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Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Background: Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2 R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2 R1051Q and VEGFR2 WT may occur and could explain, at least in part, how VEGFR2 R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2 R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2 WT ., Methods: We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity., Results: Membrane protein biotinylation reveals that VEGFR2 WT is more exposed on the cell membrane with respect to VEGFR2 R1051Q . The imaging analyses show the ability of VEGFR2 WT to form heterodimers with VEGFR2 R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2 WT /VEGFR2 R1051Q occurs, VEGFR2 WT shows reduced lateral motility and a minor pool of mobile fraction., Conclusions: This study demonstrates that active VEGFR2 R1051Q can affect the membrane behavior of the VEGFR2 WT .

Targeting aromatase deprives ER + breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER + breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long-chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor-treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER + breast cancers.

The activation loop (A-loop) of kinases, a key regulatory region, is recurrently mutated in several kinase proteins in cancer resulting in dysregulated kinase activity and response to kinase inhibitors. FGFR1 receptor tyrosine kinase represents an important oncogene and therapeutic target for solid and hematological tumors. Here we investigate the biochemical and molecular effects of D647N mutation lying in the A-loop of FGFR1. When expressed in normal and tumoral in vitro cell models, FGFR1 D647N is phosphorylated also in the absence of ligands, and this is accompanied by the activation of intracellular signaling. The expression of FGFR1 D647N significantly increases single and collective migration of cancer cells in vitro and in vivo, when compared to FGFR1 WT . FGFR1 D647N expression exacerbates the aggressiveness of cancer cells, increasing their invasiveness in vitro and augmenting their pro-angiogenic capacity in vivo. Remarkably, the D647N mutation significantly increases the sensitivity of FGFR1 to the ATP-competitive inhibitor Erdafitinib suggesting the possibility that this mutation could become a specific target for the development of new inhibitors. Although further efforts are warranted for an exhaustive description of the activation mechanisms, for the identification of more specific inhibitors and for confirming the clinical significance of mutated FGFR1 D647N , overall our data demonstrate that the D647N substitution of FGFR1 is a novel pro-oncogenic activating mutation of the receptor that, when found in cancer patients, may anticipate good response to erdafitinib treatment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mitola Stefania and Michela Corsini reports financial support was provided by Italian Association for Cancer Research. Silvia Codenotti and Elisabetta Grillo reports financial support was provided by Umberto Veronesi Foundation., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Adipose tissue (AT) is a highly active and plastic endocrine organ. It secretes numerous soluble molecules known as adipokines, which act locally to AT control the remodel and homeostasis or exert pleiotropic functions in different peripheral organs. Aberrant production or loss of certain adipokines contributes to AT dysfunction associated with metabolic disorders, including obesity. The AT plasticity is strictly related to tissue vascularization. Angiogenesis supports the AT expansion, while regression of blood vessels is associated with AT hypoxia, which in turn mediates tissue inflammation, fibrosis and metabolic dysfunction. Several adipokines can regulate endothelial cell functions and are endowed with either pro- or anti-angiogenic properties. Here we address the role of adipokines in the regulation of angiogenesis. A better understanding of the link between adipokines and angiogenesis will open the way for novel therapeutic approaches to treat obesity and metabolic diseases., Competing Interests: Conflict of interest All authors declare no Conflict of Interest that are directly or indirectly related to the work submitted for publication., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Embryonic stem cells give rise to teratomas when injected in vivo in experimental animal models. The characterization, the manipulation, and the breaking off of this specific characteristic are doubtlessly the last frontier for the applications of stem cells in translational medicine. Moreover, the urgency to adapt to new scientific demands drives the researcher to find alternative and faster models for testing the teratogenic properties of embryonic stem cells. Here, we compare the emerging model of the chick embryo chorioallantoic membrane (CAM) to the murine model, which represents the gold standard procedure for teratogenesis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Humans are transforming natural habitats into managed urban green areas and impervious surfaces at an unprecedented pace. Yet the effects of human presence per se on animal life-history traits are rarely tested. This is particularly true in cities, where human presence is often indissociable from urbanisation itself. The onset of the SARS-CoV-2 outbreak, along with the resulting lockdown restrictions, offered a unique, "natural experiment" to investigate wildlife responses to a sudden reduction in human activity. We analysed four years of avian breeding data collected in a European capital city to test whether lockdown measures altered nestbox occupancy and life-history traits in terms of egg laying date, incubation duration and clutch size in two urban adapters: great tits ( Parus major ) and blue tits ( Cyanistes caeruleus ). Lockdown measures, which modulated human presence, did not influence any of the life-history traits investigated. In contrast, the interaction between year and tree cover, a distinct ecological attribute of the urban space, was positively associated with clutch size, a key avian life-history and reproductive trait. This highlights the importance of inter-year variation and habitat quality over human activity on urban wildlife reproduction. We discuss our results in the light of other urban wildlife studies carried out during the pandemic, inviting the scientific community to carefully interpret all lockdown-associated shifts in biological traits., Supplementary Information: The online version contains supplementary material available at 10.1007/s11252-022-01309-5., Competing Interests: Conflicts of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2022.)

