Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice

The presence of skeletal hypomineralization was confirmed in mice lacking the gene for bone alkaline phosphatase, ie, the tissue-non-specific isozyme of alkaline phosphatase (TNAP). In this study, a detailed characterization of the ultrastructural localization, the relative amount and ultrastructural morphology of bone mineral was carried out in tibial growth plates and in subjacent metaphyseal bone of 10-day-old TNAP knockout mice. Alizarin red staining, microcomputerized tomography (micro CT), and FTIR imaging spectroscopy (FT-IRIS) confirmed a significant overall decrease of mineral density in the cartilage and bone matrix of TNAP-deficient mice. Transmission electron microscopy (TEM) showed diminished mineral in growth plate cartilage and in newly formed bone matrix. High resolution TEM indicated that mineral crystals were initiated, as is normal, within matrix vesicles (MVs) of the growth plate and bone of TNAP-deficient mice. However, mineral crystal proliferation and growth was inhibited in the matrix surrounding MVs, as is the case in the hereditary human disease hypophosphatasia. These data suggest that hypomineralization in TNAP-deficient mice results primarily from an inability of initial mineral crystals within MVs to self-nucleate and to proliferate beyond the protective confines of the MV membrane. This failure of the second stage of mineral formation may be caused by an excess of the mineral inhibitor pyrophosphate (PPi) in the extracellular fluid around MVs. In normal circumstances, PPi is hydrolyzed by the TNAP of MVs' outer membrane yielding monophosphate ions (Pi) for incorporation into bone mineral. Thus, with TNAP deficiency a buildup of mineral-inhibiting PPi would be expected at the perimeter of MVs.     

Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.     

Histologic and ultrastructural studies on the mineralization process in hypophosphatasia

Chondroosseous tissue from six infants with infantile hypophosphatasia and six control infants were studied by light, transmission, and scanning electron microscopy. Alkaline phosphatase histochemical reaction of the growth plate was studied in two infants and was greatly reduced when compared to two control infants. Hypertrophic chondrocytes were increased in number with persisting cartilage islets in the metaphysis. In five of the six cases studied, chondrocytes and intercartilagenous intercellular chondroid matrix appeared ultrastructurally normal. Matrix vesicle distribution was similar to that of control subjects, but they were associated with few mineral crystals. In two infants, the matrix vesicles were alkaline phosphatase nonreactive. In the calcifying zone of the growth plate and in the newly formed metaphyseal trabecular bone, cartilagenous calcospherites often were small and the orientation of crystals was nonradial when compared to that of control infants. The mineralization of diaphyseal bone appeared normal. It seems that matrix vesicles are present in hypophosphatasia and that the impaired mineralization of cartilage is due primarily to the deficiency of alkaline phosphatase. In spite of the lack of alkaline phosphatase, secondary mineralization of bone which is not mediated by matrix vesicles was normal.     

Molecular effects of the tissue-nonspecific alkaline phosphatase gene polymorphism (787T > C) associated with bone mineral density

Based on studies of hypophosphatasia, which is a systemic skeletal disorder resulting from tissuenonspecific alkaline phosphatase (TNSALP) deficiency, TNSALP was suggested to be indispensable for bone mineralization. Recently, we demonstrated that there was a significant difference in bone mineral density (BMD) among haplotypes, which was lowest among TNSALP (787T [Tyr-246Tyr]) homozygotes, highest among TNSALP (787T > C [Tyr246His]) homozygotes, and intermediate among heterozygotes. To analyze protein translated from the TNSALP gene 787T > C, we performed the biosynthesis of TNSALPs using TNSALP cDNA expression vectors. TNSALP (787T) and TNSALP (787T > C) were synthesized similarly as a high-mannose-type 66-kDa form, becoming an 80-kDa form. Expression of the human 787T > C TNSALP gene using the cultured mouse marrow stromal cell line ST2 demonstrated that the protein translated from 787T > C exhibited an ALP-specific activity similarly to that of 787T. Interestingly, the Km value for TNSALP in ST2 cells transfected with the 787T > C TNSALP gene was decreased significantly compared to that of cells carrying the 787T gene (P < 0.01). These results suggest that the significant difference in Km values between the proteins translated from 787T > C and 787T may contribute to regulatory effects on bone metabolism.     

