Fibroblast growth factors (FGFs) are a family of heparin-binding growth factors. FGFs exert their pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. Their activity is modulated by a variety of free and extracellular matrix-associated molecules. Also, the cross-talk among FGFs, vascular endothelial growth factors (VEGFs), and inflammatory cytokines/chemokines may play a role in the modulation of blood vessel growth in different pathological conditions, including cancer. Indeed, several experimental evidences point to a role for FGFs in tumor growth and angiogenesis. This review will focus on the relevance of the FGF/FGF receptor system in adult angiogenesis and its contribution to tumor vascularization. #

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Outer membrane protein A (OmpA) is a conserved major component of the outer membrane of Enterobacteriaceae. Here, we report that OmpA from Klebsiella pneumoniae (KpOmpA) activates macrophages and dendritic cells (DCs) in a TLR2-dependent way. However, TLR2 does not account for binding of KpOmpA to innate immune cells. KpOmpA binds the scavenger receptors (SRs) LOX-1 and SREC-I, but not other members of the same family. LOX-1 colocalizes and cooperates with TLR2 in triggering cellular responses. The TLR2-activated functional program includes production of the long pentraxin PTX3, a soluble pattern recognition receptor involved in resistance against diverse pathogens. PTX3, in turn, binds KpOmpA but does not affect recognition of this microbial moiety by cellular receptors. KpOmpA-elicited in vivo inflammation is abrogated in TLR2(-/-) mice and significantly reduced in PTX3(-/-) mice. Thus, SR-mediated KpOmpA recognition and TLR2-dependent cellular activation set in motion a nonredundant PTX3-mediated humoral amplification loop of innate immunity.

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein-positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/ tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis. [Cancer Res 2007;67(7):2927-31]

Nerve growth factor (NGF) has important functions during embryonic development and on various tissues and organs under normal and pathological conditions during the extrauterine life. RT-PCR analysis and immunological methods demonstrate that human umbilical vein endothelial cells (HUVECs) express the NGF receptors trkA(NGFR) and p75NTR. NGF treatment caused a rapid phosphorylation of trkA(NGFR) in HUVECs, determining a parallel increase of phosphorylated ERK1/2. Accordingly, NGF induced a significant increase in HUVEC proliferation that was abolished by the trkA(NGFR) inhibitor K252a. Also, HUVECs express significant levels of NGF under standard culture conditions that were up-regulated during serum starvation. Endogenous NGF was responsible for the basal levels of trkA(NGFR) and ERK1/2 phosphorylation observed in untreated HUVEC cultures. Finally, NGF exerted a potent, direct, angiogenic activity in vivo when delivered onto the chorioallantoic membrane of the chicken embryo. The data indicate that NGF may play an important role in blood vessel formation in the nervous system and in several pathological processes, including tumors and inflammatory diseases. Unraveling mechanisms of NGF-dependent angiogenesis could provide valuable tools for novel therapeutic approaches in antiangiogenic therapy.

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

The bone morphogenic protein antagonist gremlin is expressed during embryonic development and under different pathologic conditions, including cancer. Gremlin is a proangiogenic protein belonging to the cystine-knot superfamily that includes transforming growth factor-␤ proteins and the angiogenic vascu- IntroductionThe bone morphogenic protein (BMP) antagonist gremlin 1 induces angiogenesis in a BMP-independent manner by binding to as-yetunidentified endothelial cell (EC) membrane receptors and activating multiple tyrosine kinase-dependent intracellular signaling pathways in ECs. 2,3 Gremlin is produced by human tumors 4,5 and is expressed by fibroblast growth factor-2 (FGF2)-activated ECs and tumor endothelium. 2 Thus, gremlin may play paracrine/autocrine roles in tumor neovascularization. The identification of the EC receptors activated by gremlin has so far been unsuccessful.Vascular endothelial growth factor receptor-2 (VEGFR2) is the major proangiogenic tyrosine kinase receptor expressed by ECs and is activated by different members of the vascular endothelial growth factor (VEGF) family. 6 Both gremlin and VEGFs belong to the cystine-knot protein superfamily, 7 suggesting possible structural and/or functional similarities among these proangiogenic factors. On this basis, we investigated the capacity of gremlin to interact with and activate VEGFR2. The results demonstrate that gremlin binds and activates VEGFR2, leading to VEGFR2-dependent angiogenic responses in vitro and in vivo. Methods Ligand-receptor interaction assaysInteraction of VEGF-A and gremlin (R&D Systems) with the immobilized extracellular domain of VEGFR2 (sVEGFR2; Calbiochem) was analyzed by surface plasmon resonance (BIAcore Inc) and by competitive enzymelinked immunosorbent assay (ELISA). VEGFR2 interaction on the EC surface was characterized by cross-linking experiments, whereas VEGFR2 dimerization was assessed by fluorescence resonance energy transfer analysis. In vitro angiogenic assaysMotility and 3-dimensional gel invasion assays were performed on human, murine, and bovine ECs. 3 When indicated, ECs were stably transfected with a pcDNA3.1 expression vector harboring the mouse VEGFR2 complementary DNA.

Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) based on the implantation of a gelatin sponge on the top of the growing CAM on day 8 of development. After implantation, the sponge is treated with a stimulator of blood vessel formation in the absence or presence of an angiogenesis inhibitor. On day 12, blood vessels that are growing into the sponge are counted at macroscopic and microscopic levels. The estimated timeline for carrying out this protocol is 10 d. The presence of a vascular network in the CAM requires a careful analysis to distinguish new capillaries from pre-existing ones. This limitation does not occur in the avascular cornea assay, which may also take advantage of different genetic backgrounds when carried out in transgenic or knockout mice. Nevertheless, the gelatin sponge-CAM assay is simple, inexpensive and suitable for large-scale screening.