Mammalian tumor xenografts induce neovascularization in zebrafish embryos.

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein–positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/ tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis

Arousal effects on Fitness-to-Drive assessment: algorithms and experiments

Several elements can affect the drivers' behaviour while they are performing driving activities. Ranging from visual to cognitive distractions, emotions and other drivers' conditions (that could emerge from biometric data, such as temperature, heartbeat, pressure, etc.) can play a significant role, performing as a factor that can increase drivers' response time. This could be crucial in avoiding dangerous situations and in deciding and performing actions that could influence the happening of car accidents. This paper introduces the concept of the "Fitness-to-Drive" index and aims to evaluate how the arousal effects can influence the drivers' status. The paper presents some experimental evaluations we have conducted on a driver simulator, discussing the obtained results

Development of Lumped Element Kinetic Inductance Detectors for the W-Band

We are developing a Lumped Element Kinetic Inductance Detector (LEKID) array able to operate in the W-band (75-110 GHz) in order to perform ground-based Cosmic Microwave Background (CMB) and mm-wave astronomical observations. The W-band is close to optimal in terms of contamination of the CMB from Galactic synchrotron, free-free, and thermal interstellar dust. In this band, the atmosphere has very good transparency, allowing interesting ground-based observations with large (>30 m) telescopes, achieving high angular resolution (<0.4 arcmin). In this work we describe the startup measurements devoted to the optimization of a W-band camera/spectrometer prototype for large aperture telescopes like the 64 m SRT (Sardinia Radio Telescope). In the process of selecting the best superconducting film for the LEKID, we characterized a 40 nm thick Aluminum 2-pixel array. We measured the minimum frequency able to break CPs (i.e. $h\nu=2\Delta\left(T_{c}\right)=3.5k_{B}T_{c}$) obtaining $\nu=95.5$ GHz, that corresponds to a critical temperature of 1.31 K. This is not suitable to cover the entire W-band. For an 80 nm layer the minimum frequency decreases to 93.2 GHz, which corresponds to a critical temperature of 1.28 K; this value is still suboptimal for W-band operation. Further increase of the Al film thickness results in bad performance of the detector. We have thus considered a Titanium-Aluminum bi-layer (10 nm thick Ti + 25 nm thick Al, already tested in other laboratories), for which we measured a critical temperature of 820 mK and a cut-on frequency of 65 GHz: so this solution allows operation in the entire W-band.Comment: 16th International Workshop on Low Temperature Detectors, Grenoble 20-24 July 2015, Journal of Low Temperature Physics, Accepte

Heparan Sulfate Proteoglycans Mediate the Angiogenic Activity of the Vascular Endothelial Growth Factor Receptor-2 Agonist Gremlin.

OBJECTIVE: Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. METHODS AND RESULTS: Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase(1/2) and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. CONCLUSIONS: HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist

Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the panendothelial cell markers murine CD31 and vascular endothelial-cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or b-actin mRNA levels. RTqPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammation-related genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of nonendothelial/ inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF

Rapid and accurate simultaneous determination of abamectin and ivermectin in bovine milk by high performance liquid chromatography with fluorescence detection

An analytical method using high performance liquid chromatography with fluorescence detection for the simultaneous determination of abamectin and ivermectin in bovine milk was developed and validated. The best recovery results were achieved by using acetonitrile for extraction of the compounds followed by solid phase extraction in cartridges containing C18 for the purification of the extract. Pre-column derivatization was accomplished with N-methylimidazole and trifluoroacetic anhydride. The method limit of detection (LOD) values for abamectin and ivermectin were 0.10 and 0.14 µg L-1 and the limit of quantification (LOQ) values were 0.18 and 0.36 µg L-1, respectively. The recoveries were from 75 to 101%, with RSD values lower than 10%. The LOD and LOQ values are lower than the maximum residue limits (MRLs) in milk established by Codex Alimentarius, European Union and the Brazilian legislation

Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine.

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus

The broad-spectrum anti-DNA virus agent cidofovir inhibits lung metastasis of virus-independent, FGF2-driven tumors.

The FDA-approved anti-DNA virus agent cidofovir (CDV) is being evaluated in phase II/III clinical trials for the treatment of human papillomavirus (HPV)-associated tumors. However, previous observations had shown that CDV also inhibits the growth of vascular tumors induced by fibroblast growth factor-2 (FGF2)-transformed FGF2-T-MAE cells. Here, we demonstrate that CDV inhibits metastasis induced by FGF2-driven, virus-independent tumor cells. Pre-treatment of luciferase-expressing FGF2-T-MAE cells with CDV reduced single cell survival and anchorage-independent growth in vitro and lung metastasis formation upon intravenous inoculation into SCID mice. This occurred in the absence of any effect on homing of FGF2-T-MAE cells to the lungs and on the growth of subconfluent cell cultures or subcutaneous tumors in mice. Accordingly, CDV protected against lung metastasis when given systemically after tumor cell injection. Lung metastases in CDV-treated mice showed reduced Ki67 expression and increased nuclear accumulation of p53, indicating that CDV inhibits metastasis by affecting single cell survival properties. The anti-metastatic potential of CDV was confirmed on B16-F10 melanoma cells, both in zebrafish embryos and mice. These findings suggest that CDV may have therapeutic potential as an anti-metastatic agent and warrants further study to select those tumor types that are most likely to benefit from CDV therapy

Real-life appraisal on blood pressure targets achievement in adult outpatients at high cardiovascular risk

Background and aim: Although hypertension guidelines highlight the benefits of achieving the recommended blood pressure (BP) targets, hypertension control rate is still insufficient, mostly in high or very high cardiovascular (CV) risk patients. Thus, we aimed to estimate BP control in a cohort of patients at high CV risk in both primary and secondary prevention. Methods and results: A single-center, cross-sectional study was conducted by extracting data from a medical database of adult outpatients aged 40–75 years, who were referred to our Hypertension Unit, Rome (IT), for hypertension assessment. Office BP treatment targets were defined according to 2018 ESC/ESH guidelines as: a)&lt;130/80 mmHg in individuals aged 40–65 years; b)&lt;140/80 mmHg in subjects aged &gt;65 years. Primary prevention patients with SCORE &lt;5% were considered to be at low-intermediate risk, whilst individuals with SCORE ≥5% or patients with comorbidities were defined to be at very high risk. Among 6354 patients (47.2% female, age 58.4 ± 9.6 years), 4164 (65.5%) were in primary prevention with low-intermediate CV risk, 1831 (28.8%) in primary prevention with high-very high CV risk and 359 (5.6%) in secondary prevention. In treated hypertensive outpatients, uncontrolled hypertension rate was significantly higher in high risk primary prevention than in low risk primary prevention and secondary prevention patients (18.4% vs 24.4% vs. 12.5%, respectively; P &lt; 0.001). In high risk primary prevention diabetic patients only 10% achieved the recommended BP targets. Conclusions: Our data confirmed unsatisfactory BP control among high-risk patients, both in primary and secondary prevention, and suggest the need for a more stringent BP control policies in these patients