During angiogenic remodeling, Ang-1, the ligand of Tie2 tyrosine kinase, is involved in vessel sprouting and stabilization through unclear effects on nascent capillaries and mural cells. In our study, we hypothesized that the Ang-1/Tie2 system could cross-talk with integrins, and be influenced by the dynamic interactions between extracellular matrix and endothelial cells (ECs). Here, we show that α5β1 specifically sensitizes and modulates Tie2 receptor activation and signaling, allowing EC survival at low concentrations of Ang-1 and inducing persistent EC motility. Tie2 and α5β1 interact constitutively; α5β1 binding to fibronectin increases this association, whereas Ang-1 stimulation recruits p85 and FAK to this complex. Furthermore, we demonstrate that Ang-1 is able to mediate selectively α5β1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and α5β1 receptors that could cross-talk when Tie2/α5β1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that α5β1, but not αvβ3 activation, is essential to Ang-1–dependent angiogenesis in vivo.

IntroductionAngiogenesis is a crucial event in embryonic development and plays a critical role in many pathologic processes. 1 Sprouting of new blood vessels from pre-existing ones is a multistep process that requires digestion of extracellular matrix, and migration and proliferation of endothelial cells (ECs). 1 Finally, cells change their shape and fold up to form capillaries that are surrounded by pericytes, necessary for the cells' stabilization. 2 A cascade of molecules whose biologic activities are partially overlapped drives this process. Among them, vascular endothelial growth factor (VEGF) and angiopoietin (Ang) families have restricted activities on ECs. Through VEGF receptor-1 and -2, VEGFs primarily regulate migration, proliferation, and survival of ECs. By analysis of genetic and pathologic models, Ang-1, a ligand of Tie-2 receptor, is instead supposed to stabilize EC networks, presumably by stimulating interactions between ECs and pericytes. [3][4][5] In vitro experiments show that Ang-1 causes ECs to form capillary networks 6 and sprouts from preassembled spheroids. 7 Ang-1 induces EC chemotaxis 8,9 and chemokinesis, 10,11 which require the activation of the downstream effector phosphoinositide 3-OH kinase (PI 3-kinase) and the phosphorylation of focal adhesion kinase and paxillin. 10,12 Such morphogenetic events are dependent on cell motility and shape rearrangement, which imply dramatic changes in cytoskeletal dynamics; this suggests that Ang-1 could be directly implicated in the regulation of the motility machinery in migrating cells.Rho guanosine triphosphatases (Rho GTPases) are major regulators of cell polarization and motility. In fibroblasts, Cdc42 mediates filipodia extension, Rac1 lamellipodia formation and membrane ruffling, and RhoA stress fiber formation. 13 Besides Rac1, RhoA is involved in ruffling as well. 14 Nevertheless, considering that GTPase activities have antagonist effects in the regulation of different steps of cell motility 15 and discrete subcellular localization, 16-18 the above model seems to be more complex. Recent evidence suggests that Rho GTPases participate to the EC signaling pathways triggered by angiogenic inducers [19][20][21] and, more in general, regulate biologic responses requiring changes in EC shape. [22][23][24][25] Here we analyze the spatiotemporal modulation and localization of Rac1 and RhoA during EC locomotion induced by Ang-1 and we investigate the motility of ECs carrying RhoA and Rac1 dominant-negative molecules. Furthermore we studied the signaling pathway from Tie-2 receptor to these enzymes through PI 3-kinase activity. Materials and methods ReagentsRecombinant Ang-1 was produced in a baculovirus expression system as described and was able to phosphorylate Tie-2 in ECs. 11 Glutathione-Stransferase (GST)-Pak 1B binding domain (GST-PBD), GST-rhotekin binding domain (GST-RBD), and GST-Wiskott-Aldrich syndrome protein (WASP) binding domain (GST-WBD) fusion proteins were purified as described. 26 GST-PBD, GST-RBD, and GST-WBD bind the bound form o...

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Background: Several fractal and non-fractal parameters have been considered for the quantitative assessment of the vascular architecture, using a variety of test specimens and of computational tools. The fractal parameters have the advantage of being scale invariant, i.e. to be independent of the magnification and resolution of the images to be investigated, making easier the comparison among different setups and experiments.