Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Viral Vascular Endothelial Growth Factors Vary Extensively in Amino Acid Sequence, Receptor-binding Specificities, and the Ability to Induce Vascular Permeability yet Are Uniformly Active Mitogens

2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês

10.1074/jbc.m301194200

ISSN

1083-351X

Autores

Lyn M. Wise, Norihito Ueda, Nicola H. Dryden, Stephen B. Fleming, Carol Caesar, Sally Roufail, Marc G. Achen, Steven A. Stacker, Andrew A. Mercer,

Tópico(s)

Virus-based gene therapy research

Resumo

Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding. Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding. Members of the vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VHD, VEGF homology domain; hVEGF-DΔNΔC, human VEGF-D VHD; mVEGF164, mouse VEGF isoform 164; ORFVNZ10VEGF, VEGF from Orf virus strain NZ10; ORFVNZ2VEGF, VEGF from Orf virus strain NZ2; ORFVNZ7VEGF, VEGF from Orf virus strain NZ7; ORFVD1701VEGF, VEGF from Orf virus strain D1701; PCPVVR634VEGF, VEGF from Pseudocowpox virus strain VR634; BSA, bovine serum albumin; HMVEC, human microvascular endothelial cells; IL, interleukin; NP-1, neuropilin-1; PlGF, placenta growth factor; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; NR, non-reducing; R, reducing. family of proteins have emerged as major regulators of the formation of new blood vessels during vasculogenesis and angiogenesis (reviewed in Refs. 1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar, 2Robinson C.J. Stringer S.E. J. Cell Sci. 2001; 114: 853-865Crossref PubMed Google Scholar, 3Stacker S.A. Achen M.G. Growth Factors. 1999; 17: 1-11Crossref PubMed Scopus (51) Google Scholar). These proteins play critical roles during embryogenesis and in the normal adult, during wound healing and in pathological conditions such as tumor formation. The original member of the family, VEGF-A, also known as vascular permeability factor, was characterized as a mitogen for vascular endothelial cells (4Ferrara N. Henzel W.J. Biochem. Biophys. Res. Commun. 1989; 161: 851-858Crossref PubMed Scopus (2023) Google Scholar, 5Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar) and a potent inducer of vascular permeability (6Senger D.R. Galli S.J. Dvorak A.M. Perruzzi C.A. Harvey V.S. Dvorak H.F. Science. 1983; 219: 983-985Crossref PubMed Scopus (3428) Google Scholar). Subsequently, the discovery and characterization of other proteins, related in structure and receptor specificity to VEGF-A have been reported. Currently, the mammalian VEGF family includes VEGF-A, placenta growth factor (PlGF) (7Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar), VEGF-B (8Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar), VEGF-C (9Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 1751Crossref PubMed Scopus (393) Google Scholar), and VEGF-D (10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar). VEGF family members are, in general, secreted glycoproteins, which form either disulphide-linked or non-covalent homodimers (5Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar, 7Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar, 8Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar, 9Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 1751Crossref PubMed Scopus (393) Google Scholar, 10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar). The common structural feature of the VEGF family is the cystine knot motif, which is located in the most highly conserved region of these proteins designated the VEGF homology domain (VHD) (11McDonald N.Q. Hendrickson W.A. Cell. 1993; 73: 421-424Abstract Full Text PDF PubMed Scopus (477) Google Scholar). VEGF family members are ligands for a number of mammalian tyrosine kinase receptors: VEGFR-1 (flt-1) (12de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar), VEGFR-2 (flk-1/KDR) (13Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (673) Google Scholar), and VEGFR-3 (flt-4) (9Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 1751Crossref PubMed Scopus (393) Google Scholar). It appears that VEGFR-1 plays a role in vascular endothelial cell differentiation and migration, possibly by acting as a ligand-binding molecule, sequestering VEGF-A from VEGFR-2 signaling (14Shibuya M. Int. J. Biochem. Cell Biol. 2001; 33: 409-420Crossref PubMed Scopus (217) Google Scholar). VEGFR-2 is involved in vascular endothelial cell mitogenesis (13Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (673) Google Scholar), and VEGFR-3 is involved in the regulation of the lymphatic vasculature (15Taipale J. Makinen T. Arighi E. Kukk E. Karkkainen M. Alitalo K. Curr. Top. Microbiol. Immunol. 