Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Modulation of Angiogenesis by a Tetrameric Tripeptide That Antagonizes Vascular Endothelial Growth Factor Receptor 1

2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês

10.1074/jbc.m806607200

ISSN

1083-351X

Autores

Salvatore Ponticelli, Daniela Marasco, Valeria Tarallo, Romulo Albuquerque, Stefania Mitola, Atsunobu Takeda, Jean-Marie Stassen, Marco Presta, Jayakrishna Ambati, Menotti Ruvo, Sandro De Falco,

Tópico(s)

Chemokine receptors and signaling

Resumo

Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC50 ∼ 10 ;m). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role. Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC50 ∼ 10 ;m). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role. A complicated tuning of several growth factor families and related receptors regulates the formation of new vessels (1Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Crossref PubMed Scopus (3298) Google Scholar). Among these players, the activation of two vascular endothelial growth factor (VEGF) 6The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR-1 or -2, VEGF receptor 1 or 2; Flt-1, fms-related tyrosine kinase 1; sFlt-1, soluble Flt-1; PlGF, placental growth factor; Flk-1, fetal liver kinase receptor 1; KDR, kinase domain region; CTF, capillary-like tube formation; CAM, chorioallantoic membrane; CNV, corneal neovascularization; EC, endothelial cells; HUVEC, human umbilical vein endothelial cells; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; Fmoc, N-(9-fluorenyl)methoxycarbonyl; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; HPLC, high pressure liquid chromatography. receptors, Flt-1 and VEGF receptor 2 (Flk-1 in mouse, KDR in human), represents a crucial event in both physiological and pathological angiogenesis (2Olsson A.K. Dimberg A. Kreuger J. Claesson-Welsh L. Nat. Rev. Mol. Cell Biol. 2006; 7: 359-371Crossref PubMed Scopus (2479) Google Scholar, 3Carmeliet P. Nature. 2005; 438: 932-936Crossref PubMed Scopus (2840) Google Scholar, 4Ferrara N. Kerbel R.S. Nature. 2005; 438: 967-974Crossref PubMed Scopus (2273) Google Scholar). Three members of the VEGF family involved in angiogenesis bind and activate VEGF receptors; VEGF-A binds both Flt-1 and KDR receptors, whereas two other VEGF family members, VEGF-B and placental growth factor (PlGF), specifically bind Flt-1. The activity of VEGF-A is crucial both in physiological and in pathological angiogenesis, whereas that of PlGF and VEGF-B appears to be restricted to pathological conditions (5De Falco S. Gigante B. Persico M.G. Trends Cardiovasc. Med. 2002; 12: 241-246Crossref PubMed Scopus (112) Google Scholar, 6Shibuya M. Angiogenesis. 2006; 9 (discussion 231): 225-230Crossref PubMed Scopus (312) Google Scholar). Phenotype analysis of "knock out" mice and in vivo biochemical interaction studies strongly suggest that the inhibition of Flt-1 activity constitutes an alternative target for therapeutic modulation of angiogenesis as well as inflammatory disorders and metastatic process (7Luttun A. Tjwa M. Carmeliet P. Ann. N. Y. Acad. Sci. 2002; 979: 80-93Crossref PubMed Scopus (188) Google Scholar, 8Kaplan R.N. Rafii S. Lyden D. Cancer Res. 2006; 66: 11089-11093Crossref PubMed Scopus (503) Google Scholar). Plgf null mice (9Carmeliet P. Moons L. Luttun A. Vincenti V. Compernolle V. De Mol M. Wu Y. Bono F. Devy L. Beck H. Scholz D. Acker T. Di-Palma 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: 575-583Crossref PubMed Scopus (1403) Google Scholar), Vegf-B null mice (10Bellomo D. Headrick J.P. Silins G.U. Paterson C.A. Thomas P.S. Gartside M. Mould A. Cahill M.M. Tonks I.D. Grimmond S.M. Townson S. Wells C. Little M. Cummings M.C. Hayward