C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138⁺ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14⁺CD16⁺ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.

Topoisomerase I inhibitors down-regulate HIF-1α leading to tumor growth inhibition, but only while maintaining sustained levels of drug exposure. EZN-2208, a multi-arm 40 kDa pegylated, releasable SN38-drug conjugate, provides higher, longer lasting exposure of tumors to SN38 in contrast to SN38 that is released from CPT-11. EZN-2208 also consistently has greater antitumor activity than CPT-11 in a variety of solid and hematological tumor models. In this report, the ability of PEG-SN38 to down-regulate HIF-1α and its downstream targets, in a more potent, sustained manner compared with CPT-11 was examined. To do so, U251 glioma xenografts that stably expressed a hypoxia response element-dependent luciferase reporter gene were implanted in mice. After treatment it was found that EZN-2208 induced potent, sustained HIF-1α down-regulation (37% at 48 h and 83% at 120 h) in the tumors, whereas CPT-11 caused only minor, transient HIF-1α down-regulation. In addition, EZN-2208 down-regulated mRNA levels of HIF-1α targeted genes (MMP2, VEGF1, Glut1, Glut3 and TGFβ1). Further, western blot analyses of xenograft tumors demonstrated that EZN-2208 had significantly more effect than CPT-11 in down-regulating HIF-1α, VEGF, Glut1 and MMP2 protein levels. Significant down-regulation of HIF-1α and VEGF proteins translated to EZN-2208's superior anti-angiogenic activity compared with CPT-11, confirmed by microvessel density reduction in a chorioallantoic membrane assay and in CD-31 immunohistochemistry studies. Additional studies done with matrigel implants devoid of tumor cells show that EZN-2208 significantly inhibits angiogenesis while CPT-11 has little or no effect. It is concluded that the superior antitumor activity of EZN-2208 compared with CPT-11 is attributed, in part, to an anti-angiogenic effect. Ongoing clinical Phase I and Phase II studies will assess safety and efficacy of EZN-2208.

The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1.

Previous data report that fibroblast growth factor-2 (FGF-2)-derived peptide FREG potently inhibits FGF-2-dependent angiogenesis in vitro and in vivo. Here, we show that FREG inhibits up to 70% in vitro growth and invasion/migration of smooth muscle and melanoma cells. Such inhibition is mediated by platelet-derived growth factor-receptor-α (PDGF-Rα); in fact, proliferation and migration were restored upon PDGF-Rα neutralization. Further experiments demonstrated that FREG interacts with PDGF-Rα both in vitro and in vivo and stimulates its phosphorylation. We have previously shown that overexpressing PDGF-Rα strongly inhibits melanoma growth in vivo; we, therefore, hypothesized that PDGF-Rα agonists may represent a novel tool to inhibit melanoma growth in vivo. To support this hypothesis, FREG was inoculated intravenously (i.v.) in a mouse melanoma model and markedly inhibited pulmonary metastases formation. Immunohistochemical analyses showed less proliferation, less angiogenesis, and more apoptosis in metastasized lungs upon FREG treatment, as compared to untreated controls. Finally, in preliminary acute toxicity studies, FREG showed no toxicity signs in healthy animals, and neither microscopic nor macroscopic toxicity at the liver, kidney, and lungs level. Altogether, these data indicate that FREG systemic treatment strongly inhibits melanoma metastases development and indicate for the first time that agonists of PDGF-Rα may control melanoma both in vitro and in vivo.

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.

Adenosquamous carcinoma of the pancreas (ASCP) is an uncommon variant of exocrine pancreatic malignancies, characterized by a histological admixture of adenomatous and squamous cell elements. This cancer is characterized by a poorly differentiated histology and a poorer clinical outcome compared to pancreatic ductal adenocarcinoma (PDAC). Unlike PDAC, that is characterized by a low microvascular density (MVD) and collapsed vasculature, no data are available about angiogenesis in ASPC. Immunohistochemical evaluation of MVD and trypatse-positive mast cells (MCs) were performed on a single case of ASCP compared to PDAC. Moreover, the levels of angiopoietin-1 and -2 (Ang-1, Ang-2), receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie-2), vascular endothelial growth factor A (VEGFA), hypoxia-inducible factor 1 alpha (HIF1A), miR-21-5p, miR-181a-5p, miR-122-5p, and miR-27a-3p were evaluated by real-time PCR. Higher number of tryptase-positive MCs and MVD are observed in the ASCP case compared to PDAC one. Lower levels of miR-122-5p and higher expression of VEGFA, HIF1A and Ang-2 genes were observed in ASCP. Furthermore, lower Ang-1 and Tie-2 transcript levels and higher increases of miR-21-5p, miR27a-3p and miR-181a-5p levels were found in the rarest form of pancreatic carcinoma. Our data demonstrate an important angiogenic activity in ASCP with a putative role of miR-21-5p, miR-181a-5p, miR-122-5p and miR-27a-3p in the regulation of this process.

