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Differential Expression of Extracellular Proteins is correlated with Angiogenesis in vitro


Strains of bovine aortic endothelial cells, grown on plastic under conventional culture conditions and in the absence of growth factor supplementation, exhibited a sprouting phenotype and a predisposition toward the formation of cords and tubular structures. We examined endothelial cells at different stages of tube formation. Analysis of metabolically labeled proteins showed that the synthesis of type I collagen was initiated in sprouting cells and during the formation of tubular structures. SPARC (secreted protein, acidic and rich in cysteine) a Ca2(+)-binding protein associated with cellular shape change and morphogenetic processes (Sage H, Vernon RB, Funk SE, Everitt EA, Angello J: J Cell Biol 109:341, 1989), was upregulated during spontaneous tube formation. Levels of messenger RNA for type I collagen and SPARC corroborated the stage-specific increases observed for these proteins. Differential levels of transcription were apparent in multilayered cells directly involved in tube formation, in comparison with cells comprising either the tubes or the confluent monolayers at a distance from the tubes. Analysis of DNA synthesis indicated that multilayered sprouting cells in the proximity of the endothelial tubes were actively proliferating, whereas cells that had been incorporated into tubes showed low levels of DNA synthesis. Immunolabeling studies revealed a dense accumulation of SPARC and type I collagen in the cytoplasm of cells that were situated near the growing tubes. Two other secreted proteins, type III collagen and thrombospondin, were expressed constitutively by subconfluent cultures and were increased in those cells contributing to tube formation. We propose that type I collagen and SPARC are specifically related to the angiogenesis-like phenomenon displayed by bovine aortic endothelial cells in vitro. Type I collagen might facilitate the active migration of endothelial cells, or the stabilization of the resulting tubes, with SPARC directing the re-organization and dynamic assembly of the tubular network.

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