N.K. Kay G.F. Circ. Res. 2000; 86: E29-35Crossref PubMed Google Scholar), and mice engineered to express a truncated form of Flt-1 lacking the tyrosine kinase domain (11Hiratsuka S. Minowa O. Kuno J. Noda T. Shibuya M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9349-9354Crossref PubMed Scopus (896) Google Scholar) are borne at Mendelian frequency and are healthy and fertile. However, pathological angiogenesis in the adult is impaired in all three mouse models. Moreover, Flt-1 blockade by neutralizing anti-Flt-1 monoclonal antibody (mAb) strongly reduces the neovascularization in tumors as well as in models of ischemic retinopathy and age-related macular degeneration (9Carmeliet P. Moons L. Luttun A. Vincenti V. Compernolle V. De Mol M. Wu Y. Bono F. Devy L. Beck H. Scholz D. Acker T. Di-Palma 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: 575-583Crossref PubMed Scopus (1403) Google Scholar, 12Lyden D. Hattori K. Dias S. Costa C. Blaikie P. Butros L. Chadburn A. Heissig B. Marks W. Witte L. Wu Y. Hicklin D. Zhu Z. Hackett N.R. Crystal R.G. Moore M.A. Hajjar K.A. Manova K. Benezra R. Rafii S. Nat. Med. 2001; 7: 1194-1201Crossref PubMed Scopus (1696) Google Scholar, 13Rakic J.M. Lambert V. Devy L. Luttun A. Carmeliet P. Claes C. Nguyen L. Foidart J.M. Noel A. Munaut C. Investig. Ophthalmol. Vis. Sci. 2003; 44: 3186-3193Crossref PubMed Scopus (273) Google Scholar, 14Wu 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: 6573-6584Crossref PubMed Scopus (142) Google Scholar). Recently it has been reported that neutralizing mAb anti-PlGF is able to inhibit tumor angiogenesis with an efficacy comparable with that observed blocking VEGF/Flk-1 pathway (15Fischer 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: 463-475Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar). In contrast to KDR, which is predominantly expressed by endothelial cells (ECs), expression of Flt-1 has been detected and functionally demonstrated also in smooth muscle cells (16Wang H. Keiser J.A. Circ. Res. 1998; 83: 832-840Crossref PubMed Scopus (399) Google Scholar), in monocyte-macrophage cells (17Barleon B. Sozzani S. Zhou D. Weich H.A. Mantovani A. Marme D. Blood. 1996; 87: 3336-3343Crossref PubMed Google Scholar), and in bone marrow stem/progenitor-derived cells (12Lyden D. Hattori K. Dias S. Costa C. Blaikie P. Butros L. Chadburn A. Heissig B. Marks W. Witte L. Wu Y. Hicklin D. Zhu Z. Hackett N.R. Crystal R.G. Moore M.A. Hajjar K.A. Manova K. Benezra R. Rafii S. Nat. Med. 2001; 7: 1194-1201Crossref PubMed Scopus (1696) Google Scholar). The activation of Flt-1 is not only crucial for ECs stimulation during the neoangiogenesis process (18Errico M. Riccioni T. Iyer S. Pisano C. Acharya K.R. Persico M.G. De Falco S. J. Biol. Chem. 2004; 279: 43929-43939Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Nishi J. Minamino T. Miyauchi H. Nojima A. Tateno K. Okada S. Orimo M. Moriya J. Fong G.H. Sunagawa K. Shibuya M. Komuro I. Circ. Res. 2008; 103: 261-268Crossref PubMed Scopus (46) Google Scholar) but also plays a fundamental role in the stabilization of neovessels through the recruitment of smooth muscle cells (16Wang H. Keiser J.A. Circ. Res. 1998; 83: 832-840Crossref PubMed Scopus (399) Google Scholar), in the recruitment and differentiation of monocyte-macrophage cells (17Barleon B. Sozzani S. Zhou D. Weich H.A. Mantovani A. Marme D. Blood. 1996; 87: 3336-3343Crossref PubMed Google Scholar, 20Clauss M. Weich H. Breier G. Knies U. Rockl W. Waltenberger J. Risau W. J. Biol. Chem. 1996; 271: 17629-17634Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 21Sawano A. Iwai S. Sakurai Y. Ito M. Shitara K. Nakahata T. Shibuya M. Blood. 