Publications by authors named "Stefania Mitola"

77 Publications

Specific targeting of the KRAS mutational landscape in myeloma as a tool to unveil the elicited anti-tumor activity.

Blood 2021 Jun 2. Epub 2021 Jun 2.

University of Milan, Milan, Italy.

The multiple myeloma (MM) mutational landscape has identified alterations in KRAS as the most recurring somatic variant. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying that KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We have used AZD4785, a potent and selective antisense oligonucleotide (ASO) which selectively targets and down-regulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche; and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, confirming KRAS as a driver and a therapeutic target in MM.
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http://dx.doi.org/10.1182/blood.2020010572DOI Listing
June 2021

Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cells Activation and Infectability Amplifying the Angiogenic Process.

Infect Immun 2021 May 24. Epub 2021 May 24.

Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Italy.

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of , the mechanisms by which induces EC activation are not completely clear, as well as the possible contribution of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections exerting antimicrobial properties but also acting as a shelter for pathogens.Here we delved into the role of MSCs as reservoir of and modulator of EC functions. readily infected MSCs and survived in perinuclear bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of EGFR, TLR2 and NOD1, proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors VEGF, FGF-7, MMP-9, PIGF, serpin E1, TSP-1, uPA, IL-6, PDGF-D, CCL5 and CXCL8. Supernatants from -infected MSCs increased the susceptibility of ECs to infection and enhanced EC proliferation, invasion and reorganization in tube-like structures.Altogether, these results candidate MSCs as a still underestimated niche for persistent infection and reveal a MSC-EC crosstalk that may contribute to exacerbate bacterial-induced angiogenesis and granuloma formation.
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http://dx.doi.org/10.1128/IAI.00141-21DOI Listing
May 2021

H-ferritin suppression and pronounced mitochondrial respiration make Hepatocellular Carcinoma cells sensitive to RSL3-induced ferroptosis.

Free Radic Biol Med 2021 Jun 20;169:294-303. Epub 2021 Apr 20.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy. Electronic address:

Ferroptosis is a form of regulated cell death dependent on iron, reactive oxygen species and characterized by the accumulation of lipid peroxides. It can be experimentally initiated by chemicals, such as erastin and RSL3, that modulate GPX4 activity, the cellular antioxidant machinery that avert lipid peroxidation. The study aimed to investigate mitochondrial respiration and ferritin function as biomarkers of ferroptosis sensitivity of HepG2 and HA22T/VGH, two Hepatocellular Carcinoma (HCC) cell line models. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, labile iron levels were determined using Calcein-AM fluorescence microscopy, ferritin, glutathione and lipid peroxidation were assayed with commercially available kits. The Seahorse assay was used to investigate mitochondrial function in the cells. The study shows that highly differentiated HepG2 cells were more sensitive to RSL3-induced ferroptosis than the poorly differentiated HA22T/VGH (HCC) cell line (RSL3 IC 0.07 μM in HepG2 vs 0.3 μM in HA22T/VGH). Interestingly, HepG2 exhibited higher mitochondrial respiration and lower glycolytic activity than HA22T/VGH and were more sensitive to RSL3-induced ferroptosis, indicating a mitochondrial-specific mechanism of action of RSL3. Interestingly, iron metabolism seems to be involved in this different sensitivity, specifically, the downregulation of H-ferritin (but not of L-subunit), makes HA22T/VGH more sensitive toward both RSL3-and iron-induced ferroptosis. Hence only the H-ferritin seems involved in the protection from this cell death process.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.04.024DOI Listing
June 2021

Expression of activated VEGFR2 by R1051Q mutation alters the energy metabolism of Sk-Mel-31 melanoma cells by increasing glutamine dependence.

Cancer Lett 2021 Jun 17;507:80-88. Epub 2021 Mar 17.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, 25123, Italy. Electronic address:

Vascular endothelial growth factor receptor 2 (VEGFR2) activating mutations are emerging as important oncogenic driver events. Understanding the biological implications of such mutations may help to pinpoint novel therapeutic targets. Here we show that activated VEGFR2 via the pro-oncogenic R1051Q mutation induces relevant metabolic changes in melanoma cells. The expression of VEGFR2 leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2. Furthermore, activated VEGFR2 augments the dependence on glutamine (Gln) of melanoma cells, thus increasing Gln uptake and their sensitivity to Gln deprivation and to inhibitors of glutaminase, the enzyme initiating Gln metabolism by cells. Overall, these results highlight Gln addiction as a metabolic vulnerability of tumors harboring the activating VEGFR2 mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2.
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http://dx.doi.org/10.1016/j.canlet.2021.03.007DOI Listing
June 2021

Alpha-Synuclein in the Regulation of Brain Endothelial and Perivascular Cells: Gaps and Future Perspectives.

Front Immunol 2021 19;12:611761. Epub 2021 Feb 19.

