Osteopontin overexpression inhibits in vitro re-endothelialization via integrin engagement.
The extracellular matrix protein osteopontin (OPN) plays a nonredundant role in atherosclerosis and restenosis. Here we investigated the impact of OPN up-regulation in an in vitro model of re-endothelialization after mechanical injury of the endothelial cell monolayer. Murine aortic endothelial (MAE) cells interact via alpha(v) integrins with the integrin-binding Arg-Gly-Asp OPN sequence and adhere to immobilized OPN. On this basis, MAE cells were stably transfected with a wild-type OPN cDNA (OPN-MAE cells), with an OPN mutant lacking the Arg-Gly-Asp sequence (DeltaRGD-OPN-MAE cells), or with vector alone (mock-MAE cells). When compared with mock-MAE and DeltaRGD-OPN-MAE cells, OPN-MAE cells showed a reduced sprouting activity in fibrin gel, a reduced motility in a Boyden chamber assay, and a reduced capacity to repair the wounded monolayer. Accordingly, OPN-MAE cells at the edge of the wound were unable to form membrane ruffles, to reorganize their cytoskeleton, and to activate the focal adhesion kinase and the small GTPase Rac1, key regulators of the cell entry into the first phase of the cell migration cycle. Accordingly, wounded OPN-MAE cells failed to activate the intracellular signals RhoA and ERK1/2, involved in the later phases of the cell migration cycle. Also, parental MAE cells showed reduced re-endothelialization after wounding when seeded on immobilized OPN and exhibited increased adhesiveness to OPN-enriched extracellular matrix. In conclusion, OPN up-regulation impairs re-endothelialization by inhibiting the first phase of the cell migration cycle via alpha(v) integrin engagement by the extracellular matrix-immobilized protein. This may contribute to the adverse effects exerted by OPN in restenosis and atherosclerosis.
Leali
Daria
D
Unit of General Pathology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 25123 Brescia, Italy.
Moroni
Emanuela
E
Bussolino
Federico
F
Presta
Marco
M
D000818:Animals; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology; D002461:Cell Line, Transformed; D002465:Cell Movement; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism; D005337:Fibrin; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006083:Graft Occlusion, Vascular / Q000378:metabolism / Q000473:pathology; D039564:Integrin alphaV / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D009479:Neuropeptides / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein
D039564:Integrin alphaV; D009479:Neuropeptides; C484602:Rac1 protein, mouse; D011994:Recombinant Proteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D005337:Fibrin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein