The FGF/FGFR system in the physiopathology of the prostate gland.

Fibroblast growth factors (FGFs) are a family of proteins possessing paracrine, autocrine, or endocrine functions in a variety of biological processes, including embryonic development, angiogenesis, tissue homeostasis, wound repair, and cancer. Canonical FGFs bind and activate tyrosine kinase FGF receptors (FGFRs), triggering intracellular signaling cascades that mediate their biological activity. Experimental evidence indicates that FGFs play a complex role in the physiopathology of the prostate gland that ranges from essential functions during embryonic development to modulation of neoplastic transformation. The use of ligand- and receptor-deleted mouse models has highlighted the requirement for FGF signaling in the normal development of the prostate gland. In adult prostate, the maintenance of a functional FGF/FGFR signaling axis is critical for organ homeostasis and function, as its disruption leads to prostate hyperplasia and may contribute to cancer progression and metastatic dissemination. Dissection of the molecular landscape modulated by the FGF family will facilitate ongoing translational efforts directed toward prostate cancer therapy.

Proteomic Analysis Highlights the Impact of the Sphingolipid Metabolizing Enzyme ß-Galactosylceramidase on Mitochondrial Plasticity in Human Melanoma.

Mitochondrial plasticity, marked by a dynamism between glycolysis and oxidative phosphorylation due to adaptation to genetic and microenvironmental alterations, represents a characteristic feature of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations have shown that the lysosomal sphingolipid-metabolizing enzyme ß-galactosylceramidase (GALC) exerts pro-oncogenic functions in melanoma. Here, mining the cBioPortal for a Cancer Genomics data base identified the top 200 nuclear-encoded genes whose expression is negatively correlated with GALC expression in human melanoma. Their categorization indicated a significant enrichment in Gene Ontology terms and KEGG pathways related to mitochondrial proteins and function. In parallel, proteomic analysis by LC-MS/MS of two GALC overexpressing human melanoma cell lines identified 98 downregulated proteins when compared to control mock cells. Such downregulation was confirmed at a transcriptional level by a Gene Set Enrichment Analysis of the genome-wide expression profiling data obtained from the same cells. Among the GALC downregulated proteins, we identified a cluster of 42 proteins significantly associated with GO and KEGG categorizations related to mitochondrion and energetic metabolism. Overall, our data indicate that changes in GALC expression may exert a significant impact on mitochondrial plasticity in human melanoma cells.

Halting the FGF/FGFR axis leads to antitumor activity in Waldenström macroglobulinemia by silencing MYD88.

The human fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb because of disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule (NSC12). Wide-transcriptome profiling confirmed inhibition of FGFR signaling in NSC12-treated WM cells; unveiling a significant inhibition of MYD88 was also confirmed at the protein level. Importantly, the NSC12-dependent silencing of MYD88 was functionally active, as it led to inhibition of MYD88-driven pathways, such as BTK and SYK, as well as the MYD88-downstream target HCK. Of note, both canonical and noncanonical NF-κB cascades were downregulated in WM cells upon NSC12 treatment. Functional sequelae exerted by NSC12 in WM cells were studied, demonstrating significant inhibition of WM cell growth, induction of WM cell apoptosis, halting MAPK, JAK/STAT3, and PI3K-Akt pathways. Importantly, NSC12 exerted an anti-WM effect even in the presence of bone marrow microenvironment, both in vitro and in vivo. Our studies provide the evidence for using NSC12 as a specific FGF/FGFR system inhibitor, thus representing a novel therapeutic strategy in WM.

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

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

Zebra-Sphinx: Modeling Sphingolipidoses in Zebrafish.

Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches.

The Pro-Oncogenic Sphingolipid-Metabolizing Enzyme ß-Galactosylceramidase Modulates the Proteomic Landscape in BRAF(V600E)-Mutated Human Melanoma Cells.

ß-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing ß-galactosyl moieties from ß-galactosylceramide and ß-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.

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

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.

Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

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.

ß-Galactosylceramidase Deficiency Causes Upregulation of Long Pentraxin-3 in the Central Nervous System of Krabbe Patients and Twitcher Mice.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a neurodegenerative sphingolipidosis caused by genetic deficiency of lysosomal ß-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 (Galctwi/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 Galctwi/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/Galctwi/twi mice when compared to Galctwi/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.

Long pentraxin 3: A novel multifaceted player in cancer.

Since its discovery in 1992, long pentraxin 3 (PTX3) has been characterized as soluble patter recognition receptor, a key player of the innate immunity arm with non-redundant functions in pathogen recognition and inflammatory responses. As a component of the extra-cellular matrix milieu, PTX3 has been implicated also in wound healing/tissue remodeling, cardiovascular diseases, fertility, and infectious diseases. Consequently, PTX3 levels in biological fluids have been proposed as a fluid-phase biomarker in different pathological conditions. In the last decade, experimental evidences have shown that PTX3 may exert a significant impact also on different aspects of cancer biology, including tumor onset, angiogenesis, metastatic dissemination and immune-modulation. However, it remains unclear whether PTX3 acts as a good cop or bad cop in cancer. In this review, we will summarize and discuss the scientific literature data focusing on the role of PTX3 in experimental and human tumors, including its putative translational implications.

Caffeine Inhibits Direct and Indirect Angiogenesis in Zebrafish Embryos.

In this study, we report the effects of caffeine on angiogenesis in zebrafish embryos both during normal development and after exposure to Fibroblast Growth Factor 2 (FGF2). As markers of angiogenesis, we measured the length and width of intersegmental vessels (ISVs), performed whole-mount in situ hybridization with fli1 and cadh5 vascular markers, and counted the number of interconnecting vessels (ICVs) in sub-intestinal venous plexus (SIVP). In addition, we measured angiogenesis after performing zebrafish yolk membrane (ZFYM) assay with microinjection of fibroblast growth factor 2 (FGF2) and perivitelline tumor xenograft assay with microinjection of tumorigenic FGF2-overexpressing endothelial (FGF2-T-MAE) cells. The results showed that caffeine treatment causes a shortening and thinning of ISVs along with a decreased expression of the vascular marker genes and a decrease in the number of ICVs in the SIVP. Caffeine was also able to block angiogenesis induced by exogenous FGF2 or FGF2-producing cells. Overall, our results are suggestive of the inhibitory effect of caffeine in both direct and indirect angiogenesis.

VEGF-Independent Activation of Müller Cells by the Vitreous from Proliferative Diabetic Retinopathy Patients.

Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.

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

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-A165 (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.

Inhibition of the FGF/FGFR System Induces Apoptosis in Lung Cancer Cells via c-Myc Downregulation and Oxidative Stress.

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.

VEGFR2 activation mediates the pro-angiogenic activity of BMP4.

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.

Quantification of 3D Brain Microangioarchitectures in an Animal Model of Krabbe 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.

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

Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase ß-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.

PTX3 Modulates Neovascularization and Immune Inflammatory Infiltrate in a Murine Model of Fibrosarcoma.

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

N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) inhibits the angiogenic activity of heparin-binding growth factors.

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-A165 with no effect on the activity of the non-heparin-binding VEGF-A121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A165, thus competing for heparin interaction and preventing the binding of VEGF-A165 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.

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

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-NH2 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.