2001; 97: 785-791Crossref PubMed Scopus (418) Google Scholar, 22Luttun 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: 831-840Crossref PubMed Scopus (951) Google Scholar) and, ultimately, in the reconstitution of hematopoiesis promoting the recruitment of Flt-1-positive cells from bone marrow microenvironment (23Hattori K. Heissig B. Wu Y. Dias S. Tejada R. Ferris B. Hicklin D.J. Zhu Z. Bohlen P. Witte L. Hendrikx J. Hackett N.R. Crystal R.G. Moore M.A. Werb Z. Lyden D. Rafii S. Nat. Med. 2002; 8: 841-849Crossref PubMed Scopus (561) Google Scholar). Furthermore, Flt-1 activation is decisive in the recruitment of bone marrow-derived endothelial cells and hematopoietic precursors in tumor angiogenesis (12Lyden D. Hattori K. Dias S. Costa C. Blaikie P. Butros L. Chadburn A. Heissig B. Marks W. Witte L. Wu Y. Hicklin D. Zhu Z. Hackett N.R. Crystal R.G. Moore M.A. Hajjar K.A. Manova K. Benezra R. Rafii S. Nat. Med. 2001; 7: 1194-1201Crossref PubMed Scopus (1696) Google Scholar) as well as in inflammatory disorders (22Luttun 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: 831-840Crossref PubMed Scopus (951) Google Scholar). More recently, it has been shown that Flt-1-positive hematopoietic bone marrow progenitors are involved in the establishment of premetastatic niche and that an anti-Flt-1 mAb completely prevents metastatic process (24Kaplan 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: 820-827Crossref PubMed Scopus (2514) Google Scholar). Flt-1 receptor also exists as an alternatively spliced soluble form (sFlt-1) (25Kendall R.L. Thomas K.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10705-10709Crossref PubMed Scopus (1204) Google Scholar) that represents one of the most potent physiological inhibitors of VEGFs activity. Indeed, it is expressed during embryonic development, where it regulates the availability of VEGF and, as recently reported, in the adults, it plays a pivotal role to maintain corneal avascularity (26Ambati B.K. Nozaki M. Singh N. Takeda A. Jani P.D. Suthar T. Albuquerque R.J. Richter E. Sakurai E. Newcomb M.T. Kleinman M.E. Caldwell R.B. Lin Q. Ogura Y. Orecchia A. Samuelson D.A. Agnew D.W. St Leger J. Green W.R. Mahasreshti P.J. Curiel D.T. Kwan D. Marsh H. Ikeda S. Leiper L.J. Collinson J.M. Bogdanovich S. Khurana T.S. Shibuya M. Baldwin M.E. Ferrara N. Gerber H.P. De Falco S. Witta J. Baffi J.Z. Raisler B.J. Ambati J. Nature. 2006; 443: 993-997Crossref PubMed Scopus (572) Google Scholar). Collectively, these data strongly indicate Flt-1 as an ideal target for fighting a number of major diseases (7Luttun A. Tjwa M. Carmeliet P. Ann. N. Y. Acad. Sci. 2002; 979: 80-93Crossref PubMed Scopus (188) Google Scholar). In the effort to identify new molecules able to selectively bind Flt-1 and neutralize its activity, we screened a random combinatorial tetrameric tripeptide library built using non-natural amino acids, using a competitive ELISA-based assay (27De Falco S. Ruvoletto M.G. Verdoliva A. Ruvo M. Raucci A. Marino M. Senatore S. Cassani G. Alberti A. Pontisso P. Fassina G. J. Biol. Chem. 2001; 276: 36613-36623Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The peptide mixtures composing the library were utilized as competitors of the PlGF/Flt-1 binding, and the most active component was isolated following an iterative process (28Marino M. Ruvo M. De Falco S. Fassina G. Nat. Biotechnol. 2000; 18: 735-739Crossref PubMed Scopus (62) Google Scholar, 29Verdoliva A. Marasco D. De Capua A. Saporito A. Bellofiore P. Manfredi V. Fattorusso R. Pedone C. Ruvo M. ChemBioChem. 