compromise reproduction in mice aged 6 months and older. The 1-year survival 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 and soluble inhibitor of pathological mineralization that is backed-up by other genetic factors rendering mouse strains prone to or resistant to dystrophic calcification. Integrative genomics of the so-called Dyscalc locus led to the identification of Abcc6 as the major gene determining dystrophic cardiac calcification.158Abcc6-deficient mice have soft tissue calcifications develop, like D2-Ahsg−/− mice, albeit they are less extensive in both localization and extent. Interestingly, Abcc6 deficiency is associated with reduced plasma fetuin-A levels and calcifications can be partially corrected by overexpressing fetuin-A,159 suggesting that fetuin-A acts downstream or in concert with Abcc6. Despite heavy early-onset and life-long progressing dystrophic calcification in Abcc6−/− and especially in D2- Ahsg−/− mice, true osteogenesis involving clearly identifiable osteochondrocytic cells has never been reported in these mouse models of calcification. This casts doubt on the now popular hypothesis that pathological calcification is always a form of ectopic osteogenesis. More likely, both genes seem to be involved in systemic mineral homeostasis and transport. Therefore, we have termed fetuin-A a mineral chaperone mediating the solubilization, transport, and elimination from circulation of otherwise insoluble minerals, much like apolipoproteins help in lipid transport and metabolism.104
Because fetuin-A is highly expressed in bone and accounts for 25% of all noncollagen proteins of bone (noncollagenous proteins),102 we examined bone growth and remodeling phenotypes in mixed background 129,B6-Ahsg−/− mice.47 The skeletal structure of these mice appeared normal at birth, but abnormalities were observed in adult 129,B6-Ahsg−/− mice. Maturation of growth plate chondrocytes was impaired, and femurs lengthened more slowly between 3 and 18 months of age. Previously, it had been found that fetuin-A is a soluble TGF-β/BMP-binding protein controlling cytokine access to membrane signaling receptors.45,46,160 Hence, the altered bone phenotype was explained in terms of failure to block TGF-β–dependent signaling in osteoblastic cells. Mice lacking fetuin-A displayed growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis.47
In view of the mineralization regulation effected by fetuin-A, we revisited the bone phenotype of pure-bred B6-Ahsg−/− mice. These mice are unaffected by the secondary hyperparathyroidism and osteoporosis typical of D2-Ahsg−/− mice.103 We essentially reproduced all major findings of the previous study performed in 129,B6-Ahsg−/− mice.47 The mice displayed normal trabecular bone mass in the spine but increased cortical thickness in the femur. Bone composition and mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. The long bones, especially femora, were severely stunted in B6-Ahsg−/− mice compared to wild-type littermates, resulting in increased biomechanical stability. In addition, we determined increased mineral content in the growth plates of B6-Ahsg−/− long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix, a site of vigorous physiological mineralization. We thus demonstrated that growth plate chondrocytes are prone to “pathological calcification” in the absence of fetuin-A, and that active mineralization inhibition is a necessity for proper long bone growth, at least in mice.
The fact that three of the most potent anticalcification compounds, matrix GLA protein,157 pyrophosphate,161 and fetuin-A, all operate in and around growth plate chondrocytes suggests that this cell type is physiologically extremely prone to premature calcification. The pathomechanism of osteogenic vascular calcification likewise critically requires chondrogenic, but not necessarily osteoblastic, differentiation of vascular cells.162–164 It will be interesting to revisit the influence of fetuin-A alone and in combination on chondrocyte mineralization in a way similar to that previously performed with primary osteoblasts19 and smooth muscle cells65 as models of physiological and pathological mineralization, respectively.
Clinical Epidemiology of Serum Fetuin-A Levels and Polymorphisms
Approaching the clinical situation in humans and based on the outlined experiments, it is strongly suggested to generally view serum fetuin-A levels in combination with serum albumin as an indicator of nutritional state, as another negative acute phase protein, and as an indicator of overall hepatic protein synthesis, especially in dialysis patients. Patients in advanced stages of chronic kidney disease (CKD) clinically have the most serious cardiovascular and soft tissue calcifications develop, more than any other population, and it is now well-understood that the individual magnitude of calcification significantly corresponds with impaired survival in CKD.165–167 Furthermore, CKD patients tend to be in a state of malnutrition and microinflammation or macroinflammation, or both, in which downregulation of proteins such as fetuin-A may be expected.168
When the biochemical properties of fetuin-A became more and more apparent, it was a straightforward approach to observe the relationship between serum fetuin-A levels and outcomes in large dialysis populations. In a cohort of >300 hemodialysis patients, the lowest tertile of serum fetuin-A levels was associated with significantly increased all-cause and cardiovascular mortality.89 Fetuin-A was also found to be inversely correlated with C-reactive protein levels, emphasizing its nature as a negative acute phase reactant. These data were subsequently confirmed, demonstrating mortality risk prediction by fetuin-A deficiency in nearly 300 incident dialysis patients (including patients using peritoneal dialysis).169 In this study, a specific fetuin-A gene polymorphism (Thr256Ser) was shown to predict particularly low fetuin-A levels and to be associated with an adverse prognosis compared to patients carrying alternative polymorphisms. Hypoalbuminemia was strongly correlated with fetuin-A deficiency, suggesting the expected involvement in the malnutrition-inflammation-atherosclerosis syndrome in clinical fetuin-A deficiency. In a pure cohort of prevalent peritoneal dialysis patients, fetuin-A deficiency was also shown to be linked to features of the malnutrition-inflammation-atherosclerosis syndrome (low albumin, elevated C-reactive protein) and to cardiovascular events and mortality, respectively.88 Moreover, this study demonstrated an association between low fetuin-A levels and the magnitude of valvular calcification, emphasizing the hypothesized link between progressive calcification and impaired outcomes. In a smaller cohort of hemodialyis patients, a significant association between coronary calcification and fetuin-A deficiency was shown.170 Taken together, these observations indicate that fetuin-A deficiency may be an important inflammation-related link between cardiovascular calcification and mortality in patients using dialysis.
In patients with normal renal function as well as in predialysis CKD patients, the available information on the relationship between fetuin-A levels, the degree of calcification, and mortality is less clear. Recently, we have shown in the first prospective longitudinal study that low baseline serum levels of fetuin-A are associated with the increase of aortic valve calcification in 77 patients.171 However, in nearly 1000 patients from the Heart and Soul study focusing on patients at cardiovascular risk, mostly without renal dysfunction, high fetuin-A levels were found to be strongly associated with hyperlipidemia and features of the metabolic syndrome, but not with hard outcome parameters.172 In patients with diabetes mellitus spanning CKD stages 1 to 4, the magnitude of coronary artery calcification correlated with increased rather than with decreased fetuin-A levels.173 This finding may be specific for a pure diabetic cohort in which a different pattern of calcium-regulatory systems may be implicated and in which downregulation of fetuin-A may be partially compensated or masked by overnutrition. However, these data may imply that fetuin-A upregulation initially acts as a systemic defense mechanism (early warning system) trying to protect from or counteract against vascular calcifications in their early stages. Such an interpretation is indirectly supported by immunohistochemical findings showing strong fetuin-A deposition, but not synthesis, in areas of vascular calcification65,170 and may be better-understood once the metabolism of fetuin-A mineral complexes (calciprotein monomers, calciprotein particles/fetuin–mineral complexes) and concomitant fetuin-A serum depletion is known. A recent study suggested that serum measurements of fetuin-A have to take into account that mineral-bound fetuin-A may compete with free fetuin-A, and that centrifuge sedimentation of serum samples may considerably influence certain assay readouts but not others.142
When the calcification burden increases beyond a certain point in chronic long-term CKD, compensatory systems such as fetuin-A release may finally become exhausted and consecutive fetuin-A deficiency may start a vicious cycle of even more progressive extraosseous calcification and fetuin-A consumption. If this hypothesis of initial adaptive fetuin-A overproduction and later exhaustion of the system is correct, then future research should attempt to identify factors influencing fetuin-A expression and secretion.