Perinatal hypophosphatasia: radiology, pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene.

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)-related defective bone mineralization due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA-based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term.     

Correlations of genotype and phenotype in hypophosphatasia.

Hypophosphatasia, a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical expression. The disease is due to various mutations in the tissue-non-specific alkaline phosphatase ( TNSALP ) gene. We report here the use of clinical data, site-directed mutagenesis and computer-assisted modelling to propose a classification of 32 TNSALP gene mutations found in 23 European patients, 17 affected with lethal hypophosphatasia and six with non-lethal hypophosphatasia. Transfection studies of the missense mutations found in non-lethal hypophosphatasia showed that six of them allowed significant residual in vitro enzymatic activity, suggesting that these mutations corresponded to moderate alleles. Each of the six patients with non-lethal hypophosphatasia carried at least one of these alleles. The three-dimensional model study showed that moderate mutations were not found in the active site, and that most of the severe missense mutations were localized in crucial domains such as the active site, the vicinity of the active site and homodimer interface. Some mutations appeared to be organized in clusters on the surface of the molecule that may represent possible candidates for regions interacting with the C-terminal end involved in glycosylphosphatidylinositol (GPI) attachment or with other dimers to form tetramers. Finally, our results show a good correlation between clinical forms of the disease, mutagenesis experiments and the three-dimensional structure study, and allowed us to clearly distinguish moderate alleles from severe alleles. They also confirm that the extremely high phenotypic heterogeneity observed in patients with hypophosphatasia was due mainly to variable residual enzymatic activities allowed by missense mutations found in the human TNSALP gene.     

Comparison of Matrix Vesicles Derived from Normal and Osteoarthritic Human Articular Cartilage

Articular cartilage matrix vesicles (MVs) from normal human adult articular cartilage were examined for protein and enzyme content and biomineralizing capacity for comparison to MVs derived from human osteoarthritic (OA) cartilage. Femoral condylar and tibial plateau cartilage from each of 9 healthy donors ages 17–37y was enzymatically digested and serially ultracentrifuged to pellet MVs at 3 × 10⁶ g-min. MV protein content, nucleoside triphosphate pyrophospho hydrolase (NTPPPH) specific activity (SA) and capacity for ⁴⁵Ca precipitation were determined. MV precipitated mineral was examined using Fourier transform infrared spectroscopy (FTIR). Normal human cartilage yields 50% less MV pro-tein/g cartilage than OA cartilage (p <. 01). Normal human articular MVs possess 30–70x higher NTPPPH S A than cell-free digest. Mean NTPPPH S As of MVs derived from normal human cartilage are 3x higher than that of OA MVs (p <. 05) and normal MV NTPPPH SA appears to decrease with age (p <. 01). Normal human MVs support significantly higher calcium precipitation/mg MV protein in both ATP-dependent (p <. 01) and -independent (p =. 05) systems. The FTIR spectrum of MV mineral generated in the presence of ATP strongly resembles the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectrum of MV mineral generated without ATP resembles that of carbonate-substituted apatite (AP). The fact that isolated MVs from normal cartilage generate pathologically relevant crystal phases in vitro implies that matrix integrity and substrate availability may be crucial factors in the control of pathologic biomineralization.     

A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia

Hypophosphatasia is a rare heritable inborn error of metabolism characterized by abnormal bone mineralization associated with a deficiency of alkaline phosphatase. The clinical expression of hypophosphatasia is highly variable, ranging from death in utero to pathologic fractures first presenting in adulthood. We investigated the tissue-nonspecific alkaline phosphatase (TNSALP) gene from a Japanese female patient with hypophosphatasia. By a quantitative polymerase chain reaction (PCR) method, the amount of TNSALP mRNA appeared to be almost equal to that in normal individuals. Gene analysis clarified that the hypophosphatasia originated from a missense mutation and a nucleotide deletion. The missense mutation, a C ? T transition at position 1041 of cDNA, results in an amino acid change from Leu to Phe at codon 272, which has not yet been reported. The previously reported deletion of T at 1735 causes a frame shift mutation downstream from Leu at codon 503. Family analysis showed that the mutation 1041T and the deletion 1735T had been inherited from the proband's father and mother, respectively. An expression experiment revealed that the mutation 1041T halved the expression of alkaline phosphatase activity. Using homology analysis, the Leu-272 was confirmed to be highly conserved in other mammals.     