Division of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

Misfolded proteins, inflammation, and vascular alterations are common pathological hallmarks of neurodegenerative diseases. Alpha-synuclein is a small synaptic protein that was identified as a major component of Lewy bodies and Lewy neurites in the brain of patients affected by Parkinson's disease (PD), Lewy body dementia (LBD), and other synucleinopathies. It is mainly involved in the regulation of synaptic vesicle trafficking but can also control mitochondrial/endoplasmic reticulum (ER) homeostasis, lysosome/phagosome function, and cytoskeleton organization. Recent evidence supports that the pathological forms of α-synuclein can also reduce the release of vasoactive and inflammatory mediators from endothelial cells (ECs) and modulates the expression of tight junction (TJ) proteins important for maintaining the blood-brain barrier (BBB). This hints that α-synuclein deposition can affect BBB integrity. Border associated macrophages (BAMs) are brain resident macrophages found in association with the vasculature (PVMs), meninges (MAMs), and choroid plexus (CPMs). Recent findings indicate that these cells play distinct roles in stroke and neurodegenerative disorders. Although many studies have addressed how α-synuclein may modulate microglia, its effect on BAMs has been scarcely investigated. This review aims at summarizing the main findings supporting how α-synuclein can affect ECs and/or BAMs function as well as their interplay and effect on other cells in the brain perivascular environment in physiological and pathological conditions. Gaps of knowledge and new perspectives on how this protein can contribute to neurodegeneration by inducing BBB homeostatic changes in different neurological conditions are highlighted.
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http://dx.doi.org/10.3389/fimmu.2021.611761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933041PMC
February 2021

The Claudin-Low Subtype of High-Grade Serous Ovarian Carcinoma Exhibits Stem Cell Features.

Cancers (Basel) 2021 Feb 22;13(4). Epub 2021 Feb 22.

Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

Claudin-low cancer (CL) represents a rare and biologically aggressive variant of epithelial tumor. Here, we identified a claudin-low molecular profile of ovarian high-grade serous carcinoma (HGSOC), which exhibits the main characteristics of the homonym breast cancer subtype, including low epithelial differentiation and high mesenchymal signature. Hierarchical clustering and a centroid based algorithm applied to cell line collection expression dataset labeled 6 HGSOC cell lines as CL. These have a high energy metabolism and are enriched in CD44/CD24 mesenchymal stem-like cells expressing low levels of cell-cell adhesion molecules (claudins and E-Cadherin) and high levels of epithelial-to-mesenchymal transition (EMT) induction transcription factors (Zeb1, Snai2, Twist1 and Twist2). Accordingly, the centroid base algorithm applied to large retrospective collections of primary HGSOC samples reveals a tumor subgroup with transcriptional features consistent with the CL profile, and reaffirms EMT as the dominant biological pathway functioning in CL-HGSOC. HGSOC patients carrying CL profiles have a worse overall survival when compared to others, likely to be attributed to its undifferentiated/stem component. These observations highlight the lack of a molecular diagnostic in the management of HGSOC and suggest a potential prognostic utility of this molecular subtyping.
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http://dx.doi.org/10.3390/cancers13040906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926503PMC
February 2021

Simultaneously characterization of tumoral angiogenesis and vasculogenesis in stem cell-derived teratomas.

Exp Cell Res 2021 Mar 21;400(2):112490. Epub 2021 Jan 21.

Department of Molecular and Translational Medicine, Via Branze 39, 25123, Brescia, University of Brescia, Italy; Laboratory for Preventive and Personalized Medicine (MPP Lab), University of Brescia, Italy. Electronic address:

Tumor neovascularization may occur via both angiogenic and vasculogenic events. In order to investigate the vessel formation during tumor growth, we developed a novel experimental model that takes into account the differentiative and tumorigenic properties of Embryonic Stem cells (ESCs). Leukemia Inhibitory Factor-deprived murine ESCs were grafted on the top of the chick embryo chorionallantoic membrane (CAM) in ovo. Cell grafts progressively grew, forming a vascularized mass within 10 days. At this stage, the grafts are formed by cells with differentiative features representative of all three germ layers, thus originating teratomas, a germinal cell tumor. In addition, ESC supports neovascular events by recruiting host capillaries from surrounding tissue that infiltrates the tumor mass. Moreover, immunofluorescence studies demonstrate that perfused active blood vessels within the tumor are of both avian and murine origin because of the simultaneous occurrence of angiogenic and vasculogenic events. In conclusion, the chick embryo ESC/CAM-derived teratoma model may represent a useful approach to investigate both vasculogenic and angiogenic events during tumor growth and for the study of natural and synthetic modulators of the two processes.
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http://dx.doi.org/10.1016/j.yexcr.2021.112490DOI Listing
March 2021

Genetic perturbation of IFN-α transcriptional modulators in human endothelial cells uncovers pivotal regulators of angiogenesis.

Comput Struct Biotechnol J 2020 2;18:3977-3986. Epub 2020 Dec 2.

Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV - IRCCS, via Gattamelata 64, 35128 Padova, Italy.

Interferon-α (IFN-α) comprises a family of 13 cytokines involved in the modulation of antiviral, immune, and anticancer responses by orchestrating a complex transcriptional network. The activation of IFN-α signaling pathway in endothelial cells results in decreased proliferation and migration, ultimately leading to suppression of angiogenesis. In this study, we knocked-down the expression of seven established or candidate modulators of IFN-α response in endothelial cells to reconstruct a gene regulatory network and to investigate the antiangiogenic activity of IFN-α. This genetic perturbation approach, along with the analysis of interferon-induced gene expression dynamics, highlighted a complex and highly interconnected network, in which the angiostatic chemokine C-X-C Motif Chemokine Ligand 10 (CXCL10) was a central node targeted by multiple modulators. IFN-α-induced secretion of CXCL10 protein by endothelial cells was blunted by the silencing of Signal Transducer and Activator of Transcription 1 (STAT1) and of Interferon Regulatory Factor 1 (IRF1) and it was exacerbated by the silencing of Ubiquitin Specific Peptidase 18 (USP18). sprouting assay, which mimics angiogenesis, confirmed STAT1 as a positive modulator and USP18 as a negative modulator of IFN-α-mediated sprouting suppression. Our data reveal an unprecedented physiological regulation of angiogenesis in endothelial cells through a tonic IFN-α signaling, whose enhancement could represent a viable strategy to suppress tumor neoangiogenesis.
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http://dx.doi.org/10.1016/j.csbj.2020.11.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734228PMC
December 2020

In Situ DNA/Protein Interaction Assay to Visualize Transcriptional Factor Activation.

