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Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Motivation Inflammation is part of the complex function that addresses harmful stimuli, and the first phase of wound healing (WH), which guarantees living systems’ homeostasis. Deviances from physiology make inflammation turn acute (sepsis, 11M death/y) or chronic (non-communicable diseases, 41M death/y). Therefore, tackling inflammation is a key priority. We recently proposed (Maturo et al., 2020) to revise the conventional inflammatory pathway (innate immune response) to include WH (expanded inflammatory pathway). Methods We manually identified the Reactome pathways that include all reactions and species relevant to WH. Cytoscape was then used to perform the union of the SBML converted pathways, with the largest connected component being retained (732 nodes, 13.944 edges). The same was done for the innate immune response (R-HSA-168249.8) with 487 nodes, 11.744 edges. We then focused on: NF-kB (fundamental hub in all inflammatory reactions), TNF-α (renown target of inflammatory diseases) and RAC1 (key player in mechanotransduction events of WH). Results Preliminary topological results highlight the stability of closeness centrality, i.e. all molecules preserve their efficiency in spreading information. Conversely, betweenness centrality is stable for NF-kB (0.068), confirming NF-kB relevance, while halving its (very low) value in the expanded pathway for TNF-α (from 2.85E-06 to 1.29E-06). This indicates that the ability to bridge different parts of the graphs is less effective if we consider inflammation as an expanded concept, possibly contributing to explain the many side effects of anti-TNF-α therapies. Interestingly, RAC1 presents stable betweenness (from 0.094 to 0.093), comparable to NF-kB, supporting the hypothesis that WH-leveraging therapies could act on a relevant and stable target, so far neglected (Nardini et al., 2016). References Maturo, M.G., Soligo, M., Gibson, G., Manni, L., Nardini, C., 2020. The greater inflammatory pathway-high clinical potential by innovative predictive, preventive, and personalized medical approach. EPMA J 11, 1–16. https://doi.org/10.1007/s13167-019-00195-w Nardini, C., Devescovi, V., Liu, Y., Zhou, X., Lu, Y., Dent, J.E., 2016. Systemic Wound Healing Associated with local sub-Cutaneous Mechanical Stimulation. Sci. Rep. 6. https://doi.org/10.1038/srep3904

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.

Superhydrophobic surfaces display an extraordinary repulsion to water and water-based solutions. This effect emerges from the interplay of intrinsic hydrophobicity of the surface and its morphology. These surfaces have been established for a long time and have been studied for decades. The increasing interest in recent years has been focused towards applications in many different fields and, in particular, biomedical applications. In this paper, we review the progress achieved in the last years in the fabrication of regularly patterned superhydrophobic surfaces in many different materials and their exploitation for the manipulation and characterization of biomaterial, with particular emphasis on the issues affecting the yields of the fabrication processes and the quality of the manufactured devices.

We report on the detection of an angiogenic molecule Vascular Endothelial Growth Factor (VEGF) in different biological matrices by means of a new integrated biosensing platform exploiting the properties of Bloch surface waves. The new platform takes advantage of a tandem configuration, in which both label-free and enhanced fluorescence detection are implemented. Specifically designed one dimensional photonic crystals were deposited directly on disposable and low cost plastic biochips. A direct sandwich immunoassay was used to detect VEGF in buffer, cell culture supernatant and human plasma at low concentration (ng/mL). The platform enabled the detection of VEGF in all three matrices with high resolution, fast turnaround time (30 min) and in close agreement with the results of enzyme linked immunosorbent assays.

Receptor tyrosine kinases (RTKs) inhibitors&rsquo, activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.