2005; 6: 1242-1253Crossref PubMed Scopus (42) Google Scholar). The biological activity of the selected peptide has then been assessed in several in vitro and in vivo assays demonstrating that it is a highly stable and selective Flt-1 binder able to suppress the receptor activation. Synthesis of Combinatorial Tetrameric Tripeptide Library and Analogues of 4-23-5 Peptide—The peptide library was prepared using all commercially available amino acids and resins purchased from Chem-Impex International (Wood Dale, IL) and from Novabiochem (Laufelfingen, CH). As 30 different blocks were utilized (supplemental Table S1), a theoretical number of 27,000 peptides was generated, split in 30 separate pools of 900 peptides each. All residues were 9-fluorenylmethoxycarbonyl (Fmoc)-derivatized (>99%) and were utilized without any further purification. Amino acids in the d configuration and others bearing trifluoroacetic acid-stable side chain protections were chosen to increase the library chemical diversity and, at the same time, to introduce resistance to enzyme degradation (30Bracci L. Falciani C. Lelli B. Lozzi L. Runci Y. Pini A. De Montis M.G. Tagliamonte A. Neri P. J. Biol. Chem. 2003; 278: 46590-46595Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) for in vitro and in vivo applications. The library was chemically synthesized following the Fmoc methodology (31Fields G.B. Noble R.L. Int. J. Pept. Protein Res. 1990; 35: 161-214Crossref PubMed Scopus (2336) Google Scholar), and sequence randomization was achieved applying the portioning-mixing process as reported elsewhere (28Marino M. Ruvo M. De Falco S. Fassina G. Nat. Biotechnol. 2000; 18: 735-739Crossref PubMed Scopus (62) Google Scholar) (see also the supplemental Experimental Procedures). Other molecules, such as the monomeric, dimeric, and trimeric tripeptide variants, as well as the Ala-scanning peptides (where the monomers were systematically changed to alanine), were similarly prepared using suitable protecting groups. The cyclic dimeric variant was prepared as described elsewhere (29Verdoliva A. Marasco D. De Capua A. Saporito A. Bellofiore P. Manfredi V. Fattorusso R. Pedone C. Ruvo M. ChemBioChem. 2005; 6: 1242-1253Crossref PubMed Scopus (42) Google Scholar). Iterative Deconvolution of Tetrameric Tripeptide Library—Recombinant PlGF, VEGF-A, VEGF-B, VEGFR-1/Fc chimera, VEGFR-2/Fc chimera of human and mouse origin, and normal or biotinylated antibodies anti-PlGF, anti-VEGF-A, and anti-VEGF-B were purchased from R&D Systems (Minneapolis, MN). At every step of the deconvolution process, peptide pools were tested with a molar excess of 1,000-fold (calculated on each single peptide) over PlGF (1.3 × 10-10 m). Following the identification of active pools, dose-dependent inhibition experiments using 500-, 1,000-, 1,500-, and 2,000-fold excess were performed to confirm the inhibitory capacity. The first active pool identified was the 4-X-X (X indicates randomized positions) where "4" identified the amino acid d-glutamic acid (d-Glu, supplemental Table S1). This pool was resynthesized in 30 subpools each composed of 30 peptides and submitted to the second screening round that allowed the identification of the subpool 23 (4-23-X). The number "23" identified the amino acid l-cysteine(S-benzyl) (supplemental Table S1). Finally, the 30 single peptides composing the 4-23-X pool were synthesized and submitted to the final screening. The peptide 4-23-5, where the number "5" identified the amino acid l-cyclohexylalanine (supplemental Table S1), was the unique molecule showing inhibitory activity. ELISA-based Assays—The competitive ELISA-based assay (27De Falco S. Ruvoletto M.G. Verdoliva A. Ruvo M. Raucci A. Marino M. Senatore S. Cassani G. Alberti A. Pontisso P. Fassina G. J. Biol. Chem. 2001; 276: 36613-36623Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) for the screening of the peptide library and for dose-dependent experiments was performed by