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The Biflavonoid Amentoflavone Inhibits Neovascularization Preventing the Activity of Proangiogenic Vascular Endothelial Growth Factors*

Open AccessPublished:April 06, 2011DOI:https://doi.org/10.1074/jbc.M110.186239
      The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low μm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.

      Introduction

      In the past few years, it has been demonstrated that the proangiogenic members of VEGF family, VEGF-A, VEGF-B, and placental growth factor (PlGF),
      The abbreviations used are: PlGF-1, placental growth factor 1; VEGFR-1, VEGF receptor 1; CTF, capillary-like tube formation; HUVEC, human umbilical vein endothelial cell; AF, amentoflavone; RP, reverse phase.
      play a central role in the modulation of pathological angiogenesis (
      • Ferrara N.
      • Gerber H.P.
      • LeCouter J.
      ,
      • Carmeliet P.
      ). VEGFs accomplish their action interacting with two tyrosine kinase receptors, VEGFR-1 and VEGFR-2, also known as Flt-1 and KDR. Pathological neovascularization is a condition associated to many complex diseases such as cancer, atherosclerosis, arthritis, diabetic retinopathy, and age-related macular degeneration (
      • Carmeliet P.
      ,
      • De Falco S.
      • Gigante B.
      • Persico M.G.
      ).
      VEGF-A is the most potent proangiogenic factor known showing a crucial stimulating activity on endothelial cells. Indeed, it is the target of the first U. S. Food and Drug Administration-approved antiangiogenic drugs for the therapy of cancer and age-related macular degeneration. Bevacizumab (Avastin), a monoclonal antibody anti-VEGF-A, has been approved for the therapy of metastatic colorectal cancer in combination with chemotherapy (
      • Ferrara N.
      • Hillan K.J.
      • Novotny W.
      ), whereas pegaptanib (Macugen), an anti-VEGF-A PEGylated aptamer (
      • Gragoudas E.S.
      • Adamis A.P.
      • Cunningham Jr., E.T.
      • Feinsod M.
      • Guyer D.R.
      ), and ranibizumab (Lucentis), a Fab fragment of bevacizumab (
      • Rosenfeld P.J.
      • Brown D.M.
      • Heier J.S.
      • Boyer D.S.
      • Kaiser P.K.
      • Chung C.Y.
      • Kim R.Y.
      ), have been approved for treatment of age-related macular degeneration. Although VEGF-A-targeted therapy has improved cancer survival, many patients are refractory to VEGF-A-targeted therapy, and most patients who initially respond develop resistance (
      • Ellis L.M.
      • Hicklin D.J.
      ).
      More recently, increasing attention has been addressed to other members of VEGF network involved in angiogenesis modulation. Indeed, the block of VEGFR-1 (
      • Wu Y.
      • Zhong Z.
      • Huber J.
      • Bassi R.
      • Finnerty B.
      • Corcoran E.
      • Li H.
      • Navarro E.
      • Balderes P.
      • Jimenez X.
      • Koo H.
      • Mangalampalli V.R.
      • Ludwig D.L.
      • Tonra J.R.
      • Hicklin D.J.
      ), or of its specific ligand PlGF (
      • Fischer C.
      • Jonckx B.
      • Mazzone M.
      • Zacchigna S.
      • Loges S.
      • Pattarini L.
      • Chorianopoulos E.
      • Liesenborghs L.
      • Koch M.
      • De Mol M.
      • Autiero M.
      • Wyns S.
      • Plaisance S.
      • Moons L.
      • van Rooijen N.
      • Giacca M.
      • Stassen J.M.
      • Dewerchin M.
      • Collen D.
      • Carmeliet P.
      ,
      • Van de Veire S.
      • Stalmans I.
      • Heindryckx F.
      • Oura H.
      • Tijeras-Raballand A.
      • Schmidt T.
      • Loges S.
      • Albrecht I.
      • Jonckx B.
      • Vinckier S.
      • Van Steenkiste C.
      • Tugues S.
      • Rolny C.
      • De Mol M.
      • Dettori D.
      • Hainaud P.
      • Coenegrachts L.
      • Contreres J.O.
      • Van Bergen T.
      • Cuervo H.
      • Xiao W.H.
      • Le Henaff C.
      • Buysschaert I.
      • Kharabi Masouleh B.
      • Geerts A.
      • Schomber T.
      • Bonnin P.
      • Lambert V.
      • Haustraete J.
      • Zacchigna S.
      • Rakic J.M.
      • Jiménez W.