Methods Protoc 2020 Nov 21;3(4). Epub 2020 Nov 21.

Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

The chick embryo chorioallantoic membrane (CAM) represents a powerful in vivo model to study several physiological and pathological processes including inflammation and tumor progression. Nevertheless, the possibility of deepening the molecular processes in the CAM system is biased by the absence/scarcity of chemical and biological reagents, designed explicitly for avian species. This is particularly true for transcriptional factors, proteinaceous molecules that regulate various cellular responses, including proliferation, survival, and differentiation. Here, we propose a detailed antibody-independent protocol to visualize the activation and nuclear translocation of transcriptional factors in cells or in tissues of different animal species. As a proof of concept, DNA/cAMP response element-binding protein (CREB) interaction was characterized on the CAM tissue using oligonucleotides containing the palindromic binding sequence of CREB. Scrambled oligonucleotides were used as controls. In situ DNA/protein interaction protocol is a versatile method that is useful for the study of transcription factors in the cell and tissue of different origins.
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http://dx.doi.org/10.3390/mps3040080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720131PMC
November 2020

A novel variant of VEGFR2 identified by a pan-cancer screening of recurrent somatic mutations in the catalytic domain of tyrosine kinase receptors enhances tumor growth and metastasis.

Cancer Lett 2021 01 6;496:84-92. Epub 2020 Oct 6.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, 25123, Italy; Laboratory for Preventive and Personalized Medicine (MPP Lab), University of Brescia, 25123, Italy. Electronic address:

In cancer genomics, recurrence of mutations in gene families that share homologous domains has recently emerged as a reliable indicator of functional impact and can be exploited to reveal the pro-oncogenic effect of previously uncharacterized variants. Pan-cancer analyses of mutation hotspots in the catalytic domain of a subset of tyrosine kinase receptors revealed that two infrequent mutations of VEGFR2 (R1051Q and D1052N) recur in analogous proteins and correlate with reduced patient survival. Functional validation showed that both R1051Q and D1052N mutations increase the enzymatic activity of VEGFR2. The expression of VEGFR2 potentiates the PI3K/Akt signaling axis in cancer cells, increasing their tumorigenic potential in vitro and in vivo. In addition, it confers to cancer cells an increased sensitivity to the VEGFR2-targeted tyrosine kinase inhibitor Linifanib. In the context of an efficacious application of anti-cancer targeted therapies, these findings indicate that the screening for uncharacterized mutations, like VEGFR2, may help to predict patient prognosis and drug response, with significant clinical implications.
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http://dx.doi.org/10.1016/j.canlet.2020.09.027DOI Listing
January 2021

Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions.

Front Mol Biosci 2020 7;7:195. Epub 2020 Aug 7.

Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy.

The set-up of an advanced imaging experiment requires a careful selection of suitable labeling strategies and fluorophores for the tagging of the molecules of interest. Here we provide an experimental workflow to allow evaluation of fluorolabeling performance of the chemical tags target of phosphopantetheinyl transferase enzymes (PPTases), once inserted in the sequence of different proteins of interest. First, S6 peptide tag was fused to three different single-pass transmembrane proteins (the tyrosine receptor kinases TrkA and VEGFR2 and the tumor necrosis factor receptor p75NTR), providing evidence that all of them can be conveniently albeit differently labeled. Moreover, we chose the S6-tagged TrkA construct to test eight different organic fluorophores for the PPTase labeling of membrane receptors in living cells. We systematically compared their non-specific internalization when added to a S6-tag negative cell culture, the percentage of S6-TrkA expressing cells effectively labeled and the relative mean fluorescence intensity, their photostability upon conjugation, and ratio of specific (cellular) versus background (glass-adhered) signal. This allowed to identify which fluorophores are actually recommended for these labeling reactions. Finally, we compared the PPTase labeling of a purified, YBBR-tagged Nerve Growth Factor with two differently charged organic dyes. We detected some batch-to-batch variability in the labeling yield, regardless of the fluorophore used. However, upon purification of the fluorescent species and incubation with living primary DRG neurons, no significant difference could be appreciated in both internalization and axonal transport of the labeled neurotrophins.
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http://dx.doi.org/10.3389/fmolb.2020.00195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426934PMC
August 2020

Low Expression of Claudin-7 as Potential Predictor of Distant Metastases in High-Grade Serous Ovarian Carcinoma Patients.

Front Oncol 2020 4;10:1287. Epub 2020 Aug 4.

