Application of the mineral-binding protein fetuin-A for the detection of calcified lesions.

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

Post-translational modifications glycosylation and phosphorylation of the major hepatic plasma protein fetuin-A are associated with CNS inflammation in children.

Fetuin-A is a liver derived plasma protein showing highest serum concentrations in utero, preterm infants, and neonates. Fetuin-A is also present in cerebrospinal fluid (CSF). The origin of CSF fetuin-A, blood-derived via the blood-CSF barrier or synthesized intrathecally, is presently unclear. Fetuin-A prevents ectopic calcification by stabilizing calcium and phosphate as colloidal calciprotein particles mediating their transport and clearance. Thus, fetuin-A plays a suppressive role in inflammation. Fetuin-A is a negative acute-phase protein under investigation as a biomarker for multiple sclerosis (MS). Here we studied the association of pediatric inflammatory CNS diseases with fetuin-A glycosylation and phosphorylation. Paired blood and CSF samples from 66 children were included in the study. Concentration measurements were performed using a commercial human fetuin-A/AHSG ELISA. Of 60 pairs, 23 pairs were analyzed by SDS-PAGE following glycosidase digestion with PNGase-F and Sialidase-AU. Phosphorylation was analyzed in 43 pairs by Phos-TagTM acrylamide electrophoresis following alkaline phosphatase digestion. Mean serum and CSF fetuin-A levels were 0.30 ± 0.06 mg/ml and 0.644 ± 0.55 µg/ml, respectively. This study showed that serum fetuin-A levels decreased in inflammation corroborating its role as a negative acute-phase protein. Blood-CSF barrier disruption was associated with elevated fetuin-A in CSF. A strong positive correlation was found between the CSF fetuin-A/serum fetuin-A quotient and the CSF albumin/serum albumin quotient, suggesting predominantly transport across the blood-CSF barrier rather than intrathecal fetuin-A synthesis. Sialidase digestion showed increased asialofetuin-A levels in serum and CSF samples from children with neuroinflammatory diseases. Desialylation enhanced hepatic fetuin-A clearance via the asialoglycoprotein receptor thus rapidly reducing serum levels during inflammation. Phosphorylation of fetuin-A was more abundant in serum samples than in CSF, suggesting that phosphorylation may regulate fetuin-A influx into the CNS. These results may help establish Fetuin-A as a potential biomarker for neuroinflammatory diseases.

Rapid calcification propensity testing in blood using a temperature controlled microfluidic polymer chip.

Phosphate toxicity is a major threat to cardiovascular health in chronic kidney disease. It is associated with oxidative stress, inflammation and the accumulation of calcium phosphate commonly known as calcification in soft tissues leading to functional disorders of blood vessels. An improved calcification propensity test for the assessment of phosphate toxicity was developed, which measures the velocity of calcium phosphate mineralization from colloidal precursors in vitro. This so called T50 test measures the transformation from a primary into a secondary form of nanosized colloidal plasma protein-calcium phosphate particles known as calciprotein particles. The T50 test in its previous form required a temperature controlled nephelometer and several hours of continuous measurement, which precluded rapid bed side testing. We miniaturized the test using microfluidic polymer chips produced by ultrasonic hot embossing. A cartridge holder contained a laser diode for illumination, light dependent resistor for detection and a Peltier element for thermo control. Increasing the assay temperature from 37°C to 75°C reduced the T50 test time 36-fold from 381 ± 10 min at 37°C to 10.5 ± 0.3 min at 75°C. Incorporating sputtered micro mirrors into the chip design increased the effective light path length, and improved signal-to-noise ratio 9-fold. The speed and reproducibility of the T50 chip-based assay run at 75°C suggest that it may be suitable for rapid measurements, preferably in-line in a dialyser or in a portable microfluidic analytic device with the chip inserted as a disposable cartridge.

Nanoparticle-based test measures overall propensity for calcification in serum.

Vascular and soft tissue calcification contributes to cardiovascular morbidity and mortality in both the general population and CKD. Because calcium and phosphate serum concentrations are near supersaturation, the balance of inhibitors and promoters critically influences the development of calcification. An assay that measures the overall propensity for calcification to occur in serum may have clinical use. Here, we describe a nanoparticle-based assay that detects, in the presence of artificially elevated calcium and phosphate concentrations, the spontaneous transformation of spherical colloidal primary calciprotein particles (CPPs) to elongate crystalline secondary CPPs. We used characteristics of this transition to describe the intrinsic capacity of serum to inhibit the precipitation of calcium and phosphate. Using this assay, we found that both the sera of mice deficient in fetuin-A, a serum protein that inhibits calcification, and the sera of patients on hemodialysis have reduced intrinsic properties to inhibit calcification. In summary, we developed a nanoparticle-based test that measures the overall propensity for calcification in serum. The clinical use of the test requires evaluation in a prospective study.