      • Noël A.
      • Giacca M.
      • Colle I.
      • Foidart J.M.
      • Tobelem G.
      • Morales-Ruiz M.
      • Vilar J.
      • Maxwell P.
      • Vinores S.A.
      • Carmeliet G.
      • Dewerchin M.
      • Claesson-Welsh L.
      • Dupuy E.
      • Van Vlierberghe H.
      • Christofori G.
      • Mazzone M.
      • Detmar M.
      • Collen D.
      • Carmeliet P.
      ), is sufficient to strongly inhibit pathological angiogenesis in experimental models of pathologies such as cancer, atherosclerosis, arthritis, ocular neovascular diseases, and metastasis formation (
      • Carmeliet P.
      • Moons L.
      • Luttun A.
      • Vincenti V.
      • Compernolle V.
      • De Mol M.
      • Wu Y.
      • Bono F.
      • Devy L.
      • Beck H.
      • Scholz D.
      • Acker T.
      • DiPalma T.
      • Dewerchin M.
      • Noel A.
      • Stalmans I.
      • Barra A.
      • Blacher S.
      • Vandendriessche T.
      • Ponten A.
      • Eriksson U.
      • Plate K.H.
      • Foidart J.M.
      • Schaper W.
      • Charnock-Jones D.S.
      • Hicklin D.J.
      • Herbert J.M.
      • Collen D.
      • Persico M.G.
      ,
      • Luttun A.
      • Tjwa M.
      • Moons L.
      • Wu Y.
      • Angelillo-Scherrer A.
      • Liao F.
      • Nagy J.A.
      • Hooper A.
      • Priller J.
      • De Klerck B.
      • Compernolle V.
      • Daci E.
      • Bohlen P.
      • Dewerchin M.
      • Herbert J.M.
      • Fava R.
      • Matthys P.
      • Carmeliet G.
      • Collen D.
      • Dvorak H.F.
      • Hicklin D.J.
      • Carmeliet P.
      ,
      • Rakic J.M.
      • Lambert V.
      • Devy L.
      • Luttun A.
      • Carmeliet P.
      • Claes C.
      • Nguyen L.
      • Foidart J.M.
      • Noël A.
      • Munaut C.
      ,
      • Kaplan R.N.
      • Riba R.D.
      • Zacharoulis S.
      • Bramley A.H.
      • Vincent L.
      • Costa C.
      • MacDonald D.D.
      • Jin D.K.
      • Shido K.
      • Kerns S.A.
      • Zhu Z.
      • Hicklin D.
      • Wu Y.
      • Port J.L.
      • Altorki N.
      • Port E.R.
      • Ruggero D.
      • Shmelkov S.V.
      • Jensen K.K.
      • Rafii S.
      • Lyden D.
      ,
      • Gigante B.
      • Morlino G.
      • Gentile M.T.
      • Persico M.G.
      • De Falco S.
      ), indicating how fine-tuning of the availability of VEGFs and related receptors is required for a correct angiogenesis during pathological conditions. This is also confirmed by data indicating that the up-regulation of PlGF expression provides a tumor escape strategy to anti-VEGF-A therapy (
      • Van de Veire S.
      • Stalmans I.
      • Heindryckx F.
      • Oura H.
      • Tijeras-Raballand A.
      • Schmidt T.
      • Loges S.
      • Albrecht I.
      • Jonckx B.
      • Vinckier S.
      • Van Steenkiste C.
      • Tugues S.
      • Rolny C.
      • De Mol M.
      • Dettori D.
      • Hainaud P.
      • Coenegrachts L.
      • Contreres J.O.
      • Van Bergen T.
      • Cuervo H.
      • Xiao W.H.
      • Le Henaff C.
      • Buysschaert I.
      • Kharabi Masouleh B.
      • Geerts A.
      • Schomber T.
      • Bonnin P.
      • Lambert V.
      • Haustraete J.
      • Zacchigna S.
      • Rakic J.M.
      • Jiménez W.
      • Noël A.
      • Giacca M.
      • Colle I.
      • Foidart J.M.
      • Tobelem G.
      • Morales-Ruiz M.
      • Vilar J.
      • Maxwell P.
      • Vinores S.A.
      • Carmeliet G.
      • Dewerchin M.
      • Claesson-Welsh L.
      • Dupuy E.
      • Van Vlierberghe H.
      • Christofori G.
      • Mazzone M.
      • Detmar M.
      • Collen D.
      • Carmeliet P.
      ,
      • Cao Y.
