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Background: The COVID-19 pandemic and control measures may have had an impact on unpleasant emotions experimented during the lockdown (LD). This may have increased the number of hours spent online and could have impacted the quality of the enacted behavior, in terms of loss of control of Internet use. In this online survey, we were interested in measure how much loss of control was perceived regarding online gambling, online shopping, the fruition of online pornographic content and web navigation. Design and methods: The online survey was carried out during the COVID-19 pandemic in the post-lockdown and 1232 subjects participated in the survey. In the participating sample, healthcare workers (HW) were 43.1% of the sample, of which 18.7% were directly involved in the Coronavirus emergency, and 52.3% of the sample is not a HW. Only 0.6% of the sample gambled online and 37.5% of those reported losing control of their gambling mode. Most of the sample shopped online during the LD (70.1%), but only 7.2% of those lost control by buying and/or spending more than what they had set themselves. Results: Significant data emerged showing that those who lost control while online shopping also lost control regarding the amount of time spent online (p

Krabbe disease is a sphingolipidosis characterized by the genetic deficiency of the acid hydrolase β-galactosylceramidase (GALC). Most of the studies concerning the biological role of GALC performed on Krabbe patients and Galc -deficient twitcher mice (an authentic animal model of the disease) indicate that the pathogenesis of this disorder is the consequence of the accumulation of the neurotoxic GALC substrate β-galactosylsphingosine (psychosine), ignoring the possibility that this enzyme may exert a wider biological impact. Indeed, limited information is available about the effect of GALC downregulation on the cell lipidome in adult and developing organisms. The teleost zebrafish ( Danio rerio ) has emerged as a useful platform to model human genetic diseases, including sphingolipidoses, and two GALC co-orthologs have been identified in zebrafish ( galca and galcb ). Here, we investigated the effect of the competitive and irreversible GALC inhibitor β-galactose-cyclophellitol (GCP) on the lipid profile of zebrafish embryos. Molecular modelling indicates that GCP can be sequestered in the catalytic site of the enzyme and covalently binds human GALC, and the zebrafish Galca and Galcb proteins in a similar manner. Accordingly, GCP inhibits the β-galactosylceramide hydrolase activity of zebrafish in vitro and in vivo , leading to significant alterations of the lipidome of zebrafish embryos. These results indicate that the lack of GALC activity deeply affects the lipidome during the early stages of embryonic development, and thereby provide insights into the pathogenesis of Krabbe disease., Competing Interests: 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., (© 2024 The Authors.)

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosylceramide and β-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF -mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF -mutated human melanoma.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal β-galactosylceramidase ( GALC ), characterized by neuroinflammation and demyelination of the central (CNS) and peripheral nervous system. The acute phase protein long pentraxin-3 (PTX3) is a soluble pattern recognition receptor and a regulator of innate immunity. Growing evidence points to the involvement of PTX3 in neurodegeneration. However, the expression and role of PTX3 in the neurodegenerative/neuroinflammatory processes that characterize GLD remain unexplored. Here, immunohistochemical analysis of brain samples from Krabbe patients showed that macrophages and globoid cells are intensely immunoreactive for PTX3. Accordingly, Ptx3 expression increases throughout the course of the disease in the cerebrum, cerebellum, and spinal cord of GALC-deficient twitcher ( Galc twi/twi ) mice, an authentic animal model of GLD. This was paralleled by the upregulation of proinflammatory genes and M1-polarized macrophage/microglia markers and of the levels of PTX3 protein in CNS and plasma of twitcher animals. Crossing of Galc twi/twi mice with transgenic PTX3 overexpressing animals ( hPTX3 mice) demonstrated that constitutive PTX3 overexpression reduced the severity of clinical signs and the upregulation of proinflammatory genes in the spinal cord of P35 hPTX3 / Galc twi/twi mice when compared to Galc twi/twi littermates, leading to a limited increase of their life span. However, this occurred in the absence of a significant impact on the histopathological findings and on the accumulation of the neurotoxic metabolite psychosine when evaluated at this late time point of the disease. In conclusion, our results provide the first evidence that PTX3 is produced in the CNS of GALC-deficient Krabbe patients and twitcher mice. PTX3 may exert a protective role by reducing the neuroinflammatory response that occurs in the spinal cord of GALC-deficient animals.

