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.

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 J. Cell. Mol. Med. Vol 13, No 8B, 2009 pp. 2083-2108 Materials and methods Reagents and cellsRecombinant human FGF2 was purified as previously described [18]. Ketoprofen and hydrocortisone were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Recombinant M3 protein was expressed in the baculovirus system and purified by affinity chromatography [19]. Murine lung microvascular endothelial cells (1G11 cells) [20] were obtained from A. Vecchi (Istituto Scientifico Humanitas, Rezzato, Milan, Italy) and cultured in Dulbecco modified Eagle medium (DMEM) containing 20% inactivated foetal bovine serum (FBS Real-time PCR analysisTwo-step quantitative RT-PCR (qRT-PCR)

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.

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 ¼ 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. Mol Cancer Ther; 10(9); 1600-10. Ó2011 AACR.

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 gremlin C141A monomers. Gremlin C141A monomer retains a BMP antagonist activity similar to the wild-type dimer, but is devoid of a significant angiogenic capacity. Notably, we found that gremlin C141A mutant engages VEGFR2 in a non-productive manner, thus acting as receptor antagonist. Accordingly, both gremlin C141A and wild-type monomers inhibit angiogenesis driven by dimeric gremlin or VEGF-A 165 . Moreover, by acting as a VEGFR2 antagonist, gremlin C141A 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.