      ).
      It appears evident that due to the strict biochemical and functional relationship between VEGFs and related receptors (
      • Tarallo V.
      • Vesci L.
      • Capasso O.
      • Esposito M.T.
      • Riccioni T.
      • Pastore L.
      • Orlandi A.
      • Pisano C.
      • De Falco S.
      ), the development of a new generation of agents able to target contemporarily more than one member of the VEGFs might amplify the antiangiogenic response. This would overcome some of the difficulties associated with current angiogenesis inhibitors, representing an advantage in terms of therapeutic outcome (
      • Tarallo V.
      • Vesci L.
      • Capasso O.
      • Esposito M.T.
      • Riccioni T.
      • Pastore L.
      • Orlandi A.
      • Pisano C.
      • De Falco S.
      ,
      • Fischer C.
      • Mazzone M.
      • Jonckx B.
      • Carmeliet P.
      ).
      From this perspective, we have performed a screening plan of small molecule collections consisting of >100 extracts from plants used in the traditional medicine collected in various areas of the world. Medicinal plants have become extremely important in drug discovery for the treatment of human diseases, and their secondary metabolites have proven to be the most reliable source of new and effective anticancer agents (
      • Newman D.J.
      • Cragg G.M.
      • Snader K.M.
      ). Moreover, the management of small molecules still offers numerous advantages over biotherapeutics because they can be more easily and cheaply produced. In addition small molecules are more stable and generally free of contaminants of biological origin and may offer more opportunity for delivery.
      The screenings have been performed using competitive dose-dependent ELISA assays for PlGF-1 and VEGF-A interaction with high affinity receptor VEGFR-1 (
      • Ponticelli S.
      • Braca A.
      • De Tommasi N.
      • De Falco S.
      ,
      • Ponticelli S.
      • Marasco D.
      • Tarallo V.
      • Albuquerque R.J.
      • Mitola S.
      • Takeda A.
      • Stassen J.M.
      • Presta M.
      • Ambati J.
      • Ruvo M.
      • De Falco S.
      ). The chloroform/methanol extract from leaves of the Malian plant C. senegalensis showed the highest inhibiting activity and its subsequent bioassay-guided fractionation led to the isolation and characterization of amentoflavone (AF) as bioactive constituent able to inhibit the interaction of either PlGF-1 or VEGF-A with VEGFR-1. AF belongs to a unique class of naturally occurring biflavonoids (
      • Kim H.P.
      • Park H.
      • Son K.H.
      • Chang H.W.
      • Kang S.S.
      ).
      The binding properties and the antiangiogenic activity of the selected compound have been assessed in in vitro and cell-based assays. The data presented here demonstrate that AF specifically binds to VEGFs, preventing the activation of both VEGFR-1 and VEGFR-2. It is able to significantly inhibit growth and neoangiogenesis in two different tumor models, an orthotropic model of melanoma and a xenograft model of colon carcinoma.

      DISCUSSION

      Flavonoids are a class of natural compounds belonging to the large polyphenolic family widely distributed in the plant kingdom displaying a variety of biological effects like antioxidant, antiviral, and anti-inflammatory activities. In the past few years, the interest on these compounds has increased because they displayed potentiality not only as cancer-chemopreventive but also as cancer therapeutic natural agents. It has been reported that these compounds are able to modulate proteins activity acting on multiple key elements of signal transduction pathways involved in cell proliferation, differentiation, apoptosis, in angiogenesis, and inflammation, determining cancer growth inhibition. However, these mechanisms of action have not been fully characterized, and many features remain to be elucidated (
      • Yang C.S.
      • Landau J.M.
      • Huang M.T.
      • Newmark H.L.
      ,
      • Albini A.
      • Noonan D.M.
      • Ferrari N.
      ,
      • Ramos S.
      ).
      Here, we have reported the screening of a large collection of plant extracts based on the idea to identify small molecules able to prevent the initial event needed for the proangiogenic activity of the VEGF family members, the interaction of VEGF-A, PlGF-1, and VEGF-B with VEGFR-1 and VEGFR-2 receptors.
      As a result, we were able to isolate AF from an extract of the Malian plant C. senegalensis as an antiangiogenic bioactive molecule. Indeed, we have demonstrated that AF is able to bind VEGF-A and PlGF-1 preventing the interaction and consequent phosphorylation of VEGFR-1 and VEGFR-2, as well as endothelial cell migration and CTF induced by either protein. It also inhibits the in vitro VEGF-B/VEGFR-1 interaction. Interestingly, AF did not inhibit the activity of other homodimeric proteins involved in neovessel formation and stabilization such as PDGF, structurally related to VEGFs and FGF because it fails to block in vitro PDGF-B/PDGFR-β interaction and FGF-2-induced chemotaxis and CTF. These results indicate how AF possesses a binding specificity for VEGF family members acting at the low micromolar concentration range, therefore widely distant from its cytotoxic concentration.