Unit of Biostatistics and Bioinformatics, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

High-grade serous ovarian carcinoma (HGSOC) usually spreads directly into the peritoneal cavity following a transcoelomic dissemination route, although distant hematogenous metastasis exist and have been reported. However, no tumor markers can currently predict the risk of distant metastases in HGSOC. Claudins, belonging to tight-junction proteins, are dysregulated in HGSOC and functionally related to cancer progression. Here we analyzed claudin-3, -4, and -7 expression as potential markers of distant metastases. Using quantitative RT-PCR and immunohistochemistry we assessed the expression of claudins in primary HGSOC tissues, normal ovarian, and normal fallopian tube epithelia and correlated it with clinicopathological features, including the site of metastasis and the route of dissemination. Gene set enrichment analysis was performed on microarray-generated gene expression data to investigate key pathways in patients with distant metastases. We found the overall expression level of claudin-3, -4, and -7 mRNA decreased in HGSOC compared to normal tubal epithelium, currently considered the potential site of origin of many HGSOC. The reduced expression of claudin-7 is significantly associated with the development of distant metastases ( = 0.016), mainly by hematogenous route ( = 0.025). In patients with diminished expression of claudin-7, immunohistochemical staining revealed a heterogeneous pattern of membranous staining with discontinuous expression of claudin-7 along the cell border, indicative of a dischoesive architecture. The estimated reduction in the probability of distant disease is of 39% per unit increase in the level of claudin-7 ( = 0.03). Genes involved in epithelial to mesenchymal transition, hypoxia, and angiogenesis processes resulted strongly associated to hematogenous recurrence. Our data suggest a potential role of claudin-7 in discriminating distant metastatic events in HGSOC patients. The quantification of its expression levels could be a useful tool to identify patient deserving a personalized follow-up in terms of clinical and radiological assessment.
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http://dx.doi.org/10.3389/fonc.2020.01287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417514PMC
August 2020

Usefulness of melatonin as complementary to chemotherapeutic agents at different stages of the angiogenic process.

Sci Rep 2020 03 16;10(1):4790. Epub 2020 Mar 16.

Department of Physiology and Pharmacology, School of Medicine, University of Cantabria and Instituto de Investigación Sanitaria Valdecilla (IDIVAL), 39011, Santander, Spain.

Chemotherapeutics are sometimes administered with drugs, like antiangiogenic compounds, to increase their effectiveness. Melatonin exerts antitumoral actions through antiangiogenic actions. We studied if melatonin regulates the response of HUVECs to chemotherapeutics (docetaxel and vinorelbine). The inhibition that these agents exert on some of the processes involved in angiogenesis, such as, cell proliferation, migratory capacity or vessel formation, was enhanced by melatonin. Regarding to estrogen biosynthesis, melatonin impeded the negative effect of vinorelbine, by decreasing the activity and expression of aromatase and sulfatase. Docetaxel and vinorelbine increased the expression of VEGF-A, VEGF-B, VEGF-C, VEGFR-1, VEGFR-3, ANG1 and/or ANG-2 and melatonin inhibited these actions. Besides, melatonin prevented the positive actions that docetaxel exerts on the expression of other factors related to angiogenesis like JAG1, ANPEP, IGF-1, CXCL6, AKT1, ERK1, ERK2, MMP14 and NOS3 and neutralized the stimulating actions of vinorelbine on the expression of FIGF, FGFR3, CXCL6, CCL2, ERK1, ERK2, AKT1, NOS3 and MMP14. In CAM assay melatonin inhibited new vascularization in combination with chemotherapeutics. Melatonin further enhanced the chemotherapeutics-induced inhibition of p-AKT and p-ERK and neutralized the chemotherapeutics-caused stimulatory effect on HUVECs permeability by modifying the distribution of VE cadherin. Our results confirm that melatonin blocks proangiogenic and potentiates antiangiogenic effects induced by docetaxel and vinorelbine enhancing their antitumor effectiveness.
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http://dx.doi.org/10.1038/s41598-020-61622-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076026PMC
March 2020

D-Peptide analogues of Boc-Phe-Leu-Phe-Leu-Phe-COOH induce neovascularization via endothelial N-formyl peptide receptor 3.

Angiogenesis 2020 08 9;23(3):357-369. Epub 2020 Mar 9.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1-FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (L-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A (VEGF). Here we show that the all-D-enantiomer of L-BOC2 (D-BOC2) is devoid of any VEGF antagonist activity. At variance, D-BOC2, as well as the D-FLFLF and succinimidyl (Succ)-D-FLFLF (D-Succ-F3) D-peptide variants, is endowed with a pro-angiogenic potential. In particular, the D-peptide D-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of D-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by D-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, D-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of D-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.
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http://dx.doi.org/10.1007/s10456-020-09714-0DOI Listing
August 2020

β-Galactosylceramidase Deficiency Causes Bone Marrow Vascular Defects in an Animal Model of Krabbe Disease.

Int J Mol Sci 2019 Dec 30;21(1). Epub 2019 Dec 30.

Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase β-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in mutant mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that CD31/VEGFR2 SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.
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http://dx.doi.org/10.3390/ijms21010251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982065PMC
December 2019

Molecular insight on the altered membrane trafficking of TrkA kinase dead mutants.

Biochim Biophys Acta Mol Cell Res 2020 02 21;1867(2):118614. Epub 2019 Nov 21.

Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy; Department of Pharmacy, University of Pisa, Pisa, Italy. Electronic address:

We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking.
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http://dx.doi.org/10.1016/j.bbamcr.2019.118614DOI Listing
February 2020

Natural Histogel-Based Bio-Scaffolds for Sustaining Angiogenesis in Beige Adipose Tissue.

Cells 2019 11 18;8(11). Epub 2019 Nov 18.

Department of Molecular and Translational Medicine, University of Brescia, 25121 Brescia, Italy.