1999; 237: 85-96Crossref PubMed Scopus (67) Google Scholar, 16Stacker S.A. Caesar C. Baldwin M.E. Thornton G.E. Williams R.A. Prevo R. Jackson D.G. Nishikawa S. Kubo H. Achen M.G. Nat. Med. 2001; 7: 186-191Crossref PubMed Scopus (1063) Google Scholar). VEGFR-1 binds VEGF-A, VEGF-B, and PlGF (12de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar, 17Park J.E. Chen H.H. Winer J. Houck K.A. Ferrara N. J. Biol. Chem. 1994; 269: 25646-25654Abstract Full Text PDF PubMed Google Scholar, 18Olofsson B. Korpelainen E. Pepper M.S. Mandriota S.J. Aase K. Kumar V. Gunji Y. Jeltsch M.M. Shibuya M. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11709-11714Crossref PubMed Scopus (450) Google Scholar). VEGFR-2 binds VEGF-A, VEGF-C, and VEGF-D (9Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 1751Crossref PubMed Scopus (393) Google Scholar, 10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar, 13Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (673) Google Scholar). VEGFR-3 binds VEGF-C and VEGF-D (9Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 1751Crossref PubMed Scopus (393) Google Scholar, 10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar). In addition, the neuronal cell guidance receptors, neuropilin-1 (NP-1) and neuropilin-2, have recently been discovered to interact with VEGF-A, PlGF, and VEGF-B and are thought to act as co-receptors that enhance binding to the VEGFRs (19Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 20Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar, 21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar, 22Fuh G. Garcia K.C. de Vos A.M. J. Biol. Chem. 2000; 275: 26690-26695Abstract Full Text Full Text PDF PubMed Google Scholar). The role of the VEGFRs in the induction of vascular permeability is, however, unclear. We and others (21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar, 23Lyttle D.J. Fraser K.M. Fleming S.B. Mercer A.A. Robinson A.J. J. Virol. 1994; 68: 84-92Crossref PubMed Google Scholar, 24Ogawa S. Oku A. Sawano A. Yamaguchi S. Yazaki Y. Shibuya M. J. Biol. Chem. 1998; 273: 31273-31282Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 25Meyer M. Clauss M. Lepple-Wienhues A. Waltenberger J. Augustin H.G. Ziche M. Lanz C. Buttner M. Rziha H.J. Dehio C. EMBO J. 1999; 18: 363-374Crossref PubMed Scopus (413) Google Scholar, 26Mercer A.A. Wise L.M. Scagliarini A. McInnes C.J. Buettner M. Rhiza H.J. McCaughan C.A. Fleming S.B. Ueda N. Nettleton P.F. J. Gen. Virol. 2002; 83: 2845-2855Crossref PubMed Scopus (53) Google Scholar, 27Ueda N. Wise L.M. Stacker S.A. Fleming S.B. Mercer A.A. Virology. 2003; 305: 298-309Crossref PubMed Scopus (39) Google Scholar) have recently reported a group of poxvirus-derived VEGF-like factors. These proteins have significant homology to VEGF-A and are encoded by Orf virus and Pseudocowpox virus. Orf virus and Pseudocowpox virus are distinct species within the genus Parapoxvirus, and infect sheep and goats and cattle, respectively, but both readily infect humans (28Haig D.M. Mercer A.A. Vet. Res. 1998; 29: 311-326PubMed Google Scholar, 29Mercer A. Fleming S. Robinson A. Nettleton P. Reid H. Arch. Virol. Suppl. 1997; 13: 25-34PubMed Google Scholar). The resulting lesions in the skin demonstrate extensive vascular dilation, dermal edema, and proliferation of endothelial cells (28Haig D.M. Mercer A.A. Vet. Res. 1998; 29: 311-326PubMed Google Scholar, 30Groves R.W. Wilson-Jones E. MacDonald D.M. J. Am. Acad. Dermatol. 1991; 25: 706-711Abstract Full Text PDF PubMed Scopus (100) Google Scholar). We have shown that the disruption of the VEGF-like gene in Orf virus strain NZ2 markedly reduces these characteristics of infection (31Savory L.J. Stacker S.A. Fleming S.B. Niven B.E. Mercer A.A. J. Virol. 2000; 74: 10699-10706Crossref PubMed Scopus (112) Google Scholar). Comparisons of the VEGF-like genes from 24 isolates of Orf virus have revealed a surprising degree of sequence variation between isolates (26Mercer A.A. Wise L.M. Scagliarini A. McInnes C.J. Buettner M. Rhiza H.J. McCaughan C.A. Fleming S.B. Ueda N. Nettleton P.F. J. Gen. Virol. 