      In vivo, AF is able to inhibit VEGF-induced chorioallantoic membrane neovascularization. Finally, we evaluated its anti-tumor effects on two different cancer experimental models: an orthotropic model of melanoma and a xenograft model of colon carcinoma. AF, daily delivered intraperitoneally at 50 mg/kg, was able to inhibit tumor growth and associated neoangiogenesis in a significant manner in both models.
      AF belongs to a unique class of naturally occurring biflavonoids and is able to exert the general properties of flavonoids (
      • Cholbi M.R.
      • Paya M.
      • Alcaraz M.J.
      ,
      • Kim H.K.
      • Son K.H.
      • Chang H.W.
      • Kang S.S.
      • Kim H.P.
      ,
      • Lin Y.M.
      • Flavin M.T.
      • Schure R.
      • Chen F.C.
      • Sidwell R.
      • Barnard D.L.
      • Huffman J.H.
      • Kern E.R.
      ,
      • Ma S.C.
      • But P.P.
      • Ooi V.E.
      • He Y.H.
      • Lee S.H.
      • Lee S.F.
      • Lin R.C.
      ). More recently, its antiangiogenic effect has been described in an assay of tumor-directed capillary formation using a melanoma model generated via subcutaneous injection of B16-F10 melanoma cells. AF delivered simultaneously with tumor cell inoculation for 5 days, determined a significant inhibition of tumor directed neovessels, as observed 9 days after cell injection. The authors reported an altered proinflammatory circulating cytokines production as well as reduction of circulating VEGF-A as possible explanation of AF action (
      • Guruvayoorappan C.
      • Kuttan G.
      ).
      These results may be fully accounted for by considering the mechanism of action that we have discovered, which has never been reported before. Indeed, the AF ability to interact with proangiogenic VEGF family members preventing their binding to VEGF receptors is crucial for the observed inhibition of new vessel formation, as direct effect on endothelial cells proliferation, migration, and differentiation, in which both proangiogenic VEGF growth factors and VEGF receptors are involved.
      Moreover, inflammation is strictly associated with tumor angiogenesis and growth and is able to sustain neoangiogenesis. PlGF and VEGF-A play a crucial role in the recruitment of inflammatory cells at neoangiogenic site (
      • Tarallo V.
      • Vesci L.
      • Capasso O.
      • Esposito M.T.
      • Riccioni T.
      • Pastore L.
      • Orlandi A.
      • Pisano C.
      • De Falco S.
      ), mainly interacting with VEGFR-1 expressed on their surfaces (
      • Clauss M.
      • Weich H.
      • Breier G.
      • Knies U.
      • Röckl W.
      • Waltenberger J.
      • Risau W.
      ,
      • Sawano A.
      • Iwai S.
      • Sakurai Y.
      • Ito M.
      • Shitara K.
      • Nakahata T.
      • Shibuya M.
      ), as demonstrated by the inhibition of F4/80 positive cells recruitment observed in AF treated melanoma. This reduction may be responsible, almost in part, for the reported decrease of general proinflammatory circulating cytokines. Moreover, this reduction, together with reduced tumor growth, also explains the decrease of circulating VEGF-A because it is mainly produced by tumor cells and by recruited myeloid cells such as monocyte macrophages. Nonetheless, we cannot exclude that AF may exert its action also modulating proinflammatory cytokines by alternative pathways.
      From a structural point of view, AF is an apigenin dimer. Many data have been reported on the antiangiogenic and antitumoral action of apigenin, in particular for its ability to interfere with hypoxia-inducible factor-1α activity and expression (
      • Osada M.
      • Imaoka S.
      • Funae Y.
      ,
      • Fang J.
      • Xia C.
      • Cao Z.
      • Zheng J.Z.
      • Reed E.
      • Jiang B.H.
      ). Interestingly, neither apigenin nor related molecules as quercetin, naringenin, and catechin are able to interact with VEGFs, as assessed by surface plasmon resonance assays. Consequently, the binding properties and activity of AF strictly depends on their own structural features and not on general flavonoid characteristics.