Most research on urban avian ecology has focused on population- and community-level phenomena, whereas fewer studies have examined how urbanization affects individual behavioral responses to a sudden and novel stimulus, and how those translate to fitness. We measured between-individual variation in provisioning latency in two urban adapters - great tits and blue tits - in response to an infrared camera installed in the nestbox, encountered when offspring in the nest were at the peak of food demand (9-10-days old). For each nestbox, we quantified urbanization as intensity in human activity, distance to road and proportion of impervious surface area. In both species, provisioning latency increased closer to roads. Moreover, increased provisioning latency when exposed to a novel object was associated with higher reproductive success in great tits whose nestboxes were surrounded by high amounts of impervious surface. In contrast, increased provisioning latency was consistently associated with lower reproductive success in blue tits. Our results suggest that provisioning latency changes in relation to the environment surrounding the nest, and may be context- and species-specific when exposed to a novel stimulus, such as a novel object in the nest. To better understand the role of initial behavioral responses towards novelty across an individual's lifetime and, ultimately, its impact on fitness in the urban mosaic, further research explicitly testing different behavioral responses across the entire breeding cycle in wild model systems is needed., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Solid waste pollution (garbage discarded by humans, such as plastic, metal, paper) has received increased attention given its importance as a global threat to biodiversity. Recent studies highlight how animals incorporate anthropogenic materials into their life-cycle, for example in avian nest construction. While increasingly monitored in natural areas, the influence of solid waste pollution on wildlife has been seldom explored in the urban habitat. There is limited data on the relationship between anthropogenic solid waste pollution, nest design, and reproductive success in an urban context. We address this knowledge gap (i) by investigating the presence of environmental solid waste pollution in the breeding habitats of great tits Parus major and blue tits Cyanistes caeruleus reproducing in a gradient of urbanisation, and (ii) by quantifying (ii) the contribution of different anthropogenic materials in their nests. We further examine potential drivers of solid waste pollution by inferring three distinct properties of the urban space: environmental solid waste pollution on the ground, human presence, and the intensity of urbanisation (e.g impervious surfaces) in nestbox vicinity. Finally, (iii) we explore the relationship between anthropogenic nest materials and reproductive success. We found that environmental solid waste pollution was positively associated with human presence and urbanisation intensity. There was also a positive relationship between increased human presence and the amount of anthropogenic materials in great tit nests. Interestingly, in both species, anthropogenic nest materials covaried negatively with nest materials of animal origin (fur and feathers). We suggest that fur and feathers - key insulating materials in nest design - may be scarcer in areas with high levels of human presence, and are consequently replaced with anthropogenic nest materials. Finally, we report a negative relationship between anthropogenic nest materials and blue tit reproductive success, suggesting species-specific vulnerability of urban birds to solid waste pollution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Urbanisation is a major anthropogenic perturbation presenting novel ecological and evolutionary challenges to wild populations. Symbiotic microorganisms residing in the gastrointestinal tracts (gut) of vertebrates have mutual connections with host physiology and respond quickly to environmental alterations. However, the impact of anthropogenic changes and urbanisation on the gut microbiota remains poorly understood, especially in early development. To address this knowledge gap, we characterised the gut microbiota of juvenile great tits (Parus major) reared in artificial nestboxes and in natural cavities in an urban mosaic, employing two distinct frameworks characterising the urban space. Microbial diversity was influenced by cavity type. Alpha diversity was affected by the amount of impervious surface surrounding the breeding location, and positively correlated with tree cover density. Community composition differed between urban and rural sites: these alterations covaried with sound pollution and distance to the city centre. Overall, the microbial communities reflect and are possibly influenced by the heterogeneous environmental modifications that are typical of the urban space. Strikingly, the choice of framework and environmental variables characterising the urban space can influence the outcomes of such ecological studies. Our results open new perspectives to investigate the impact of microbial symbionts on the adaptive capacity of their hosts., (© 2022. The Author(s).)