Clearance of fetuin-A--containing calciprotein particles is mediated by scavenger receptor-A.

RATIONALE: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. OBJECTIVE: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. METHODS AND RESULTS: In mice, fetuin-A-containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO-deficient mice. CONCLUSIONS: We could demonstrate that fetuin-A-containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.

Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation.

Recent research findings postulate that adipocytes and endothelial cells (EC) may share a common progenitor. However, the interlinking pathways between adipose tissue and endothelium, and the differentiation potential of cells to convert from one tissue into the other via progenitor cells have not been elucidated and are therefore the focus of this study. Stromal vascular fraction (SVF) cells were isolated from liposuction aspirates or excised adipose tissue and separated into CD31+ and CD31- populations by magnet-assisted cell sorting. Differentiation to fat tissue was induced in both CD31 fractions after expansion by insulin, dexamethasone, isobutylmethylxanthine, triiodothyronine, pioglitazone, and transferrin. Differentiation was assayed enzymatically and by cell counting. Maturation to endothelium was performed with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 plus 2% fetal calf serum, and confirmed by flow cytometry and tube formation assays on Matrigel. Our results show that the SVF contains a CD31-, S100+ cell type that can differentiate into adipocytes and EC. The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. These findings demonstrate the potency of SVF cells to perform both adipogenic and endothelial differentiation. Further, they reveal the plasticity of mature cells of mesenchymal origin to undergo conversion from endothelium to adipose tissue and vice versa.

CCAAT enhancer binding protein beta and hepatocyte nuclear factor 3beta are necessary and sufficient to mediate dexamethasone-induced up-regulation of alpha2HS-glycoprotein/fetuin-A gene expression.

Alpha2HS-glycoprotein/fetuin-A (Ahsg) is a serum protein preventing soft tissue calcification. In trauma and inflammation, Ahsg is down-regulated and therefore considered a negative acute phase protein. Enhancement of Ahsg expression as a protective serum protein is desirable in several diseases including tissue remodelling after trauma and infection, kidney and heart failure, and cancer. Using reporter gene assays in hepatoma cells combined with electrophoretic mobility shift assays we determined that dexamethasone up-regulates hepatic Ahsg. A steroid response unit at position -146/-119 within the mouse Ahsg promoter mediates the glucocorticoid-induced increase of Ahsg mRNA. It binds the hepatocyte nuclear factor 3beta and CCAAT enhancer binding protein beta (C/EBP-beta). The up-regulation is mediated indirectly via glucocorticoid hormone-induced transcriptional up-regulation in C/EBP-beta protein. A high degree of sequence identity in mouse, rat and human Ahsg promoters suggests that the promoter is similarly up-regulated by dexamethasone in all three species. Therefore, our findings suggest that glucocorticoids may be used to enhance the level of Ahsg protein circulating in serum.

Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A.

Fetuins are serum proteins with diverse functions including the regulation of osteogenesis and inhibition of unwanted mineralization. Besides the alpha2-Heremans and Schmid glycoprotein/fetuin-A, the recently identified fetuin-B is a second member of the fetuin family [Olivier, Soury, Risler, Smih, Schneider, Lochner, Jouzeau, Fey and Salier (1999) Genomics 57, 352-364; Olivier, Soury, Ruminy, Husson, Parmentier, Daveau and Salier (2000) Biochem. J. 350, 589-597], which belongs to the cystatin superfamily. We compared the expressions of fetuin-B and fetuin-A at the RNA level and established that both genes are most highly expressed in liver tissue. Like fetuin-A, fetuin-B mRNA is also highly expressed in tongue and placenta tissues. We demonstrated for the first time that fetuin-B is also expressed at the protein level in sera and several organs of mouse, rat and human. We isolated contiguous genomic clones containing both fetuin-B and fetuin-A genes, indicating that these genes are closely linked at the genome level. The close proximity of both these genes may explain our observation that fetuin-B expression was decreased in fetuin-A-deficient mice. Unlike fetuin-A, the amount of fetuin-B protein in human serum varied with gender and was higher in females than in males. Functional analysis revealed that fetuin-B, similarly to fetuin-A, is an inhibitor of basic calcium phosphate precipitation, albeit less active when compared with fetuin-A. Therefore fetuin-B may have a function that is partly overlapping, if not identical, with the function of fetuin-A.