RAPID COMMUNICATIONS A Unique Family of Endothelial Cell Polypeptide Mitogens: The Antigenic and Receptor Cross-Reactivity of Bovine Endothelial Cell Growth Factor, Brain-derived Acidic Fibroblast Growth Factor, and Eye-derived Growth Factor-II

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [1251]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pl brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II (EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [1251] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGFII is potentiated by the glycosylaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors. Several growth factors of peptidic nature have been isolated and purified from bovine neural tissue (1, 4-6, 9, 10, 14-19, 23, 24). These polypeptides share common biological properties since they are all potent mitogens for murine BALB/c 3T3 cells in vitro. In addition, several of these polypeptide mitogens stimulate endothelial cell proliferation in vitro (1, 5, 6, 11, 13, 16, 18, 19, 23, 24) and may play an important role in homeostatic and pathophysiological processes involving the vascular tree (21). These growth factors include polypeptides of cationic nature such as basic isoelectric point (pI) ~ fibroblast growth factor (FGF) (4, 10), chondrosarcoma-de1 Abbreviations used in this paper." BAEC, bovine aortic endothelial cells; DME-BSA, Dulbecco's modified Eagle's medium, pH 7.4, containing 0.1% bovine serum albumin and 50 mM HEPES; ECGF, endothelial cell growth factor; EDGF-II, eye-derived growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; HDGF, hypothalamus-derived growth factor; HUVEC, human umbilical vein endothelial cell; HGF, heparin-binding growth factor; pI, isoelectric point; RDGF, retina-derived growth factor. rived growth factor (23), and basic pI hypothalamus-derived growth factor-I (HDGF), as well as polypeptide mitogens with anionic isoelectric points. These acidic growth factors include endothelial cell growth factor (ECGF) (18, 19), acidic pI brainderived FGF (9, 24), eye-derived growth factor-II (EDGF-II) (1-3), retina-derived growth factor (RDGF) (6), a-heparinbinding growth factor (a-HGF) (18), and acidic pI HDGF (15). Radioreceptor and immunological assays have recently been developed for the acidic polypeptide, ECGF (20, 22). The availability of purified growth factors and these characterization methods prompted us to determine whether these endothelial cell mitogens share common immunological and cell receptor recognition features. We now report that both acidic FGF and EDGF-II compete with ECGF for receptor occupancy and cross-react with monoclonal antibodies prepared against ECGF. Furthermore, the mitogenic activity of the acidic polypeptide growth factors acidic FGF and EDGFII on endothelial cells is potentiated by the glycosaminoglycan, heparin, a biological attribute unique to ECGF (22). THE JOURNAL OF CELL BIOLOGY • VOLUME 101 OCTOBER 1985 1623-1626 1623 © The Rockefeller University Press 0021-9525/85/1011623/04 $1.00 on July 7, 2017 jcb.rress.org D ow nladed fom MATERIALS AND METHODS Reagents: Epidermal growth factor (EGF) was purified from male mouse submaxillary glands by reverse-phase high pressure liquid chromatography as previously described (22). Basic FGF, purified to homogeneity from bovine pituitary glands (4) was a generous gift from Dr. Denis Gospodarowicz and ECGF (M, 20,000) and acidic FGF (M, =17,000) was purified from bovine brain by procedures previously described (20, 24) (Burgess, W. H., T. Mehlman, R. Friesel, W. Johnson and T. Maciag, J. Biol. Chem., In press). Purified preparations of EDGF-II were obtained from bovine retina by extraclion and Afti-Blue chromatography as described (3), followed by elution of EDGF-II from heparin-Sepharose between 0.9 and I. l M NaCl (20). Murine monoclonal antibodies and rabbit polyclonal antisera against ECGF were prepared as previously described (20, 22). The monocional antibodies H9, an IgM(k), and H 15 an IgG2(k) are neutralizing antibodies for ECGF (22). Heparin and bovine serum albumin (BSA) were obtained from Armour Pharmaceutical Co. (Tarrytown, NY) and heparin-Sepharose 6B was purchased from Pharmacia Fine Chemicals (Piseataway, N J). Cells: Human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) were established from primary cultures. HUVEC were grown on human fibroncetin--coated (5 #g/cm 2) (Armour Pharmaceutical Co.) cell culture dishes in Dulbecco's modified Eagle's medium (DME) containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 2 ng/ml ECGF containing 20 tzg heparin/ml. BAEC were propagated in DMEM containing 10% fetal bovine serum. The endothelial cell character of the BAEC and HUVEC populations was confirmed by the presence of human Factor VIII:Ag and membrane anglotensin-converting enzyme (monoclonal antibody against angiotensin-converting enzyme kindly provided by Dr. R. Auerbach) as previously described (20). Cell Proliferation Assay: The low seed density human endothelial cell proliferation assay was performed with HUVEC as previously described (2 l, 25). Briefly, HUVEC were seeded ( lO a cells/cm 2) on human fibronectin (5 ug/cm 2) in 96-well cluster dishes (Costar, Cambridge, MA) in DME supplemented with 10% FBS. Mitogens were added for 3 d at 37"C prior to protein determination as described in Results. The incorporation of [aH]methylthymidine (New England Nuclear, Boston, MA) into DNA in quiescent BAEC was performed as previously described for routine lung capillary endothelial cells