The endothelin A receptor and epidermal growth factor receptor signaling converge on β-catenin to promote ovarian cancer metastasis

Aims: Endothelin A receptor (ETAR) and epidermal growth factor receptor (EGFR) cross-talk enhances the metastatic potential of epithelial ovarian cancer (EOC) cells activating different pathways, including β-catenin signalling. Here, we evaluated β-catenin as one of ETAR/EGFR downstream pathway in the invasive behaviour of EOC cells and their therapeutic potential to co-target ETAR and EGFR. Main methods: The phosphorylation status and interactions of different proteins were analysed by immunoblotting and immunoprecipitation. Reporter activity and RT-PCR was used for evaluation of β-catenin transcriptional activity and gene expression. Functional effects were evaluated by gelatin zymography and cell invasion assays. An orthotopic model of metastatic human EOC in mice was used for in vivo studies. Key findings: In EOC cell lines, ET-1 induced Src-dependent EGFR transactivation, causing tyrosine (Y) phosphorylation of β-catenin at the residue Y654, its dissociation from E-cadherin complexes and the accumulation as an active form. This pool of Tyr-β-catenin relocalised to the nucleus promoting its transcriptional activity, and the expression of its target genes, such as MMP-2. At functional level, ET-1 and EGFR circuits enhanced protease activity and cell invasion. All these effects were significantly inhibited by the ETAR antagonist, zibotentan, or EGFR inhibitor, gefitinib, and are completely blocked by co-addition of both drugs. In vivo, zibotentan treatment significantly inhibited metastases, associated with reduced expression and activation of MMPs and active β-catenin, especially when combined with gefitinib. Significance: Altogether these findings provide additional support to the potential use of ETAR and EGFR blockade as a new therapeutic opportunity for EOC treatment. © 2012 Elsevier Inc

β-Arrestin 1 is required for endothelin-1-induced NF-κB activation in ovarian cancer cells

Aims In epithelial ovarian cancer (EOC), activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) signalling is linked to many tumor promoting effects, such as proliferation, angiogenesis, invasion and metastasis. These effects are dependent by the activation of critical signalling pathways, such as MAPK, Akt, and β-catenin, through specific cytosolic and nuclear scaffolding functions of β-arrestin 1 (β-arr1). Here, we have assessed the potential role of ET-1/ETAR in promoting NF-κB signalling in EOC cells through β-arr-1 recruitment. Main methods We used cultured HEY EOC cells cultured in the presence or absence of ET-1 and the ETAR antagonist BQ123. The phosphorylation of p65 and Iκ-Bα was evaluated by immunoblotting analysis. The interaction between p65 and β-arr1 was evaluated by immunoprecipitation experiments in nuclear extracts. NF-κB promoter activity was evaluated by transfection with NF-κB-driven luciferase reporter construct. Assessment of the function of β-arr1 was achieved by β-arr1 silencing with shRNA and expression of β-arr1-FLAG expression vector. Key findings In EOC cells, ET-1 promotes the phosphorylation of p65 subunit and the cytoplasmic inhibitor IκB that in turn led to increased NF-κB transcriptional activity. These effects were inhibited by the use of BQ123, as well as by β-arr-1 silencing, suggesting that ET-1 through ETAR promotes the recruitment of β-arr1 to regulate NF-κB signalling. Moreover, the nuclear physical interaction between p65 and β-arr1 indicates a nuclear function of β-arr-1 in ETAR-driven NF-κB transcriptional activity. Significance Altogether these findings reveal a previously unrecognized pathway that depends on β-arr1 to sustain NF-κB signalling in response to ETAR activation in ovarian cancer

Endothelin-1 cooperates with hypoxia to induce vascular-like structures through vascular endothelial growth factor-C, -D and -A in lymphatic endothelial cells.

Abstract Aims Lymphangiogenesis refers to the formation of new lymphatic vessels and is thought to constitute conduits for the tumor cells to metastasize. We previously demonstrated that endothelin (ET)-1 through its binding with ETB receptor (ET B R) expressed on lymphatic endothelial cells (LEC), induced cell growth and invasiveness. Since vascular endothelial growth factor (VEGF)-A/-C/-D, and hypoxia play key role in lymphatic differentiation, in this study we investigated the involvement of these growth factors and hypoxia in ET-1-induced lymphangiogenesis. Main methods Real time PCR and ELISA were used to quantify VEGF-A/-C/-D. LEC morphological differentiation was analyzed by tube formation assay on Matrigel. Key findings Hypoxia, as well as ET-1, induced an increase in VEGF-A/-C and -D expression that was reduced in the presence of a selective ET B R antagonist, BQ788, and enhanced when ET-1 was administered under hypoxic conditions. We analyzed the role of hypoxia on LEC morphological differentiation, and found that hypoxia increased the formation of vascular-like structures on Matrigel and that in combination with ET-1 this effect was markedly enhanced. The use of specific antibodies neutralizing VEGF-A, or recombinant VEGFR-3/(Flt-4)/Fc that block VEGF-C/-D, inhibited the effect of ET-1 as well that of hypoxia. Significance These results demonstrated that ET-1 and hypoxia act, at list in part, through VEGF to induce lymphangiogenic events and that these two stimuli may cooperate to induce VEGF-A/-C/-D expression and lymphatic differentiation. These data further support the role of ET-1 as potent lymphangiogenic factor that relies on the interplay with hypoxic microenvironment and with VEGF family members

