VEGF-Mediated Signal Transduction in Tumor Angiogenesis

The vascular endothelial growth factor-A (VEGF) plays a crucial role in tumor angiogenesis. Through its primary receptor VEGFR-2, VEGF exerts the activity of a multitasking cytokine, which is able to stimulate endothelial cell survival, invasion and migration into surrounding tissues, proliferation, as well as vascular permeability and inflammation. The core components of VEGF signaling delineate well-defined intracellular routes. However, the whole scenario is complicated by the fact that cascades of signals converge and branch at many points in VEGF signaling, thus depicting a complex signal transduction network that is also finely regulated by different mechanisms. In this chapter, we present a careful collection of the best-characterized VEGF-induced signal transduction pathways, attempting to offer an overview of the complexity of VEGF signaling in the context of tumor angiogenesis

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

Electrostimulation of a 3D in vitro skin model to activate wound healing

The aim of the work is to propose a methodology for the stimulation of a 3D in vitro skin model to activate wound healing. The presented work is in the frame of the national research project, CronXCov, “Checking the CHRONIC to prevent COVID-19”, devoted to understand how physiologic and inflamed skin on chip 3D models evolve upon a range of physical (e.g., electrical, mechanical, optical) stimulations, over time. Thanks to the 3D modelling, using Next Generation Sequencing and the network medicine frame of analysis to process the data, we will systematically characterize the effects of the applied stimuli, offering new insight for the exploitation of wound healing

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

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

Effect on albumin and fibronectin adsorption of silver doping via ionic exchange of a silica-based bioactive glass

Protein adsorption is a crucial step in the life of biomaterials for bone application, such as bioactive glasses. The investigation of adsorption mechanisms is a difficult task per se, which is even more complex on bioactive glasses due to surface reactivity. Here, the effect of silver doping by ionic exchange on the interaction of a silica-based bioactive glass with albumin and fibronectin, serum proteins related to osseointegration, is reported. The presence of silver does not change relevant surface properties such as topography, surface energy, wettability, or surface ζ potential. Nevertheless, the interactions with proteins are much different. The presence of silver significantly increases the adsorption of albumin and fibronectin and leads to a higher loss of secondary structure compared to the undoped surface, as a consequence of the interactions and bonding between silver and thiols in the cysteine residues. Selectivity of silver-doped glass is discovered: Ag enhances more adsorption and dena- turation of albumin since it has more cysteines than fibronectin. It is also here observed that due to the formation of a hydrated silica gel layer during adsorption, proteins are not only present on the surface of the bioactive glasses, but also embedded inside the surface reaction laye

In Vitro Synovial Membrane 3D Model Developed by Volumetric Extrusion Bioprinting

Background: Synovial tissue plays a fundamental role in inflammatory processes. Therefore, understanding the mechanisms regulating healthy and diseased synovium functions, as in rheumatic diseases, is crucial to discovering more effective therapies to minimize or prevent pathological progress. The present study aimed at developing a bioartificial synovial tissue as an in vitro model for drug screening or personalized medicine applications using 3D bioprinting technology. (2) Methods: The volumetric extrusion technique has been used to fabricate cell-laden scaffolds. Gelatin Methacryloyl (GelMA), widely applied in regenerative medicine and tissue engineering, was selected as a bioink and combined with an immortalized cell line of fibroblast-like synoviocytes (K4IM). (3) Results: Three different GelMA formulations, 7.5–10–12.5% w/v, were tested for the fabrication of the scaffold with the desired morphology and internal architecture. GelMA 10% w/v was chosen and combined with K4IM cells to fabricate scaffolds that showed high cell viability and negligible cytotoxicity for up to 14 days tested by Live & Dead and lactate dehydrogenase assays. (4) Conclusions: We successfully 3D bioprinted synoviocytes-laden scaffolds as a proof-of-concept (PoC) towards the fabrication of a 3D synovial membrane model suitable for in vitro studies. However, further research is needed to reproduce the complexity of the synovial microenvironment to better mimic the physiological condition