In the treatment of obesity and its related disorders, one of the measures adopted is weight reduction by controlling nutrition and increasing physical activity. A valid alternative to restore the physiological function of the human body could be the increase of energy consumption by inducing the browning of adipose tissue. To this purpose, we tested the ability of Histogel, a natural mixture of glycosaminoglycans isolated from animal Wharton jelly, to sustain the differentiation of adipose derived mesenchymal cells (ADSCs) into brown-like cells expressing UCP-1. Differentiated cells show a higher energy metabolism compared to undifferentiated mesenchymal cells. Furthermore, Histogel acts as a pro-angiogenic matrix, induces endothelial cell proliferation and sprouting in a three-dimensional gel in vitro, and stimulates neovascularization when applied in vivo on top of the chicken embryo chorioallantoic membrane or injected subcutaneously in mice. In addition to the pro-angiogenic activity of Histogel, also the ADSC derived beige cells contribute to activating endothelial cells. These data led us to propose Histogel as a promising scaffold for the modulation of the thermogenic behavior of adipose tissue. Indeed, Histogel simultaneously supports the acquisition of brown tissue markers and activates the vasculature process necessary for the correct function of the thermogenic tissue. Thus, Histogel represents a valid candidate for the development of bioscaffolds to increase the amount of brown adipose tissue in patients with metabolic disorders.
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http://dx.doi.org/10.3390/cells8111457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912328PMC
November 2019

VEGFR2 activation mediates the pro-angiogenic activity of BMP4.

Angiogenesis 2019 11 30;22(4):521-533. Epub 2019 Jul 30.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

The Bone Morphogenetic Protein 4 (BMP4) regulates multiple biological processes, including vascular development and angiogenesis. Here, we investigated the role of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in mediating the angiogenic activity of BMP4. BMP4 induces a rapid relocation and phosphorylation of VEGFR2 on the endothelial cell membrane. These effects occur in the absence of a direct interaction of BMP4 and/or BMP receptors with VEGFR2. At variance, BMP4, by interacting with the BMPRI-II hetero-complex, induces c-Src phosphorylation which, in turn, activates VEGFR2, leading to an angiogenic response. Accordingly, the BMPR inhibitor dorsomorphin prevents c-Src activation and specific inhibition of c-Src significantly reduces downstream VEGFR2 phosphorylation and the angiogenic activity exerted by BMP4 in a chick embryo chorioallantoic membrane assay. Together, our data indicate that the pro-angiogenic activity exerted by BMP4 in endothelial cells is mediated by a BMPR-mediated intracellular transactivation of VEGFR2 via c-Src.
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http://dx.doi.org/10.1007/s10456-019-09676-yDOI Listing
November 2019

Atypical Chemokine Receptor 3 Generates Guidance Cues for CXCL12-Mediated Endothelial Cell Migration.

Front Immunol 2019 15;10:1092. Epub 2019 May 15.

Unit of Experimental Oncology and Immunology, Department of Molecular and Translational Medicine, School of Medicine, University of Brescia, Brescia, Italy.

Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the zebrafish ortholog in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that is a CXCL12-binding ortholog of human . is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of becomes restricted to the basal part of the somites. After knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker . In addition, morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for in endothelial cell guidance, CXCL12 gradient generated by expression in CHO cell transfectants guides human endothelial cell migration in an cell co-culture chemotaxis assay. Our results demonstrate that plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12.
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http://dx.doi.org/10.3389/fimmu.2019.01092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529557PMC
September 2020

Silencing of pantothenate kinase 2 reduces endothelial cell angiogenesis.

Mol Med Rep 2018 Nov 13;18(5):4739-4746. Epub 2018 Sep 13.

Section of Biotechnology, Department of Molecular and Translational Medicine, University of Brescia, I‑25123 Brescia, Italy.

Coenzyme A (CoA) is an essential cofactor of cellular metabolism that is involved in ~4% of cellular reactions. Its de novo production relies on five subsequent enzymatic steps, starting with the phosphorylation of vitamin B5. Pantothenate kinase 2 (PANK2) and coenzyme A synthase (COASY) catalyze the first and last steps of this pathway. Mutations in these genes lead to severe and progressive movement disorders, with neurodegeneration and iron accumulation in the basal ganglia, known as PANK2‑ and COASY protein‑associated neurodegeneration, respectively. Given the ubiquitous role of CoA in cellular metabolism, it is still not clear why patients carrying PANK2 and COASY mutations develop almost exclusively neurological symptoms. Important clues are the energetic profile of neural cells as well as the high levels of PANK2 expression in the brain; however, other features may contribute to this selective tissue vulnerability. Notably, when pank2 or coasy expression was suppressed in zebrafish evident perturbation of neuronal development was observed, as well as severe defects in vasculature formation. Supplementation of CoA to fish water prevented the appearance of the phenotype, thereby confirming the specific connection with the availability of the metabolic cofactor. The present study investigated the associations between PANK2 defects and angiogenesis in a mammalian setting, and revealed that PANK2 expression was required for normal angiogenetic properties of human umbilical vein endothelial cells.
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http://dx.doi.org/10.3892/mmr.2018.9480DOI Listing
November 2018

Claudin3 is localized outside the tight junctions in human carcinomas.

Oncotarget 2018 Apr 6;9(26):18446-18453. Epub 2018 Apr 6.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.

Claudin3 is an integral component of the tight junction proteins in polarized epithelia. The expression of claudin3 was assessed in epithelial-derived tumors using Oncomine database. To determine the gene alteration during carcinogenesis, copy number alterations and mutations of claudin3 were evaluated using cBioPortal database. Claudin3 is overexpressed in several tumors including gynecological, bladder, breast and prostate carcinomas. 38% of the 163 evaluated studies show mutations and/or amplification of claudin3. 3D reconstruction of tissue samples following immunofluorescence analysis clearly demonstrated that, unlike in healthy tissues, claudin3 is mislocalized and unengaged in the formation of tight junction in tumor samples. These data strongly support the evaluation of unengaged claudin3 as a target for the development of novel diagnostic probes, optical approaches for real time detection of tumoral tissues during surgery, and target therapeutic drugs.
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http://dx.doi.org/10.18632/oncotarget.24858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915084PMC
April 2018

Multi-physics interactions drive VEGFR2 relocation on endothelial cells.