Fetuin-A Consumption and Downregulation in Calciphylaxis/Calcific Uremic Arteriolopathy
Perhaps the most intriguing experimental example of fetuin-A consumption was published by Price et al,118 who demonstrated transient increases of total serum calcium up to 10 mmol/L by high-dose vitamin D treatment in rats within a few hours and a return to normocalcemia within 1 day. A concomitant loss of half the serum fetuin-A that formed a high-molecular-weight fetuin–mineral complex was observed, and it was probably cleared by the reticuloendothelial system or deposited in the bone or in extraskeletal sites.115 A potential clinical example of fetuin-A consumption and exhaust may be calciphylaxis (calcific uremic arteriolopathy), a rare but potentially life-threatening syndrome characterized by progressive and painful skin ulcerations associated with media calcification of medium and small cutaneous arterial vessels.174,175 Calciphylaxis primarily affects patients using dialysis or after renal transplantation. We reported fetuin-A deficiency with very low serum levels in a case series of calciphylaxis patients.103 To demonstrate the functional calcification inhibitory capacity of fetuin-A deficiency, we used sera from calcific uremic arteriolopathy patients in an ex vivo [45]CaCl2 radioisotope assay, which enables quantification of serum-induced inhibition of a calcium phosphate precipitation. These fetuin-A–deficient calcific uremic arteriolopathy sera were significantly less effective at inhibiting calcium phosphate crystal formation than sera from healthy subjects with appropriate fetuin-A concentrations. This lack of efficacy could be reversed by the addition of purified fetuin-A to the calciphylaxis sera in quantities restoring normal serum levels. In this context, it remained unclear whether calcific uremic arteriolopathy was initially triggered by a lack of fetuin-A in the circulation, whether the system was already exhausted by a major attempt to counteract this fulminant calcification process, or whether levels were low secondary to a calciphylaxis-induced systemic inflammatory reaction.
Conclusions
Although we have focused solely on fetuin-A, we do not advertise fetuin-A as the “holy grail” of mineralization research. We are fully aware that fetuin-A plays its role toward the very end of pathological mineralization, when most of the damage is done and mineralization is imminent that would not occur in the presence of potent mineralization inhibitors like pyrophosphate and matrix GLA protein, and in the absence of local inflammation, cell death, destruction of matrix, and so on. Figure 6 concatenates major principles of pathological mineralization known to date and puts fetuin-A in perspective. Fetuin-A serves as a mineral chaperone, a carrier protein facilitating transport and clearing of potentially proinflammatory and procalcific cargo (waste). The mechanistic and functional analogy between lipoproteins and fetuin-A–based calciprotein particles is obvious. No matter what the preferred mechanism of atherosclerotic lesion calcification is, deranged mineral homeostasis, dyslipidemia, compromised scavenging and debris clearing, inflammation, apoptosis, matrix mineralization, and osteogenesis are all known pundits (“partners in crime”), and it seems like fetuin-A counters many of them and thus is a highly pleomorphic protein and is truly a systemic regulator of mineralization.
Figure 6. Calcification-related genes “at work” in vascular smooth muscle cells (VSMC) undergoing metaplasia. After “transdifferentiation” into mineralizing VSMC, the cells elaborate markers of the osteogenic/chondrogenic lineage. The sodium/phosphate cotransporter Pit-1 mediates phosphate transport into the cell. Elevated phosphate levels in the cytoplasm upregulate expression of Runx2/Cbfa-1, an osteogenic transcription factor. In addition, hyperphosphatemia enhances production of apoptotic bodies and matrix vesicles that nucleate vascular mineral deposition. The transforming growth factor (TGF)-β/TGF-like cytokine BMP-7 maintains the contractile phenotype (via Smad 6/similiar to mothers against decapentaplegic 6 and Smad 7 signaling) and BMP-2 and TGF-β1 enhance the osteogenic phenotype. Extracellular calcium is transported into matrix vesicles by Ca2+ channel-forming annexins II, V, and VI. Calcium enhances the phosphate-dependent osteogenic differentiation by upregulation of Pit-1 expression. Pyrophosphate (PP) acts as an inhibitor of basic calcium phosphate crystal growth. The concentration of PP is controlled by nucleotide pyrophosphatase/phosphotransferase-1 (ENPP1), which generates PP, the PP transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP), which cleaves PP. When cells fail to properly handle a high mineral load because of elevated extracellular calcium phosphate, especially in the absence of extracellular fetuin-A, they will succumb to apoptosis.65 Apoptotic bodies (AB) containing high amounts of mineral-like matrix vesicles readily mineralize and form a potent nidus for further extracellular matrix calcification. Unlike matrix vesicle-mediated mineralization, AB-mediated mineralization does not require alkaline phosphatase and annexins. In addition, phosphatidylserine (PS) is localized on opposite sides of the plasma membrane of matrix 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 thus reduces calcium-induced apoptosis in VSMC. BMPR-I indicates BMP receptor-I; MGP, matrix GLA protein; NTP, nucleotide triphosphate; NMP, nucleotide monophosphate; OPN, osteopontin; SM-MHC, smooth muscle myosin heavy chain; TNF-α, tumor necrosis factor-α. Illustration credit: Cosmocyte/Ben Smith.
Sources of Funding
This work was funded by the
Disclosures
None.
Non-standard Abbreviations and Acronyms
| α2-HS-glycoprotein | α2-Heremans Schmid glycoprotein/fetuin-A |
| AB | Apoptotic body, membrane enclosed fragments of cells undergoing apoptosis |
| AFP | Alpha-fetoprotein |
| AHSG | Genetic symbol for α2-HS-glycoprotein |
| ANK | Ankylosing spondylitis gene, mutation of this gene causes spondyloarthritis |
| BCP | Basic calcium phosphate (octacalcium phosphate or 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-cleaving enzyme |
| EPIC | European Prospective Investigation into Cancer and Nutrition study involving 27 548 subjects |
| FABP | Fatty acid binding protein |
| FMC | Fetuin mineral complex |
| HMGB1 | High-mobility group protein B1, a proinflammatory nuclear protein |
| HRG | Histidine-rich glycoprotein, a fetuin-related protein |
| KNG | Kininogen, a fetuin-related protein |
| LPS | Lipopolysaccharide, a strong inflammatory agent from bacterial cell walls |
| MGP | Matrix GLA protein |
| MO | Monocyte |
| MIA | Malnutrition-inflammation-atherosclerosis syndrome |
| MMP | Matrix metalloproteinase |
| MP | Macrophage |
| MV | Matrix vesicles, mineral-laden vesicles believed 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 inhibitor and anticalcification agent |
| PS | Phosphatidylserine, membrane phospholipid exposed on the cell surface during apoptosis |
| Runx2/Cbfa-1 | Runt-related transcription factor 2/core binding factor 1, an osteoblastic transcription factor |
| SANS | Small-angle neutron scattering, an analytical technique to determine molecular structure |
| SAXS | Small angle x-ray scattering, an analytical technique to study crystal growth at high resolution |
| Smad 6 | Similar to mothers against decapentaplegic 6, an intracellular signaling protein |
| SM-MHC | Smooth muscle myosin heavy chain |
| TGF-β | Transforming growth factor beta |
| TNAP | Tissue nonspecific alkaline phosphatase, cleaves pyrophosphate PP |
| TNF | Tumor necrosis factor |
| VSMC | Vascular smooth muscle cell |
Footnotes
This article is part of a thematic series on Pathobiology of Calcific Vasculopathy and Valvulopathy, which included the 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 [Circ Res. 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
References
- 1.
Pedersen KO . Fetuin, a new globulin isolated from serum. Nature.1944; 154: 575. CrossrefGoogle Scholar - 2.
Heremans JF . Les Globulines Sériques du Système Gamma.Brussels: Arscia, 1960.Google Scholar - 3.
Schmid K, Bürgi W . Preparation and properties of the human plasma Ba-alpha2-glycoproteins. Biochim Biophys Acta.1961; 47: 440– 453. CrossrefMedlineGoogle Scholar - 4.