2002; 83: 2845-2855Crossref PubMed Scopus (53) Google Scholar). The majority of VEGF-like genes were similar to that of Orf virus strain NZ2. Although these could be grouped as NZ2-like VEGFs, they showed levels of amino acid identity (average between isolates of 86.1%) more reminiscent of that seen between species of poxviruses than inter-strain variation. A few isolates carried genes identical or very nearly identical to the VEGF-like gene from Orf virus strain NZ7, which shares only 43.6% amino acid identity with the VEGF encoded by strain NZ2 (ORFVNZ2VEGF). The VEGF from another species of parapoxvirus, Pseudocowpox virus, strain VR634, shares only 41.4 and 60.8% amino acid identity with ORFVNZ2VEGF and ORFVNZ7VEGF, respectively (27Ueda N. Wise L.M. Stacker S.A. Fleming S.B. Mercer A.A. Virology. 2003; 305: 298-309Crossref PubMed Scopus (39) Google Scholar). The extent of sequence variation seen between members of the viral subfamily raises the possibility that these sequence differences might give rise to functional variations. We and others reported previously that the VEGFs from Orf virus strains NZ2, NZ7, and D1701 and that from Pseudocowpox virus strain VR634 share some of the properties of mammalian VEGF (21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar, 24Ogawa S. Oku A. Sawano A. Yamaguchi S. Yazaki Y. Shibuya M. J. Biol. Chem. 1998; 273: 31273-31282Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 25Meyer M. Clauss M. Lepple-Wienhues A. Waltenberger J. Augustin H.G. Ziche M. Lanz C. Buttner M. Rziha H.J. Dehio C. EMBO J. 1999; 18: 363-374Crossref PubMed Scopus (413) Google Scholar, 27Ueda N. Wise L.M. Stacker S.A. Fleming S.B. Mercer A.A. Virology. 2003; 305: 298-309Crossref PubMed Scopus (39) Google Scholar). However, the viral VEGFs differ from the cellular family members in their distinct receptor binding profile recognizing VEGFR-2 but not VEGFR-1 or VEGFR-3. In addition, the VEGF from Orf virus strain NZ2 has been shown to bind NP-1 (21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar). The functional studies to date have each examined only a single viral VEGF and differences in the protein expression and assay systems used in the different studies make direct comparisons difficult. In this study we quantitatively compared five of the more divergent variants of the viral VEGFs to determine whether this variation in sequence translates to quantitative differences in receptor-binding specificity and biological activity. Cloning—DNA fragments containing nucleotides 4–398, 4–398, and 4–406 of the VEGF-like genes of Orf virus strains NZ10, D1701, and NZ7, respectively, were prepared by PCR using viral DNA as the templates (23Lyttle D.J. Fraser K.M. Fleming S.B. Mercer A.A. Robinson A.J. J. Virol. 1994; 68: 84-92Crossref PubMed Google Scholar, 26Mercer A.A. Wise L.M. Scagliarini A. McInnes C.J. Buettner M. Rhiza H.J. McCaughan C.A. Fleming S.B. Ueda N. Nettleton P.F. J. Gen. Virol. 2002; 83: 2845-2855Crossref PubMed Scopus (53) Google Scholar). These fragments were inserted into the pEFBOS-I-FLAG expression vector (C. MacFarlane, Walter and Eliza Hall Institute, Melbourne) (32Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar) immediately upstream from the DNA sequence encoding the FLAG octapeptide (Scientific Imaging Systems). The proteins derived from Orf virus strains NZ2, NZ10, D1701, and NZ7 were designated ORFVNZ2VEGF, ORFVNZ10VEGF, ORFVD1701VEGF, and ORFVNZ7VEGF, respectively. Also available were equivalent vectors, containing nucleotides 4–401 from Orf virus strain NZ2 (21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar), nucleotides 4–458 from Pseudocowpox virus strain VR634 (27Ueda N. Wise L.M. Stacker S.A. Fleming S.B. Mercer A.A. Virology. 2003; 305: 298-309Crossref PubMed Scopus (39) Google Scholar), and nucleotides 4–576 of mouse VEGF164 (33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). XbaI fragments from the vectors described above, containing the VEGF coding regions and FLAG octapeptide, were subcloned into the mammalian expression vector, pAPEX-3 (34Evans M.J. Hartman S.L. Wolff D.W. Rollins S.A. Squinto S.P. J. Immunol. Methods. 1995; 184: 123-138Crossref PubMed Scopus (42) Google Scholar). Protein synthesis from these vectors would therefore give rise to secreted polypeptides that were tagged with the FLAG octapeptide at their C termini. The proteins derived from Pseudocowpox virus and mouse VEGF, were designated PCPVVR634VEGF and mVEGF164, respectively. An equivalent vector, pVDAPEXΔNΔC, containing amino acids 93–201 from human VEGF-D (10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar) expresses a truncated secreted VEGF-D polypeptide with the FLAG octapeptide at its N terminus. This protein was designated hVEGF-DΔNΔC. Expression and Purification—FLAG-tagged proteins were expressed by transfection of 293EBNA cells, with