      Data generated by cross-linking and limited proteolysis clearly indicated the capability of AF to interact in the area of PlGF-1 involved in the recognition of the receptor. Indeed, the residue Gln-27 highlighted with limited proteolysis analysis is involved in polar interaction with VEGFR-1 (
      • Iyer S.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Maglione D.
      • Battisti M.
      • Tucci M.
      • Persico M.G.
      • Acharya K.R.
      ). The relevance of this residue in the PlGF-1·VEGFR-1 complex stabilization was demonstrated previously also with mutagenesis studies, which indicated that its mutation in Ala was sufficient to decrease to 50% the PlGF-1 binding activity to the receptor (
      • Errico M.
      • Riccioni T.
      • Iyer S.
      • Pisano C.
      • Acharya K.R.
      • Persico M.G.
      • De Falco S.
      ). Among the residues of the peptide-(94–110) evidenced by UV cross-linking, the residue Tyr-100 is involved in both van der Waals and polar interactions with VEGFR-1. Mutagenesis studies indicated that its mutation in Ala, as well as that of the residue Pro-98, determined a decrease of 25 or of 50% of PlGF-1 binding activity to the receptor, respectively (Table 2) (
      • Errico M.
      • Riccioni T.
      • Iyer S.
      • Pisano C.
      • Acharya K.R.
      • Persico M.G.
      • De Falco S.
      ,
      • Iyer S.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Maglione D.
      • Battisti M.
      • Tucci M.
      • Persico M.G.
      • Acharya K.R.
      ).
      To better understand the mechanism of interaction between AF and PlGF-1, these data have been used to generate a preliminary model of PlGF-1·AF complex using the docking approach (Fig. 6C) (
      • Lengauer T.
      • Rarey M.
      ,
      • van Dijk A.D.
      • Boelens R.
      • Bonvin A.M.
      ). The atomic coordinates and three-dimensional structure of recombinant PlGF-1 homodimer used for calculations were based on crystallographic structure of PlGF-1 (Protein Data Bank code 1FZV) (
      • Iyer S.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Maglione D.
      • Battisti M.
      • Tucci M.
      • Persico M.G.
      • Acharya K.R.
      ).
      Figure thumbnail gr6
      FIGURE 6Docking analysis of PlGF-1·amentoflavone complex. Shown is a surface plot of PlGF-1 showing the residues involved in Flt-1 recognition in green (A), the peptide-(94–110), identified by UV cross-linking analysis, and the residues Gln-27 and Arg-32, evidenced with limited proteolysis analysis, highlighted in blue and yellow, respectively (B). C, surface plot of the model of the PlGF-1·AF complex showing the lowest binding energy. Red indicates the area within 6 Å from the ligand. A high overlapping with blue and green residues was observed. D, magnification of interaction area between AF and PlGF-1. The PlGF-1 residues of the two monomers (green and cyan) involved in AF interaction are indicated. AF and side chains of PlGF-1 residues involved in contacts are represented as sticks with colored atoms (white, hydrogen; red, oxygen; blue, nitrogen). E, the PlGF-1·VEGFR-1 D2 complex (Protein Data Bank code 1RV6) is shown with the two monomers of PlGF-1 homodimer represented in green and cyan, and the VEGFR-1 D2 domain is represented in yellow, whereas the PlGF-1 residues and corresponding side chains at 6 Å from AF inhibitor are highlighted in red.
      PlGF-1 residues Pro-25, Phe-26, and Gln-27 from one monomer and Gln-88, Arg-97, Pro-98, Ser-99, and Tyr-100 from the other monomer all localized in the area involved in VEGFR-1 recognition (Fig. 6, D and E), were found at 6 Å from the inhibitor (
      • Errico M.
      • Riccioni T.
      • Iyer S.
      • Pisano C.
      • Acharya K.R.
      • Persico M.G.
      • De Falco S.
      ,
      • Iyer S.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Maglione D.
      • Battisti M.
      • Tucci M.
      • Persico M.G.
      • Acharya K.R.
      ,
      • Christinger H.W.
      • Fuh G.
      • de Vos A.M.
      • Wiesmann C.
      ). According to this model, the interaction between PlGF-1 and AF is mainly hydrophobic (Table 2). Docking of the nonactive flavonoid catechin performed with identical parameters showed an interaction involving a protein region clearly different from that observed for AF and not implicated in the VEGFR-1 recognition (supplemental Fig. S9).