Schultze HE, Heide K, Haupt H . Charakterisierung eines niedermolekularen a2-Mukoids aus Humanserum. Naturwiss.1962; 49: 15– 17. CrossrefGoogle Scholar - 5.
Heiss A, DuChesne A, Denecke B, Grötzinger J, Yamamoto K, Renné T, Jahnen-Dechent W . Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J Biol Chem.2003; 278: 13333– 13341. CrossrefMedlineGoogle Scholar - 6.
Jahnen-Dechent W, Trindl A, Godovac-Zimmermann J, Müller-Esterl W . Posttranslational processing of human alpha 2-HS glycoprotein (human fetuin). Evidence for the production of a phosphorylated single-chain form by hepatoma cells. Eur J Biochem.1994; 226: 59– 69. CrossrefMedlineGoogle Scholar - 7.
Nawratil P, Lenzen S, Kellermann J, Haupt H, Schinke T, Müller-Esterl W, Jahnen-Dechent W . Limited proteolysis of human alpha2-HS glycoprotein/fetuin. Evidence that a chymotryptic activity can release the connecting peptide. J Biol Chem.1996; 271: 31735– 31741. CrossrefMedlineGoogle Scholar - 8.
Edge AS, Spiro RG . Presence of an O-glycosidically linked hexasaccharide in fetuin. J Biol Chem.1987; 262: 16135– 16141. CrossrefMedlineGoogle Scholar - 9.
Bendiak B, Harris-Brandts M, Michnick SW, Carver JP, Cumming DA . Separation of the complex asparagine-linked oligosaccharides of the glycoprotein fetuin and elucidation of three triantennary structures having sialic acids linked only to galactose residues. Biochemistry.1989; 28: 6491– 6499. CrossrefMedlineGoogle Scholar - 10.
Denecke B, Gräber S, Schäfer C, Heiss A, Wöltje M, Jahnen-Dechent W . Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A. Biochem J.2003; 376: 135– 145. CrossrefMedlineGoogle Scholar - 11.
Ohnishi T, Nakamura O, Arakaki N, Miyazaki H, Daikuhara Y . Effects of cytokines and growth factors on phosphorylated fetuin biosynthesis by adult rat hepatocytes in primary culture. Biochem Biophys Res Comm.1994; 200: 598– 605. CrossrefMedlineGoogle Scholar - 12.
Ohnishi T, Nakamura O, Arakaki N, Daikuhara Y . Effect of phosphorylated rat fetuin on the growth of hepatocytes in primary culture in the presence of human hepatocyte-growth factor. Evidence that phosphorylated fetuin is a natural modulator of hepatocyte-growth factor. Eur J Biochem.1997; 243: 753– 761. CrossrefMedlineGoogle Scholar - 13.
Haglund AC, Ek B, Ek P . Phosphorylation of human plasma alpha2-Heremans-Schmid glycoprotein (human fetuin) in vivo. Biochem J.2001; 357: 437– 445. CrossrefMedlineGoogle Scholar - 14.
Memoli B, De Bartolo L, Favia P, Morelli S, Lopez LC, Procino A, Barbieri G, Curcio E, Giorno L, Esposito P, Cozzolino M, Brancaccio D, Andreucci VE, d'Agostino R, Drioli E . Fetuin-A gene expression, synthesis and release in primary human hepatocytes cultured in a galactosylated membrane bioreactor. Biomaterials.2007; 28: 4836– 4844. CrossrefMedlineGoogle Scholar - 15.
Kübler D, Gosenca D, Wind M, Heid H, Friedberg I, Jahnen-Dechent W, Lehmann WD . Proteolytic processing by matrix metalloproteinases and phosphorylation by protein kinase CK2 of fetuin-A, the major globulin of fetal calf serum. Biochimie.2007; 89: 410– 418. CrossrefMedlineGoogle Scholar - 16.
Hortin GL, Schilling M, Graham JP . Inhibitors of the sulfation of proteins, glycoproteins, and proteoglycans. Biochem Biophys Res Comm.1988; 150: 342– 348. CrossrefMedlineGoogle Scholar - 17.
Auberger P, Falquerho L, Contreres JO, Pages G, Le Cam G, Rossi B, Le Cam A . Characterization of a natural inhibitor of the insulin receptor tyrosine kinase: cDNA cloning, purification, and anti-mitogenic activity. Cell.1989; 58: 631– 640. CrossrefMedlineGoogle Scholar - 18.
Mathews ST, Chellam N, Srinivas PR, Cintron VJ, Leon MA, Goustin AS, Grunberger G . Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor. Mol Cell Endocrinol.2000; 164: 87– 98. CrossrefMedlineGoogle Scholar - 19.
Schinke T, Amendt C, Trindl A, Pöschke O, Müller-Esterl W, Jahnen-Dechent W . The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis. J Biol Chem.1996; 271: 20789– 20796. CrossrefMedlineGoogle Scholar - 20.
Berg T, Ose T, Ose L, Tolleshaug H . Intracellular degradation of 125I-labelled asialo-glycoproteins in rat hepatocytes: effect of leupeptin on subcellular distribution of asialo-fetuin. Int J Biochem.1981; 13: 253– 259. CrossrefMedlineGoogle Scholar - 21.
Lodish HF . Recognition of complex oligosaccharides by the multi-subunit asialoglycoprotein receptor. Trends Biochem Sci.1991; 16: 374– 377. CrossrefMedlineGoogle Scholar - 22.
Spiess M . The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry.1990; 29: 10009– 10018. CrossrefMedlineGoogle Scholar - 23.
Nie Z . Fetuin: its enigmatic property of growth promotion. Am J Physiol.1992; 263: C551– C562. CrossrefMedlineGoogle Scholar - 24.
Hinkle P . Efraim Racker, 1913–1991. J Bioenerg Biomembr.1992; 24: 517– 519. CrossrefMedlineGoogle Scholar - 25.
Kornberg A . Ten commandments: lessons from the enzymology of DNA replication. J Bacteriol.2000; 182: 3613– 3618. CrossrefMedlineGoogle Scholar - 26.
Olivier E, Soury E, Ruminy P, Husson A, Parmentier F, Daveau M, Salier JP . Fetuin-B, a second member of the fetuin family in mammals. Biochem J.2000; 350: 589– 597. CrossrefMedlineGoogle Scholar - 27.
Jethwaney D, Lepore T, Hassan S, Mello K, Rangarajan R, Jahnen-Dechent W, Wirth D, Sultan AA . Fetuin-A, a hepatocyte-specific protein that binds Plasmodium berghei thrombospondin-related adhesive protein: a potential role in infectivity. Infect Immun.2005; 73: 5883– 5891. CrossrefMedlineGoogle Scholar - 28.
Fisher H, Puck T, Sato G . Molecular growth requirements of single mammalian cells: the action of fetuin in promoting cell attachment to glass. Proc Natl Acad Sci U S A.1958; 44: 4– 10. CrossrefMedlineGoogle Scholar - 29.
Tajirian T, Dennis JW, Swallow CJ . Regulation of human monocyte proMMP-9 production by fetuin, an endogenous TGF-beta antagonist. J Cell Physiol.2000; 185: 174– 183. CrossrefMedlineGoogle Scholar - 30.
Leite-Browning ML, McCawley LJ, Choi OH, Matrisian LM, Ochieng J . Interactions of alpha2-HS-glycoprotein (fetuin) with MMP-3 and murine squamous cell carcinoma cells. Int J Oncol.2002; 21: 965– 971. MedlineGoogle Scholar - 31.
Hedrich J, Lottaz D, Meyer K, Yiallouros I, Jahnen-Dechent W, Stöcker W, Becker-Pauly C . Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases. Biochemistry.2010; 49: 8599– 8607. CrossrefMedlineGoogle Scholar - 32.
Mellgren RL, Huang X . Fetuin A stabilizes m-calpain and facilitates plasma membrane repair. J Biol Chem.2007; 282: 35868– 35877. CrossrefMedlineGoogle Scholar - 33.