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).     

Effects of pyrophosphate on desmal and endochondral mineralization and TNAP activity in organoid culture

During endochondral and desmal osteogenesis, mineralization of bone and cartilage matrix requires an appropriate solubility product of calcium and phosphate, collagen as a nucleator and deactivation of inhibitors, in order to prevent heterotopic calcification. In the 1960s, Fleisch and coworkers detected pyrophosphate (PPi) as an inhibitor of hydroxyapatite crystal growth, which should be removed by cleavage to tissue non-specific alkaline phosphatase (TNAP) activity. This theory had been established by basic experiments performed with collagen gels and demineralized matrices. In order to investigate the effect of PPi on matrix mineralization in bone and cartilage, calcium content and TNAP activity were measured in organoid cultures of mouse calvarial osteoblasts and limb bud cartilage after treatment with PPi and/or levamisole. In organoid cultures, bone and cartilage develop in a clear histotypical manner. PPi did not induce mineralization. Beta-glycerophosphate (β-GP) and inorganic phosphate (Pi) induced mineralization which could be significantly reduced by PPi. Levamisole, an inhibitor of TNAP, also reduced mineralization; the combination with PPi was additive. TNAP activity was increased after treatment with PPi and levamisole in both osteoblast and cartilage cultures. Mineralization induced by β-GP and Pi decreased TNAP activity in the osteoblast but not in cartilage organoid culture. In this culture system, PPi reduced mineralization as predicted by Fleisch's theory. Indications of cleavage of PPi were indirectly found because inhibition of hydrolysis of PPi by levamisole further reduced mineralization, probably due to the higher amounts of PPi available for binding to hydroxyapatite.     

Evidence of a founder effect for the tissue-nonspecific alkaline phosphatase (TNSALP) gene E174K mutation in hypophosphatasia patients

Hypophosphatasia is a rare inborn error of metabolism characterised by defective bone mineralisation caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The clinical expression of the disease is highly variable, ranging from stillbirth with a poorly mineralised skeleton to pathologic skeletal fractures which develop in late adulthood only. This clinical heterogeneity is due to the strong allelic heterogeneity in the TNSALP gene. We found that mutation E174K is the most frequent in Caucasian patients, and that it was carried by 31% of our patients with mild hypophosphatasia. Because the mutation was found in patients of various geographic origins, we investigated whether it had a unique origin or rather multiple origins due to recurrence of de novo mutations. Three intragenic polymorphisms, S93S, 472+12delG and V505A, were genotyped in patients carrying E174K and in normal unrelated individuals. Our results show that all the E174K mutations are carried by a common ancestral haplotype, also found at low frequency in normal and hypophosphatasia chromosomes. We conclude that the TNSALP gene E174K mutation is the result of a relatively ancient ancestral mutation that occurred on a single chromosome in the north of Western Europe and spread throughout the rest of Europe and into the New World as a result of human migration.     

Crystallization pathways in bone

Many biomineralization processes involve the sequestering of ions by cells and their translocation through the cells to the final deposition site. In many invertebrate crystallization pathways the cells deposit an initial highly disordered mineral phase with intracellular vesicles, and this mineral is subsequently transported into the final deposition site outside the cell. As this initial mineral phase is metastable, it can easily dissolve or crystallize during sample preparation and examination. A cryogenic electron microscopy study of the forming fin bone of a zebra fish strain with continuously growing fins shows that the cells responsible for bone tissue formation do have mineral-bearing intracellular vesicles and that the mineral phase is a highly disordered calcium phosphate. We also show that globules of disordered calcium phosphate are present in the extracellular collageneous matrix and that they are not membrane bound. Close to the mineralization front these globules appear to penetrate into the collagen fibrils where they crystallize to form mature bone. This crystallization pathway is similar to pathways observed in invertebrates, and it differs from the matrix vesicle pathway documented for a variety of vertebrate mineralizing tissues as the extracellular mineral globules are not membrane bound.     