      Docking results furtherly support the role of PlGF-1 residues highlighted with cross-linking and limited proteolysis analyses. In addition, the three residues Pro-25, Phe-26, and Gln-27 are part of the PlGF-1 α1-helix, a structural element crucial for the stabilization of PlGF-1 dimer (
      • Iyer S.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Maglione D.
      • Battisti M.
      • Tucci M.
      • Persico M.G.
      • Acharya K.R.
      ,
      • Christinger H.W.
      • Fuh G.
      • de Vos A.M.
      • Wiesmann C.
      ). Moreover, the residues Phe-26 and Gln-88 are involved in van der Waals interactions with the VEGFR-1 receptor, whereas mutagenesis of Pro-25 in Ala determined a 25% reduction of PlGF-1 binding activity to the receptor (Table 2 and supplemental Fig. S10) (
      • Errico M.
      • Riccioni T.
      • Iyer S.
      • Pisano C.
      • Acharya K.R.
      • Persico M.G.
      • De Falco S.
      ,
      • Iyer S.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Maglione D.
      • Battisti M.
      • Tucci M.
      • Persico M.G.
      • Acharya K.R.
      ).
      Altogether, these data suggest how AF is able to interact with PlGF-1 residues that play a crucial role in receptor recognition. Moreover, additional structural studies are needed to identify the residues of VEGF-A and VEGF-B involved in AF binding to correctly evaluate how this interaction may affect the receptor recognition.
      In conclusion, the data here reported demonstrate that AF possesses antiangiogenic activity due to its ability to selectively bind proangiogenic VEGF family members, preventing their interaction with VEGF receptors. Therefore, we propose that AF may be considered as a promising new chemical scaffold to develop, by medicinal chemistry approaches, powerful new small molecules for the inhibition of pathological neoangiogenesis.

      Acknowledgments

      We thank Professor D. Collen, Chairman of the D. Collen Research Foundation, for support; Vincenzo Mercadante and all the staff of IGB animal house for technical assistance; Andrea Lorentzen for assistance in acquisition of data on the 4800 instrument; and Anna Maria Aliperti for manuscript editing.

      REFERENCES

        • Ferrara N.
        • Gerber H.P.
        • LeCouter J.
        Nat. Med. 2003; 9: 669-676
        • Carmeliet P.
        Nature. 2005; 438: 932-936
        • De Falco S.
        • Gigante B.
        • Persico M.G.
        Trends Cardiovasc. Med. 2002; 12: 241-246
        • Ferrara N.
        • Hillan K.J.
        • Novotny W.
        Biochem. Biophys. Res. Commun. 2005; 333: 328-335
        • Gragoudas E.S.
        • Adamis A.P.
        • Cunningham Jr., E.T.
        • Feinsod M.
        • Guyer D.R.
        N. Engl. J. Med. 2004; 351: 2805-2816
        • Rosenfeld P.J.
        • Brown D.M.
        • Heier J.S.
        • Boyer D.S.
        • Kaiser P.K.
        • Chung C.Y.
        • Kim R.Y.
        N. Engl. J. Med. 2006; 355: 1419-1431
        • Ellis L.M.
        • Hicklin D.J.
        Nat. Rev. Cancer. 2008; 8: 579-591
        • Wu Y.
        • Zhong Z.
        • Huber J.
        • Bassi R.
        • Finnerty B.
        • Corcoran E.
        • Li H.
        • Navarro E.
        • Balderes P.
        • Jimenez X.
        • Koo H.
        • Mangalampalli V.R.
        • Ludwig D.L.
        • Tonra J.R.
        • Hicklin D.J.
        Clin Cancer Res. 2006; 12: 6573-6584
        • Fischer C.
        • Jonckx B.
        • Mazzone M.
        • Zacchigna S.
        • Loges S.
        • Pattarini L.
        • Chorianopoulos E.
        • Liesenborghs L.
        • Koch M.
        • De Mol M.
        • Autiero M.
        • Wyns S.
        • Plaisance S.
        • Moons L.
        • van Rooijen N.
        • Giacca M.
        • Stassen J.M.
        • Dewerchin M.
        • Collen D.
        • Carmeliet P.
        Cell. 2007; 131: 463-475
        • Van de Veire S.
        • Stalmans I.
        • Heindryckx F.
        • Oura H.
        • Tijeras-Raballand A.
        • Schmidt T.
        • Loges S.
        • Albrecht I.
        • Jonckx B.
        • Vinckier S.
        • Van Steenkiste C.
        • Tugues S.
        • Rolny C.
        • De Mol M.
        • Dettori D.
        • Hainaud P.