Nakamura O, Kazi JA, Ohnishi T, Arakaki N, Shao Q, Kajihara T, Daikuhara Y . Effects of rat fetuin on stimulation of bone resorption in the presence of parathyroid hormone. Biosci Biotechnol Biochem.1999; 63: 1383– 1391. CrossrefMedlineGoogle Scholar - 34.
Kundranda MN, Henderson M, Carter KJ, Gorden L, Binhazim A, Ray S, Baptiste T, Shokrani M, Leite-Browning ML, Jahnen-Dechent W, Matrisian LM, Ochieng J . The serum glycoprotein fetuin-A promotes Lewis lung carcinoma tumorigenesis via adhesive-dependent and adhesive-independent mechanisms. Cancer Res.2005; 65: 499– 506. CrossrefMedlineGoogle Scholar - 35.
Leite-Browning ML, McCawley LJ, Jahnen-Dechent W, King L, Matrisian L, Ochieng J . Alpha 2-HS glycoprotein (fetuin-A) modulates murine skin tumorigenesis. Int J Oncol.2004; 25: 319– 324. MedlineGoogle Scholar - 36.
Kumbla L, Bhadra S, Subbiah MT . Multifunctional role for fetuin (fetal protein) in lipid transport. FASEB J.1991; 5: 2971– 2975. CrossrefMedlineGoogle Scholar - 37.
Cayatte AJ, Kumbla L, Subbiah MT . Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin. J Biol Chem.1990; 265: 5883– 5888. CrossrefMedlineGoogle Scholar - 38.
Kumbla L, Cayatte AJ, Subbiah MT . Association of a lipoprotein-like particle with bovine fetuin. FASEB J.1989; 3: 2075– 2080. CrossrefMedlineGoogle Scholar - 39.
Dasgupta S, Bhattacharya S, Biswas A, Majumdar SS, Mukhopadhyay S, Ray S, Bhattacharya S . NF-kappaB mediates lipid-induced fetuin-A expression in hepatocytes that impairs adipocyte function effecting insulin resistance. Biochem J.2010; 429: 451– 462. CrossrefMedlineGoogle Scholar - 40.
Furuhashi M, Hotamisligil GS . Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov.2008; 7: 489– 503. CrossrefMedlineGoogle Scholar - 41.
Skelton SK, Wong KH . Simple, efficient purification of filamentous hemagglutinin and pertussis toxin from Bordetella pertussis by hydrophobic and affinity interaction. J Clin Microbiol.1990; 28: 1062– 1065. CrossrefMedlineGoogle Scholar - 42.
Chong P, Klein M . Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography. Biochem Cell Biol.1989; 67: 387– 391. CrossrefMedlineGoogle Scholar - 43.
Grossbard ML, Lambert JM, Goldmacher VS, Blättler WA, Nadler LM . Correlation between in vivo toxicity and preclinical in vitro parameters for the immunotoxin anti-B4-blocked ricin. Cancer Res.1992; 52: 4200– 4207. MedlineGoogle Scholar - 44.
Cawley DB, Simpson DL, Herschman HR . Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes. Proc Natl Acad Sci U S A.1981; 78: 3383– 3387. CrossrefMedlineGoogle Scholar - 45.
Demetriou M, Binkert C, Sukhu B, Tenenbaum HC, Dennis JW . Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist. J Biol Chem.1996; 271: 12755– 12761. CrossrefMedlineGoogle Scholar - 46.
Binkert C, Demetriou M, Sukhu B, Szweras M, Tenenbaum HC, Dennis JW . Regulation of osteogenesis by fetuin. J Biol Chem.1999; 274: 28514– 28520. CrossrefMedlineGoogle Scholar - 47.
Szweras M, Liu D, Partridge EA, Pawling J, Sukhu B, Clokie C, Jahnen-Dechent W, Tenenbaum HC, Swallow CJ, Grynpas MD, Dennis JW . alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling. J Biol Chem.2002; 277: 19991– 19997. CrossrefMedlineGoogle Scholar - 48.
Swallow CJ, Partridge EA, Macmillan JC, Tajirian T, DiGuglielmo GM, Hay K, Szweras M, Jahnen-Dechent W, Wrana JL, Redston M, Gallinger S, Dennis JW . alpha2HS-glycoprotein, an antagonist of transforming growth factor beta in vivo, inhibits intestinal tumor progression. Cancer Res.2004; 64: 6402– 6409. CrossrefMedlineGoogle Scholar - 49.
Zhang M, Caragine T, Wang H, Cohen PS, Botchkina G, Soda K, Bianchi M, Ulrich P, Cerami A, Sherry B, Tracey KJ . Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response. J Exp Med.1997; 185: 1759– 1768. CrossrefMedlineGoogle Scholar - 50.
Wang H, Zhang M, Bianchi M, Sherry B, Sama A, Tracey KJ . Fetuin (alpha2-HS-glycoprotein) opsonizes cationic macrophagedeactivating molecules. Proc Natl Acad Sci U S A.1998; 95: 14429– 14434. CrossrefMedlineGoogle Scholar - 51.
Wang H, Zhang M, Soda K, Sama A, Tracey K . Fetuin protects the fetus from TNF. Lancet.1997; 350: 861– 862. CrossrefMedlineGoogle Scholar - 52.
Dziegielewska KM, Andersen NA . The fetal glycoprotein, fetuin, counteracts ill-effects of the bacterial endotoxin, lipopolysaccharide, in pregnancy. Biol Neonate.1998; 74: 372– 375. CrossrefMedlineGoogle Scholar - 53.
Ombrellino M, Wang H, Yang H, Zhang M, Vishnubhakat J, Frazier A, Scher LA, Friedman SG, Tracey KJ . Fetuin, a negative acute phase protein, attenuates TNF synthesis and the innate inflammatory response to carrageenan. Shock.2001; 15: 181– 185. CrossrefMedlineGoogle Scholar - 54.
Wang H, Li W, Zhu S, Li J, D'Amore J, Ward MF, Yang H, Wu R, Jahnen-Dechent W, Tracey KJ, Wang P, Sama AE . Peripheral administration of fetuin-A attenuates early cerebral ischemic injury in rats. J Cereb Blood Flow Metab.2010; 30: 493– 504. CrossrefMedlineGoogle Scholar - 55.
Li W, Zhu S, Li J, Huang Y, Rongrong , Zhou , Fan X, Yang H, Gong X, Eissa NT, Jahnen-Dechent W, Wang P, Tracey KJ, Sama AE, Wang H . A hepatic protein, fetuin-A, occupies a protective role in lethal systemic inflammation.PLoS One.2011: 1– 31.Google Scholar - 56.
Chertov OYu, Ermolaeva MV, Satpaev DK, Saschenko LP, Kabanova OD, Lukanidin EM, Lukjianova TI, Redchenko IV, Blishchenko LYu, Gnuchev NV . Inhibitory effect of calf fetuin on the cytotoxic activity of LAK cell-derived factors and tumor necrosis factor. Immunol Lett.1994; 42: 97– 100. CrossrefMedlineGoogle Scholar - 57.
Terkeltaub RA, Santoro DA, Mandel G, Mandel N . Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals. Evidence that serum alpha 2-HS glycoprotein is a potent and specific crystal-bound inhibitor. Arthritis Rheum.1988; 31: 1081– 1089. CrossrefMedlineGoogle Scholar - 58.
Pazár B, Ea HK, Narayan S, Kolly L, Bagnoud N, Chobaz V, Roger T, Lioté F, So A, Busso N . Basic calcium phosphate crystals induce monocyte/macrophage IL-1β secretion through the NLRP3 inflammasome in vitro. J Immunol.2011; 186: 2495– 2502. CrossrefMedlineGoogle Scholar - 59.
Nadra I, Boccaccini AR, Philippidis P, Whelan LC, McCarthy GM, Haskard DO, Landis RC . Effect of particle size on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages. Atherosclerosis.2008; 196: 98– 105. CrossrefMedlineGoogle Scholar - 60.