Chemogenic versus biogenic synthesis of Selenium nanoparticles: a structural characterization

Among the plethora of available metal- and metalloid-based nanomaterials (NMs), selenium nanostructures (SeNSs) are one of the most interesting from an applicative perspective due to their intermediate properties between metals and non-metals, as well as their high biocompatibility. In this regard, the capability of microorganisms to biotransform toxic Se-oxyanions – i.e., selenite (SeO32-) and selenate (SeO42-) – into their less bioavailable elemental forms [Se(0)], mostly generating Se nanoparticles (SeNPs), represents as a useful and green alternative over chemogenic synthesis allowing to obtain highly thermodynamically stable NMs. However, their structural characterization, in terms of biomolecules and interactions stabilizing the biogenic colloidal solution, is still a black hole in the microbial nanotechnology field, impairing the exploitation of biogenic SeNP full potential. Here, a parallel characterization between biogenic and chemogenic SeNPs was carried out through Fourier Transform Infrared spectroscopy in Attenuated Total Reflectance (ATR-FTIR) mode, Nuclear Magnetic Resonance (NMR) spectroscopy, and Density Functional Theory (DFT) calculations, to better understand which functional groups, hence biomolecules, contribute the most to the stabilization of biogenic SeNPs

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50–75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC

Endothelin-1 regulates hypoxia-inducible factor-1α and -2α stability through prolyl hydroxylase domain 2 inhibition in human lymphatic endothelial cells

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Artificial Intelligence Application to Screen Abdominal Aortic Aneurysm Using Computed tomography Angiography

The aim of our study is to validate a totally automated deep learning (DL)-based segmentation pipeline to screen abdominal aortic aneurysms (AAA) in computed tomography angiography (CTA) scans. We retrospectively evaluated 73 thoraco-abdominal CTAs (48 AAA and 25 control CTA) by means of a DL-based segmentation pipeline built on a 2.5D convolutional neural network (CNN) architecture to segment lumen and thrombus of the aorta. The maximum aortic diameter of the abdominal tract was compared using a threshold value (30 mm). Blinded manual measurements from a radiologist were done in order to create a true comparison. The screening pipeline was tested on 48 patients with aneurysm and 25 without aneurysm. The average diameter manually measured was 51.1 ± 14.4 mm for patients with aneurysms and 21.7 ± 3.6 mm for patients without aneurysms. The pipeline correctly classified 47 AAA out of 48 and 24 control patients out of 25 with 97% accuracy, 98% sensitivity, and 96% specificity. The automated pipeline of aneurysm measurements in the abdominal tract reported a median error with regard to the maximum abdominal diameter measurement of 1.3 mm. Our approach allowed for the maximum diameter of 51.2 ± 14.3 mm in patients with aneurysm and 22.0 ± 4.0 mm in patients without an aneurysm. The DL-based screening for AAA is a feasible and accurate method, calling for further validation using a larger pool of diagnostic images towards its clinical use

Cardiac involvement in systemic sclerosis: identification of high-risk patient profiles in different patterns of clinical presentation

Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by widespread microvascular damage, dysregulation of fibroblasts with collagen overproduction and excessive fibrosis of the skin and internal organs, as well as complex immune system abnormalitie\u2026

Double role of HMTA in ZnO nanorods grown by chemical bath deposition

ZnO nanorods (NRs) grown by chemical bath deposition (CBD) are among the most promising semiconducting nanostructures currently investigated for a variety of applications. Still, contrasting experimental results appear in the literature on the microscopic mechanisms leading to high aspect ratio and vertically aligned ZnO NRs. Here, we report on CBD of ZnO NRs using Zn nitrate salt and hexamethylenetetramine (HMTA), evidencing a double role of HMTA in the NRs growth mechanism. Beyond the well-established pH buffering activity, HMTA is shown to introduce a strong steric hindrance effect, biasing growth along the c-axis and ensuring the vertical arrangement. This twofold function of HMTA should be taken into account for avoiding detrimental phenomena such as merging or suppression of NRs, which occur at low HMTA concentration