Background: Adipose tissue plays a pivotal role in the development and progression of the metabolic syndrome which along with its complications is an epidemic of the 21st century. Irisin is an adipo-myokine secreted mainly by skeletal muscle and targeting, among others, adipose tissue. In brown adipose tissue it upregulates uncoupling protein-1 (UCP1) which is responsible for mitochondrial non-shivering thermogenesis., Methods: Here we analyzed the effects of irisin on the metabolic activity of 3T3-L1 derived adipocytes through a mitochondrial flux assay. We also assessed the effects of irisin on the intracellular signaling through Western Blot. Finally, the gene expression of ucp1 and lipolytic genes was examined through RT-qPCR., Results: Irisin affects mitochondrial respiration and lipolysis in a time-dependent manner through the regulation of PI3K-AKT pathway. Irisin also induces the expression of UCP1 and the regulation of NF-κB, and CREB and ERK pathways., Conclusion: Our data supports the role of irisin in the induction of non-shivering thermogenesis, the regulation of energy expenditure and lipolysis in adipocytes., General Significance: Irisin may be an attractive therapeutic target in the treatment of obesity and related metabolic disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fibroblast growth factor receptors (FGFRs) are recurrently altered by single nucleotide variants (SNVs) in many human cancers. The prevalence of SNVs in FGFRs depends on the cancer type. In some tumors, such as the urothelial carcinoma, mutations of FGFRs occur at very high frequency (up to 60%). Many characterized mutations occur in the extracellular or transmembrane domains, while fewer known mutations are found in the kinase domain. In this study, we performed a bioinformatics analysis to identify novel putative cancer driver or therapeutically actionable mutations of the kinase domain of FGFRs. To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) of different kinases as a predictor of functional impact. By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations of FGFRs which recur in other protein kinases. By revealing unanticipated correspondence with known variants, we were able to infer their functional effects, as alterations clustering on similar residues in analogous proteins have a high probability to elicit similar effects. As FGFRs represent an important class of oncogenes and drug targets, our study opens the way for further studies to validate their driver and/or actionable nature and, in the long term, for a more efficacious application of precision oncology., (Copyright © 2021. Published by Elsevier B.V.)

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

The tremendous number of cancer variants that can be detected by NGS analyses has required the development of computational approaches to prioritize mutations on the basis of their biological and clinical significance. Standard strategies take a gene-centric approach to the problem, allowing exclusively the identification of highly frequent variants. On the contrary, protein domain (PD)-based approaches allow to identify functionally relevant low frequency variants by searching for mutations that recur on analogous residues across homologous proteins (i.e. containing the same PD). Such approaches enable to transfer information about the effects and druggability from one known mutation to unknown ones. Here we describe how PD-based strategies work, and discuss how they could be exploited for mutation prioritization. The principle that mutations clustered on specific residues of PDs have the same functional consequences and are therapeutically actionable in a similar manner could help the choice of patient-specific targeted drugs, eventually improving the management of cancer patients., (Copyright © 2021 Elsevier B.V. All rights reserved.)

