Tumor necrosis factor-alpha promotes in vitro calcification of vascular cells via the cAMP pathway.
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BACKGROUND: Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-alpha (TNF-alpha), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-alpha in in vitro vascular calcification. METHODS AND RESULTS: A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-alpha, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-alpha for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF-alpha for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A-specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase activity, and mineralization induced by TNF-alpha. Consistent with this, the intracellular cAMP level was elevated after TNF-alpha treatment. Electrophoretic mobility shift assay demonstrated that TNF-alpha enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation. CONCLUSIONS: These results suggest that TNF-alpha enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway. (+info)
Abnormal localisation and hyperclustering of (alpha)(V)(beta)(3) integrins and associated proteins in Src-deficient or tyrphostin A9-treated osteoclasts.
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The non-receptor tyrosine kinase Src was shown to be essential for osteoclast function in vivo. We have previously reported that engagement of (alpha)(v)(beta)(3) integrin in osteoclasts induces tyrosine phosphorylation and activation of the adhesion kinase PYK2 and the adaptor protein p130(Cas) in a Src-dependent manner. The objective of this study was to analyse the role of c-Src in the (alpha)(v)(beta)(3) integrin-dependent recruitment of signalling and cytoskeletal molecules in osteoclasts during bone resorption. Using prefusion osteoclasts (pOCs) obtained from cocultures of osteoblasts and spleen cells isolated from Src(-/-) mice or their normal littermates, we found: (1) similar expression levels and ligand binding affinities of (alpha)(v)(beta)(3) integrins in Src(-/-) and Src(+/?) pOCs, (2) reduced adhesion and spreading of Src(-/-) pOCs, (3) defective organisation of the microfilament proteins, F-actin, vinculin and paxillin, and of PYK2 and p130(Cas) in the sealing zone of Src(-/-)OCLs, and (4) hyperclustering of (alpha)(v)(beta)(3) integrins together with microfilament and signalling proteins in the basal membrane of Src-deficient OCLs. In normal OCLs, the tyrosine kinase inhibitor tyrphostin A9 inhibits actin ring formation, bone resorption and tyrosine phosphorylation of several proteins, including c-Src. Furthermore, tyrphostin A9 induced similar hyperclustering of (alpha)(v)(beta)(3) integrins in osteoclasts as observed in Src(-/-) OCLs. Taken together, these findings suggest that normal localisation of (alpha)(v)(beta)(3) and recruitment of its downstream effectors to the appropriate compartments of the osteoclast during resorption depend on Src kinase activity. (+info)
The correlation between immune rejection and osteoinduction of allogeneic bone grafting.
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OBJECTIVE: To evaluate the relationship between the immune rejection and the osteoinductive potential of bone allograft. METHODS: Allogeneic and syngeneic fresh bone, autolyzed antigen-extracted bone, bone matrix gelatin and demineralized bone matrix were implanted into the muscle of mice, and immunological tests, histological observation and alkaline phosphatase assay were performed. RESULTS: Three and 6 weeks after implantation, all kinds of allogeneic implants activated immune rejection, among them, fresh bone induced the most vigorous immune rejection and bone matrix gelatin caused the weakest response. Allogeneic autolyzed antigen-extracted bone, bone matrix gelatin and demineralized bone matrix inhibited proliferation of the lymphocytes in vitro and bone matrix gelatin had the most powerful inhibiting effect. Both allogeneic and syngeneic autolyzed antigen-extracted bone, bone matrix gelatin, and demineralized bone matrix induced heterotopic osteogenesis in vivo and bone matrix gelatin had the best osteoinductive capacity. CONCLUSION: There is a negative correlation between immune rejection to bone allograft and osteoinductive capacity of the graft. (+info)
A molecular analysis of matrix remodeling and angiogenesis during long bone development.
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The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a 'molecular map' of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias. (+info)
Expression and localization of activin receptors during endochondral bone development.
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The expression and localization of activins (dimeric protein of inhibin beta subunit) and activin receptors in skeletal tissue were examined. RT-PCR revealed that cultured chondrocytes expressed mRNAs of inhibin/activin betaA and four activin receptors (two type I (ActRI and ActRIB) and two type II (ActRII and ActRIIB)). Immunohistochemical analyses showed that activin betaA, ActRI and ActRII were localized in proliferating chondrocytes and osteoblasts in tibiae of neonatal rats, and in implants of demineralized bone matrix, a well-established model of ectopic bone formation. The immunoreactivities of osteoblasts were decreased with aging in the tibiae and with progressing endochondral bone development in the implants. The strong expression of ActRI was also detected in hypertrophic chondrocytes both in the tibial growth plate and in the implants, whereas immunoreactive ActRII was lower in hypertrophic chondrocytes. Western blot analysis also showed that immunoreactive ActRI, migrating at 52 kDa, was detected only in the implants on days 9 and 11, the period of conversion from cartilage to bone. In view of the sharing of type II receptors between activins and bone morphogenetic proteins (BMPs), our findings suggest that activin/BMP activity involves in bone modeling, especially during active chondro- and osteogenesis and during the conversion from cartilage to bone. (+info)
Transforming growth factor beta-1 stimulates articular chondrocyte elaboration of matrix vesicles capable of greater calcium pyrophosphate precipitation.
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Objective To determine the role of transforming growth factor beta1 (TGFbeta) in early calcium pyrophosphate formation by measuring its effects on articular chondrocyte matrix vesicle (MV) formation, specific activity of the inorganic pyrophosphate(PPi)-generating enzyme nucleoside triphosphate pyrophospho-hydrolase (NTPPPH) and biomineralization capacity. Methods MV elaborated from mature porcine chondrocyte monolayers+/-TGFbeta were compared for protein content, NTPPPH activity, and ATP-dependent biomineralization. Precipitation of calcium pyrophosphate mineral phases by MV was determined by a radiometric assay and by Fourier transform infrared spectroscopy (FTIR). Results MV from monolayers exposed to TGFbeta were enriched in NTPPPH activity compared to MV from control monolayers (P< 0.01) and precipitated more calcium/mg MV protein than controls (P+info)
Calcification of rachitic rat cartilage in vitro by extracellular matrix vesicles.
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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. (+info)
Osteoblastic differentiation of periosteum-derived cells is promoted by the physical contact with the bone matrix in vivo.
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The periosteum contains osteoprogenitors that differentiate to osteoblasts in bone growth or repair. Our previous studies suggested the hypothesis that the physical contact of the periosteum with the bone matrix is requisite for the differentiation of osteoblasts. To test the hypothesis, the present study was designed to investigate how the contact between the periosteum and the bone matrix influences the osteoblastic differentiation of periosteal cells with establishing a new experimental model in vivo. Differentiation of osteoblasts was assessed by gene expression of type I collagen, osteocalcin and bone sialoprotein using in situ hybridization. A barrier was designed to prevent periosteal cells from contacting the bone matrix using the membrane filter. The membrane filter was inserted surgically between the surface of rat parietal bone and the periosteum after being punched out with pin holes. Periosteal cells were allowed to contact with the bone surface only through the pin holes. The pin hole was filled with cells derived from the periosteum 1 week after inserting the filter. Differentiation of osteoblasts in week 2 and noticeable bone formation in week 3 were identified on the bone surface only under the pin hole but not under the filter. The present study demonstrated that the physical contact with the bone matrix promotes osteoblastic differentiation of periosteum-derived cells in vivo. (+info)