Purpose: Limb intensive care unit (ICU)-acquired weakness (ICUAW) and ICU acquired diaphragm weakness (DW) occur frequently in mechanically ventilated (MV) patients; their coexistence in cooperative and uncooperative patients is unknown. This study was designed to (1) describe the co-occurrence of the two conditions (2) evaluate the impact of ICUAW and DW on the ventilator-free days (VFDs) at 28 days and weaning success, and (3) assess the correlation between maximal inspiratory pressure (MIP) and thickening fraction (TFdi) in patients with DW., Methods: This prospective pilot study was conducted in a single-center on 73 critically ill MV patients. Muscle weakness was defined as a Medical Research Council score < 48 in cooperative patients or a bilateral mean simplified peroneal nerve test < 5.26 mV in uncooperative patients. Diaphragm dysfunction was defined as MIP < 30 cm H 2 O or as a TFdi < 29%. Weaning success was defined according to weaning according to a new definition (WIND)., Results: Fifty-seven patients (78%) had ICUAW and 59 (81%) had DW. The coexistence of the two conditions occurred in 48 patients (65%), without association (χ 2 = 1.06, p = 0.304). In the adjusted analysis, ICUAW was independently related to VFDs at 28-days (estimate difference 6 days, p = 0.016), and WIND (OR of 3.62 for having WIND different than short weaning), whereas DW was not. The linear mixed model showed a significant but weak correlation between MIP and TFdi ( p < 0.001)., Conclusion: This pilot study is the first to explore the coexistence of ICUAW and DW in both cooperative and uncooperative patients; a lack of association was found between DW and ICUAW when considering both cooperative and uncooperative patients. We found a strong correlation between ICUAW but not DW with the VFDs at 28 days and weaning success. A future larger study is warranted in order to confirm our results, and should also investigate the use of transdiaphragmatic twitch pressure measurement during bilateral anterior magnetic phrenic nerve stimulation for the diagnosis of DW., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bertoni, Piva, Beretta, Bongiovanni, Contarino, Artigas, Ceresoli, Marchesi, Falappi, Belleri, Goffi, Pozzi, Rasulo and Latronico.)

β-galactosylceramidase (GALC) is a lysosomal enzyme that removes β-galactose from β-galactosylceramide, leading to the formation of the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth by acting as an oncosuppressive or oncogenic enzyme depending on the different experimental approaches, in vitro versus in vivo observations, preclinical versus clinical findings, and tumor type investigated. This review will recapitulate and discuss the contrasting experimental evidence related to the impact of GALC on the biological behavior of cancer and stromal cells and its contribution to tumor progression., (Copyright © 2021. Published by Elsevier B.V.)

