Manuela Almeida

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro.

Osteopontin is an Arg-Gly-Asp-containing acidic phosphoprotein recently shown to be upregulated in vascular smooth muscle during rat arterial neointima formation and in human atherosclerotic plaques. Functional studies showed that osteopontin promoted adhesion of both cultured aortic endothelial cells and aortic smooth muscle cells. Adhesion of vascular cells to osteopontin was dose dependent and half maximal when solutions containing 7 and 30 nmol/L osteopontin were used to coat wells for endothelial and smooth muscle cells, respectively. Smooth muscle cells adherent to osteopontin were spread after 60 minutes, whereas endothelial cells remained round, although flattened, at this time point but were spread at 90 minutes. Cell spreading on osteopontin was accompanied by the formation of focal adhesion plaques. A newly developed anti-osteopontin antibody completely inhibited adhesion of both cell types to osteopontin but not to fibronectin or vitronectin. In addition, the peptide GRGDSP blocked adhesion to osteopontin, suggesting that integrins mediate Arg-Gly-Asp-dependent adhesion. Indeed, an antibody against the alpha v beta 3 integrin neutralized adhesion of both endothelium and smooth muscle cells to osteopontin by approximately 50%, demonstrating that alpha v beta 3 is one osteopontin receptor on vascular cells. Osteopontin also promoted the migration of smooth muscle cells in a Boyden-type chamber, with half-maximal effects observed at 77 nmol/L osteopontin. Checkerboard analysis demonstrated that this stimulus was chemotactic in nature. Our findings suggest that osteopontin may be functionally important as an adhesive and chemotactic molecule for vascular cells, particularly when levels of osteopontin are dramatically increased, as is the case after arterial angioplasty and in atherosclerotic plaques.

Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification.

Ectopic calcification, the abnormal calcification of soft tissues, can have severe clinical consequences especially when localized to vital organs such as heart valves, arteries, and kidneys. Recent observations suggest that ectopic calcification, like bone biomineralization, is an actively regulated process. These observations have led a search for molecular determinants of ectopic calcification. A candidate molecule is osteopontin (OPN), a secreted phosphoprotein invariantly associated with both normal and pathological mineral deposits. In the present study, OPN was found to be a natural inhibitor of ectopic calcification in vivo. Glutaraldehyde-fixed aortic valve leaflets showed accelerated and fourfold to fivefold greater calcification after subcutaneous implantation into OPN-null mice compared to wild-type mice. In vitro and in vivo studies suggest that OPN not only inhibits mineral deposition but also actively promotes its dissolution by physically blocking hydroxyapatite crystal growth and inducing expression of carbonic anhydrase II in monocytic cells and promoting acidification of the extracellular milieu. These findings suggest a novel mechanism of OPN action and potential therapeutic approach to the treatment of ectopic calcification.

Osteopontin expression in cardiovascular diseases.

Adhesive interactions are recognized requirements for cellular proliferation, migration and differentiation during normal morphogenesis as well as disease. By differential cloning, osteopontin was identified as an adhesive protein upregulated during vascular remodeling and neointima formation in both rat models and human vascular diseases including atherosclerosis and restenosis. In functional studies, purified osteopontin promoted adhesion, focal contact formation, and migration of vascular smooth muscle and endothelial cells. Utilizing neutralizing antibodies, three integrin-type receptors, alpha v beta 3, alpha v beta 1, and alpha v beta 5 were found to support cellular adhesion to osteopontin. In contrast, only cells containing the alpha v beta 3 integrin could migrate towards an osteopontin gradient, demonstrating for the first time that different functions of osteopontin are mediated via distinct receptors. These results suggest a model whereby osteopontin, via its integrin-type receptors, contributes to vascular remodeling during development and disease by facilitating smooth muscle migration and simultaneously promoting endothelial coverage of the affected area.

Neutralizing Antibodies Directed Against Osteopontin Inhibit Rat Carotid Neointimal Thickening After Endothelial Denudation

Osteopontin is an arginine-glycine-aspartate-containing acidic glycoprotein with adhesive and migratory activities in vitro. We previously showed that osteopontin was highly expressed in injured rat arteries as well as in human atherosclerotic plaques. In contrast, uninjured blood vessels make very little osteopontin. In this report, we have investigated the role of osteopontin in rat neointima formation using neutralizing antibodies. Rats were treated with either nonimmune or antiosteopontin antibody and subjected to endothelial denudation of the carotid artery by using a balloon catheter. Two weeks after injury, intimal areas and cell numbers were significantly decreased (33% and 31%, respectively) in the antiosteopontin group compared with the nonimmune IgG group. No differences in carotid medial areas or cell numbers were observed. Intimal and medial replication rates, as measured by continuous bromodeoxyuridine infusion during the final week of the experimental protocol, were not significantly different between the two groups. No gross histological changes were noted in the intimas formed in the presence of either neutralizing or nonimmune antibody. In addition, no difference in early carotid medial cell replication rate was observed when antibodies were infused for 4 days after angioplasty. These data demonstrate for the first time a functional role for osteopontin in the process of carotid neointimal thickening in vivo and suggest that osteopontin plays an active role in the remodeling processes important for human atherosclerotic and restenotic lesion development.