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...
Vascular endothelial growth factor (VEGF) is a potent and selective mitogen for endothelial cells that is angiogenic in vivo and induced by hypoxia. A homologous protein, placenta growth factor (PlGF), is also reported to be mitogenic for endothelial cells in culture. The rat GS-9L glioma cell line produces not only VEGF homodimers but also PlGF homodimers and a novel heterodimer composed of VEGF and PlGF subunits. All three dimeric forms were purified to apparent homogeneity, and their structures and mitogenic activities were compared. VEGF.PlGF heterodimers are vascular endothelial cell mitogens nearly as potent as VEGF homodimers. Therefore, some of the biological activities attributed to VEGF homodimers might be mediated by VEGF.PlGF heterodimers. In contrast, pure PlGF homodimers are mitogenic for endothelial cells only at high, possibly non-physiologic concentrations; thus the biological relevance of their mitogenic activity for these cells is not obvious. However, the existence of not only homodimers but also heterodimers clearly extends the similarity between the VEGF/PlGF and the homologous platelet-derived growth factor systems.
Chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) in the presence of cytochalasin B stimulates the release of leukotriene B4 (LTB4), superoxide (02) The influx and activation of neutrophils [PMN (polymorphonuclear leukocytes)] are essential for induction of inflammatory processes (1, 2). The chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) and products of complement activation such as Ca recruit and activate the PMN to release active oxygen species and undergo degranulation (2,3). Such actions in concert with increased levels of prostaglandin E2 (PGE2) and prostacyclin (PGI2) lead to the cardinal symptoms of acute inflammation (3)(4)(5). Leukotriene B4 (LTB4), a product of the 5-lipoxygenase pathway, is itself a potent chemoattractant for PMN (6) and promotes their accumulation and adherence to the vascular endothelium proximal to the area of inflammation (3,7). Whether these actions of LTB4 supplement or mediate the effects of other chemoattractants remains unknown.Pharmacological levels of PGs inhibit superoxide (02) production induced by fMet-Leu-Phe in the presence of cytochalasin B (2) by an action purported to depend on increased cyclic AMP production (8). A similar cyclic AMP-dependent inhibition of lysosomal enzyme release has been observed by using large amounts of PGs (2, 8). Pharmacological levels of PGI2 (2.8 X 10-6 M) have also been reported to cause a partial inhibition (45%) of LTB4 release from human PMN activated with serum-treated zymosan (9).The studies in this report indicate that fMet- was obtained from J. Rokach (Merck Frosst) (10); MMM-I-135 (10,10-difluoro-13-dehydroprostacyclin) was a gift from J. Fried (University of Chicago) (11).The incubation buffer contained 130 mM NaCl, 5.5 mM KCl, 0.6 mM Na2HPO4, 0.6 mM NaH2PO4, 10 mM glucose, 1.0 mM CaC12, and 25 mM Tris HCl adjusted to pH 7.4 (12). Preparation of Cells. Rat PMN were obtained by peritoneal lavage with 40 ml of 0.9% NaCl/10 mM EDTA, pH 7.0, 16-24 hr after aseptic induction (13). PMN were recovered by centrifugation and the pellet was washed once in calcium-free incubation buffer, subjected to lysis buffer (14) for 5 min, rewashed in incubation buffer, and resuspended at a concentration of 5 x 106 cells per ml.Human peripheral PMN were isolated from fresh citrated human blood as described (15) and lysed as were the rat cells.Both preparations were >95% PMN.Incubation Conditions. Two-milliliter aliquots of PMN suspension were preincubated with calcium for 3 min at 37°C and then PGs or solvent was added for 5 min. Cytochalasin B (5 ,ug/ ml) was next added for 5 min, followed by addition of 10 ,uM fMet-Leu-Phe for 5 min. Dimethyl sulfoxide was at a final concentration of 0.7%. The incubations were terminated by centrifugation at 260 X g for 5 min at 0°C. The supernatants were analyzed for (5S)-5-hydroxy-6,8,11,14-icosatetraenoic acid (5-HETE), LTB4, and enzymes. Cell pellets were assayed for cyclic AMP.
A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of~'1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction.This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with bloodvessel growth and maintenance. genes (1-7). All of these FGFs, with the exception of the int-2 and FGF-6 gene products, which have not been isolated, have been shown to be potent vascular endothelial cell mitogens in vitro. Furthermore, exogenously applied aFGF (8) and bFGF (9) induce angiogenesis in vivo. An angiogenic platelet-derived endothelial cell growth factor (PD-ECGF), a monomeric 45-kDa protein, has also been purified and structurally characterized. This protein, like aFGF and bFGF, lacks a secretory leader sequence and so is presumably released by lysis of platelets (10,11 We have identified and purified a vascular endothelial cell mitogen that is not only produced but also apparently secreted by cultured cells derived from a chemically induced rat glioma (13). This glioma-derived vascular endothelial cell growth factor (GD-VEGF) is structurally unrelated to the previously well-characterized mitogens for large-vessel endothelium. Pure GD-VEGF approaches the specific mitogenic activity of FGFs for cultured human umbilical vein endothelial (HUVE) cells. MATERIALS AND METHODSMitogenic Assays. HUVE cells were isolated and maintained as described (14). The cells were plated in gelatincoated 48-well tissue culture dishes (Costar) at 6000 cells per well in 400 ,ul of medium 199 (GIBCO) containing 15 mM Hepes buffer and supplemented with 20% heat-inactivated fetal bovine serum (Hazelton Research Products, Reston, VA) and antibiotics (penicillin G, 100 units/ml; streptomycin sulfate, 100lg/ml; amphotericin B, 0.25 ,ug/ml; GIBCO). Samples to be assayed were added at the time of cell plating and the tissue culture dishes were incubated at 37°C under 5% CO2. After a 12-hr incubation, 0.64 ,Ci of [methyl-3H]-thymidine (20 Ci/mmol; New England Nuclear; 1 Ci = 37 GBq) was added per well and the dishes were incubated for an additional 60 hr. The cells were then washed with Hanks' ...
Human acidic fibroblast growth factor (aFGF) is a potent broad-spectrum mitogen that contains three Cys residues within its monomeric structure. We have found that site-directed mutants in which any one of these Cys residues is converted to serine remain highly active, although variably dependent on heparin, so none of the three possible intramolecular disulfide bonds that can be formed are required for mitogenic activity. Furthermore, a dispensable disulfide bond that might stabilize the active conformation is not present since all three Cys residues are accessible to chemical modification in recombinant as well as brain-derived aFGFs. Finally, formation of a disulfide bond between the two Cys residues conserved among all seven known members of the FGF family results in a virtually inactive product that can subsequently be reactivated by reduction. Thus, despite the extracellular function of aFGF, its Cys residues do not form intramolecular disulfide bonds in the active conformation.
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