Cosetta Ravelli

Gremlin is a novel agonist of the major proangiogenic receptor VEGFR2.

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-β proteins and the angiogenic vascular endothelial growth factors (VEGFs). Here, we demonstrate that gremlin binds VEGF receptor-2 (VEGFR2), the main transducer of VEGF-mediated angiogenic signals, in a bone morphogenic protein-independent manner. Similar to VEGF-A, gremlin activates VEGFR2 in endothelial cells, leading to VEGFR2-dependent angiogenic responses in vitro and in vivo. Gremlin thus represents a novel proangiogenic VEGFR2 agonist distinct from the VEGF family ligands with implications in vascular development, angiogenesis-dependent diseases, and tumor neovascularization.

Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of human immunodeficiency virus type 1 tat and gp120 proteins

ABSTRACT Human immunodeficiency virus (HIV) Tat and gp120 intriguingly share the feature of being basic peptides that, once released by HIV+ cells, bind to polyanionic heparan sulfate proteoglycans (HSPGs) on target uninfected cells, contributing to the onset of AIDS-associated pathologies. To identify multitarget anti-HIV prodrugs, we investigated the gp120 and Tat antagonist potentials of a series of polyanionic synthetic sulfonic acid polymers (SSAPs). Surface plasmon resonance revealed that SSAPs inhibit with a competitive mechanism of action the binding of Tat and gp120 to surface-immobilized heparin, an experimental condition that resembles binding to cellular HSPGs. Accordingly, SSAPs inhibited HSPG-dependent cell internalization and the transactivating activity of Tat. Little is known about the binding of free gp120 to target cells. Here, we identified two classes of gp120 receptors expressed on endothelial cells, one of which was consistent with an HSPG-binding, low-affinity/high-capacity receptor that is inhibited by free heparin. SSAPs inhibited the binding of free gp120 to endothelial cells, as well as its capacity to induce apoptosis in the same cells. In all the assays, poly(4-styrenesulfonic acid) (PSS) proved to be the most potent antagonist of Tat and gp120. Accordingly, PSS bound both proteins with high affinity. In conclusion, SSAPs represent an interesting class of compounds that bind both gp120 and Tat and inhibit their HSPG-dependent cell surface binding and pathological effects. As these activities contribute to both AIDS progression and associated pathologies, SSAPs can be considered prototypic molecules for the development of multitarget drugs for the treatment of HIV infection and AIDS-associated pathologies.

Heparan Sulfate Proteoglycans Mediate the Angiogenic Activity of the Vascular Endothelial Growth Factor Receptor-2 Agonist Gremlin

Objective—Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. Methods and Results—Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase1/2 and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. Conclusion—HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist.

Involvement of αvβ3 integrin in gremlin-induced angiogenesis

Abstractαvβ3 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 αvβ3 and extracellular matrix components on EC activation induced by gremlin. Gremlin triggers VEGFR2 phosphorylation and cell motility in ECs adherent to the αvβ3 ligand fibrinogen but not in ECs adherent to type-I collagen or fibronectin. Also, gremlin and VEGF-A stimulate the formation of VEGFR2/αvβ3 integrin complexes as shown by co-immunoprecipitation experiments and fluorescence resonance energy transfer analysis of β3-ECFP/VEGFR2-EYFP co-transfected ECs. Accordingly, anti-β3 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 αvβ3 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.

Angiopoietin-1 mediates the proangiogenic activity of the bone morphogenic protein antagonist Drm

Recent observations have shown that Drm, a member the Dan family of bone morphogenic protein (BMP) antagonists, induces endothelial cell (EC) sprouting in vitro and angiogenesis in vivo by interacting with signaling EC receptors in a BMP-independent manner. Here, recombinant Drm (rDrm) up-regulates angiopoientin-1 (Ang-1) expression in EC without affecting Ang-2 and Tie-2 receptor expression. Ang-1 up-regulation is mediated by the activation of the transcription factor NF-kappaB. Specific inhibition of Ang-1 activity by anti-Ang-1 antibodies, soluble Tie-2 receptor, or Ang-1 siRNA transfection significantly reduced the rDrm-mediated sprouting of EC in three-dimensional fibrin and type I collagen gels. In addition, Ang-1 antagonists inhibited the angiogenic activity exerted by rDrm in the chick embryo chorioallantoic membrane. Taken together, the data indicate that the proangiogenic activity of Drm is mediated by the activation of an Ang-1-dependent autocrine loop of stimulation in EC.