While there are increasing examples of phenotypic and genotypic differences between urban and non-urban populations of plants and animals, few studies identified the mechanisms explaining those dissimilarities. The characterization of the urban landscape, which can only be achieved by measuring variability in relevant environmental factors within and between cities, is a keystone prerequisite to understand the effects of urbanization on wildlife. Here, we measured variation in bird exposure to metal pollution within 8 replicated urbanization gradients and within 2 flagship bird species in urban evolutionary ecology: the blue tit (Cyanistes caeruleus) and the great tit (Parus major). We report on a highly significant, positive linear relationship between the magnitude of urbanization-inferred as either tree cover, impervious surface cover, or an urbanization score computed from several environmental variables, and copper, zinc and lead concentrations in bird feathers. The reverse relationship was measured in the case of mercury, while cadmium and arsenic did not vary in response to the urbanization level. This result, replicated across multiple cities and two passerine species, strongly suggests that copper, zinc, lead and mercury pollution is likely to trigger the emergence of parallel responses at the phenotypic and/or genotypic level between urban environments worldwide., (© 2021. The Author(s).)

Foxp3 + T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients., Competing Interests: Declaration of interests T.R.M. is a founder and shareholder in Monopteros Therapeutics, Inc. This commercial relationship is unrelated to this study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 R1051Q leads to higher energy metabolism and ATP production compared to control cells expressing VEGFR2 WT . Furthermore, activated VEGFR2 R1051Q 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 R1051Q mutation and suggest novel therapeutic approaches for those patients harboring activating mutations of VEGFR2., (Copyright © 2021. Published by Elsevier B.V.)

During multiple myeloma (MM) progression the activation of the angiogenic process represents a key step for the formation of the vascular niche, where different stromal components and neoplastic cells collaborate and foster tumor growth. Among the different pro-angiogenic players, Fibroblast Growth Factor 2 (FGF2) plays a pivotal role in BM vascularization occurring during MM progression. Long Pentraxin 3 (PTX3), a natural FGF antagonist, is able to reduce the activation of stromal components promoted by FGF2 in various in vitro models. An increased FGF/PTX3 ratio has also been found to occur during MM evolution, suggesting that restoring the "physiological" FGF/PTX3 ratio in plasma cells and BM stromal cells (BMSCs) might impact MM. In this work, taking advantage of PTX3-inducible human MM models, we show that PTX3 produced by tumor cells is able to restore a balanced FGF/PTX3 ratio sufficient to prevent the activation of the FGF/FGFR system in endothelial cells and to reduce the angiogenic capacity of MM cells in different in vivo models. As a result of this anti-angiogenic activity, PTX3 overexpression causes a significant reduction of the tumor burden in both subcutaneously grafted and systemic MM models. These data pave the way for the exploitation of PTX3-derived anti-angiogenic approaches in MM.

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., (Copyright © 2021. Published by Elsevier Inc.)

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.

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 R1051Q 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 R1051Q , may help to predict patient prognosis and drug response, with significant clinical implications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. β-Galactosylceramidase (GALC) removes β-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb , a zebrafish ortholog of human GALC , affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc -silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3 , which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy. SIGNIFICANCE: Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that β-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target., (©2020 American Association for Cancer Research.)

Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy (PDR). The pathogenesis of PDR is complex and involves vascular, inflammatory, and neuronal mechanisms. Several structural and molecular alterations associated to PDR are related to the presence of inflammation that appears to play a non-redundant role in the neovascular response that characterizes the retina of PDR patients. Vascular endothelial growth factor (VEGF) blockers have evolved over time for the treatment of retinal neovascularization. However, several limitations to anti-VEGF interventions exist. Indeed, the production of other angiogenic factors and pro-inflammatory mediators may nullify and/or cause resistance to anti-VEGF therapies. Thus, appropriate experimental models are crucial for dissecting the mechanisms leading to retinal neovascularization and for the discovery of more efficacious anti-angiogenic/anti-inflammatory therapies for PDR patients. This review focuses on the tight cross talk between angiogenesis and inflammation during PDR and describe how the chick embryo chorioallantoic membrane (CAM) assay may represent a cost-effective and rapid in vivo tool for the study of the relationship between neovascular and inflammatory responses elicited by the vitreous humor of PDR patients and for the screening of novel therapeutic agents., (Copyright © 2020 Rezzola, Loda, Corsini, Semeraro, Annese, Presta and Ribatti.)