Epidermal growth factor receptor (EGFR) and its ligand heparin-binding EGF-like growth factor (HB-EGF) sustain endothelial cell proliferation and angiogenesis in solid tumors, but little is known about the role of HB-EGF-EGFR signaling in bone marrow angiogenesis and multiple myeloma (MM) progression. We found that bone marrow endothelial cells from patients with MM express high levels of EGFR and HB-EGF, compared with cells from patients with monoclonal gammopathy of undetermined significance, and that overexpressed HB-EGF stimulates EGFR expression in an autocrine loop. We also found that levels of EGFR and HB-EGF parallel MM plasma cell number, and that HB-EGF is a potent inducer of angiogenesis in vitro and in vivo. Moreover, blockade of HB-EGF-EGFR signaling, by an anti-HB-EGF neutralizing antibody or the EGFR inhibitor erlotinib, limited the angiogenic potential of bone marrow endothelial cells and hampered tumor growth in an MM xenograft mouse model. These results identify HB-EGF-EGFR signaling as a potential target of anti-angiogenic therapy, and encourage the clinical investigation of EGFR inhibitors in combination with conventional cytotoxic drugs as a new therapeutic strategy for MM.

Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS).

Fibrosarcoma is an aggressive subtype of soft tissue sarcoma categorized in infantile/congenital-type and adult-type. Fibrosarcoma cells and its surrounding immune inflammatory infiltrates overexpress or induce the expression of fibroblast growth factor-2 (FGF-2) that have a crucial role in tumor progression and angiogenesis. The inflammation-associated long pentraxin 3 (PTX3) was found to reduce FGF-2-mediated angiogenesis, but its role on fibrosarcoma immune inflammatory infiltrate is still unknown. In this study, we have evaluated the PTX3 activity on immune infiltrating mast cells, macrophages and T-lymphocytes by immunohistochemistry on murine MC-TGS17-51 fibrosarcoma cells and on transgenic TgN(Tie2-hPTX3) mouse. In these fibrosarcoma models we found a reduced neovascularization and a significant decrease of inflammatory infiltrate. Indeed, we show that PTX3 reduces the level of complement 3 (C3) deposition reducing fibrosarcoma progression. In conclusion, we hypothesize that targeting fibrosarcoma microenvironment by FGF/FGFR inhibitors may improve treatment outcome.

Interactions of malignant multiple myeloma (MM) plasma cells (MM-cells) with the microenvironment control MM-cell growth, survival, drug-resistance and dissemination. As in MM microvascular density increases in the bone marrow (BM), we investigated whether BM MM endothelial cells (MMECs) control disease progression via the junctional adhesion molecule A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MMECs in 111 newly diagnosed (NDMM) and 201 relapsed-refractory (RRMM) patients compared to monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Elevated membrane expression of JAM-A on MMECs predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MMECs increased angiogenesis whereas its inhibition impaired angiogenesis and MM growth in 2D and 3D in vitro cell culture and chorioallantoic membrane-assays. To corroborate these findings, we treated MM bearing mice with JAM-A blocking mAb and demonstrated impaired MM progression corresponding to decreased MM-related vascularity. These findings support JAM-A as an important mediator of MM progression through facilitating MM-associated angiogenesis. Collectively, elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for patient survival in both NDMM and RRMM. Blocking JAM-A restricts angiogenesis in vitro, in embrio and in vivo and represents a suitable druggable molecule to halt neoangiogenesis and MM progression.

The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galactosylceramide and from other sphingolipids. GALC deficiency is responsible for globoid cell leukodystrophy (GLD), or Krabbe's disease, an early lethal inherited neurodegenerative disorder characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system (CNS). The poor outcome of current clinical treatments calls for novel model systems to investigate the biological impact of GALC down-regulation and for the search of novel therapeutic strategies in GLD. Zebrafish (Danio rerio) represents an attractive vertebrate model for human diseases. Here, lysosomal GALC activity was demonstrated in the brain of zebrafish adults and embryos. Accordingly, we identified two GALC co-orthologs (named galca and galcb) dynamically co-expressed in CNS during zebrafish development. Both genes encode for lysosomal enzymes endowed with GALC activity. Single down-regulation of galca or galcb by specific antisense morpholino oligonucleotides results in a partial decrease of GALC activity in zebrafish embryos that was abrogated in double galca/galcb morphants. However, no psychosine accumulation was observed in galca/galcb double morphants. Nevertheless, double galca/galcb knockdown caused reduction and partial disorganization of the expression of the early neuronal marker neuroD and an increase of apoptotic events during CNS development. These observations provide new insights into the pathogenesis of GLD, indicating that GALC loss-of-function may have pathological consequences in developing CNS independent of psychosine accumulation. Also, they underscore the potentiality of the zebrafish system in studying the pathogenesis of lysosomal neurodegenerative diseases, including GLD.