Sci Rep 2017 12 1;7(1):16700. Epub 2017 Dec 1.

Università degli Studi di Brescia, DMMT, Department of Molecular and Translational Medicine, Brescia, 25123, Italy.

Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) is a pro-angiogenic receptor, expressed on endothelial cells (ECs). Although biochemical pathways that follow the VEGFR2 activation are well established, knowledge about the dynamics of receptors on the plasma membrane remains limited. Ligand stimulation induces the polarization of ECs and the relocation of VEGFR2, either in cell protrusions or in the basal aspect in cells plated on ligand-enriched extracellular matrix (ECM). We develop a mathematical model in order to simulate the relocation of VEGFR2 on the cell membrane during the mechanical adhesion of cells onto a ligand-enriched substrate. Co-designing the in vitro experiments with the simulations allows identifying three phases of the receptor dynamics, which are controlled respectively by the high chemical reaction rate, by the mechanical deformation rate, and by the diffusion of free receptors on the membrane. The identification of the laws that regulate receptor polarization opens new perspectives toward developing innovative anti-angiogenic strategies through the modulation of EC activation.
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http://dx.doi.org/10.1038/s41598-017-16786-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711959PMC
December 2017

Tumor angiogenesis revisited: Regulators and clinical implications.

Med Res Rev 2017 11 23;37(6):1231-1274. Epub 2017 Jun 23.

Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium.

Since Judah Folkman hypothesized in 1971 that angiogenesis is required for solid tumor growth, numerous studies have been conducted to unravel the angiogenesis process, analyze its role in primary tumor growth, metastasis and angiogenic diseases, and to develop inhibitors of proangiogenic factors. These studies have led in 2004 to the approval of the first antiangiogenic agent (bevacizumab, a humanized antibody targeting vascular endothelial growth factor) for the treatment of patients with metastatic colorectal cancer. This approval launched great expectations for the use of antiangiogenic therapy for malignant diseases. However, these expectations have not been met and, as knowledge of blood vessel formation accumulates, many of the original paradigms no longer hold. Therefore, the regulators and clinical implications of angiogenesis need to be revisited. In this review, we discuss recently identified angiogenesis mediators and pathways, new concepts that have emerged over the past 10 years, tumor resistance and toxicity associated with the use of currently available antiangiogenic treatment and potentially new targets and/or approaches for malignant and nonmalignant neovascular diseases.
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http://dx.doi.org/10.1002/med.21452DOI Listing
November 2017

Role of Autophagy in HIV-1 Matrix Protein p17-Driven Lymphangiogenesis.

J Virol 2017 08 27;91(16). Epub 2017 Jul 27.

Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy

AIDS-related lymphomas (ARLs) are expected to increase in the future since combined antiretroviral therapy (cART) enhances the life expectancy of HIV-1-infected (HIV) patients but does not affect the occurrence of ARLs to the same extent as that of other tumors. Lymphangiogenesis is essential in supporting growth and metastatic spreading of ARLs. HIV-1 does not infect the neoplastic B cells, but HIV-1 proteins have been hypothesized to play a key role in sustaining a prolymphangiogenic microenvironment in lymphoid organs. The HIV-1 matrix protein p17 is detected in blood and accumulates in the germinal centers of lymph nodes of HIV patients under successful cART. The viral protein displays potent lymphangiogenic activity and This is, at least in part, mediated by the secretion of the lymphangiogenic factor endothelin-1, suggesting that activation of a secretory pathway sustains the lymphangiogenic activity of p17. Here, we show that the p17 lymphangiogenic activity occurs on human lymph node-derived lymphatic endothelial cells (LN-LECs) under stress conditions only and relies entirely on activation of an autophagy-based pathway. In fact, induction of autophagy by p17 promotes lymphangiogenesis, whereas pharmacological and genetic inhibition of autophagy inhibits p17-triggered lymphangiogenesis. Similarly, the vasculogenic activity of p17 was totally inhibited in autophagy-incompetent mice. Our findings reveal a previously unrecognized role of autophagy in lymphangiogenesis and open the way to identify novel treatment strategies aimed at inhibiting aberrant tumor-driven lymphangiogenesis in HIV patients. AIDS-related lymphomas (ARLs) are the most common malignancies in HIV-1-infected (HIV) patients after the introduction of combined antiretroviral therapy (cART). Lymphangiogenesis is of critical importance in sustaining growth and metastasis of ARLs. Indeed, enhanced lymphangiogenesis occurs in the lymph nodes of HIV patients under successful cART. The HIV-1 matrix protein p17 is detected in blood and accumulates in the lymph node germinal centers even in the absence of virus replication. Several findings suggest a key role for p17 as a microenvironmental factor capable of promoting lymphangiogenesis. Here, we show that p17 promotes lymphangiogenesis of human lymph node-derived lymphatic endothelial cells (LN-LECs). The lymphangiogenic activity of p17 is sustained by an autophagy-based pathway that enables LN-LECs to release prolymphangiogenic factors into the extracellular microenvironment. Our findings indicate that specific targeting of autophagy may provide an important new tool for treating ARLs.
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http://dx.doi.org/10.1128/JVI.00801-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533914PMC
August 2017

Inflammation and N-formyl peptide receptors mediate the angiogenic activity of human vitreous humour in proliferative diabetic retinopathy.