coating on 96-well plates a recombinant form of Fc/Flt-1 at 0.5 ;g/ml, 100 ;l/well (the same volume was used for all subsequent steps), 16 h at room temperature. The plate was then blocked for 3 h at room temperature with 1% bovine serum albumin (BSA), and a recombinant form of PlGF at 5 ng/ml concentration in PBS containing 0.1% BSA, 5 mm EDTA, 0.004% Tween 20 (PBET) was added and incubated for 1 h at 37 °C followed by 1 h at room temperature. A biotinylated anti-human PlGF polyclonal antibody diluted in PBET at 300 ng/ml was added to the wells and incubated for 1 h at 37 °C followed by 1 h at room temperature. A solution containing an avidin and biotinylated horseradish peroxidase macromolecular complex was prepared as suggested by the manufacturer (Vectastain elite ABC kit, Vector Laboratories, Burlingame, CA) and added to the wells and incubated for 1 h at room temperature followed by the horseradish peroxidase substrate composed of 1 mg/ml ortho-phenylenediamine in 50 mm citrate phosphate buffer, pH 5, 0.006% of H2O2, incubated for 40 min in the dark at room temperature. The reaction was blocked by adding 30 ;l/well of 4 n H2SO4, and the absorbance was measured at 490 nm on a microplate reader (BenchMark, Bio-Rad). Peptide pools or single peptides dissolved in DMSO were properly diluted and added to the wells along with ligand. For dose-dependent experiments performed with VEGF-A and VEGF-B, 10 ng/ml recombinant proteins and 500 ng/ml polyclonal antibody, anti-human VEGF-A or anti-human VEGF-B, were used. 4-23-5 and control peptides were used at concentrations ranging between 1.56 and 50.0 ;m. Using the same ELISA-based assay, the inhibitory capacities of alanine-scan peptides and structurally related analogues (supplemental Table S3) were investigated in both PlGF and VEGF interaction with Flt-1 receptor. Analogues were used at 50 ;m, whereas the 4-23-5 peptide was used at 12.5 ;m. To evaluate the binding of the 4-23-5 peptide to Flt-1 receptor, the peptide or the negative controls were coated at 20 ;m. for 16 h at 4 °C. A recombinant form of extracellular domains of human and mouse Flt-1 receptors fused to human Fc in PBET was added to the wells and incubated for 1 h at 37 °C followed by 30 min at room temperature. Goat anti-human Fc antibody (Jackson ImmunoResearch, West Grove, PA) and the secondary donkey anti-goat hypoxanthine-guanine phosphoribosyltransferase antibody (Santa Cruz Biotechnology, Santa Cruz, CA), both at 1:1000 dilution, were added and incubated for 1 h at room temperature. Finally, plates were developed as described above. To evaluate the binding of 4-23-5 to VEGFR-2, recombinant human Fc/KDR and mouse Fc/Flk-1 were used. To evaluate the ability of PlGF and VEGF-A to compete the interaction of Flt-1 with the synthetic ligand, 4-23-5 was coated at 20 ;m, and the human Fc/Flt-1 receptor was used at a fixed concentration (125 pm). The dose-dependent competition was performed using PlGF and VEGF-A at concentrations ranging between 0.05 and 6.25 nm. Stability Assay—The selected 4-23-5 peptide was dissolved in neat DMSO at a 10 mg/ml concentration. The sample was then serially diluted in PBS, pH 7.3, containing 10% fetal calf serum to obtain 100 ;g/ml solutions and incubated at 37 °C for 168 h. 10-;l aliquots (1.0 ;g of total peptide) were removed after every hour within the first 12 h and subsequently at 24, 72, 120, and 168 h. Samples were immediately centrifuged for 5 min at 14,000 rpm and analyzed by reverse phase-HPLC after discarding the pellet. To closely check the peptide concentration within samples, a reference curve was obtained by analyzing, under the same conditions, different amounts of the pure compound dissolved in DMSO (the peptide solubility in