Interactions of multiple myeloma (MM) cells with endothelial cells (ECs) enhance angiogenesis and MM progression. Here, we investigated the role of Notch signaling in the cross talk between ECs and MM cells enabling angiogenesis. MMECs showed higher expression of Jagged1/2 ligands, of activated Notch1/2 receptors, and of Hes1/Hey1 Notch target genes than ECs from monoclonal gammopathy of undetermined significance patients, suggesting that homotypic activation of Notch pathway occurs in MM. MM cells co-cultured with MMECs triggered Notch activation in these cells through a cell-to-cell contact-dependent way via Jagged1/2, resulting in Hes1/Hey1 overexpression. The angiogenic effect of Notch pathway was analyzed through Notch1/2·siRNAs and the γ-secretase inhibitor MK-0752 by in vitro (adhesion, migration, chemotaxis, angiogenesis) and in vivo (Vk12598/C57B/6 J mouse model) studies. Activated Notch1/2 pathway was associated with the overangiogenic MMEC phenotype: Notch1/2 knockdown or MK-0752 treatment reduced Hes1/Hey1 expression, impairing in vitro angiogenesis of both MMECs alone and co-cultured with MM cells. MM cells were unable to restore angiogenic abilities of treated MMECs, proving that MMEC angiogenic activities closely rely on Notch pathway. Furthermore, Notch1/2 knockdown affected VEGF/VEGFR2 axis, indicating that the Notch pathway interferes with VEGF-mediated control on angiogenesis. MK-0752 reduced secretion of proangiogenic/proinflammatory cytokines in conditioned media, thus inhibiting blood vessel formation in the CAM assay. In the Vk12598/C57B/6 J mouse, MK-0752 treatment restrained angiogenesis by reducing microvessel density. Overall, homotypic and heterotypic Jagged1/2-mediated Notch activation enhances MMECs angiogenesis. Notch axis inhibition blocked angiogenesis in vitro and in vivo, suggesting that the Notch pathway may represent a novel therapeutic target in MM.

Cognitive deficit has been identified in one third of patients affected by Duchenne Muscular Dystrophy, primarily attributed to loss of the short Dp71 dystrophin, the major brain dystrophin isoform. In this study, we investigated for the first time the Dp71 and Dp71-associated proteins cellular localization and expression in human neurons obtained by differentiation from induced pluripotent stem cell line of a patient affected by cognitive impairment. We found structural and molecular alterations in both pluripotent stem cell and derived neurons, reduced Dp71 expression, and a Ca2+ cytoplasmic overload in neurons coupled with increased expression of the SERCA2 pump in the dystrophic neurons. These results suggest that the reduction of Dp71 protein in the Duchenne muscular dystrophy neurons leads to alterations in SERCA2 and to elevated cytosolic Ca2+ concentration with consequent potential disruption of the dystrophin proteins and Dp71-associated proteins.

Glucose-6-phosphate isomerase (GPI), also known as phosphoglucose isomerase, was initially identified as the second glycolytic enzyme that catalyzes the interconversion of glucose-6-phosphate to fructose-6-phosphate. Later studies demonstrated that GPI was the same as the autocrine motility factor (AMF), and that it mediates its biological effects through the interaction with its surface receptor (AMFR/gp78). In this study, we assessed the role of GPI/AMF as a prognostic factor for clear cell renal cell carcinoma (ccRCC) cancer-specific (CSS) and progression-free survival (PFS). In addition, we evaluated the expression and localization of GPI/AMF and AMFR, using tissue microarray-based immunohistochemistry (TMA-IHC), indirect immunofluorescence (IF), and confocal microscopy analysis.Primary renal tumor and nonneoplastic tissues were collected from 180 patients who underwent nephrectomy for ccRCC. TMA-IHC and IF staining showed an increased signal for both GPI and AMFR in cancer cells, and their colocalization on plasma membrane. Kaplan-Meier curves showed significant differences in CSS and PFS among groups of patients with high versus low GPI expression. In particular, patients with high tissue levels of GPI had a 5-year survival rate of 58.8%, as compared to 92.1% for subjects with low levels (P < 0.0001). Similar findings were observed for PFS (56.8% vs 93.3% at 5 years). At multivariate analysis, GPI was an independent adverse prognostic factor for CSS (HR = 1.26; P = 0.001), and PFS (HR = 1.16; P = 0.01).In conclusion, our data suggest that GPI could serve as a marker of ccRCC aggressiveness and a prognostic factor for CSS and PFS.

