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      Endothelial Cells Modulate Osteogenesis in Calcifying Vascular Cells

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          Abstract

          The potential role of vascular endothelium in atherosclerotic calcification is unknown. Endothelial cells (EC) express bone morphogenetic proteins (BMP), and EC-conditioned medium is osteoinductive in marrow stromal cells. To test whether EC are osteoinductive in vascular cells, we used calcifying vascular cells (CVC) that form nodules and mineralize in vitro. We established a coculture model with EC grown opposite CVC on membranes coated with collagen I or collagen IV, both of which are expressed in atherosclerotic lesions. On collagen I, EC did not alter CVC nodule formation, calcification or expression of the osteogenic marker Cbfa1, the chondrogenic marker collagen IX or smooth muscle cell α-actin. However, on collagen IV, EC abolished nodule formation and calcification, and expression of cell markers decreased, suggesting dedifferentiation. Matrix GLA protein (MGP), also expressed in atherosclerotic lesions, was added to CVC in coculture. Unexpectedly, MGP enhanced Cbfa1 expression in CVC on both collagen I and IV. The enhancement was most apparent on collagen IV, where calcification also increased. However, MGP did not restore nodule formation on collagen IV, suggesting that nodule formation and cell differentiation are separate processes. The effect of EC on CVC calcification was suppressed by noggin, an inhibitor of BMP activity, and in part mimicked by replacement of EC by BMP-2. Our results support a role for endothelium in vascular calcification, modulated by collagens and MGP.

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          Most cited references 10

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          Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188.

          Vascular endothelial growth factor (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF120 isoform (VEGF120/120 mice). Neonatal VEGF120/120 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone growth and a 34% enlargement of the hypertrophic chondrocyte zone of the growth plate. Surprisingly, embryonic hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte differentiation were significantly decreased in VEGF120/120 bones. Furthermore, inhibition of all VEGF isoforms in cultures of embryonic cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-1/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF120/120 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF164 and/or VEGF188 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.
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            Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification.

            Osteopontin (OPN) is a non-collagenous, glycosylated phosphoprotein associated with biomineralization in osseous tissues, as well as ectopic calcification. We previously reported that osteopontin was co-localized with calcified deposits in atherosclerotic lesions, and that osteopontin potently inhibits calcium deposition in a human smooth muscle cell (HSMC) culture model of vascular calcification. In this report, the role of phosphorylation in osteopontin's mineralization inhibitory function was examined. The ability of OPN to inhibit calcification completely depended on post-translational modifications, since bacteria-derived recombinant OPN did not inhibit HSMC mineralization. Following casein kinase II treatment, phosphorylated OPN (P-OPN) dose-dependently inhibited calcification of HSMC cultured in vitro about as effectively as native OPN. The inhibitory effect of osteopontin depended on the extent of phosphorylation. To determine the specific structural domains of OPN important for inhibition of calcification, we compared OPN fragments (N-terminal, C-terminal, and full-length), and compared the inhibitory effect of both phosphorylated and non-phosphorylated fragments. While none of the non-phosphorylated OPN fragments effected calcification, P-OPN caused dose dependent inhibition of HSMC calcification. P-OPN was treated with alkaline phosphatase to create dephosphorylated OPN. Dephosphorylated OPN did not have an inhibitory effect on calcification. The expression of OPN mRNA and P-OPN secretion by HSMC were decreased in a time-dependent manner during culture calcification. These results indicate that phosphorylation is required for the inhibitory effect of OPN on HSMC calcification, and that regulation of OPN phosphorylation represents one way in which mineralization may be controlled by cells.
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              Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: implications for fracture healing.

              The endothelium is a metabolically active secretory tissue, capable of responding to a wide array of environmental stimuli. Hypoxia and vascular endothelial growth factor (VEGF) are two components of the putative fracture microenvironment. This study investigated the role of hypoxia and VEGF on endothelial cell activation as it relates to the bone repair process. It was hypothesized that endothelial cells may have an important osteogenic role in fracture healing through the production of bone morphogenetic protein-2 (BMP-2), an osteogenic cytokine at the fracture site. Therefore, BMP-2 mRNA and protein expression in endothelial cells under hypoxia and/or VEGF treatment was studied. The authors observed a 2-fold to 3-fold up-regulation of BMP-2 mRNA expression in bovine capillary endothelial cells and human microvascular endothelial cells stimulated with hypoxia or rhVEGF. Furthermore, the combined effects of hypoxia and rhVEGF appeared to be additive on BMP-2 mRNA expression in bovine capillary endothelial cells. Actinomycin D and cycloheximide studies suggested that the increased mRNA expression was transcriptionally regulated. BMP-2 protein expression was up-regulated after 24 and 48 hours of treatment with either hypoxia or rhVEGF in bovine capillary endothelial cells. Surprisingly, the data suggest that endothelial cells may play not only an angiogenic role but also an osteogenic role by a direct stimulation of the osteoblasts, through the enhanced expression of a potent osteogenic factor, BMP-2, at the fracture site.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2004
                April 2004
                21 April 2004
                : 41
                : 2
                : 193-201
                Affiliations
                aDivision of Cardiology, David Geffen School of Medicine at UCLA, bMolecular Biology Institute, UCLA, Los Angeles, Calif., USA
                Article
                77394 J Vasc Res 2004;41:193–201
                10.1159/000077394
                15031603
                © 2004 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 7, References: 37, Pages: 9
                Categories
                Research Paper

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