Introduction: Rapid environmental change driven by urbanization offers a unique insight into the adaptive potential of urban-dwelling organisms. Urban-driven phenotypic differentiation is increasingly often demonstrated, but the impact of urbanization (here modelled as the percentage of impervious surface (ISA) around each nestbox) on offspring developmental rates and subsequent survival remains poorly understood. Furthermore, the role of selection on urban-driven phenotypic divergence was rarely investigated to date., Methods and Results: Data on nestling development and body mass were analysed in a gradient of urbanization set in Warsaw, Poland, in two passerine species: great tits ( Parus major ) and blue tits ( Cyanistes caeruleus ). Increasing levels of impervious surface area (ISA) delayed the age of fastest growth in blue tits. Nestling body mass was also negatively affected by increasing ISA 5 and 10 days after hatching in great tits, and 10 and 15 days in blue tits, respectively. High levels of ISA also increased nestling mortality 5 and 10 days after hatching in both species. An analysis of selection differentials performed for two levels of urbanization (low and high ISA) revealed a positive association between mass at day 2 and survival at fledging., Discussion: This study confirms the considerable negative impact of imperviousness-a proxy for urbanization level-on offspring development, body mass and survival, and highlights increased selection on avian mass at hatching in a high ISA environment., (© 2020 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

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 ( p = 0.016), mainly by hematogenous route ( p = 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 ( p = 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., (Copyright © 2020 Romani, Zizioli, Silvestri, Ardighieri, Bugatti, Corsini, Todeschini, Marchini, D'Incalci, Zanotti, Ravaggi, Facchetti, Gambino, Odicino, Sartori, Santin, Mitola, Bignotti and Calza.)

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 165 (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.

Glioblastoma (GBM) is the most malignant brain tumor of adults and is characterized by extensive cell dissemination within the brain parenchyma and enhanced angiogenesis. Effective preclinical modeling of these key features suffers from several shortcomings. Aim of this study was to determine whether modulating the expression of extracellular matrix (ECM) modifiers in proneural (PN) and mesenchymal (MES) cancer stem cells (CSCs) and in conventional glioma cell lines (GCLs) might improve tumor invasion and vascularization. To this end, we selected secreted, acidic and rich in cysteine-like 1 (SPARCL1) as a potential mediator of ECM remodeling in GBM. SPARCL1 transcript and protein expression was assessed in PN and MES CSCs as well as GCLs, in their xenografts and in patient-derived specimens by qPCR, WB and IHC. SPARCL1 expression was then enforced in both CSCs and GCLs by lentiviral-based transduction. The effect of SPARCL1 gain-of-function on microvascular proliferation, microglia activation and advanced imaging features was tested in intracranial xenografts by IHC and MRI and validated by chorioallantoic membrane (CAM) assays. SPARCL1 expression significantly enhanced the infiltrative and neoangiogenic features of PN and MES CSC/GCL-induced tumors, with the concomitant activation of inflammatory responses associated with the tumor microenvironment, thus resulting in experimental GBMs that reproduced both the parenchymal infiltration and the increased microvascular density, typical of GBM. Overall, these results indicate that SPARCL1 overexpression might be instrumental for the generation of CSC-derived preclinical models of GBM in which the main pathognomonic hallmarks of GBMs are retrievable, making them suitable for effective preclinical testing of therapeutics., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A 165 with no effect on the activity of the non-heparin-binding VEGF-A 121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A 165 , thus competing for heparin interaction and preventing the binding of VEGF-A 165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors.

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 2 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.

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., Competing Interests: The authors declare there are no relevant conflicts of interest to disclose.

