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Osteopontin and β3 Integrin Are Coordinately Expressed in Regenerating Endothelium In Vivo and Stimulate Arg-Gly-Asp–Dependent Endothelial Migration In Vitro

Osteopontin is an Arg-Gly-Asp (RGD)–containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the αvβ3 integrin, to endothelial regenerative processes by using both in vivo and in vitro models. In vivo, uninjured rat arterial endothelium had undetectable levels of osteopontin and β3-integrin mRNA by in situ hybridization. After balloon catheter denudation, osteopontin mRNA levels correlated temporally and spatially with active endothelial proliferation and migration, with the highest levels observed at the wound edge between 8 hours and 2 weeks after injury, declining to uninjured levels at 6 weeks, when regeneration was complete. Osteopontin protein levels, as determined by immunocytochemistry, paralleled the time course of mRNA expression. Likewise, β3-integrin mRNA and protein levels were substantially elevated in regenerating endothelial cells but were not detectable in uninjured or healed endothelium. In vitro, rat smooth muscle cell–derived and bacterial expressed mouse recombinant osteopontins both stimulated the adhesion and directed migration of bovine aortic endothelial cells through interactions with the αvβ3 receptor. Structural mutants of osteopontin confirmed the importance of the RGD domain for both adhesion and migration of endothelial cells through αvβ3. These data suggest important roles for osteopontin and β3 integrin in regenerating endothelium.

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An improved method for the quantitation of cellular migration: Role of αvβ3 integrin in endothelial and smooth muscle cell migration

The present study was undertaken to develop a simple and improvedmethod for the accurate quantitation of cellular migration and to examinethe role of agrvbeta3 integrins in different cellular migration. Using our newly developed micro-volume chemotaxis assay, we developed an improved quantitative method to measure in vitro chemotaxis of smooth muscle or endothelial cells toward different extracellular matrix proteins. The convenience in setup and counting of migrated cells using this method allows for large capacity screening and for various research applications with other cells as well. The signal to noise ratios were in the range of 10/1, along with about 10–20% intra- or inter-assay variabilities. Using this method, we have determined that either vitronectin at 0.4 µg/well or osteopontin at 0.4 µg/well are selective agrvbeta3 chemoattractants for endothelial or smooth muscle cells (0.5 × 105 cells/well). Additionally, a selective agrvbeta3 small molecule peptiddomimetic, monoclonal antibody LM609, or an anti-beta3 (agrvbeta3/agrIIbeta3) anti-body, c7E3 demonstrated maximal inhibition of cellular migration toward vitronectin or osteopontin. These data suggest the potential utility of this method in assessing the role of various mechanisms in cellular migration and also suggests the potential implication of an agrvbeta3 antagonist in blocking pathological processes involving endothelial or smooth muscle cell adhesion/migration.

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