Long Pentraxin-3 Inhibits Epithelial–Mesenchymal Transition in Melanoma Cells

During melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an epithelial–mesenchymal transition (EMT) process associated with the acquisition of an invasive, prometastatic phenotype. The fibroblast growth factor-2 (FGF2)/FGF receptor (FGFR) system plays a pivotal role in melanoma, leading to autocrine/paracrine induction of tumor cell proliferation and angiogenesis. Long pentraxin-3 (PTX3) interacts with FGF2, and other FGF family members, inhibiting FGF-dependent neovascularization and tumor growth. Here, PTX3 protein and the PTX3-derived acetylated pentapeptide Ac-ARPCA-NH2 inhibit FGF2-driven proliferation and downstream FGFR signaling in murine melanoma B16-F10 cells. Moreover, human PTX3-overexpressing hPTX_B16-F10 cells are characterized by the reversed transition from a mesenchymal to an epithelial-like appearance, inhibition of cell proliferation, loss of clonogenic potential, reduced motility and invasive capacity, downregulation of various mesenchymal markers, and upregulation of the epithelial marker E-cadherin. Accordingly, PTX3 affects cell proliferation and EMT transition in human A375 and A2058 melanoma cells. Also, hPTX_B16-F10 cells showed a reduced tumorigenic and metastatic activity in syngeneic C57BL/6 mice. In conclusion, PTX3 inhibits FGF/FGFR-driven EMT in melanoma cells, hampering their tumorigenic and metastatic potential. These data represent the first experimental evidence about a nonredundant role of the FGF/FGFR system in the modulation of the EMT process in melanoma and indicate that PTX3 or its derivatives may represent the basis for the design of novel therapeutic approaches in FGF/FGFR-dependent tumors, including melanoma. Mol Cancer Ther; 12(12); 2760–71. ©2013 AACR.

Cyclic Adenosine Monophosphate-Response Element–Binding Protein Mediates the Proangiogenic or Proinflammatory Activity of Gremlin

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.

Substrate-Immobilized HIV-1 Tat Drives VEGFR2/α v β 3 –Integrin Complex Formation and Polarization in Endothelial Cells

Objective—The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation. Methods and Results—Immobilized Tat induces actin cytoskeleton organization, formation of &agr;v&bgr;3 integrin+focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/&agr;v&bgr;3 coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of &agr;v&bgr;3 integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK1/2 activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK1/2 pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs. Conclusion—Substrate-immobilized Tat causes VEGFR2/&agr;v&bgr;3 complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK1/2 phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk.

Vascular disrupting activity of combretastatin analogues

Tubulin binding agents (TBAs) are drugs commonly used in cancer therapy as antimitotics. In the last years it has been described that TBAs, like combretastatin A-4 (CA-4), present also vascular disrupting activity and among its derivatives we identified three analogues endowed with potent microtubule depolymerizing activity, higher than that of the lead compound. In this paper we have investigated the anti-vascular activity of these derivatives. We tested the anti-angiogenic effects in human umbilical endothelial cells (HUVEC) and in vivo in chick chorioallantoic membrane assay (CAM), and in a syngeneic tumor mouse model. The three molecules, compound 1: 1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-1,2,4-triazole; compound 2: (1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetrazole, compound-3 (4-amino-2-p-tolylaminothiazol-5-yl)-(3,4,5-trimethoxyphenyl)-methanone) showed a moderate effect on the growth of HUVEC cells at concentrations below 200nM. At lower concentrations (5-20nM), in particular compound 2, they induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the alteration of the microfilaments network. Moreover, they also increased permeability of HUVEC cells in a time dependent manner. In addition, compounds 1 and 3, as well as the reference compound CA-4, inhibited VEGF-induced phosphorylation of VE-cadherin and in addition compound 3 prevented the VEGF-induced phosphorylation of FAK. In CAM assay, both compounds 2 and 3 efficiently counteracted the strong angiogenic response induced by bFGF, even at the lowest concentration used (1pmol/egg). Moreover in a syngenic mouse model, compounds 1-3 after a single i.p. injection (30mg/kg), showed a stronger reduction of microvascular density. Altogether our results identified these derivatives as potential new vascular disrupting agents candidates.

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist

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.