Asfotase Alfa in Perinatal/Infantile-Onset and Juvenile-Onset Hypophosphatasia: A Guide to Its Use in the USA

Subcutaneous asfotase alfa (Strensiq?), a first-in-class bone-targeted human recombinant tissue-nonspecific alkaline phosphatase (TNSALP) replacement therapy, is approved in the USA for the treatment of patients with perinatal/infantile- or juvenile-onset hypophosphatasia (HPP). In clinical trials, asfotase alfa was an effective and generally well tolerated treatment for perinatal/infantile- and juvenile onset-HPP through at least 3 and 5 years’ treatment, respectively. Relative to untreated age-matched, juvenile-onset-HPP historical control cohorts, survival and ventilation-free survival were significantly prolonged in asfotase alfa-treated patients, consequent to preceding improvements in bone mineralization.     

Denaturing gradient gel electrophoresis analysis of the tissue nonspecific alkaline phosphatase isoenzyme gene in hypophosphatasia

Hypophosphatasia, a heritable form of rickets/osteomalacia, was first described in 1948. The biochemical hallmark, subnormal alkaline phosphatase (ALP) activity in serum, reflects a generalized disturbance involving the tissue-nonspecific isoenzyme of ALP (TNSALP). Deactivating mutations in the gene that encodes TNSALP have been reported in patients worldwide. Nevertheless, hypophosphatasia manifests an extraordinary range of clinical severity spanning death in utero to merely premature loss of adult teeth. There is no known medical treatment. To delineate the molecular pathology which explains the disease variability and to clarify the pattern(s) of inheritance for mild cases of hypophosphatasia, we developed comprehensive mutational analysis of TNSALP. High efficiency of mutation detection was possible by denaturing gradient gel electrophoresis (DGGE). Primers and conditions were established for all TNSALP coding exons (2-12) and adjacent splice sites so that the amplicons incorporated a GC clamp on one end. For each amplicon, the optimum percentage denaturant was determined by perpendicular DGGE. In 19 severely affected pediatric subjects (having perinatal or infantile hypophosphatasia or early presentation during childhood) from among our large patient population, we detected 2 TNSALP mutations each in 16 patients (84%) as expected for autosomal recessive disease. For 2 patients (11%), only 1 TNSALP mutation was detected by DGGE. However, one subject (who died from perinatal hypophosphatasia) had a large deletion as the second mutation. In the other (with infantile hypophosphatasia), no additional mutation was detected by DNA sequencing of all protein-coding exons. Possibly, she too has a deletion. For the final patient, with unclassifiable hypophosphatasia (5%), we detected only a single mutation which has been reported to cause relatively mild autosomal dominant disease; the other allele appeared to be intact. Hence, DGGE analysis was 100% efficient in detecting mutations in the coding exons and adjacent splice sites of TNSALP in this group of severely affected patients but, as expected, failed to detect a large deletion. To date, at least 78 different TNSALP mutations (in about 70 hypophosphatasia patients) have been reported globally. In our large subset of severely affected patients, we identified 8 novel TNSALP mutations (Ala34Ser, Val111Met, Delta G392, Thr117His, Arg206Gln, Gly322Arg, Leu397Met, and Gly409Asp) and 1 new TNSALP polymorphism (Arg135His) furthering the considerable genotypic variability of hypophosphatasia.     