Objective: During neovessel formation, angiogenic growth factors associate with the extracellular matrix. These immobilized factors represent a persistent stimulus for the otherwise quiescent endothelial cells (ECs), driving directional EC migration and proliferation and leading to new blood vessel growth. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main mediator of angiogenesis. Although VEGFR2 signaling has been deeply characterized, little is known about its subcellular localization during neovessel formation. Aim of this study was the characterization and molecular determinants of activated VEGFR2 localization in ECs during neovessel formation in response to matrix-immobilized ligand., Approach and Results: Here we demonstrate that ECs stimulated by extracellular matrix-associated gremlin, a noncanonical VEGFR2 ligand, are polarized and relocate the receptor in close contact with the angiogenic factor-enriched matrix both in vitro and in vivo. GM1 (monosialotetrahexosylganglioside)-positive planar lipid rafts, β3 integrin receptors, and the intracellular signaling transducers focal adhesion kinase and RhoA (Ras homolog gene family, member A) cooperate to promote VEGFR2 long-term polarization and activation., Conclusions: A ligand anchored to the extracellular matrix induces VEGFR2 polarization in ECs. Long-lasting VEGFR2 relocation is closely dependent on lipid raft integrity and activation of β3 integrin pathway. The study of the endothelial responses to immobilized growth factors may offer insights into the angiogenic process in physiological and pathological conditions, including cancer, and for a better engineering of synthetic tissue scaffolds to blend with the host vasculature., (© 2015 The Authors.)

Angiogenesis, the process of new blood vessels formation, plays a key role in different physiological and pathological conditions and it is considered a promising target for the development of new anti-inflammatory and anti-tumor therapies. Several assays have been developed to mimic the angiogenic process in vitro and in vivo. Here we propose a technique for the quantification of the pro-angiogenic or anti-angiogenic responses induced by different molecules when implanted in vivo on the chick embryo chorioallantoic membrane (CAM). At day 11 of development CAM is completely vascularized and neo-vessels induced by exogenous molecules converge radially to the implant. Our algorithm is an effective and rapid tool to characterize molecules endowed with proor anti-angiogenic effects by means of the quantification of the vessels present in the CAM macroscopic images. Based on conventional and dedicated image morphology tools, the proposed technique is able to discriminate radial from non-radial vessels, excluding the last ones from the count.

Vascular endothelial growth factor (VEGF) blockers have been developed for the treatment of proliferative diabetic retinopathy (PDR), the leading cause of visual impairments in the working-age population in the Western world. However, limitations to anti-VEGF therapies may exist because of the local production of other proangiogenic factors that may cause resistance to anti-VEGF interventions. Thus, novel therapeutic approaches targeting additional pathways are required. Here, we identified a sulfated derivative of the Escherichia coli polysaccharide K5 [K5-N,OS(H)] as a multitarget molecule highly effective in inhibiting VEGF-driven angiogenic responses in different in vitro, ex vivo, and in vivo assays, including a murine model of oxygen-induced retinopathy. Furthermore, K5-N,OS(H) binds a variety of heparin-binding angiogenic factors upregulated in PDR vitreous humor besides VEGF, thus inhibiting their biological activity. Finally, K5-N,OS(H) hampers the angiogenic activity exerted in vitro and in vivo by human vitreous fluid samples collected from patients with PDR. Together, the data provide compelling experimental evidence that K5-N,OS(H) represents an antiangiogenic multitarget molecule with potential implications for the therapy of pathologic neovessel formation in the retina of patients with PDR., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Fibroblast growth factor-8b (FGF8b) affects the epithelial/stromal compartments of steroid hormone-regulated tumors by exerting an autocrine activity on cancer cells and a paracrine pro-angiogenic function, thus contributing to tumor progression. The FGF8b/FGF receptor (FGFR) system may therefore represent a target for the treatment of steroid hormone-regulated tumors. The soluble pattern recognition receptor long pentraxin-3 (PTX3) binds various FGFs, including FGF2 and FGF8b, thus inhibiting the angiogenic and tumorigenic activity of androgen-regulated tumor cells. Nevertheless, the complex/proteinaceous structure of PTX3 hampers its pharmacological exploitation. In this context, the acetylated pentapeptide Ac-ARPCA-NH2 (ARPCA), corresponding to the N-terminal amino acid sequence PTX3(100-104), was identified as a minimal FGF2-binding peptide able to antagonize the biological activity of FGF2. Here, we demonstrate that ARPCA binds FGF8b and inhibits its capacity to form FGFR1-mediated ternary complexes with heparan sulphate proteoglycans. As a FGF8b antagonist, ARPCA inhibits FGFR1 activation and signalling in endothelial cells, hampering the angiogenic activity exerted in vitro and in vivo by FGF8b. Also, ARPCA suppresses the angiogenic and tumorigenic potential of prototypic androgen/FGF8b-dependent Shionogi 115 mammary carcinoma cells and of androgen/FGF8b/FGF2-dependent TRAMP-C2 prostate cancer cells. In conclusion, ARPCA represents a novel FGF8b antagonist with translational implications for the therapy of steroid hormone-regulated tumors.