        • Coenegrachts L.
        • Contreres J.O.
        • Van Bergen T.
        • Cuervo H.
        • Xiao W.H.
        • Le Henaff C.
        • Buysschaert I.
        • Kharabi Masouleh B.
        • Geerts A.
        • Schomber T.
        • Bonnin P.
        • Lambert V.
        • Haustraete J.
        • Zacchigna S.
        • Rakic J.M.
        • Jiménez W.
        • Noël A.
        • Giacca M.
        • Colle I.
        • Foidart J.M.
        • Tobelem G.
        • Morales-Ruiz M.
        • Vilar J.
        • Maxwell P.
        • Vinores S.A.
        • Carmeliet G.
        • Dewerchin M.
        • Claesson-Welsh L.
        • Dupuy E.
        • Van Vlierberghe H.
        • Christofori G.
        • Mazzone M.
        • Detmar M.
        • Collen D.
        • Carmeliet P.
        Cell. 2010; 141: 178-190
        • Carmeliet P.
        • Moons L.
        • Luttun A.
        • Vincenti V.
        • Compernolle V.
        • De Mol M.
        • Wu Y.
        • Bono F.
        • Devy L.
        • Beck H.
        • Scholz D.
        • Acker T.
        • DiPalma T.
        • Dewerchin M.
        • Noel A.
        • Stalmans I.
        • Barra A.
        • Blacher S.
        • Vandendriessche T.
        • Ponten A.
        • Eriksson U.
        • Plate K.H.
        • Foidart J.M.
        • Schaper W.
        • Charnock-Jones D.S.
        • Hicklin D.J.
        • Herbert J.M.
        • Collen D.
        • Persico M.G.
        Nat. Med. 2001; 7: 575-583
        • Luttun A.
        • Tjwa M.
        • Moons L.
        • Wu Y.
        • Angelillo-Scherrer A.
        • Liao F.
        • Nagy J.A.
        • Hooper A.
        • Priller J.
        • De Klerck B.
        • Compernolle V.
        • Daci E.
        • Bohlen P.
        • Dewerchin M.
        • Herbert J.M.
        • Fava R.
        • Matthys P.
        • Carmeliet G.
        • Collen D.
        • Dvorak H.F.
        • Hicklin D.J.
        • Carmeliet P.
        Nat. Med. 2002; 8: 831-840
        • Rakic J.M.
        • Lambert V.
        • Devy L.
        • Luttun A.
        • Carmeliet P.
        • Claes C.
        • Nguyen L.
        • Foidart J.M.
        • Noël A.
        • Munaut C.
        Invest. Ophthalmol. Vis. Sci. 2003; 44: 3186-3193
        • Kaplan R.N.
        • Riba R.D.
        • Zacharoulis S.
        • Bramley A.H.
        • Vincent L.
        • Costa C.
        • MacDonald D.D.
        • Jin D.K.
        • Shido K.
        • Kerns S.A.
        • Zhu Z.
        • Hicklin D.
        • Wu Y.
        • Port J.L.
        • Altorki N.
        • Port E.R.
        • Ruggero D.
        • Shmelkov S.V.
        • Jensen K.K.
        • Rafii S.
        • Lyden D.
        Nature. 2005; 438: 820-827
        • Gigante B.
        • Morlino G.
        • Gentile M.T.
        • Persico M.G.
        • De Falco S.
        FASEB J. 2006; 20: 970-972
        • Cao Y.
        Sci. Signal. 2009; 2: re1
        • Tarallo V.
        • Vesci L.
        • Capasso O.
        • Esposito M.T.
        • Riccioni T.
        • Pastore L.
        • Orlandi A.
        • Pisano C.
        • De Falco S.
        Cancer Res. 2010; 70: 1804-1813
        • Fischer C.
        • Mazzone M.
        • Jonckx B.
        • Carmeliet P.
        Nat. Rev. Cancer. 2008; 8: 942-956
        • Newman D.J.
        • Cragg G.M.
        • Snader K.M.
        J. Nat. Prod. 2003; 66: 1022-1037
        • Ponticelli S.
        • Braca A.
        • De Tommasi N.
        • De Falco S.
        Planta Med. 2008; 74: 401-406
        • Ponticelli S.
        • Marasco D.
        • Tarallo V.
        • Albuquerque R.J.
        • Mitola S.
        • Takeda A.
        • Stassen J.M.
        • Presta M.
        • Ambati J.
        • Ruvo M.
        • De Falco S.
