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.

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Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of ␤-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs ␤-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis.

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...

A one-dimensional photonic crystal (1DPC) based on a planar stack of dielectric layers is used as an optical transducer for biosensing, upon the coupling of TE-polarized Bloch Surface Waves (BSW). The structure is tailored with a polymeric layer providing a chemical functionality facilitating the covalent binding of orienting proteins needed for a subsequent grafting of antibodies in an immunoassay detection scheme. The polymeric layer is impregnated with Cy3 dye, in such a way that the photonic structure can exhibit an emissive behavior. The BSW-coupled fluorescence shift is used as a means for detecting refractive index variations occurring at the 1DPC surface, according to a label-free concept. The proposed working principle is successfully demonstrated in real-time tracking of protein G covalent binding on the 1DPC surface within a fluidic cell.

BackgroundEnhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.MethodsWe investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.ResultsTrabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.ConclusionsPARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0652-5) contains supplementary material, which is available to authorized users.

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During angiogenic remodelling in embryo and adult life, endothelial cells lining blood vessel walls dynamically modify their integrin-mediated adhesive contacts with the surrounding extracellular matrix. However, besides regulating cell adhesion and migration, integrins dynamically participate in a network with soluble molecules and their receptors. Angiogenesis is characterized by opposing autocrine and paracrine loops of growth factors and semaphorins that regulate the activation of integrins on the endothelial surface through tyrosine kinase receptors (TKR) and the neuropilin/plexin system. Moreover, pro- and anti-angiogenic factors can directly bind integrins and regulate endothelial cell behaviour. This review summarizes the recent progress in understanding the reciprocal interactions between integrins, TKR, and semaphorin receptors.