Purpose: Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina., Methods: Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used., Results: UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays., Conclusions: The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Objective: Angiogenesis and inflammation are closely related processes. Gremlin is a novel noncanonical vascular endothelial growth factor receptor-2 (VEGFR2) ligand that induces a proangiogenic response in endothelial cells (ECs). Here, we investigated the role of the cyclic adenosine monophosphate-response element (CRE)-binding protein (CREB) in mediating the proinflammatory and proangiogenic responses of ECs to gremlin., Approach and Results: Gremlin induces a proinflammatory response in ECs, leading to reactive oxygen species and cyclic adenosine monophosphate production and the upregulation of proinflammatory molecules involved in leukocyte extravasation, including chemokine (C-C motif) ligand-2 (Ccl2) and Ccl7, chemokine (C-X-C motif) ligand-1 (Cxcl1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Accordingly, gremlin induces the VEGFR2-dependent phosphorylation, nuclear translocation, and transactivating activity of CREB in ECs. CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to monocyte/macrophage adhesion to ECs and their extravasation. All these effects are inhibited by EC transfection with a dominant-negative CREB mutant or with a CREB-binding protein-CREB interaction inhibitor that competes for CREB/CRE binding. Also, both recombinant gremlin and gremlin-expressing tumor cells induce proinflammatory/proangiogenic responses in vivo that are suppressed by the anti-inflammatory drug hydrocortisone. Similar effects were induced by the canonical VEGFR2 ligand VEGF-A165., Conclusions: Together, the results underline the tight cross-talk between angiogenesis and inflammation and demonstrate a crucial role of CREB activation in the modulation of the VEGFR2-mediated proinflammatory/proangiogenic response of ECs to gremlin.

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

α(v)β(3) integrin modulates pro-angiogenic endothelial cell (EC) responses following vascular endothelial growth factor receptor-2 (VEGFR2) engagement. The bone morphogenic protein antagonist gremlin is a novel non-canonical VEGFR2 ligand that promotes the acquisition of a pro-angiogenic phenotype in ECs. Here we investigated the role of α(v)β(3) and extracellular matrix components on EC activation induced by gremlin. Gremlin triggers VEGFR2 phosphorylation and cell motility in ECs adherent to the α(v)β(3) ligand fibrinogen but not in ECs adherent to type-I collagen or fibronectin. Also, gremlin and VEGF-A stimulate the formation of VEGFR2/α(v)β(3) integrin complexes as shown by co-immunoprecipitation experiments and fluorescence resonance energy transfer analysis of β(3)-ECFP/VEGFR2-EYFP co-transfected ECs. Accordingly, anti-β(3) antibodies block the angiogenic activity exerted by gremlin or VEGF-A in vitro, ex vivo and in vivo. The results demonstrate a non-redundant role for α(v)β(3) in gremlin-induced angiogenesis and emphasize its contribution to the formation of functional multi-molecular VEGFR2 complexes responsible for the neovascularization events triggered by canonical and non-canonical pro-angiogenic VEGFR2 ligands.