Nadra I, Mason JC, Philippidis P, Florey O, Smythe CD, McCarthy GM, Landis RC, Haskard DO . Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification?Circ Res.2005; 96: 1248– 1256. LinkGoogle Scholar - 61.
Ewence AE, Bootman M, Roderick HL, Skepper JN, McCarthy G, Epple M, Neumann M, Shanahan CM, Proudfoot D . Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ Res.2008; 103: 28– 34. LinkGoogle Scholar - 62.
Ea HK, Uzan B, Rey C, Lioté F . Octacalcium phosphate crystals directly stimulate expression of inducible nitric oxide synthase through p38 and JNK mitogen-activated protein kinases in articular chondrocytes. Arthritis Res Ther.2005; 7: R915– 926. CrossrefMedlineGoogle Scholar - 63.
Halverson PB, Derfus BA . Calcium crystal-induced inflammation. Curr Opin Rheumatol.2001; 13: 221– 224. CrossrefMedlineGoogle Scholar - 64.
Halverson PB, Greene A, Cheung HS . Intracellular calcium responses to basic calcium phosphate crystals in fibroblasts. Osteoarth Cartilage.1998; 6: 324– 329. CrossrefMedlineGoogle Scholar - 65.
Reynolds JL, Skepper JN, McNair R, Kasama T, Gupta K, Weissberg PL, Jahnen-Dechent W, Shanahan CM . Multifunctional roles for serum protein fetuin-a in inhibition of human vascular smooth muscle cell calcification. J Am Soc Nephrol.2005; 16: 2920– 2930. CrossrefMedlineGoogle Scholar - 66.
Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O, Erlandsson-Harris H, Janson A, Kokkola R, Zhang M, Yang H, Tracey KJ . High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med.2000; 192: 565– 570. CrossrefMedlineGoogle Scholar - 67.
Zhang M, Borovikova LV, Wang H, Metz C, Tracey KJ . Spermine inhibition of monocyte activation and inflammation. Mol Med.1999; 5: 595– 605. CrossrefMedlineGoogle Scholar - 68.
Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J, Frazier A, Yang H, Ivanova S, Borovikova L, Manogue KR, Faist E, Abraham E, Andersson J, Andersson U, Molina PE, Abumrad NN, Sama A, Tracey KJ . HMG-1 as a late mediator of endotoxin lethality in mice. Science.1999; 285: 248– 251. CrossrefMedlineGoogle Scholar - 69.
Shanahan CM . Inflammation ushers in calcification: a cycle of damage and protection?Circulation.2007; 116: 2782– 2785. LinkGoogle Scholar - 70.
Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA . Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation.2006; 113: 1760– 1767. LinkGoogle Scholar - 71.
Weikert C, Stefan N, Schulze MB, Pischon T, Berger K, Joost HG, Häring HU, Boeing H, Fritsche A . Plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke. Circulation.2008; 118: 2555– 2562. LinkGoogle Scholar - 72.
Stefan N, Fritsche A, Weikert C, Boeing H, Joost HG, Häring HU, Schulze MB . Plasma fetuin-A levels and the risk of type 2 diabetes. Diabetes.2008; 57: 2762– 2767. CrossrefMedlineGoogle Scholar - 73.
Fisher E, Stefan N, Saar K, Drogan D, Schulze MB, Fritsche A, Joost HG, Häring HU, Hubner N, Boeing H, Weikert C . Association of AHSG gene polymorphisms with fetuin-A plasma levels and cardiovascular diseases in the EPIC-Potsdam study. Circ Cardiovasc Genet.2009; 2: 607– 613. LinkGoogle Scholar - 74.
Rauth G, Pöschke O, Fink E, Eulitz M, Tippmer S, Kellerer M, Häring HU, Nawratil P, Haasemann M, Jahnen-Dechent W . The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor. Eur J Biochem.1992; 204: 523– 529. CrossrefMedlineGoogle Scholar - 75.
Le Cam A, Auberger P, Falquerho L, Contreres JO, Pages G, Le Cam G, Rossi B . pp63 is very likely the rat fetuin. Cell.1992; 68: 8. CrossrefGoogle Scholar - 76.
Brown WM, Christie DL, Dziegielewska KM, Saunders NR, Yang F . The rat protein encoded by clone pp63 is a fetuin/alpha 2-HS glycoprotein-like molecule, but is it the tyrosine kinase inhibitor pp63?Cell.1992; 68: 7– 8. CrossrefMedlineGoogle Scholar - 77.
Chen H, Srinivas PR, Cong LN, Li Y, Grunberger G, Quon MJ . Alpha2-Heremans Schmid glycoprotein inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in rat adipose cells. Endocrinology.1998; 139: 4147– 4154. CrossrefMedlineGoogle Scholar - 78.
Mathews ST, Srinivas PR, Leon MA, Grunberger G . Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase. Life Sci.1997; 61: 1583– 1592. CrossrefMedlineGoogle Scholar - 79.
Mathews ST, Rakhade S, Zhou X, Parker GC, Coscina DV, Grunberger G . Fetuin-null mice are protected against obesity and insulin resistance associated with aging. Biochem Biophys Res Comm.2006; 350: 437– 443. CrossrefMedlineGoogle Scholar - 80.
Jahnen-Dechent W, Schinke T, Trindl A, Müller-Esterl W, Sablitzky F, Kaiser S, Blessing M . Cloning and targeted deletion of the mouse fetuin gene. J Biol Chem.1997; 272: 31496– 31503. CrossrefMedlineGoogle Scholar - 81.
Wong N, Blair AR, Morahan G, Andrikopoulos S . The deletion variant of nicotinamide nucleotide transhydrogenase (Nnt) does not affect insulin secretion or glucose tolerance. Endocrinology.2010; 151: 96– 102. CrossrefMedlineGoogle Scholar - 82.
Laboratory TJ . The Nnt deletion in C57BL/6J mice—questions and answers.Jackson Laboratory Newsletter.2009: 1– 2.Google Scholar - 83.
Mathews ST, Singh GP, Ranalletta M, Cintron VJ, Qiang X, Goustin AS, Jen KL, Charron MJ, Jahnen-Dechent W, Grunberger G . Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes.2002; 51: 2450– 2458. CrossrefMedlineGoogle Scholar - 84.
Falquerho L, Paquereau L, Vilarem MJ, Galas S, Patey G, Le Cam A . Functional characterization of the promoter of pp63, a gene encoding a natural inhibitor of the insulin receptor tyrosine kinase. Nucleic Acids Res.1992; 20: 1983– 1990. CrossrefMedlineGoogle Scholar - 85.
Daveau M, Christian-Davrinche , Julen N, Hiron M, Arnaud P, Lebreton JP . The synthesis of human alpha-2-HS glycoprotein is down-regulated by cytokines in hepatoma HepG2 cells. FEBS Lett.1988; 241: 191– 194. CrossrefMedlineGoogle Scholar - 86.
Lebreton JP, Joisel F, Raoult JP, Lannuzel B, Rogez JP, Humbert G . Serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant. J Clin Invest.1979; 64: 1118– 1129. CrossrefMedlineGoogle Scholar - 87.
Metry G, Stenvinkel P, Qureshi AR, Carrero JJ, Yilmaz MI, Bárány P, Snaedal S, Heimbürger O, Lindholm B, Suliman ME . Low serum fetuin-A concentration predicts poor outcome only in the presence of inflammation in prevalent haemodialysis patients. Eur J Clin Invest.2008; 38: 804– 811. CrossrefMedlineGoogle Scholar - 88.
Wang AY, Woo J, Lam CW, Wang M, Chan IH, Gao P, Lui SF, Li PK, Sanderson JE . Associations of serum fetuin-A with malnutrition, inflammation, atherosclerosis and valvular calcification syndrome and outcome in peritoneal dialysis patients. Nephrol Dial Transplant.2005; 20: 1676– 1685. CrossrefMedlineGoogle Scholar - 89.