pAPEX-3 vectors with or without VEGF inserts, using FuGENE 6 (Roche Diagnostics), and the proteins were purified by affinity chromatography with M2 (anti-FLAG, Sigma) monoclonal antibody as previously described (21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar). A mock elution sample was obtained from conditioned medium from pAPEX-3 transfected 293EBNA cells that underwent an identical purification process to the FLAG-tagged proteins. Analysis and Quantification—Purified proteins were combined with SDS-PAGE sample buffer either with or without 2% 2-mercaptoethanol, boiled, and resolved by SDS-PAGE. When required proteins were transferred to nitrocellulose and visualized by detection with an M2 (anti-FLAG) antibody as described elsewhere (21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar). Serial dilutions of control proteins of known concentration (carbonic anhydrase, BSA, and VEGF-DΔNΔC), and the purified VEGF proteins were resolved by SDS-PAGE. The proteins were then stained with Coomassie blue, and the bands were quantitated using a densitometer and the Quantity One program (Bio-Rad Laboratories). A standard curve obtained for the control protein was used to determine the amount of purified VEGF protein present in each sample. Construction of VEGFR-2-Ig Fusion Proteins—DNA fragments containing the coding region for the first four Ig-like domains of human and mouse VEGFR-2 were amplified by PCR from the templates Signal pIgplus-KDR (Y. Gunji, Haartman Institute, Helsinki, Finland) (10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar, 21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar) and VEGFR2-EX-FLAG (33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), respectively. The DNA fragments were then cloned into the expression vector pAPEX-Fc-FLAG (M. Halford, Ludwig Institute of Cancer Research, Melbourne, Australia) immediately upstream from DNA sequences encoding the Fc portion of human IgG1 and the FLAG octapeptide. Protein synthesis would, therefore, give rise to secreted FLAG-tagged VEGFR-2-Ig fusion proteins. Mitogenic Assay with Human Microvascular Endothelial Cells—Human microvascular endothelial cells (HMVEC) were seeded at 104 cells per well into a 24-well plate and were allowed to adhere. Samples of purified growth factors were serially diluted in medium with reduced serum and without growth supplements. Cells were then incubated in the media for 72 h at 37 °C, and proliferation was measured by direct counting. Miles Assay for Vascular Permeability—The Miles vascular permeability assay was performed using anesthetized guinea pigs as previously described (35Miles A.A. Miles E.M. J. Physiol. (Lond.). 1952; 118: 228-257Crossref Scopus (323) Google Scholar). Animals were given an intracardiac injection of 500 μl of 0.5% Evans blue dye in PBS to introduce the dye into the blood stream. Samples of purified growth factors were diluted in PBS and 100 μl was injected intradermally into a shaved area on the back of each animal. After 30 min, animals were sacrificed and an area of skin excised. The dye was then eluted from the skin sample in formamide and quantitated by measuring the OD at 620 nm. Controls of 20 ng/ml VEGF and PBS alone were also administered to each animal to allow normalization across animals. In Vivo Assay in Sheep Skin—Each of the purified proteins were diluted to 20 ng/μl in PBS, 100 μl of which was injected intradermally at duplicate sites within the wool-free regions of the inside hind legs of two sheep. Animals were divided into three groups of two and each group was given duplicate injections of either ORFVNZ2VEGF, ORFVNZ10VEGF, and ORFVD1701VEGF, or ORFVNZ7VEGF, PCPVVR634VEGF, and hVEGF-DΔNΔC, or the control samples, FLAG-tagged ovine IL-10, Orf virus IL-10, and a mock purification sample. PBS and mVEGF164 were also administered to each hind leg of individual animals to allow comparison between groups. Boost injections of identical samples were administered to each site after 72 h. 4 mm biopsies were taken from each site and adjacent areas of untreated skin after 9 days. Biopsies were fixed in 10% neutral buffered formalin and processed into paraffin wax. Two 4 μm, serial sections were taken ∼100 μm into the biopsy within the fixed block, followed by two more serial sections, each 100 μm apart. Paired sections were then stained with hematoxylin and eosin or used for immunohistochemical analysis of vascularization. Vascular endothelial cells were identified using a polyclonal rabbit anti-human von Willebrand factor