Diabetologia 2017 04 13;60(4):719-728. Epub 2017 Jan 13.

Department of Molecular and Translational Medicine, University of Brescia, Via Branze 39, Brescia, 25123, Italy.

Aims/hypothesis: Angiogenesis and inflammation characterise proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus. However, the impact of inflammation on the pathogenesis of PDR neovascularisation has not been elucidated. Here, we assessed the capacity of PDR vitreous fluid to induce pro-angiogenic/proinflammatory responses in endothelium and the contribution of the inflammation-related pattern recognition N-formyl peptide receptors (FPRs) in mediating these responses.

Methods: Pooled and individual pars plana vitrectomy-derived PDR vitreous fluid ('PDR vitreous') samples were assessed in endothelial cell proliferation, motility, sprouting and morphogenesis assays, and for the capacity to induce proinflammatory transcription factor activation, reactive oxygen species production, intercellular junction disruption and leucocyte-adhesion molecule upregulation in these cells. In vivo, the pro-angiogenic/proinflammatory activity of PDR vitreous was tested in murine Matrigel plug and chick embryo chorioallantoic membrane (CAM) assays. Finally, the FPR inhibitors Boc-Phe-Leu-Phe-Leu-Phe (Boc-FLFLF) and Ac-L-Arg-Aib-L-Arg-L-Cα(Me)Phe-NH tetrapeptide (UPARANT) were evaluated for their capacity to affect the biological responses elicited by PDR vitreous.

Results: PDR vitreous activates a pro-angiogenic/proinflammatory phenotype in endothelial cells. Accordingly, PDR vitreous triggers a potent angiogenic/inflammatory response in vivo. Notably, the different capacity of individual PDR vitreous samples to induce neovessel formation in the CAM correlates with their ability to recruit infiltrating CD45 cells. Finally, the FPR inhibitor Boc-FLFLF and the novel FPR antagonist UPARANT inhibit neovessel formation and inflammatory responses triggered by PDR vitreous in the CAM assay.

Conclusions/interpretation: This study provides evidence that inflammation mediates the angiogenic activity of PDR vitreous and paves the way for the development of FPR-targeting anti-inflammatory/anti-angiogenic approaches for PDR therapy.
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http://dx.doi.org/10.1007/s00125-016-4204-0DOI Listing
April 2017

Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.

Sci Rep 2016 12 1;6:38027. Epub 2016 Dec 1.

Department of Molecular and Translational Medicine, University of Brescia Medical School, Brescia, Italy.

The human immune deficiency virus type 1 (HIV-1) matrix protein p17 (p17), although devoid of a signal sequence, is released by infected cells and detected in blood and in different organs and tissues even in HIV-1-infected patients undergoing successful combined antiretroviral therapy (cART). Extracellularly, p17 deregulates the function of different cells involved in AIDS pathogenesis. The mechanism of p17 secretion, particularly during HIV-1 latency, still remains to be elucidated. A recent study showed that HIV-1-infected cells can produce Gag without spreading infection in a model of viral latency. Here we show that in Gag-expressing cells, secretion of biologically active p17 takes place at the plasma membrane and occurs following its interaction with phosphatidylinositol-(4,5)-bisphosphate and its subsequent cleavage from the precursor Gag (Pr55) operated by cellular aspartyl proteases. These enzymes operate a more complex Gag polypeptide proteolysis than the HIV-1 protease, thus hypothetically generating slightly truncated or elongated p17s in their C-terminus. A 17 C-terminal residues excised p17 was found to be structurally and functionally identical to the full-length p17 demonstrating that the final C-terminal region of p17 is irrelevant for the protein's biological activity. These findings offer new opportunities to identify treatment strategies for inhibiting p17 release in the extracellular microenvironment.
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http://dx.doi.org/10.1038/srep38027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131311PMC
December 2016

Annexin 2A sustains glioblastoma cell dissemination and proliferation.

Oncotarget 2016 Aug;7(34):54632-54649

Department of Woman and Child Health, University of Padova, Padova, IT.

Glioblastoma (GBM) is the most devastating tumor of the brain, characterized by an almost inevitable tendency to recur after intensive treatments and a fatal prognosis. Indeed, despite recent technical improvements in GBM surgery, the complete eradication of cancer cell disseminated outside the tumor mass still remains a crucial issue for glioma patients management. In this context, Annexin 2A (ANXA2) is a phospholipid-binding protein expressed in a variety of cell types, whose expression has been recently associated with cell dissemination and metastasis in many cancer types, thus making ANXA2 an attractive putative regulator of cell invasion also in GBM.Here we show that ANXA2 is over-expressed in GBM and positively correlates with tumor aggressiveness and patient survival. In particular, we associate the expression of ANXA2 to a mesenchymal and metastatic phenotype of GBM tumors. Moreover, we functionally characterized the effects exerted by ANXA2 inhibition in primary GBM cultures, demonstrating its ability to sustain cell migration, matrix invasion, cytoskeletal remodeling and proliferation. Finally, we were able to generate an ANXA2-dependent gene signature with a significant prognostic potential in different cohorts of solid tumor patients, including GBM.In conclusion, we demonstrate that ANXA2 acts at multiple levels in determining the disseminating and aggressive behaviour of GBM cells, thus proving its potential as a possible target and strong prognostic factor in the future management of GBM patients.
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http://dx.doi.org/10.18632/oncotarget.10565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342369PMC
August 2016

The Novel Antitubulin Agent TR-764 Strongly Reduces Tumor Vasculature and Inhibits HIF-1α Activation.