        J. Biol. Chem. 2008; 283: 34250-34259
        • Kim H.P.
        • Park H.
        • Son K.H.
        • Chang H.W.
        • Kang S.S.
        Arch. Pharm. Res. 2008; 31: 265-273
        • Maglione D.
        • Guerriero V.
        • Viglietto G.
        • Ferraro M.G.
        • Aprelikova O.
        • Alitalo K.
        • Del Vecchio S.
        • Lei K.J.
        • Chou J.Y.
        • Persico M.G.
        Oncogene. 1993; 8: 925-931
        • Dal Piaz F.
        • Vassallo A.
        • Lepore L.
        • Tosco A.
        • Bader A.
        • De Tommasi N.
        J. Med. Chem. 2009; 52: 3814-3828
        • Errico M.
        • Riccioni T.
        • Iyer S.
        • Pisano C.
        • Acharya K.R.
        • Persico M.G.
        • De Falco S.
        J. Biol. Chem. 2004; 279: 43929-43939
        • Marcellini M.
        • De Luca N.
        • Riccioni T.
        • Ciucci A.
        • Orecchia A.
        • Lacal P.M.
        • Ruffini F.
        • Pesce M.
        • Cianfarani F.
        • Zambruno G.
        • Orlandi A.
        • Failla C.M.
        Am J. Pathol. 2006; 169: 643-654
        • Guruvayoorappan C.
        • Kuttan G.
        Biochemistry. 2008; 73: 209-218
        • Gillet A.
        • Sanner M.
        • Stoffler D.
        • Olson A.
        Structure. 2005; 13: 483-491
        • Patel D.
        • Shukla S.
        • Gupta S.
        Int. J. Oncol. 2007; 30: 233-245
        • Renzone G.
        • Salzano A.M.
        • Arena S.
        • D'Ambrosio C.
        • Scaloni A.
        Curr. Proteomics. 2007; 4: 1-16
        • Iyer S.
        • Leonidas D.D.
        • Swaminathan G.J.
        • Maglione D.
        • Battisti M.
        • Tucci M.
        • Persico M.G.
        • Acharya K.R.
        J. Biol. Chem. 2001; 276: 12153-12161
        • Yang C.S.
        • Landau J.M.
        • Huang M.T.
        • Newmark H.L.
        Annu. Rev. Nutr. 2001; 21: 381-406
        • Albini A.
        • Noonan D.M.
        • Ferrari N.
        Clin. Cancer Res. 2007; 13: 4320-4325
        • Ramos S.
        Mol. Nutr. Food Res. 2008; 52: 507-526
        • Cholbi M.R.
        • Paya M.
        • Alcaraz M.J.
        Experientia. 1991; 47: 195-199
        • Kim H.K.
        • Son K.H.
        • Chang H.W.
        • Kang S.S.
        • Kim H.P.
        Arch. Pharm. Res. 1998; 21: 406-410
        • Lin Y.M.
        • Flavin M.T.
        • Schure R.
        • Chen F.C.
        • Sidwell R.
        • Barnard D.L.
        • Huffman J.H.
        • Kern E.R.
        Planta Med. 1999; 65: 120-125
        • Ma S.C.
        • But P.P.
        • Ooi V.E.
        • He Y.H.
        • Lee S.H.
        • Lee S.F.
        • Lin R.C.
        Biol. Pharm. Bull. 2001; 24: 311-312
        • Clauss M.
        • Weich H.
        • Breier G.
        • Knies U.
        • Röckl W.
        • Waltenberger J.
        • Risau W.
        J. Biol. Chem. 1996; 271: 17629-17634
        • Sawano A.
        • Iwai S.
        • Sakurai Y.
        • Ito M.
        • Shitara K.
        • Nakahata T.
        • Shibuya M.
        Blood. 2001; 97: 785-791
        • Osada M.
        • Imaoka S.
        • Funae Y.
        FEBS Lett. 2004; 575: 59-63
        • Fang J.
        • Xia C.
        • Cao Z.
        • Zheng J.Z.
        • Reed E.
        • Jiang B.H.
        FASEB J. 2005; 19: 342-353
        • Lengauer T.
        • Rarey M.
        Curr. Opin. Struct. Biol. 1996; 6: 402-406
        • van Dijk A.D.
        • Boelens R.
        • Bonvin A.M.
        FEBS J. 2005; 272: 293-312
        • Christinger H.W.
        • Fuh G.
        • de Vos A.M.
        • Wiesmann C.
        J. Biol. Chem. 2004; 279: 10382-10388