Ketteler M, Bongartz P, Westenfeld R, Wildberger JE, Mahnken AH, Böhm R, Metzger T, Wanner C, Jahnen-Dechent W, Floege J . Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study. Lancet.2003; 361: 827– 833. CrossrefMedlineGoogle Scholar - 90.
Ogawa W, Kasuga M . Cell signaling. Fat stress and liver resistance. Science.2008; 322: 1483– 1484. CrossrefMedlineGoogle Scholar - 91.
Hennige AM, Staiger H, Wicke C, Machicao F, Fritsche A, Häring H-U, Stefan N . Fetuin-A induces cytokine expression and suppresses adiponectin production. PLoS ONE.2008; 3: e1765. CrossrefMedlineGoogle Scholar - 92.
Schinke T, Koide T, Jahnen-Dechent W . Human histidine-rich glycoprotein expressed in SF9 insect cells inhibits apatite formation. FEBS Lett.1997; 412: 559– 562. CrossrefMedlineGoogle Scholar - 93.
Herwald H, Jahnen-Dechent W, Alla SA, Hock J, Bouma BN, Müller-Esterl W . Mapping of the high molecular weight kininogen binding site of prekallikrein. Evidence for a discontinuous epitope formed by distinct segments of the prekallikrein heavy chain. J Biol Chem.1993; 268: 14527– 14535. CrossrefMedlineGoogle Scholar - 94.
Brown WM, Dziegielewska KM, Saunders NR, Christie DL, Nawratil P, Müller-Esterl W . The nucleotide and deduced amino acid structures of sheep and pig fetuin. Common structural features of the mammalian fetuin family. Eur J Biochem.1992; 205: 321– 331. CrossrefMedlineGoogle Scholar - 95.
Hock J, Vogel R, Linke RP, Müller-Esterl W . High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies. J Biol Chem.1990; 265: 12005– 12011. CrossrefMedlineGoogle Scholar - 96.
Kellermann J, Haupt H, Auerswald E, Müller-Ester W . The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens. J Biol Chem.1989; 264: 14121– 14128. CrossrefMedlineGoogle Scholar - 97.
White H, Totty N, Panayotou G . Haemonectin, a granulocytic-cell-binding protein, is related to the plasma glycoprotein fetuin. Eur J Biochem.1993; 213: 523– 528. CrossrefMedlineGoogle Scholar - 98.
Triffitt JT, Owen ME, Ashton BA, Wilson JM . Plasma disappearance of rabbit alpha2HS-glycoprotein and its uptake by bone tissue. Calcif Tissue Res.1978; 26: 155– 161. CrossrefMedlineGoogle Scholar - 99.
Triffitt JT, Gebauer U, Ashton BA, Owen ME, Reynolds JJ . Origin of plasma alpha2HS-glycoprotein and its accumulation in bone. Nature.1976; 262: 226– 227. CrossrefMedlineGoogle Scholar - 100.
Triffitt JT . Plasma proteins present in human cortical bone: enrichment of the alpha2HS-glycoprotein. Calcif Tissue Res.1976; 22: 27– 33. MedlineGoogle Scholar - 101.
Dickson IR, Poole AR, Veis A . Localisation of plasma alpha2HS glycoprotein in mineralising human bone. Nature.1975; 256: 430– 432. CrossrefMedlineGoogle Scholar - 102.
Termine JD, Belcourt AB, Conn KM, Kleinman HK . Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem.1981; 256: 10403– 10408. CrossrefMedlineGoogle Scholar - 103.
Schäfer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, M̈uller-Esterl W, Schinke T, Jahnen-Dechent W . The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest.2003; 112: 357– 366. CrossrefMedlineGoogle Scholar - 104.
Jahnen-Dechent W, Schäfer C, Ketteler M, McKee MD . Mineral chaperones: a role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification. J Mol Med.2008; 86: 379– 389. CrossrefMedlineGoogle Scholar - 105.
Westenfeld R, Schäfer C, Krüger T, Haarmann C, Schurgers LJ, Reutelingsperger C, Ivanovski O, Drueke T, Massy ZA, Ketteler M, Floege J, Jahnen-Dechent W . Fetuin-A protects against atherosclerotic calcification in CKD. J Am Soc Nephrol.2009; 20: 1264– 1274. CrossrefMedlineGoogle Scholar - 106.
Westenfeld R, Schäfer C, Smeets R, Brandenburg VM, Floege J, Ketteler M, Jahnen-Dechent W . Fetuin-A (AHSG) prevents extraosseous calcification induced by uraemia and phosphate challenge in mice. Nephrol Dial Transplant.2007; 22: 1537– 1546. CrossrefMedlineGoogle Scholar - 107.
Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM . Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol.2004; 15: 2857– 2867. CrossrefMedlineGoogle Scholar - 108.
Olde Loohuis KM, Jahnen-Dechent W, van Dorp W . The case: milky ascites is not always chylous. Kidney Int.2010; 77: 77– 78. CrossrefMedlineGoogle Scholar - 109.
Heiss A, Eckert T, Aretz A, Richtering W, van Dorp W, Schäfer C, Jahnen-Dechent W . Hierarchical role of fetuin-A and acidic serum proteins in the formation and stabilization of calcium phosphate particles. J Biol Chem.2008; 283: 14815– 14825. CrossrefMedlineGoogle Scholar - 110.
Wald J, Wiese S, Eckert T, Jahnen-Dechent W, Richtering W, Heiss A . Structure and stability of calcium phosphate - fetuin-A colloids probed by time-resolved dynamic light scattering. Soft Matter.2011; 7: 2869– 2874. CrossrefGoogle Scholar - 111.
Heiss A, Pipich V, Jahnen-Dechent W, Schwahn D . Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on fetuin-A controlled calcification inhibition. Biophys J.2010; 99: 3989– 3995. CrossrefGoogle Scholar - 112.
Rochette CN, Rosenfeldt S, Heiss A, Narayanan T, Ballauff M, Jahnen-Dechent W . A shielding topology stabilizes the early stage protein-mineral complexes of fetuin-A and calcium phosphate: a time-resolved small-angle X-ray study. Chembiochem.2009; 10: 735– 740. CrossrefMedlineGoogle Scholar - 113.
Heiss A, Jahnen-Dechent W, Endo H, Schwahn D . Structural dynamics of a colloidal protein-mineral complex bestowing on calcium phosphate a high solubility in biological fluids. Biointerphases.2007; 2: 16– 20. CrossrefMedlineGoogle Scholar - 114.
Price PA, Williamson MK, Nguyen TM, Than TN . Serum levels of the fetuin-mineral complex correlate with artery calcification in the rat. J Biol Chem.2004; 279: 1594– 1600. CrossrefMedlineGoogle Scholar - 115.
Price PA, Lim JE . The inhibition of calcium phosphate precipitation by fetuin is accompanied by the formation of a fetuin-mineral complex. J Biol Chem.2003; 278: 22144– 22152. CrossrefMedlineGoogle Scholar - 116.
Price PA, Nguyen TM, Williamson MK . Biochemical characterization of the serum fetuin-mineral complex. J Biol Chem.2003; 278: 22153– 22160. CrossrefMedlineGoogle Scholar - 117.
Price PA, Caputo JM, Williamson MK . Bone origin of the serum complex of calcium, phosphate, fetuin, and matrix Gla protein: biochemical evidence for the cancellous bone-remodeling compartment. J Bone Miner Res.2002; 17: 1171– 1179. CrossrefMedlineGoogle Scholar - 118.
Price PA, Thomas GR, Pardini AW, Figueira WF, Caputo JM, Williamson MK . Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats. J Biol Chem.2002; 277: 3926– 3934. CrossrefMedlineGoogle Scholar - 119.
Ciftçioğlu N, McKay DS . Pathological calcification and replicating calcifying-nanoparticles: general approach and correlation. Pediatr Res.2010; 67: 490– 499. CrossrefMedlineGoogle Scholar - 120.