DMSO is >100 mg/ml). The reference curve was also used to exclude effects of sample subtraction by nonspecific binding to albumin or other serum proteins. The experiment was carried out in duplicate, and the data were reported as residual peptide in solution (in percentages) versus time. Inhibition of Flt-1 Phosphorylation and Capillary-like Tube Formation—These assays were performed as described elsewhere (18Errico M. Riccioni T. Iyer S. Pisano C. Acharya K.R. Persico M.G. De Falco S. J. Biol. Chem. 2004; 279: 43929-43939Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In phosphorylation experiments, to induce Flt-1 and KDR receptor activation, 20 ng/ml PlGF and 50 ng/ml VEGF were used on starved 293-Flt-1 or HUVEC cells. To detect phosphorylated forms of receptors in Western blot experiments, anti-pFlt-1 (R&D Systems) at 1:500 dilution and anti-pKDR (Cell Signaling, Danvers, MA) at 1:1000 dilution antibodies were used. Capillary-like tube formation were performed in 24-well culture plates. At the end of the experiment, the culture medium was replaced with PBS, and the images were captured with the inverted microscope Leica DM IRB (Wetzlar, Germany) equipped with a Leica DC 350 FX camera (original magnification ×10). Images were acquired with Leica FW 4000 software. Chicken Embryo Chorioallantoic Membrane (CAM) Assay—Alginate beads (5 ;l) containing vehicle, 150 ng/embryo of VEGF-A, or 250 ng/embryo of PlGF with or without peptides (0.25 and 0.025 nmol/embryo) were prepared as described previously (32Mitola S. Belleri M. Urbinati C. Coltrini D. Sparatore B. Pedrazzi M. Melloni E. Presta M. J. Immunol. 2006; 176: 12-15Crossref PubMed Scopus (202) Google Scholar) and placed on top of the CAM of fertilized White Leghorn chicken eggs at day 11 of incubation (6-8 eggs per experimental group). After 72 h, new blood vessels converging toward the implant were counted by two observers in a double-blind fashion under a stereomicroscope and photographed in ovo at original magnification ×5, using a STEMI SR stereomicroscope, equipped with an objective f equal to 100 mm with adapter ring 475070 (Zeiss, Germany) and a Camedia C-4040 digital camera (Olympus, Melville, NY). Images were acquired and processed using the Image-Pro Plus software. Differences among groups were tested by one-way analysis of variance using the SPSS statistical package (version 12.1, Chicago, IL). Cornea Neovascularization (CNV)—Single injections (33-gauge needle) of 0.40, 4.0, or 20 nmol of peptide 4-23-5 and of only 20 nmol of control peptide in the same volume of DMSO were carried out in the corneas of BALB/c mice (n = 3 each group). Eyes were harvested 7 days after injection, corneas were gently isolated, and immunohistochemical staining for endothelial cells was performed. Corneas were fixed in 100% acetone for 20 min, washed with PBS, 0.05% Tween 20 for 10 min four consecutive times, and blocked with 3% BSA in PBS for 48 h. The corneas were then incubated with fluorescein isothiocyanate-coupled monoclonal anti-mouse CD31 antibody (Pharmingen) at 3:1000 and rabbit anti-mouse LYVE-1 antibody (Abcam, Cambrige, UK) at 3:1000 in 3% BSA PBS solution at 4 °C for 48 h. The corneas were washed as described previously and incubated in Cy3-conjugated donkey anti-rabbit at 3:1000 in 3% BSA PBS solution for 2 h, after which they were washed and mounted with an antifading agent (Vectashield, Vector Laboratories). The corneal flat mounts were visualized with a fluorescent microscope (Nikon Eclipse TE2000-E) equipped with Nikon DXM200F camera at original magnification ×4. Images acquisitions were performed with Nikon, ACT-1 software, version 2.63. Blood vessels were identified as CD31-positive and LYVE-1-negative. Synthesis, Characterization, and Screening of the Combinatorial Peptide Library—To increase the molecular surface, the library was assembled as described previously (28Marino M. Ruvo M. De Falco S. Fassina G. Nat. Biotechnol. 