Background: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2&alpha, dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1&alpha, and HIF-2&alpha, SB3 acts by (i) up-regulating HIF-1&alpha, transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2&alpha, stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2&alpha, transcripts, (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2&alpha, transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2&alpha, transcripts. Conclusions: These data outline either HIF-2&alpha, and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

We describe the design and fabrication of biochips based on 1-D photonic crystals supporting Bloch surface waves for label-free optical biosensing. The optical properties of Bloch surface waves are studied in relation to the geometry of the photonic crystals and on the properties of the dielectric materials used for the fabrication. The planar stacks of the biochips are composed of silica, tantala, and titania that were deposited using plasma-ion-assisted evaporation under high-vacuum conditions. The biochip surfaces were functionalized by silanization, and appropriate fluidic cells were designed to operate in an automated platform. An angularly resolved ellipsometric optical sensing apparatus was assembled to carry out the sensing studies. The angular operation is obtained by a focused laser beam at a fixed wavelength and detection of the angular reflectance spectrum by means of an array detector. The results of the experimental characterization of the physical properties of the fabricated biochips show that their characteristics, in terms of sensitivity and figure of merit, match those expected from the numerical simulations. Practical application of the sensor was demonstrated by detecting a specific glycoprotein, Angio-poietin 2, that is involved in angiogenesis and inflammation processes. The protocol used for the label-free detection of Angiopoietin 2 is described, and the results of an exemplary assay, carried out at a relatively high concentration of 1 µg/ml, are given and confirm that an efficient detection can be achieved. The limit of detection of the biochips for Angiopoietin 2, based on the protocol used, is 1.5 pg/mm2 in buffer solution. The efficiency of the label-free assay is confirmed by independent measurements carried out by means of confocal fluorescence microscopy.

We describe the design and fabrication of biochips based on one dimensional photonic crystals supporting Bloch surface waves for label-free optical biosensing. The planar stacks of the biochips are composed of silica, tantala and titania that were deposited using plasma ion assisted evaporation under high vacuum conditions. The biochip surfaces were functionalized by silanization and appropriate fluidic cells were designed to operate in an automated platform. An angularly resolved optical sensing apparatus was assembled to carry out the sensing studies. The angular operation is obtained by a focused laser beam at a fixed wavelength and detection of the angular reflectance spectrum by means of an array detector. Practical application of the sensor was demonstrated by detecting a specific glycoprotein, Angiopoietin 2, that is involved in angiogenesis and inflammation processes. The protocol used for the label-free detection of Angiopoietin 2 is described and the results of an exemplary assay are given, confirming that an efficient detection can be achieved.

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.

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

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.

Background Enhancing 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. Methods We 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. Results Trabectedin 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. Conclusions PARP1 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 material The online version of this article (doi:10.1186/s12943-017-0652-5) contains supplementary material, which is available to authorized users.

The binding of fluorescein sodium salt with three kinds of commercially available bovine serum albumin (BSA) of different grades of purity was investigated at 288, 298 and 313 K by fluorescence and absorption measurements at pH 7.50. The association and dissociation constants K a and K d were determined by the quenching of BSA fluorescence in the presence of fluorescein sodium salt. The best results were obtained by fitting raw data by non-linear regression and Lineweaver–Burk equations. The modified Stern–Volmer and Scatchard plots gave less reliable data since the fitting was much more difficult. The agreement of the constants for the three sets of measurements coming from the different BSA was not as good as expected. BSA binding properties differ depending on the different BSA grades of purity. Actually, the binding constants found for the three BSAs used differed in the same set of interactions, even by keeping the experimental conditions constant. These results are a novelty in the field of BSA–ligand binding studies and should be taken into account for future binding studies using BSA. Actually, a large number of aspects should be considered including the grade of purity and the presence of BSA covalent and non-covalent dimers, trimers and oligomers in solution which can affect the goodness of the binding results.

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.

We report on a label-free time-resolved biosensing technique based on refractometric measurements. A one-dimensional photonic crystal (1-DPC) constituted by a planar stack of dielectric layers is used for supporting Bloch surface waves. These electromagnetic surface waves probe the aqueous environment within few hundreds of nanometers from the photonic crystal surface, which is functionalized with a hydrogel binding matrix. Therein, selected antibodies are grafted for the specific recognition of the tumoral biomarker Angiopoietin-1. A model bioassay is established demonstrating a detection of Antiopoiegin-1 as low as picomolar concentrations.

Dielectric multilayered structures are introduced here as building blocks of disposable chips for sensing applications. Some experimental configurations are illustrated wherein flat or patterned multilayers are employed for sensing in either label-free, spectroscopic or fluorescence operational modes. In particular, the possibility to enhance and control the emission from fluorescence markers on the chip surface is demonstrated to improve the detection limit in bio-assays for cancer diagnostics.