Raoult D, Drancourt M, Azza S, Nappez C, Guieu R, Rolain JM, Fourquet P, Campagna B, La Scola B, Mege J-L, Mansuelle P, Lechevalier E, Berland Y, Gorvel JP, Renesto P . Nanobacteria are mineralo fetuin complexes. PLoS Pathog.2008; 4: e41. CrossrefMedlineGoogle Scholar - 121.
Young JD, Martel J, Young L, Wu CY, Young A, Young D . Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis. PLoS ONE.2009; 4: e4417. CrossrefMedlineGoogle Scholar - 122.
Wu C-Y, Martel J, Young D, Young JD . Fetuin-a/albumin-mineral complexes resembling serum calcium granules and putative nanobacteria: demonstration of a dual inhibition-seeding concept. PLoS ONE.2009; 4: e8058. CrossrefMedlineGoogle Scholar - 123.
Mahamid J, Aichmayer B, Shimoni E, Ziblat R, Li C, Siegel S, Paris O, Fratzl P, Weiner S, Addadi L . Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays. Proc Natl Acad Sci U S A.2010; 107: 6316– 6321. CrossrefMedlineGoogle Scholar - 124.
Wang L, Nancollas GH . Pathways to biomineralization and biodemineralization of calcium phosphates: the thermodynamic and kinetic controls. Dalton Transactions.2009; 2665– 2672. Google Scholar - 125.
Nudelman F, Pieterse K, George A, Bomans PH, Friedrich H, Brylka LJ, Hilbers PA, de With G, Sommerdijk NA . The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater.2010; 9: 1004– 1009. CrossrefMedlineGoogle Scholar - 126.
Cölfen H . Biomineralization: A crystal-clear view. Nat Mater.2010; 9: 960– 961. CrossrefMedlineGoogle Scholar - 127.
Price PA, Toroian D, Lim JE . Mineralization by inhibitor exclusion: the calcification of collagen with fetuin. J Biol Chem.2009; 284: 17092– 17101. CrossrefMedlineGoogle Scholar - 128.
Toroian D, Price PA . The essential role of fetuin in the serum-induced calcification of collagen. Calcif Tissue Int.2008; 82: 116– 126. CrossrefMedlineGoogle Scholar - 129.
Toroian D, Lim JE, Price PA . The size exclusion characteristics of type I collagen: implications for the role of noncollagenous bone constituents in mineralization. J Biol Chem.2007; 282: 22437– 22447. CrossrefMedlineGoogle Scholar - 130.
Folk R . SEM imaging of bacteria and nannobacteria in carbonate sediments and rocks. J Sediment Res.1993; 63: 990– 999. Google Scholar - 131.
McKay DS, Gibson EK, Thomas-Keprta KL, Vali H, Romanek CS, Clemett SJ, Chillier XD, Maechling CR, Zare RN . Search for past life on Mars: possible relic biogenic activity in martian meteorite ALH84001. Science.1996; 273: 924– 930. CrossrefMedlineGoogle Scholar - 132.
Kajander EO, Ciftçioglu N . Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc Natl Acad Sci U S A.1998; 95: 8274– 8279. CrossrefMedlineGoogle Scholar - 133.
Ciftçioglu N, Björklund M, Kuorikoski K, Bergström K, Kajander EO . Nanobacteria: an infectious cause for kidney stone formation. Kidney Int.1999; 56: 1893– 1898. CrossrefMedlineGoogle Scholar - 134.
Sedivy R, Battistutti WB . Nanobacteria promote crystallization of psammoma bodies in ovarian cancer. APMIS.2003; 111: 951– 954. CrossrefMedlineGoogle Scholar - 135.
Hjelle JT, Miller-Hjelle MA, Poxton IR, Kajander EO, Ciftcioglu N, Jones ML, Caughey RC, Brown R, Millikin PD, Darras FS . Endotoxin and nanobacteria in polycystic kidney disease. Kidney Int.2000; 57: 2360– 2374. CrossrefMedlineGoogle Scholar - 136.
Miller VM, Rodgers G, Charlesworth JA, Kirkland B, Severson SR, Rasmussen TE, Yagubyan M, Rodgers JC, Cockerill FR, Folk RL, Rzewuska-Lech E, Kumar V, Farell-Baril G, Lieske JC . Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves. Am J Physiol Heart Circ Physiol.2004; 287: 1115– 1124. CrossrefMedlineGoogle Scholar - 137.
Cisar JO, Xu DQ, Thompson J, Swaim W, Hu L, Kopecko DJ . An alternative interpretation of nanobacteria-induced biomineralization. Proc Natl Acad Sci U S A.2000; 97: 11511– 11515. CrossrefMedlineGoogle Scholar - 138.
Young JD, Martel J . The rise and fall of nanobacteria. Sci Am.2010; 302: 52– 59. CrossrefMedlineGoogle Scholar - 139.
Martel J, Young JD . Purported nanobacteria in human blood as calcium carbonate nanoparticles. Proc Natl Acad Sci U S A.2008; 105: 5549– 5554. CrossrefMedlineGoogle Scholar - 140.
Young JD, Martel J, Young D, Young A, Hung CM, Young L, Chao YJ, Young J, Wu CY . Characterization of granulations of calcium and apatite in serum as pleomorphic mineralo-protein complexes and as precursors of putative nanobacteria. PLoS ONE.2009; 4: e5421. CrossrefMedlineGoogle Scholar - 141.
Matsui I, Hamano T, Mikami S, Fujii N, Takabatake Y, Nagasawa Y, Kawada N, Ito T, Rakugi H, Imai E, Isaka Y . Fully phosphorylated fetuin-A forms a mineral complex in the serum of rats with adenine-induced renal failure. Kidney Int.2009; 75: 915– 928. CrossrefMedlineGoogle Scholar - 142.
Hamano T, Matsui I, Mikami S, Tomida K, Fujii N, Imai E, Rakugi H, Isaka Y . Fetuin-mineral complex reflects extraosseous calcification stress in CKD. J Am Soc Nephrol JASN.2010; 21: 1998– 2007. CrossrefMedlineGoogle Scholar - 143.
van Oss CJ, Gillman CF, Bronson PM, Border JR . Opsonic properties of human serum alpha-2 hs glycoprotein. Immunol Commun.1974; 3: 329– 335. CrossrefMedlineGoogle Scholar - 144.
Lewis JG, André CM . Effect of human alpha 2HS glycoprotein on mouse macrophage function. Immunology.1980; 39: 317– 322. MedlineGoogle Scholar - 145.
Lewis JG, André CM . Enhancement of human monocyte phagocytic function by alpha 2HS glycoprotein. Immunology.1981; 42: 481– 487. MedlineGoogle Scholar - 146.
Thiele L, Diederichs JE, Reszka R, Merkle HP, Walter E . Competitive adsorption of serum proteins at microparticles affects phagocytosis by dendritic cells. Biomaterials.2003; 24: 1409– 1418. CrossrefMedlineGoogle Scholar - 147.
Jersmann HP, Dransfield I, Hart SP . Fetuin/alpha2-HS glycoprotein enhances phagocytosis of apoptotic cells and macropinocytosis by human macrophages. Clin Sci.2003; 105: 273– 278. CrossrefMedlineGoogle Scholar - 148.
Moghimi SM, Hunter AC . Recognition by macrophages and liver cells of opsonized phospholipid vesicles and phospholipid headgroups. Pharm Res.2001; 18: 1– 8. CrossrefMedlineGoogle Scholar - 149.
Andrade JD, Hlady V . Plasma protein adsorption: the big twelve. Ann N Y Acad Sci.1987; 516: 158– 172. CrossrefMedlineGoogle Scholar - 150.
Trinkle-Mulcahy L, Boulon S, Lam YW, Urcia R, Boisvert FM, Vandermoere F, Morrice NA, Swift S, Rothbauer U, Leonhardt H, Lamond A . Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol.2008; 183: 223– 239. CrossrefMedlineGoogle Scholar - 151.
Wajih N, Borras T, Xue W, Hutson SM, Wallin R . Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin. J Biol Chem.2004; 279: 43052– 43060. CrossrefMedlineGoogle Scholar - 152.