We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3''-amino-4''-methoxyphenyl)-1-(5'-methoxy-3',4'-methylendioxyphenyl)-2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-to-straight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.

The analysis of cancer metabolome has shown that proliferating tumor cells require a large quantities of different nutrients in order to support their high rate of proliferation. In this study we analyzed the metabolic profile of glycolysis and the pentose phosphate pathway (PPP) in human clear cell-renal cell carcinoma (ccRCC) and evaluate the role of these pathways in sustaining cell proliferation, maintenance of NADPH levels, and production of reactive oxygen species (ROS). Metabolomic analysis showed a clear signature of increased glucose uptake and utilization in ccRCC tumor samples. Elevated levels of glucose-6-phosphate dehydrogenase (G6PDH) in association with higher levels of PPP-derived metabolites, suggested a prominent role of this pathway in RCC-associated metabolic alterations. G6PDH inhibition, caused a significant decrease in cancer cell survival, a decrease in NADPH levels, and an increased production of ROS, suggesting that the PPP plays an important role in the regulation of ccRCC redox homeostasis. Patients with high levels of glycolytic enzymes had reduced progression-free and cancer-specific survivals as compared to subjects with low levels. Our data suggest that oncogenic signaling pathways may promote ccRCC through rerouting the sugar metabolism. Blocking the flux through this pathway may serve as a novel therapeutic target.

In this study we purposed an alternative method to study the angiogenic and invasive potential of neuroblastoma cell suspensions implanted on the chick embryo chorioallantoic membrane (CAM) surface. Neuroblastoma cells were seeded in Matrigel and thereafter the suspension was pipetted onto the CAM surface at day 8 of incubation inside a silicon ring previously loaded onto the CAM surface. Four days after implantation, the silicon ring was removed and the angiogenic and invasive response were studied morphologically at macroscopic and microscopic levels and by reverse transcriptase-polymerase chain reaction (RT-PCR) by using human and chicken primers for several angiogenic cytokines, namely vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), hypoxia inducible factor-2alpha (HIF-2alpha), and for an endogenous angiostatic molecule, namely endostatin. Results showed that: (1) Neuroblastoma cells induced an angiogenic response in the CAM assay comparable to that induced by FGF-2; (2) neuroblastoma cells are packed inside Matrigel or are recognizable in the CAM mesenchyme; (3) Angiogenic activity of neuroblastoma cells is associated to an high expression of the transcripts of human VEGF-A, FGF-2, ANG-1 and HIF-2alpha and to a low expression in the transcript of a human endostatin while in the control specimens there is no expression of both angiogenic and angiostatic molecules; and (4) the expression of the transcripts of the same chicken angiogenesis stimulators and inhibitor is unmodified in treated and control specimens. Overall, these data indicate that neuroblastoma cells growth on the chick CAM express characteristics of the human disease. This experimental model could be employed for further research on human tumor progression and anti-angiogenic molecules screening.

Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC.

A fraction of patients affected by Duchenne Muscular Dystrophy (DMD) shows mental disability as a consequence of neuronal and metabolic alteration. In this study, we evaluated the effect of α-methyl-prednisolone (PDN) on the expression of the angiogenic marker HIF1α, VEGFA and VEGFR-2 (FLK1) in correlation with PKC expression in the brain of mdx mouse, an experimental model of DMD. We demonstrated that HIF1α, VEGFA and FLK1 are overexpressed in the brain of dystrophic mdx mice in parallel with an increase of PKC expression and reduction of the tight junctions Occludin leading to altered angiogenesis. Moreover, we demonstrated that PDN treatment induces a significant reduction in the HIF1α, VEGF, FLK1, and PKC mRNA and proteins levels and restores Occludin expression reducing its phosphorylation pattern. Our results suggest a new mechanism of action of PDN that through PKC suppression normalizes the angiogenesis in dystrophic mdx brains.

Telocytes (TCs) are peculiar interstitial cells, characterized by their typical elongated and interconnected processes called telopodes. TCs are supposed to contribute to maintain tissue homeostasis but also to be involved in the pathophysiology of many disorders. The aim of the study was to identify TCs in pterygium, a chronic condition of bulbar conjunctiva, and to examine possible differences in TCs in terms of immunophenotype and/or localization between pterygium and normal conjunctiva, to evaluate the possible involvement of TCs in pathogenesis of pterygium.