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Macroglial Müller cells appear to play a pivotal role in the pathogenesis of iERM where they may undergo glial-to-mesenchymal transition (GMT), a transdifferentiation process characterized by the downregulation of Müller cell markers, paralleled by the upregulation of pro-fibrotic myofibroblast markers. Previous observations from our laboratory allowed the molecular identification of two major clusters of iERM patients (named iERM-A and iERM-B), iERM-A patients being characterized by less severe clinical features and a more "quiescent" iERM gene expression profile when compared to iERM-B patients. In the present work, Müller MIO-M1 cells were exposed to vitreous samples obtained before membrane peeling from the same cohort of iERM-A and iERM-B patients. The results demonstrate that iERM vitreous induces proliferation, migration, and GMT in MIO-M1 cells, a phenotype consistent with Müller cell behavior during iERM progression. However, even though the vitreous samples obtained from iERM-A patients were able to induce a complete GMT in MIO-M1 cells, iERM-B samples caused only a partial GMT, characterized by the downregulation of Müller cell markers in the absence of upregulation of pro-fibrotic myofibroblast markers. Together, the results indicate that a relationship may exist among the ability of iERM vitreous to modulate GMT in Müller cells, the molecular profile of the corresponding iERMs, and the clinical features of iERM patients., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Background: The COVID-19 pandemic and control measures may have had an impact on unpleasant emotions experimented during the lockdown (LD). This may have increased the number of hours spent online and could have impacted the quality of the enacted behavior, in terms of loss of control of Internet use. In this online survey, we were interested in measure how much loss of control was perceived regarding online gambling, online shopping, the fruition of online pornographic content and web navigation. Design and methods: The online survey was carried out during the COVID-19 pandemic in the post-lockdown and 1232 subjects participated in the survey. In the participating sample, healthcare workers (HW) were 43.1% of the sample, of which 18.7% were directly involved in the Coronavirus emergency, and 52.3% of the sample is not a HW. Only 0.6% of the sample gambled online and 37.5% of those reported losing control of their gambling mode. Most of the sample shopped online during the LD (70.1%), but only 7.2% of those lost control by buying and/or spending more than what they had set themselves. Results: Significant data emerged showing that those who lost control while online shopping also lost control regarding the amount of time spent online (p<0.001); 21.6% of the sample, reported making use of online pornographic material during LD, 4.7% of them stated that the frequency increased and 5.1% reported losing control by having spent more money or more time than what was intended. Finally, 44.7% of the sample have experienced loss of control during the web navigation. Furthermore, during the LD 67.8% of the sample reports having experienced unpleasant emotions. Of these, 8.4% state that they enacted behaviors such as online gambling, online shopping, online pornographic material viewing and web navigation to counter their negative emotions. Interestingly, we found a correlation between loss of control during web navigation and online shopping and the emotional states "upset", "scared" and "restless" (p<0.05). Conclusion: To conclude, there was no significant increase in potentially addictive behaviors, nor an increase in loss of control of these behaviors when enacted online. However, the loss of control in online shopping and web navigation was significantly correlated to the unpleasant emotional states of nervousness, fear and restlessness, whereas those who reported feeling strong and able to handle the situation experienced a lower loss of control in their web navigation. These correlations may suggest that these online behaviors may act as modulators of unpleasant emotional states., (©Copyright: the Author(s).)

Avian reovirus p17 (ARV p17) is a non-structural protein known to activate autophagy, interfere with gene transcription and induce a significant tumor cell growth inhibition in vitro and in vivo. In this study, we show that ARV p17 is capable of exerting potent antiangiogenic properties. The viral protein significantly inhibited the physiological angiogenesis of human endothelial cells (ECs) by affecting migration, capillary-like structure and new vessel formation. ARV p17 was not only able to suppress the EC physiological angiogenesis but also rendered ECs insensitive to two different potent proangiogenic inducers, such as VEGF-A and FGF-2 in the three-dimensional (3D) Matrigel and spheroid assay. ARV p17 was found to exert its antiangiogenic activity by upregulating transcription and release of the well-known tumor suppressor molecule dipeptidyl peptidase 4 (DPP4). The ability of ARV p17 to impact on angiogenesis is completely new and highlights the "two compartments" activity of the viral protein that is expected to hamper the tumor parenchymal/stromal crosstalk. The complex antitumor activities of ARV p17 open the way to a new promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1α pathways, respectively. HIF-1α silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl 2 , exogenous ET-1, or CoCl 2 plus ET-1, down-regulated HIF-1α and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Russignan, Dal Collo, Bagnato, Tamassia, Bugatti, Belleri, Lorenzi, Borsi, Bazzoni, Gottardi, Terragna, Vermi, Giacomini, Presta, Cassatella, Krampera and Tecchio.)

Idiopathic epiretinal membranes (ERMs) are fibrocellular membranes containing extracellular matrix proteins and epiretinal cells of retinal and extraretinal origin. iERMs lead to decreased visual acuity and their pathogenesis has not been completely defined. Aim of this study was to provide a molecular characterization of iERMs by gene expression analysis. To this purpose, 56 iERMs obtained by pars plana vitrectomy were analyzed for the expression levels of genes encoding biomarkers of the cellular and molecular events occurring in iERMs. RT-qPCR analysis showed significant differences in the levels of cell population, extracellular matrix and cytokine/growth factor biomarkers among the iERMs investigated. Hierarchical clustering of RT-qPCR data identified two distinct iERM clusters, Cluster B samples representing transcriptionally "activated" iERMs when compared to transcriptionally "quiescent" Cluster A specimens. Further, Cluster B could be subdivided in two subgroups, Cluster B1 iERMs, characterized by a marked glial cell activation, and Cluster B2 samples characterized by a more pro-fibrotic phenotype. Preoperative decimal best-corrected visual acuity and post-surgery inner segment/outer grading values were higher in Cluster A patients, that showed a prevalence of fovea-attached type iERMs with near-normal inner retina, than in Cluster B patients, that presented more severe clinical and spectral domain optical coherence tomography (SD-OCT) features. In conclusion, this molecular characterization has identified two major clusters of iERM specimens with distinct transcriptional activities that reflect different clinical and SD-OCT features of iERM patients. This retrospective work paves the way to prospective whole-genome transcriptomic studies to allow a molecular classification of iERMs and for the identification of molecular signature(s) of prognostic and therapeutic significance., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.)

