Eliot M. Rosen

Regulation of angiogenesis

Angiogenesis and angiogenesis inhibition: An overview.- Significance of angiogenesis in human disease.- Role of the macrophage in angiogenesis-dependent diseases.- Angiogenesis in human gliomas: Prognostic and therapeutic implications.- Regulation of angiogenesis in malignant gliomas.- Lymphangiogenesis: Mechanisms, significance and clinical implications.- Angiogenesis as a biologic and prognostic indicator in human breast carcinoma.- Molecular mechanisms of angiogenesis regulation.- Control of angiogenesis by cytokines and growth factors.- Fibroblast growth factors as angiogenesis factors: New insights into their mechanism of action.- Regulation of angiogenesis by scatter factor.- Vascular endothelial growth factor: Basic biology and clinical implications.- Vascular permeability factor/vascular endothelial growth factor: A multifunctional angiogenic cytokine.- Angiogenesis inhibition.- Angiostatin: An endogenous inhibitor of angiogenesis and of tumor growth.- Thrombospondin as a regulator of angiogenesis.- Regulation of angiogenesis by cell-matrix cell-cell and other interactions.- Regulation of capillary formation by laminin and other components of the extracellular matrix.- Hypoxia and angiogenesis in experimental tumor models: Therapeutic implications.- The role of vascular cell integrins ?v?3 and ?v?5 in angiogenesis.- Role of fibrin and plasminogen activators in repair-associated angiogenesis: In vitro studies with human endothelial cells.- Tumor angiogenesis: Functional similarities with tumor invasion.- Control of angiogenesis by the pericyte: Molecular mechanisms and significance.

Smooth muscle releases an epithelial cell scatter factor which binds to heparin

SummaryWe report that culture bovine calf aorta and human adult iliac artery smooth muscle cells release a soluble factor which causes spreading and separation of cells in normally tight, cohesive epithelial colonies, similar to the morphologic changes induced by the fibroblast-derived scatter factor (SF). Smooth muscle-derived SF was heat sensitive, trypsin labile, and nondialyzable, consistent with a protein (or proteins). Its effects on epithelium were not mimicked by a variety of proteolytic enzymes, growth factors, or hormones, and were not blocked by antiproteases or by antibodies to fibronectin and basic fibroblast growth factor. Epithelial cell proliferation was unaffected or only mildly stimulated by partially purified SF at concentrations that produced cell scattering. Both smooth muscle-and MRC5 human embryo fibroblast-derived SFs could be partially purified with similar elution patterns on a number of different chromatographic columns, including DEAE-agarose, heparin-sepharose, Bio-Rex 70, concanavalin A-sepharose, and MonoQ. SF from both sources bound tightly to heparin-sepharose, requiring 1.3 to 1.4M NaCl for elution. The morphologically obvious cell scattering effect was markedly inhibited by soluble heparin at concentrations down to 5 μg/ml, and this inhibition was prevented by protamine. These data suggest that vascular smooth muscle cells produce an epithelial cell scattering factor with properties similar to the fibroblast-produced factor, including a high affinity for heparin. Such factors are potentially important because they may represent a new class of proteins that primarily regulate cell mobility rather than growth and differentiation.

Protein factors which regulate cell motility

SummaryCell motility (i.e., movement) is an essential component of normal development, inflammation, tissue repair, angiogenesis, and tumor invasion. Various molecules can affect the motility and positioning of mammalian cells, including peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases. Recent studies have demonstrated a group of motility-stimulating proteins which do not appear to fit into any of the above categories. Examples include: 1)scatter factor (SF), a mesenchymal cell-derived protein which causes contiguous sheets of epithelium to separate into individual cells and stimulates the migration of epithelial as well as vascular endothelial cells; 2)autocrine motility factor (AMF), a tumor cell-derived protein which stimulates migration of the producer cells; and 3)migration-stimulating factor (MSF), a protein produced by fetal and cancer patient fibroblasts which stimulates penetration of three-dimensional collagen gels by non-producing adult fibroblasts. SF, AMF, and MSF are soluble and heat labile proteins with Mr of 77, 55, and 70 kd by SDS-PAGE, respectively, and may be members of a new class of cell-specific regulators of motility. Their physiologic functions have not been established, but available data suggest that they may be involved in fetal development and/or tissue repair.

Scatter factor stimulates migration of vascular endothelium and capillary-like tube formation.

Scatter factors (SFs) are heat- and trypsin-sensitive cytokines secreted by fibroblastic and vascular smooth muscle cell lines which stimulate motility of normal epithelium, carcinoma cells, and vascular endothelium. Human and mouse SFs have been purified and identified as 90 kD heterodimeric proteins consisting of heavy (58 kD) and light (31 kD) disulfide-bonded subunits. Partial amino acid sequence data from SF-derived tryptic peptides indicate marked sequence homology with hepatocyte growth factors, suggesting a common multigene family. In this chapter we describe the regulation by SF of vascular endothelial cell chemotaxis and chemokinesis; migration from microcarrier beads to flat surfaces; invasion through porous filters coated with reconstituted basement membrane; secretion of plasminogen activator; and in vitro capillary-like tube formation on a basement membrane surface.

Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells.

SummaryUsing an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C2, and H5–6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of “fast” and “slow” moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C2 and H5–6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C2 and H5–6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.

Interleukin-6 stimulates motility of vascular endothelium

Interleukin-6 (IL-6) is a cytokine which regulates host response to injury. Various preparations of recombinant human IL-6 stimulated migration of bovine brain and bovine aortic endothelial cells, with maximal responses at 100-600 ng/ml. The migration response was inhibited by anti-IL-6 monoclonal antibody. IL-6 also inhibited endothelial cell proliferation in a dose-dependent fashion. Combinations of IL-6 and tumor necrosis factor induced additive stimulation of migration. Studies with inhibitors and stimulators of various metabolic processes suggest that IL-6-induced motility: 1) does not require a pertussis toxin-sensitive G-protein, protein kinase C, or DNA synthesis; and 2) is regulated differently from the motility induced by scatter factor. A possible role for IL-6 in the regulation of physiologic angiogenesis is discussed.

Purification, characterization and mechanism of action of scatter factor from human placenta

Scatter factor (SF) causes contiguous sheets of epithelium to spread and cells to separate from each other. SF also increases the velocity, area, and reduces the circularity of individual cells. These changes are mediated in part by alterations in protein synthesis, protein phosphorylation, cytoskeletal reorganization, and cell surface components. SF has been purified from the conditioned medium of ras transformed 3T3 cells and human placenta. Sequence information suggests that SF from 3T3 cells is closely related to hepatocyte growth factor. SF is a glycoprotein, but glycosylation is not necessary for its activity. Glycosylation of target cell proteins, however, is required for SF action.

Scatter factor is a glycoprotein but glycosylation is not required for its activity

Scatter factor (SF) is a protein produced by fibroblasts, smooth muscle cells, and human placenta which scatter cohesive epithelial cell colonies and increases cellular motility. SF bound to concanavalin A and other lectins with high affinity. SF could also be stained with a glycoprotein specific stain. Incubation of producer cells (N-ras-transformed 3T3), with tunicamycin homolog A1 did not have any significant effect on the secretory activity of SF. The treatment of SF with N- and O-glycanases as well as endoglycosidase H had no effect on its activity. However, treatment of target (Madin Darby canine kidney) cells with tunicamycin A1, abolished the scattering response. These studies suggest that scatter factor is a glycoprotein, but glycosylation is not required for its secretion or activity by the producer cells; however, glycosylation of proteins in the target cells is required for SF action.

Rat Placental Hepatocyte Growth Factor/Scatter Factor: Purification, Characterization, and Developmental Regulation

Abstract In view of significant species-specific sequence differences between human and rat placental hepatocyte growth factor (HGF)/scatter factor (SF), the rat placental HGF/SF (rpSF) was purified, and its properties compared with human placental HGF/SF (hpSF). Like hpSF, rpSF scattered Madin-Darby canine kidney cells at 1-2 ng/ml and is composed of two subunits of 60 kDa and 30 kDa. Higher amounts (>50%) of uncleaved 90-kDa form was present in the HGF/SF preparations from both human and rat placentas. Rat placental SF reacts with antibodies raised against hpSF in rabbits and chickens. The SF activity when expressed per gram rat placental tissue rises rapidly up to 9 days and then levels off. When expressed per milligram tissue protein it also increases rapidly up to 9 days and then declines. The expression of HGF/SF mRNA during development parallels that of HGF/SF activity. The specific activity of HGF/SF receptor (c-met) mRNA also appears to peak at 6 days. These findings suggest that (i) in spite of significant (>10%) sequence differences between rpSF and hpSF, they exhibit similar structural, biologic, and immunologic characteristics and (ii) HGF/SF and its receptor are expressed in high amounts on Day 6 and then decline in developing placenta.

Binding of scatter factor to epithelial cell membrane protein: identification of its receptor

Binding of scatter factor (SF) to the surface protein of Madin-Darby canine kidney (MDCK) cells was investigated. The factor has a specific affinity for membrane proteins of MDCK cells and could be purified 10-20-fold using a membrane protein-affinity chromatographic procedure. The binding was pH- and salt-dependent. The factor did not bind to columns prepared with membrane proteins from non responder cells or with bovine serum albumin. Further purification to homogeneity was achieved using reverse phase and immunoaffinity chromatography. The factor dissociated into 92, 62 and 34/32 kDa bands on SDS-PAGE under reducing conditions. A 230 kDa protein band, the receptor-SF complex, was observed when radiolabeled SF was crosslinked to surface proteins of MDCK cells and the complexes were subjected to electrophoresis. The binding of radiolabeled SF to the MDCK cells was decreased in presence of excess unlabeled SF. These observations suggest that the binding of SF to surface proteins of MDCK cells is specific and occurs predominantly to a 150 kDa protein.