The increasing demand for early detection of diseases drives the efforts to develop more and more sensitive techniques to detect biomarkers in extremely low concentrations. Electromagnetic modes at the surface of one dimensional photonic crystals, usually called Bloch surface waves, were demonstrated to enhance the resolution and constitute an attractive alternative to surface plasmon polariton optical biosensors. We report on the development of Bloch surface wave biochips operating in both label-free and fluorescence modes and demonstrate their use in ovalbumin recognition assays.

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.

Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.

The need for decentralized clinical tests together with the concept of time and cost saving are pushing the development of portable, miniaturized, compact biosensors with diagnostic and prognostic purpose. Here, we propose an innovative detection system based on a Single Photon Avalanche Diode (SPAD) with high sensitivity and low noise, crucial features for an efficient chemiluminescence biosensor. The SPAD detector, having 60. ?m diameter, has a Photon Detection Efficiency higher than 55% at 425. nm and a Dark Count Rate lower than 100. Hz at room temperature. Our design allows a good optical coupling efficiency between sample and detector. A specific biofunctional surface was implemented taking advantage of aptamers, short DNA sequences having high selectivity and affinity toward their targets. We successfully detected physiological levels of Vascular Endothelial Growth Factor (VEGF), a circulating protein biomarker highly correlated with cancer. The SPAD aptasensor showed a Limit of Detection (LoD) in the pM range, stability (up to 42 days) and re-usability (up to seven cycles). This compact biosensor is therefore a promising step toward the actual use of portable microdevices in diagnostics.

Biosensors based on surface plasmons have shown to be reliable and highly sensitive platforms. Here we investigate the feasibility and the potential of an alternative platform for biological sensing based on Bloch surface waves sustained by a one-dimensional photonic crystal made of dielectric materials.

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.

Vascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.

In this paper we consider the modeling of a portion of the signal transduction pathway involved in the angiogenic process. The detailed model of this process is affected by a high level of complexity due to the functional properties that are represented and the size of its state space. To overcome these problems, we suggest approaches to simplify the detailed representation that result in models with a lower computational and structural complexity, while still capturing the overall behavior of the detailed one.The simplification process must take into account both the structural aspects and the quantitative behavior of the original model. To control the simplification from a structural point of view, we propose a set of reduction steps that maintain the invariants of the original model. To ensure the correspondence between the simplified and the original models from a quantitative point of view we use the flow equivalent method that provides a way of obtaining the parameters of the simplified model on the basis of those of the original one.To support the proposed methodology we show that a good agreement exists among the temporal evolutions of the relevant biological products in the simplified and detailed model evaluated with a large set of input parameters.

Microcantilever based oscillators have shown the possibility of highly sensitive label-free detection by allowing the transduction of a target mass into a resonant frequency shift. Most of such measurements were performed in air or vacuum environment, since immersion in liquid dramatically deteriorates the mechanical response of the sensor. Besides, the integration of microcantilever detection in a microfluidic platform appears a highly performing technological solution to exploit real time monitoring of biomolecular interactions, while limiting sample handling and promoting portability and automation of routine diagnostic tests (Point-Of-Care devices). In the present paper, we report on the realization and optimization of a microcantilever-based Lab-on-Chip, showing that microplates rather than microbeams exhibit largest mass sensitivity in liquid, while pirex rather than polymers represents the best choice for microfluidic channels. Maximum Q factor achieved was 140 (for fifth resonance mode of Pirex prototype), as our knowledge the highest value reported in literature for cantilever biosensors resonating in liquid environment without electronic feedback. Then, we proved the successfully detection of Angiopoietin-1 (a putative marker in tumor progression), showing that the related frequency shifts coming from non-specific interactions (negative controls) are roughly one order of magnitude lower than typical variations due to specific protein binding. Furthermore, we monitored the formation of antibody–antigen complex on MC surface in real-time. The proposed tool could be extremely useful for the comprehension of complex biological systems such as angiogenic machinery and cancer progression.