Raman microspectrometry of laser-reshaped rabbit auricular cartilage: preliminary study on laser-induced cartilage mineralization

Laser-assisted cartilage reshaping (LACR) is a relatively novel technique designed to noninvasively and permanently restructure cartilaginous tissue. It is believed that heat-induced stress relaxation, in which a temperature-mediated disruption of H2O binding is associated with conformational alterations in the proteoglycan and collagen-rich matrix, constitutes the underlying mechanism of LACR. Several reports have suggested that laser-mediated cartilage mineralization may contribute to the permanent shape change of laser-reshaped cartilage. In an effort to validate these results in the context of Er:glass LACR, we performed a preliminary Raman microspectrometric study to characterize the crystal deposits in laser-irradiated chondrocytes and extracellular matrix. For the first time, we identified intracellular calcium sulfate deposits and extracellular calcium phosphate (apatite) crystals in laser-reshaped rabbit auricular cartilage. Calcium carbonate deposits are localized in both irradiated and nonirradiated samples, suggesting that this mineral plays no role in conformational retention. In our discussion, we elaborate on the possible molecular and cellular mechanisms responsible for intra- and extracellular crystallization, and propose a novel hypothesis on the formation of apatite, inasmuch as the biological function of this mineral (providing structure and rigidity in bones and dental enamel) may be extrapolated to the permanent shape change of laser-irradiated cartilage.     

Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems. It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase. This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system. Mineralization was induced by supplementation of 1.8 mM of Ca(+2) and 5 mM of beta-GP or Pi. Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM. Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation. Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively. Levamisole and beta-GP did not appear to influence DNA synthesis. Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture. The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization. Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral. In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism.     

Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae

We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.     

Calcification of rachitic rat cartilage in vitro by extracellular matrix vesicles.

Growth plate cartilage from rachitic rats was studied to assess the role in calcification of extracellular matrix vesicles, which are thought to participate in the initial stage of mineralization of connective tissue. The concentration of matrix vesicles and their distribution within the longitudinal septa was found to be normal in rats made rachitic by feeding by a diet low in vitamin D and phosphate for 3 weeks after weaning. Rachitic cartilage matrix did not contain circumvesicular clusters of apatite as does normal cartilage; however, occasional vesicles did enclose one or a few apatite needles. When slices of rachitic cartilage were incubated at 37 C in a metastable calcium phosphate solution ([Ca++] times [PO SEE ARTICLE] equals 3.5 mM identical to 2), apatite formation was initiated in association with matrix vesicles. Under these conditions, mineralization was prominent in the upper hypertrophic cartilage, where matrix vesicles became encrusted with apatite after only 2 to 3 hours of incubation. Vesicular apatite accumulation was inhibited by preheating the cartilage to 60 C for 30 minutes. Measurements of 45Ca uptake by rachitic cartilage slices from metastable calcium phosphates solution also indicated inhibition of calcification by heat. Light microscopic autoradiographs showed 45Ca localization primarily in the matrix of longitudinal septa and substantiated the inhibition site of mineralization in healing rachitic cartilage. The presence of apatite within rachitic vesicles prior to heating and the inhibition of vesicle calcification by heat suggests an active, enzymatically and mediated mechanism of vesicular calcification.     

HMGB1 induces secretion of matrix vesicles which participate in microcalcification of atherosclerotic plaques

AIM: Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1( HMGB1),a cytokine associated with biomineralizing process under physiological and pathological conditions. Our study aims to evaluate whether HMGB1 induces ectopic mineralization via promoting the secretion of matrix vesicles( MVs) from macrophages. METHODS: HMGB1 was added to the medium of macrophages,the secretion of MVs in the supernatant was tested by flow cytometry analysis. The mineral deposition in calcifying medium was detected by Alizarin Red staining and von Kossa staining. Transmission electron microscopy showed the formation of hydroxyapatite crystals in MVs. Then we subcutaneous injection into mice with MVs to induce regional mineralization. RESULTS: HMGB1 significantly promoted secretion of MVs from macrophages as raveled by flow cytometry analysis. TNAP activity,considered as a marker of MVs maturation,was higher in HMGB1-induced MVs compared to the control-MVs. HMGB1-MVs also led to mineral deposition in an in vitro MVs-collagen mineralization model. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase 2( n SMase2) that involved the receptor for advanced glycation end products( RAGE) and p38MAPK( upstream of n SMase2). Inhibition of n SMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. CONCLUSIONS: HMGB1 induces MVs secretion from macrophages at least in part,via the RAGE / p38 MAPK /n SMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 may participated in the early calcification of atherosclerotic plaques.