2000; 18: 735-739Crossref PubMed Scopus (62) Google Scholar) on a peptide scaffold composed by a polylysine core (supplemental Fig. S1) (33Tam J.P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5409-5413Crossref PubMed Scopus (1206) Google Scholar). To obtain mixtures, three levels of randomization were achieved applying the portioning-mixing method (34Furka A. Sebestyen F. Asgedom M. Dibo G. Int. J. Pept. Protein Res. 1991; 37: 487-493Crossref PubMed Scopus (1075) Google Scholar, 35Lam K.S. Salmon S.E. Hersh E.M. Hruby V.J. Kazmierski W.M. Knapp R.J. Nature. 1991; 354: 82-84Crossref PubMed Scopus (1744) Google Scholar). The tetrameric library was synthesized using 29 non-natural amino acids plus glycine (supplemental Table S1) and was initially arranged in 30 pools, each identified with the N-terminal building block. Each pool contained 900 different molecules (302). The total complexity of the library was instead determined by the formula 303 = 27,000 different peptides. Peptide mixtures were obtained in high yield (about 70%) as calculated assuming an average molecular mass of 2130 atomic mass units for each library component and with an average purity of the crude products >85% (as determined on the single molecules assayed during the last screening round, not shown). The liquid chromatography-mass spectrometry analyses of some selected 30-component mixtures showed that the expected molecules were almost all equally represented and that molecular weights were in very good agreement with those calculated (not shown). Liquid chromatography-mass spectrometry analyses of single peptides also showed very good agreement between calculated and experimental molecular weights (supplemental Table S2). The screening was carried out by a competition assay, whereby the displacement of soluble recombinant PlGF from coated Flt-1 by the peptide pools was evaluated. PlGF was used in the assay as, among all the VEGF family members, it shows the highest affinity for Flt-1 (6Shibuya M. Angiogenesis. 2006; 9 (discussion 231): 225-230Crossref PubMed Scopus (312) Google Scholar). The iterative procedure consisted of three screening rounds that led to the selection of building blocks denoted as 4, 23, and 5 in positions 1, 2, and 3, respectively (supplemental Table S1). A schematic representation and the chemical structure of the selected tetrameric tripeptide, hereafter named 4-23-5, are reported in Fig. 1. Monomers 4, 23, and 5 refer to the amino acids d-glutamic acid (d-Glu), S-benzylated l-cysteine, and l-cyclohexylalanine. The selected peptide was readily resynthesized on a larger scale and purified by reverse phase HPLC. In the same ELISA assay, the purified 4-23-5 blocked the interaction of human PlGF with Flt-1 in a dose-dependent manner, with an IC50 of about 10 ;m (Fig. 2A), whereas the control tetrameric peptides 21-1-5 and 4-23-A (supplemental Table S3) did not show any effect (Fig. 2A). Inhibitory Properties of the Peptide 4-23-5—Because the binding mechanism of VEGFs members able to recognize Flt-1 is similar (18Errico M. Riccioni T. Iyer S. Pisano C. Acharya K.R. Persico M.G. De Falco S. J. Biol. Chem. 2004; 279: 43929-43939Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 36Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 37Iyer S. Leonidas D.D. Swaminathan G.J. Maglione D. Battisti M. Tucci M. Persico M.G. Acharya K.R. J. Biol. Chem. 2001; 276: 12153-12161Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 38Iyer S. Scotney P.D. Nash A.D.

Referência(s)