Microcantilever biosensors have been proposed in the last years as very sensitive mass detectors, but few works focused on the precision and specificity of such tools. We measured the repeatability and reproducibility of our cantilever-based system, proponing the combination of results coming from both the first and second mode of vibration. Then, we optimized two biodesigns (a receptor-based and an antibody-based) to the detection of Angiopoietin-1, a possible marker in tumor progression. The reported results show that our microcantilever-based system can detect Angiopoietin-1 masses of the order of few hundreds of picograms with less than 0.5% of relative uncertainty. We showed that the evaluation of the protein surface density (number of molecules per cm(2)) could reveal interesting features concerning the multimerization state of the targeted protein. We also performed negative controls (dipping the sample in PBS without proteins) and specificity tests (dipping the sample in PBS with a "false" antigen). The related frequency shifts coming from non-specific interactions were found to be at least one order of magnitude lower than typical variations due to specific protein binding. Thanks to its fine precision and optimal specificity, our microcantilever-based system can be successfully applied as a quantitative tool for systems biology studies such as the comprehension of angiogenic machinery and cancer progression.

The interaction between glutathione S -transferase and its antibody α-glutathione S -transferase (B-14) was studied using fluorescence anisotropy, subsequent to glutathione S -transferase bioconjugation with fluorescein-5-maleimide, leading to the determination of the dissociation and association binding constants, K d and K a ; good binding specificity was observed between glutathione S -transferase and the antibody B-14. The use of spectroscopic techniques, fluorescence anisotropy in particular, is a useful and favourable tool to study biochemical problems.

Understanding the role of integrins in the formation of vascular bed is important for designing new therapeutic approaches to ameliorate or inhibit pathological vascularization. Besides regulating cell adhesion and migration, integrins dynamically participate in a network with soluble molecules and their receptors. This study summarizes recent progress in the understanding of the reciprocal interactions between integrins, tyrosine kinase, and semaphorin receptors.During angiogenic remodeling, endothelial cells that line blood vessel walls dynamically modify their integrin-mediated adhesive contacts with the surrounding extracellular matrix. During angiogenesis, opposing autocrine and paracrine loops of growth factors and semaphorins regulate endothelial integrin activation and function through tyrosine kinase receptors and the neuropilin/plexins system. Moreover, proangiogenic and antiangiogenic factors can directly bind integrins and regulate endothelial cell behavior. Studies describing these intense research areas are discussed.Alteration in the balance between the angiogenic growth factors and semaphorins results in an impairment of integrin functions and could account for cardiovascular malformation and structural and functional abnormalities of the tumor vasculature.

Compared to lower metazoans, vertebrates built up an exclusively new set of adhesion-related genes involved in the tissue development and in their functions. They include a large variety of extracellular matrix proteins and their heterodimeric integrin adhesive receptors. Integrins control the adhesive state of the cell through complex molecular mechanisms. Outside-in signalling informs the cell about the extracellular matrix environment, while Inside-out signalling results in changes in integrin functional activity. In the last 10 years it has well established a reciprocal integration of signals originating from integrins and receptors for soluble growth factors. This review summarizes the current understanding of this connection in vascular endothelial cells and highlights how integrins regulate a genetic program triggered by angiogenic inducers during embryo development and in adult life.

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 alpha5beta1 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 alpha5beta1 interact constitutively; alpha5beta1 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 alpha5beta1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and alpha5beta1 receptors that could cross-talk when Tie2/alpha5beta1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that alpha5beta1, but not alphavbeta3 activation, is essential to Ang-1-dependent angiogenesis in vivo.

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.

Angiopoietin-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)

MEK and ERK are central components of the mitogen-activated protein kinase pathway. However, an accurate interaction has never been studied and accurate binding constants of the binary interaction have never been directly measured. In the present work, we studied the interaction between MEK and ERK by stopped-flow fluorescence intensity and evaluated the association and dissociation rate constants (kon and koff) from the kinetic study. We compared the results obtained by using commercial and homemade protein productions. The dissociation binding constant (Kd) value determined for the binding of MEK to ERK is in good agreement with the values obtained from the analysis of the kinase enzymatic reaction in previous in vitro studies.