antiserum (Dako). To quantitate the extent of dermal vascularization, four grids covering a combined area of 0.2 mm2 were overlaid on areas of dermis, avoiding glands and hair follicles. The side of each grid measured 0.224 mm and was divided by 10 equidistant lines. The points at which stained endothelial cells crossed the intersecting points of the grids were counted, and the areal fraction of vascularized dermis was expressed as the fraction of the intersecting points on which stained cells fell. The average areal fraction of vascularization was determined from three semi-serial sections per inoculation site and from four equivalent samples from different animals. Binding Assays with Soluble VEGFR Extracellular Domains— VEGFR-3-Ig (K. Pajusola, Haartman Institute, Helsinki, Finland) and FLAG-tagged VEGFR-2-Ig fusion proteins were expressed in 293EBNA cells. Cells were incubated for 24 h after transfection then serum-starved for 24 h in Dulbecco's modified Eagle's medium containing 0.2% BSA. The fusion proteins were precipitated from the clarified conditioned medium using protein A-Sepharose beads. Purified VEGFR-1-Ig fusion protein (R&D Systems) was also precipitated using protein A-Sepharose beads. 900 μl of conditioned medium from 293EBNA cells that had been transfected with expression plasmids encoding one of the growth factors or a vector control and then biosynthetically labeled with [35S]Cys for 6 h were combined with 100 μl of 10 X Binding Buffer (5% BSA, 0.2% Tween 20, and 10 μg/ml heparin (VEGFR-2 only)) and either anti-FLAG (M2) or VEGFR-Ig fusion protein-bound beads. After 3 h at room temperature, the Sepharose beads were washed three times with binding buffer at 4 °C. Bound proteins were released by boiling in SDS-PAGE sample buffer and resolved by SDS-PAGE under reducing conditions. Radiolabelled proteins were detected by a phosphorimaging analyzer (Molecular Dynamics) and were quantitated using the program Quantity One (Bio-Rad Laboratories). Levels of binding to the Ig fusion proteins were quantitated and expressed as a relative binding index, a ratio of the amount of labeled protein precipitated by each receptor, compared with that precipitated by anti-FLAG beads alone. Bioassays for the Binding and Cross-linking of the Extracellular Domains of VEGFR-2 and VEGFR-3—Bioassays for monitoring the binding and cross-linking of VEGFR-2 and VEGFR-3 involved the use of cell lines expressing chimeric receptors consisting of the extracellular, ligand-binding domains of mouse VEGFR-2 or human VEGFR-3 and the transmembrane and cytoplasmic domains of the erythropoietin receptor (33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 36Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (99) Google Scholar). The chimeric receptors are expressed by a Ba/F3-derived cell line that survives and proliferates in the presence of interleukin-3 (IL-3) but which dies after IL-3 deprivation. It has previously been shown that signaling from the cytoplasmic erythropoietin receptor domain of the chimeric receptors upon ligand binding is capable of rescuing these cells in the absence of IL-3, thereby allowing detection of specific ligand binding and cross-linking of the extracellular domains of these receptors (10Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1020) Google Scholar, 21Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar, 27Ueda N. Wise L.M. Stacker S.A. Fleming S.B. Mercer A.A. Virology. 2003; 305: 298-309Crossref PubMed Scopus (39) Google Scholar, 33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 36Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (99) Google Scholar, 37Baldwin M.E. Catimel B. Nice E.C. Roufail S. Hall N.E. Stenvers K.L. Karkkainen M.J. Alitalo K. Stacker S.A. Achen M.G. J. Biol. Chem. 2001; 276: 19166-19171Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 38Makinen T. Veikkola T. Mustjoki S. Karpanen T. Catimel B. Nice E.C. Wise L. Mercer A. Kowalski H. Kerjaschki D. Stacker S.A. Achen M.G. Alitalo K. EMBO J. 2001; 20: 4762-4773Crossref PubMed Scopus (675) Google Scholar). Samples of purified growth factors were serially diluted in cell culture medium deficient in IL-3. The bioassay cell lines were then incubated in the supplemented media for 48 h at 37 °C. DNA synthesis was quantitated by including 1 μCi of [3H]thymidine during the final 4 h of incubation followed by harvesting using an automated cell harvester (Tomtec®). Incorporated [3H]thymidine was measured by β-counting (Canberra Packard Top Count NXT™ scintillation

Referência(s)