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.

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

We performed a three-dimensional (3D) analysis of the microvascular network of the cerebral cortex of twitcher mice (an authentic model of Krabbe disease) using a restricted set of indexes that are able to describe the arrangement of the microvascular tree in CD31-stained sections. We obtained a near-linear graphical "fingerprint" of the microangioarchitecture of wild-type and twitcher animals that describes the amounts, spatial dispersion, and spatial relationships of adjacent classes of caliber-filtered microvessels. We observed significant alterations of the microangioarchitecture of the cerebral cortex of twitcher mice, whereas no alterations occur in renal microvessels, which is keeping with the observation that kidney is an organ that is not affected by the disease. This approach may represent an important starting point for the study of the microvascular changes that occur in the central nervous system (CNS) under different physiopathological conditions.

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 pivotal role in the physiology and pathology of the brain. Microvascular alterations have been observed in various neurodegenerative disorders, including genetic leukodystrophies. Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by β-galactosylceramidase (GALC) deficiency and characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system and peripheral tissues. Structural and functional alterations occur in the microvascular endothelium of the brain of GLD patients and twitcher mice, a murine model of the disease. In addition, increased vessel permeability and a reduced capacity to respond to proangiogenic stimuli characterize the endothelium of twitcher animals. On the one hand, these alterations may depend, at least in part, on the local and systemic angiostatic activity exerted by psychosine on endothelial cells. On the other hand, studies performed in vivo on zebrafish embryos and in vitro on human endothelial cells suggest that GALC downregulation may also lead to psychosine-independent neuronal and vascular defects. Together, experimental observations indicate that endothelial cell dysfunctions may represent a novel pathogenic mechanism in human leukodystrophies, including GLD. A better understanding of the molecular mechanisms responsible for these microvascular alterations may provide new insights for the therapy of GLD. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal β-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31(+) vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31(+) vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, confirmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis.

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.)

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.

Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Seminal studies in the early 1980 s about the angiogenic activity exerted by mammalian retinal tissue extracts on the chick embryo chorioallantoic membrane and the later discovery of vascular endothelial growth factor (VEGF) accumulation in eyes of patients with diabetic retinopathy paved the way for the development of anti-angiogenic VEGF blockers for the treatment of retinal neovascularization. Since then, numerous preclinical and clinical studies about diabetic retinopathy and other retinal disorders have opened new lines of angiogenesis inquiry, indicating that limitations to anti-VEGF therapies may exist. Moreover, the production of growth factors other than VEGF may affect the response to anti-VEGF approaches. Thus, experimental models of retinal angiogenesis remain crucial for investigating novel anti-angiogenic therapies and bringing them to patients. To this aim, in vitro and ex vivo angiogenesis assays may be suitable for a rapid screening of potential anti-angiogenic molecules before in vivo validation of the putative lead compounds. This review focuses on the different in vitro and ex vivo angiogenesis assays that have been developed over the years based on the isolation of endothelial cells from the retina of various animal species and ex vivo cultures of neonatal and adult retina explants. Also, recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryos, an in vivo animal platform experimentally analogous to in vitro/ex vivo models, may represent a novel target for the identification of angiogenesis inhibitors. When compared to in vivo assays, in vitro and ex vivo models of retina neovascularization, including zebrafish embryo, may represent cost-effective and rapid tools for the screening of novel anti-angiogenic therapeutics.