Fibroblast growth factor-8b (FGF8b) exerts nonredundant autocrine/paracrine functions in steroid hormone-regulated tumors. Previous observations had shown that the soluble pattern recognition receptor long pentraxin-3 (PTX3) is a natural selective antagonist for a restricted number of FGF family members, inhibiting FGF2 but not FGF1 and FGF4 activity. Here, we assessed the capacity of PTX3 to antagonize FGF8b and to inhibit the vascularization and growth of steroid hormone-regulated tumors. Surface plasmon resonance analysis shows that PTX3 binds FGF8b with high affinity (K(d) = 30-90 nmol/L). As a consequence, PTX3 prevents the binding of FGF8b to its receptors, inhibits FGF8b-driven ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells, and suppresses FGF8b-induced neovascularization in vivo. Also, PTX3 inhibits dihydrotestosterone (DHT)- and FGF8b-driven proliferation of androgen-regulated Shionogi 115 (S115) mouse breast tumor cells. Furthermore, DHT-treated, PTX3 overexpressing hPTX3_S115 cell transfectants show a reduced proliferation rate in vitro and a limited angiogenic activity in the chick embryo chorioallantoic membrane and murine s.c. Matrigel plug assays. Accordingly, hPTX3_S115 cells show a dramatic decrease of their tumorigenic activity when grafted in immunodeficient male mice. These results identify PTX3 as a novel FGF8b antagonist endowed with antiangiogenic and antineoplastic activity with possible implications for the therapy of hormonal tumors.

Fibroblast growth factor receptor-1 (FGFR-1) transduces proangiogenic and proliferative signals in human cancers. Thus, FGFR-1 may represent a target for the development of antiangiogenic/antineoplastic therapies. We screened a human single-chain fragment variable (scFv) antibody phage display library against the extracellular domain of the FGFR-1-IIIc isoform that harbors the FGF binding site. Several phages were isolated and tested for specificity and sensitivity, and the most promising antibody fragment RR-C2 was characterized for its biochemical and biological properties. ScFv RR-C2 specifically recognizes FGFR-1α and FGFR-1β isoforms in ELISA, Western blotting, and surface plasmon resonance analysis with a K(d) value of 300 and 144 nmol/L for the 2 receptor isoforms, respectively. The antibody fragment also recognizes FGFR-1 when the receptor is exposed on the cell surface, thus preventing the formation of the ternary complex among FGFR-1, its ligand FGF2, and cell surface heparan sulfate proteoglycans. Accordingly, scFv RR-C2 specifically inhibits FGF2-mediated mitogenic activity in endothelial cells of human, bovine, and murine origin in a nanomolar range of concentrations. Also, the antibody fragment prevents FGF2-triggered sprouting of both human umbilical vein endothelial cell spheroids and of murine endothelium from aortic rings. Finally, the antibody fragment hampers the angiogenic activity exerted both by FGF2 in the chick embryo chorioallantoic membrane assay and by S115 mouse mammary tumor cells in the Matrigel plug assay. Taken together, the data show that scFv RR-C2 recognizes and neutralizes FGFR-1 activity in different animal species, including humans, thus representing a novel tool for the development of antiangiogenic/antineoplastic therapies., (©2010 AACR.)

Fibroblast growth factor-2 (FGF2) is a potent angiogenic growth factor. Here, gene expression profiling of FGF2-stimulated microvascular endothelial cells revealed, together with a prominent pro-angiogenic profile, a pro-inflammatory signature characterized by the upregulation of pro-inflammatory cytokine/chemokines and their receptors, endothelial cell adhesion molecules and members of the eicosanoid pathway. Real-time quantitative PCR demonstrated early induction of most of the FGF2-induced, inflammation-related genes. Accordingly, chick embryo chorioallantoic membrane (CAM) and murine Matrigel plug angiogenesis assays demonstrated a significant monocyte/macrophage infiltrate in the areas of FGF2-driven neovascularization. Similar results were obtained when the conditioned medium (CM) of FGF2-stimulated endothelial cells was delivered onto the CAM, suggesting that FGF2-upregulated chemoattractants mediate the inflammatory response. Importantly, FGF2-triggered new blood vessel formation was significantly reduced in phosphatidylinositol 3-kinase-gamma null mice exhibiting defective leucocyte migration or in clodronate liposome-treated, macrophage-depleted mice. Furthermore, the viral pan-chemokine antagonist M3 inhibited the angiogenic and inflammatory responses induced by the CM of FGF2-stimulated endothelial cells and impaired FGF2-driven neovascularization in the CAM assay. These findings point to inflammatory chemokines as early mediators of FGF2-driven angiogenesis and indicate a non-redundant role for inflammatory cells in the neovascularization process elicited by the growth factor.