Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor.

Abstract

Glioma-derived vascular endothelial cell growth factor (GD-VEGF) is a 46-kDa dimeric glycoprotein mitogen with apparently greater specificity for vascular endothelial cells than the well-characterized fibroblast growth factors. The GD-VEGF cDNA sequence encodes a 190-amino acid residue subunit that is converted, by removal of an aminoterminal hydrophobic secretory leader sequence, to the mature 164-residue subunit characterized by direct amino acid sequencing. The GD-VEGF homodimeric subunit is homologous to the platelet-derived growth factor A and B chains and its oncogene homologue v-sis. We report here the complete cDNAt and amino acid sequence of rat GD-VEGF. The sequence confirms its identification as a secretory homodimeric glycoprotein and reveals an unexpected homology to platelet-derived growth factor (PDGF), a mitogen for connective tissues cells but not vascular endothelial cells from large vessels. GD-VEGF as described (5). Purity was confirmed on all samples by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Aliquots (1-2 ,.g) of the purified protein, quantitated by using an extinction coefficient based on amino acid analysis (5), were reduced and carboxymethylated with iodo[2-14C]acetic acid as described (8). The 14C-carboxymethylated GD-VEGF product was repurified on a 4.6 mm x 5 cm Vydac C4 reversed-phase HPLC column by elution at 20'C with a linear gradient of 0-67% acetonitrile in 0.1% trifluoroacetic acid over 30 min at a flow rate of 0.75 ml/min.Enzymatic Digestion and Polypeptide Purification. Reduced and carboxymethylated GD-VEGF (725 ng) was digested on the carboxyl-terminal side of most lysine and arginine residues with 30 ng of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin (Worthington) in 200 ,ul of 0.1 M ammonium bicarbonate (pH 8.3) for 6 hr at 37°C. The polypeptide digestion mixture was loaded directly on a 4.6 mm x 25 cm Vydac C18 reversed-phase column and fractionated by elution at 20°C with a 0-67% (vol/vol) linear gradient of acetonitrile in 0.1% trifluoroacetic acid over 2 hr at a flow rate of 0.75 ml/min. Polypeptide peaks were identified by monitoring A210 and individually collected.A similar digest was performed on 925 ng of carboxymethylated GD-VEGF by using 50 ng of Lys-C endoproteinase (Lys-C implies lysine specific) (Boehringer Mannheim) in 50 ,ul of 0.1 M Tris, pH 8.5/1 mM EDTA at 37°C for 8 hr. Polypeptide products, the result of cleavage on the carboxylterminal side of lysine residues, were purified as described for the tryptic digest.A final enzymatic digestion was done on 1.1 ,ug of carboxymethylated GD-VEGF by using 65 ng ofStaphylococcus aureus V8 protease (Miles). The substrate was dissolved in 5 ,ul of6 M guanidinium chloride/0.1% EDTA buffered with 0.7 M Tris to pH 7.8. The protease was added in 65 ,ul of 0.1% EDTA buffered to pH 8.0 with 0.1 M ammonium bicarbonate. The digest was incubated at 37°C for 48 hr, and the polypeptides, generated primarily by cleavage on the carboxylterminal side ofglutamic ac...

Overexpression of vascular endothelial growth factor induces cell transformation in cooperation with fibroblast growth factor 2

“…In contrast to TGF-b, which mainly acts through monocyte chemotaxis (Wahl et al, 1987), VEGF is categorized as a direct angiogenic factor, as it stimulates endothelial cell proliferation and migration in the absence of any other cell type (PloueÈ t et al, 1989). Unlike FGF-1 or FGF-2 (Jaye et al, 1986;Abraham et al, 1986), VEGF is a secreted peptide with a typical NH 2 -terminal signal sequence (Leung et al, 1989;Keck et al, 1989;Conn et al, 1990b). The mitogenic activity of VEGF is mainly restricted to vascular endothelial cells.…”

Evaluating effects of deferoxamine in a rat tibia critical bone defect model

“…11 Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific cytokine that has been shown to have mitogenic and chemotactic effects on endothelial cells in vitro. 12 Wang et al showed that HIFa promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. 13 In addition, VEGF has been shown to induce angiogenesis in many in vivo models.…”

Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors.

“…Three classes of VPF clones that have been isolated from various cDNA libraries (Keck et al, 1989;Leung et al, 1989;Tischer et al, 1989;Conn et al, 1990a;Berse, unpublished data) contain three coding region variants. Tischer et al (1991) have shown that the human VPF/VEGF gene consists of eight exons and that the three VPF/VEGF mRNAs arise by alternative splicing.…”

“…Alternatively, elicited macrophages were isolated from the peritoneal cavity 4-5 d after intraperitoneal injection of 20 ml sterile mineral oil (Hammond and Dvorak, 1972 (Keck et al, 1989;Leung et al, 1989). The guinea pig PCR product exhibits very high sequence homology (>90%) with human, as well as bovine and rat, VPF/VEGF cDNAs (Leung et al, 1989;Tischer et al, 1989;Conn et al, 1990a (5-50 ,ug) were size-fractionated on 1.2% agarose containing 6% formaldehyde and transferred to Nytran membranes (Schleicher and Schuell, Keene, NH). Hybridization was carried out for 24 h at 42°C in 50% formamide, 5X SSPE, 2.5X Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), and 10% dextran sulphate (Sambrook et al, 1989).…”

“…More recently, a structurally similar protein, identified as a heparin-binding mitogen specific for endothelial cells, was purified from the culture medium of bovine pituitary-derived folliculo-stellate cells (vascular endothelial growth factor or VEGF, Ferrara and Henzel, 1989; folliculo-stellate-derived growth factor or FSdGF, Gospodarowicz et al, 1989) and from rat glioma cells (glioma-derived vascular endothelial growth factor or GD-VEGF, Conn et al, 1990b). Protein sequencing and molecular cloning of human, bovine, and rat cDNAs (Keck et al, 1989;Leung et al, 1989;Tischer et al, 1989;Conn et al, 1990a) demonstrated that tumor VPF, pituitary VEGF/FSdGF, and glioma GD-VEGF are products of the same gene. The sequences also revealed limited but distinct homology of VPF/VEGF to plateletderived growth factor (PDGF).…”