Stable interaction between α5β1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1

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

Adaptor ShcA Protein Binds Tyrosine Kinase Tie2 Receptor and Regulates Migration and Sprouting but Not Survival of Endothelial Cells

Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1

A new computational approach to analyze human protein complexes and predict novel protein interactions

We propose a new approach to identify interacting proteins based on gene expression data. By using hypergeometric distribution and extensive Monte-Carlo simulations, we demonstrate that looking at synchronous expression peaks in a single time interval is a high sensitivity approach to detect co-regulation among interacting proteins. Combining gene expression and Gene Ontology similarity analyses enabled the extraction of novel interactions from microarray datasets. Applying this approach to p21-activated kinase 1, we validated α-tubulin and early endosome antigen 1 as its novel interactors

Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network. Adherens junctions and myosin light chain as targets of Rac1 and RhoA

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 beta-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs beta-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

Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi's sarcoma cells.

Recent evidence suggesting vascular endothelial growth factor-C (VEGF-C), which is a regulator of lymphatic and vascular endothelial development, raised the question whether this molecule could be involved in Kaposi's sarcoma (KS), a strongly angiogenic and inflammatory tumor often associated with infection by human immunodeficiency virus-1. This disease is characterized by the presence of a core constituted of three main populations of "spindle" cells, having the features of lymphatic/vascular endothelial cells, macrophagic/dendritic cells, and of a mixed macrophage-endothelial phenotype. In this study we evaluated the biological response of KS cells to VEGF-C, using an immortal cell line derived from a KS lesion (KS IMM), which retains most features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice. We show that VEGFR-3, the specific receptor for VEGF-C, is expressed by KS IMM cells grown in vitro and in vivo. In vitro, VEGF-C induces the tyrosine phosphorylation of VEGFR-2, a receptor also for VEGF-A, as well as that of VEGFR-3. The activation of these two receptors in KS IMM cells is followed by a dose-responsive mitogenic and motogenic response. The stimulation of KS IMM cells with a mutant VEGF-C unable to bind and activate VEFGR-2 resulted in no proliferative response and in a weak motogenic stimulation, suggesting that VEGFR-2 is essential in transducing a proliferative signal and cooperates with VEGFR-3 in inducing cell migration. Our data add new insights on the pathogenesis of KS, suggesting that the involvement of endothelial growth factors may not only determine KS-associated angiogenesis, but also play a critical role in controlling KS cell growth and/or migration and invasion

Quantum Dot-Based Screening Identifies F3 Peptide and Reveals Cell Surface Nucleolin as a Therapeutic Target for Rhabdomyosarcoma.

Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS

Tie-2–dependent activation of RhoA and Rac1 participates in endothelial cell motility triggered by angiopoietin-1

AbstractAngiopoietin-1 is implicated in the maturation and remodeling of the vascular network during embryo development and in adult life. Through its tyrosine kinase receptor Tie-2 it stimulates endothelial cells to migrate and change shape. Here we show that angiopoietin-1 elicits chemokinesis of endothelial cells by a phosphoinositide 3-OH kinase/son of sevenless-dependent modulation of Rac1 and RhoA. The resulting temporal events are associated with cytoskeletal rearrangements and occur in discrete zones of the cell. Endothelial cells carrying dominant-negative mutants of RhoA and Rac1 or treated with LY294002, an inhibitor of phosphoinositide 3-OH kinase, dramatically decrease their chemokinetic velocity. Taken together, these results further expand our understanding of angiopoietin-1-mediated endothelial cell motility during vascular network assembly and angiogenesis. (Blood. 2003;102:2482-2490

Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy

Pleural mesothelioma risk in the construction industry: a case-control study in Italy, 2000-2018

Objectives Workers in the construction industry have been exposed to asbestos in various occupations. In Italy, a National Mesothelioma Registry has been implemented more than 20 years ago. Using cases selected from this registry and exploiting existing control data sets, we estimated relative risks for pleural mesothelioma (PM) among construction workers. DesignCase-control study. SettingCases from the National Mesothelioma Registry (2000-2018), controls from three previous case-control studies. MethodsWe selected male PM incident cases diagnosed in 2000-2018. Population controls were taken from three studies performed in six Italian regions within two periods (2002-2004 and 2012-2016). Age-adjusted and period-adjusted unconditional logistic regression models were fitted to estimate odds ratios (OR) for occupations in the construction industry. We followed two approaches, one (primary) excluding and the other (secondary) including subjects employed in other non-construction blue collar occupations for >5 years. For both approaches, we performed an overall analysis including all cases and, given the incomplete temporal and geographic overlap of cases and controls, three time or/and space restricted sensitivity analyses. ResultsThe whole data set included 15 592 cases and 2210 controls. With the primary approach (4797 cases and 1085 controls), OR was 3.64 (2181 cases) for subjects ever employed in construction. We found elevated risks for blue-collar occupations (1993 cases, OR 4.52), including bricklayers (988 cases, OR 7.05), general construction workers (320 cases, OR 4.66), plumbers and pipe fitters (305 cases, OR 9.13), painters (104 cases, OR 2.17) and several others. Sensitivity analyses yielded very similar findings. Using the secondary approach, we observed similar patterns, but ORs were remarkably lower. ConclusionsWe found markedly increased PM risks for most occupations in the construction industry. These findings are relevant for compensation of subjects affected with mesothelioma in the construction industry