Neven E, D'Haese PC . Vascular calcification in chronic renal failure: what have we learned from animal studies?Circ Res.2011; 108: 249– 264. LinkGoogle Scholar - 153.
van den Broek FA, Beynen AC . The influence of dietary phosphorus and magnesium concentrations on the calcium content of heart and kidneys of DBA/2 and NMRI mice. Lab Anim.1998; 32: 483– 491. CrossrefMedlineGoogle Scholar - 154.
van den Broek FA, Bakker R, den Bieman M, Fielmich-Bouwman AX, Lemmens AG, van Lith HA, Nissen I, Ritskes-Hoitinga JM, van Tintelen G, van Zutphen LF . Genetic analysis of dystrophic cardiac calcification in DBA/2 mice. Biochem Biophys Res Comm.1998; 253: 204– 208. CrossrefMedlineGoogle Scholar - 155.
Brunnert SR . Morphologic response of myocardium to freeze-thaw injury in mouse strains with dystrophic cardiac calcification. Lab Anim Sci.1997; 47: 11– 18. MedlineGoogle Scholar - 156.
Merx MW, Schäfer C, Westenfeld R, Brandenburg V, Hidajat S, Weber C, Ketteler M, Jahnen-Dechent W . Myocardial stiffness, cardiac remodeling, and diastolic dysfunction in calcification-prone fetuin-A-deficient mice. J Am Soc Nephrol.2005; 16: 3357– 3364. CrossrefMedlineGoogle Scholar - 157.
Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G . Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature.1997; 386: 78– 81. CrossrefMedlineGoogle Scholar - 158.
Meng H, Vera I, Che N, Wang X, Wang SS, Ingram-Drake L, Schadt EE, Drake TA, Lusis AJ . Identification of Abcc6 as the major causal gene for dystrophic cardiac calcification in mice through integrative genomics. Proc Natl Acad Sci U S A.2007; 104: 4530– 4535. CrossrefMedlineGoogle Scholar - 159.
Jiang Q, Dibra F, Lee MD, Oldenburg R, Uitto J . Overexpression of fetuin-a counteracts ectopic mineralization in a mouse model of pseudoxanthoma elasticum (abcc6(-/-)). J Invest Dermatol.2010; 130: 1288– 1296. CrossrefMedlineGoogle Scholar - 160.
Umulis D, O'Connor MB, Blair SS . The extracellular regulation of bone morphogenetic protein signaling. Development.2009; 136: 3715– 3728. CrossrefMedlineGoogle Scholar - 161.
Harmey D, Hessle L, Narisawa S, Johnson KA, Terkeltaub R, Millán JL . Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders. Am J Pathol.2004; 164: 1199– 1209. CrossrefMedlineGoogle Scholar - 162.
Neven E, Persy V, Dauwe S, De Schutter T, De Broe ME, D'Haese PC . Chondrocyte rather than osteoblast conversion of vascular cells underlies medial calcification in uremic rats. Arterioscler Thromb Vasc Biol.2010; 30: 1741– 1750. LinkGoogle Scholar - 163.
Murshed M, McKee MD . Molecular determinants of extracellular matrix mineralization in bone and blood vessels. Curr Opin Nephrol Hypertension.2010; 19: 359– 365. CrossrefMedlineGoogle Scholar - 164.
Speer MY, Yang HY, Brabb T, Leaf E, Look A, Lin WL, Frutkin A, Dichek D, Giachelli CM . Smooth muscle cells give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries. Circ Res.2009; 104: 733– 741. LinkGoogle Scholar - 165.
Blacher J, Guerin AP, Pannier B, Marchais SJ, London GM . Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease. Hypertension.2001; 38: 938– 942. LinkGoogle Scholar - 166.
Block GA, Raggi P, Bellasi A, Kooienga L, Spiegel DM . Mortality effect of coronary calcification and phosphate binder choice in incident hemodialysis patients. Kidney Int.2007; 71: 438– 441. CrossrefMedlineGoogle Scholar - 167.
Wang AY, Wang M, Woo J, Lam CW, Li PK, Lui SF, Sanderson JE . Cardiac valve calcification as an important predictor for all-cause mortality and cardiovascular mortality in long-term peritoneal dialysis patients: a prospective study. J Am Soc Nephrol JASN.2003; 14: 159– 168. CrossrefMedlineGoogle Scholar - 168.
Stenvinkel P, Ketteler M, Johnson RJ, Lindholm B, Pecoits-Filho R, Riella M, Heimbürger O, Cederholm T, Girndt M . IL-10, IL-6, and TNF-alpha: central factors in the altered cytokine network of uremia–the good, the bad, and the ugly. Kidney Int.2005; 67: 1216– 1233. CrossrefMedlineGoogle Scholar - 169.
Stenvinkel P, Wang K, Qureshi AR, Axelsson J, Pecoits-Filho R, Gao P, Barany P, Lindholm B, Jogestrand T, Heimbürger O, Holmes C, Schalling M, Nordfors L . Low fetuin-A levels are associated with cardiovascular death: Impact of variations in the gene encoding fetuin. Kidney Int.2005; 67: 2383– 2392. CrossrefMedlineGoogle Scholar - 170.
Moe SM, Reslerova M, Ketteler M, O'neill K, Duan D, Koczman J, Westenfeld R, Jahnen-Dechent W, Chen NX . Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD). Kidney Int.2005; 67: 2295– 2304. CrossrefMedlineGoogle Scholar - 171.
Koos R, Brandenburg V, Mahnken AH, Mühlenbruch G, Stanzel S, Günther RW, Floege J, Jahnen-Dechent W, Kelm M, Kühl HP . Association of fetuin-A levels with the progression of aortic valve calcification in non-dialyzed patients. Eur Heart J.2009; 30: 2054– 2061. CrossrefMedlineGoogle Scholar - 172.
Parker BD, Schurgers LJ, Brandenburg VM, Christenson RH, Vermeer C, Ketteler M, Shlipak MG, Whooley MA, Ix JH . The associations of fibroblast growth factor 23 and uncarboxylated matrix Gla protein with mortality in coronary artery disease: the Heart and Soul Study. Ann Intern Med.2010; 152: 640– 648. CrossrefMedlineGoogle Scholar - 173.
Mehrotra R, Westenfeld R, Christenson P, Budoff M, Ipp E, Takasu J, Gupta A, Norris K, Ketteler M, Adler S . Serum fetuin-A in nondialyzed patients with diabetic nephropathy: relationship with coronary artery calcification. Kidney Int.2005; 67: 1070– 1077. CrossrefMedlineGoogle Scholar - 174.
Brandenburg VM, Cozzolino M, Ketteler M . Calciphylaxis: a still unmet challenge. J Nephrol.2011; 24: 142– 148. CrossrefMedlineGoogle Scholar - 175.
Schlieper G, Brandenburg V, Ketteler M, Floege J . Sodium thiosulfate in the treatment of calcific uremic arteriolopathy. Nat Rev Nephrol.2009; 5: 539– 543. CrossrefMedlineGoogle Scholar - 176.
Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V . The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J.1988; 7: 2593– 2599. CrossrefMedlineGoogle Scholar - 177.
Serre L, Verbree EC, Dauter Z, Stuitje AR, Derewenda ZS . The Escherichia coli malonyl-CoA:acyl carrier protein transacylase at 1.5-A resolution. Crystal structure of a fatty acid synthase component. J Biol Chem.1995; 270: 12961– 12964. CrossrefMedlineGoogle Scholar - 178.
Momma K, Izumi F . VESTA: a three-dimensional visualization system for electronic and structural analysis. J Applied Crystallography.2008; 41: 653– 658. CrossrefGoogle Scholar - 179.
Humphrey W, Dalke A, Schulten K . VMD: visual molecular dynamics. J Mol Graph.1996; 14: 33– 38. CrossrefMedlineGoogle Scholar - 180.
Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA .. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A.2001; 98: 10037– 10041. CrossrefMedlineGoogle Scholar
eLetters(0)
eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.
Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.