Sci Rep 2016 06 13;6:27886. Epub 2016 Jun 13.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia pediatrica, Università di Padova, 35128 Padova, Italy.

Tubulin binding agents (TBAs) are commonly used in cancer therapy as antimitotics. It has been described that TBAs, like combretastatin A-4 (CA-4), present also antivascular activity and among its derivatives we identified TR-764 as a new inhibitor of tubulin polymerization, based on the 2-(alkoxycarbonyl)-3-(3',4',5'-trimethoxyanilino)benzo[b]thiophene molecular skeleton. The antiangiogenic activity of TR-764 (1-10 nM) was tested in vitro on human umbilical endothelial cells (HUVECs), and in vivo, on the chick embryo chorioallantoic membrane (CAM) and two murine tumor models. TR-764 binding to tubulin triggers cytoskeleton rearrangement without affecting cell cycle and viability. It leads to capillary tube disruption, increased cell permeability, and cell motility reduction. Moreover it disrupts adherens junctions and focal adhesions, through mechanisms involving VE-cadherin/β-catenin and FAK/Src. Importantly, TR-764 is active in hypoxic conditions significantly reducing HIF-1α. In vivo TR-764 (1-100 pmol/egg) remarkably blocks the bFGF proangiogenic activity on CAM and shows a stronger reduction of tumor mass and microvascular density both in murine syngeneic and xenograft tumor models, compared to the lead compound CA-4P. Altogether, our results indicate that TR-764 is a novel TBA with strong potential as both antivascular and antitumor molecule that could improve the common anticancer therapies, by overcoming hypoxia-induced resistance mechanisms.
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http://dx.doi.org/10.1038/srep27886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904223PMC
June 2016

Vascular disrupting activity of combretastatin analogues.

Vascul Pharmacol 2016 08 25;83:78-89. Epub 2016 May 25.

Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Università di Padova, 35131 Padova, Italy. Electronic address:

Tubulin binding agents (TBAs) are drugs commonly used in cancer therapy as antimitotics. In the last years it has been described that TBAs, like combretastatin A-4 (CA-4), present also vascular disrupting activity and among its derivatives we identified three analogues endowed with potent microtubule depolymerizing activity, higher than that of the lead compound. In this paper we have investigated the anti-vascular activity of these derivatives. We tested the anti-angiogenic effects in human umbilical endothelial cells (HUVEC) and in vivo in chick chorioallantoic membrane assay (CAM), and in a syngeneic tumor mouse model. The three molecules, compound 1: 1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-1,2,4-triazole; compound 2: (1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetrazole, compound-3 (4-amino-2-p-tolylaminothiazol-5-yl)-(3,4,5-trimethoxyphenyl)-methanone) showed a moderate effect on the growth of HUVEC cells at concentrations below 200nM. At lower concentrations (5-20nM), in particular compound 2, they induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the alteration of the microfilaments network. Moreover, they also increased permeability of HUVEC cells in a time dependent manner. In addition, compounds 1 and 3, as well as the reference compound CA-4, inhibited VEGF-induced phosphorylation of VE-cadherin and in addition compound 3 prevented the VEGF-induced phosphorylation of FAK. In CAM assay, both compounds 2 and 3 efficiently counteracted the strong angiogenic response induced by bFGF, even at the lowest concentration used (1pmol/egg). Moreover in a syngenic mouse model, compounds 1-3 after a single i.p. injection (30mg/kg), showed a stronger reduction of microvascular density. Altogether our results identified these derivatives as potential new vascular disrupting agents candidates.
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http://dx.doi.org/10.1016/j.vph.2016.05.006DOI Listing
August 2016

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist.

Oncotarget 2016 Jun;7(23):35353-68

Experimental Oncology and Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, 25123, Italy.

Angiogenesis plays a key role in various physiological and pathological conditions, including inflammation and tumor growth. The bone morphogenetic protein (BMP) antagonist gremlin has been identified as a novel pro-angiogenic factor. Gremlin promotes neovascular responses via a BMP-independent activation of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2). BMP antagonists may act as covalent or non-covalent homodimers or in a monomeric form, while VEGFRs ligands are usually dimeric. However, the oligomeric state of gremlin and its role in modulating the biological activity of the protein remain to be elucidated.Here we show that gremlin is expressed in vitro and in vivo both as a monomer and as a covalently linked homodimer. Mutagenesis of amino acid residue Cys141 prevents gremlin dimerization leading to the formation of gremlinC141A monomers. GremlinC141A monomer retains a BMP antagonist activity similar to the wild-type dimer, but is devoid of a significant angiogenic capacity. Notably, we found that gremlinC141A mutant engages VEGFR2 in a non-productive manner, thus acting as receptor antagonist. Accordingly, both gremlinC141A and wild-type monomers inhibit angiogenesis driven by dimeric gremlin or VEGF-A165. Moreover, by acting as a VEGFR2 antagonist, gremlinC141A inhibits the angiogenic and tumorigenic potential of murine breast and prostate cancer cells in vivo.In conclusion, our data show that gremlin exists in multiple forms endowed with specific bioactivities and provide new insights into the molecular bases of gremlin dimerization. Furthermore, we propose gremlin monomer as a new inhibitor of VEGFR2 signalling during tumor growth.
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http://dx.doi.org/10.18632/oncotarget.9286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085234PMC
June 2016