Denis Gospodarowicz

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

Factors involved in the modulation of cell proliferation in vivo and in vitro: The role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells

SummaryThe control of proliferation of mesoderm-derived cells by EGF and FGF has been examined taking, as an example, the vascular endothelium. The mechanisms by which cell proliferation can be brought to a stop in vivo and in vitro have been reviewed.

Vascular endothelial growth factor: A new member of the platelet-derived growth factor gene family

Using applications of the polymerase chain reaction (PCR) technique, cDNA clones have been isolated encoding bovine vascular endothelial growth factor (VEGF), a mitogen with specificity for vascular endothelial cells. Analysis of the clones indicates that VEGF can exist in two forms, probably due to alternative RNA splicing. The amino acid sequences predicted from the clones also show that VEGF shares homologies of about 21% and 24% respectively with the A and B chains of human platelet-derived growth factor (PDGF), and has complete conservation of the eight cysteine residues found in both mature PDGF chains. The homology is not reflected in function, however, since the cell types responsive to VEGF are distinct from those responsive to homo- and heterodimers of the PDGF chains.

Stimulation of corneal endothelial cell proliferation in vitro by fibroblast and epidermal growth factors

Abstract Bovine corneal endothelial cell culture has been developed to study the factors controlling corneal endothelium proliferation. Cells were prepared by scraping the posterior corneal surface gently with a grooved director. The endothelial segments were then suspended in DME with 10% calf serum and 100 ng/ml of fibroblast growth factor (FGF). After 4 days, corneal endothelial cells attached to the tissue culture dish, giving rise to colonies of small, rapidly dividing cells. Fixed cultures stained with silver nitrate to accentuate the inter-cellular function revealed the closely apposed nature of the polygonal cells within the monolayer, and the ovoid nucleus positioned eccentrically in each cell. This morphology is similar to that observed with the corneal endothelium in vivo. The ultrastructure of the bovine corneal endothelial cells grown in vitro was typical of cells with high metabolic rates. Cells had numerous mitochondria, polysomes and microfilament bundles. Ultrastructure and histochemical staining of the culture with alcian green demonstrated that the cells maintained in culture produced extracellular collagenous material. Both fibroblast growth factor (FGF) and epidermal growth factor (EGF) are mitogenic in vitro for endothelial cells derived from bovine corneas. Cells maintained at low density in the presence of 5% bovine plasma divided with a doubling time of 48 hr, but addition of either EGF or FGF reduced the cell doubling time to 20–24 hr. The final cell density reached in the presence of either growth factor was ten times that of cells maintained in 5% plasma alone. In the presence of 10% calf serum, corneal endothelial cells reached a final density identical to that obtained in 1% serum plus FGF and addition of FGF to 10% serum resulted in a final density three times higher than that observed with 10% serum alone. Inclusion of FGF in DME medium containing serum permitted cloning of corneal endothelial cells from cultures seeded at low density (0·6 cells/cm 2 ). The half-maximal response for the stimulation of proliferation with EGF was seen at 1·5 × 10 −10 m and with FGF at 6 × 10 −10 m . Both EGF and FGF stimulated DNA synthesis in resting corneal endothelial cells, with EGF effective from 1·5 × 10 −15 to 1·5 × 10 −13 m and FGF effective from 7 × 10 −12 to 7 × 10 −9 m . Autoradiography demonstrated that FGF and EGF, like serum, stimulated the cell population as a whole to initiate DNA synthesis.

Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix versus plastic.

Growth of human tumor cells (hepatocarcinoma, Ewings sarcoma) on an extracellular matrix (ECM) produced by bovine corneal endothelial cells is associated with the adoption of a morphological appearance and growth properties that are not expressed when the cells are maintained on plastic. Within minutes after seeding cell aggregates onto an ECM, the aggregates attached firmly. Active cell migration leading to the formation of flattened and nonoverlapping cell clusters was subsequently observed. In contrast, no firm attachment, migratory activity or disorganization of cell aggregates was observed when the same cells were maintained on plastic. Cells seeded on ECM, instead of growing as floating or loosely attached aggregates, formed a cell monolayer composed of firmly attached, highly flattened and closely apposed epithelioid-like cells. Cell overlapping and subsequent detachment were observed only late at confluence. Cells maintained on ECM had a higher growth rate as well as a lower serum requirement than those maintained on plastic. These results demonstrate that the phenotypic expression as well as the proliferation of tumor cells can be modulated by their adhesive interaction with the extracellular matrix. Both tumor cells and normal cells of epithelial origin are more likely to resemble their in vivo counterparts when maintained on extracellular matrix than on plastic, and when so maintained can therefore provide a better model for oncogenic studies.

The angiogenic activity of the fibroblast and epidermal growth factor.

Abstract The angiogenic activities of the fibroblast and epidermal growth factors in vivo have been analyzed using the rabbit cornea as a model. The angiogenic activity of slow-release form of Elwax 40 containing either fibroblast growth factor (FGF) or epidermal growth factor (EGF) at concentrations ranging from 0·5 to 60 μg were compared. A positive response was observed with concentrations of EGF as low as 1 μg per implant. The optimal concentration causing neovascularization in 100% of the cases was 10 μg per implant. With FGF, a positive response was observed at 2·5 μg per implant and the optimal concentration causing neovascularization in 100% of the cases was 25 μg per implant. In both cases, and in contrast with implants of serum albumin, the neovascularization could be seen to progress in the absence of inflammation.

Primary structure of bovine brain acidic fibroblast growth factor (FGF)

The major anionic mitogenic polypeptide for endothelial cells, acidic fibroblast growth factor (FGF), has been purified to homogeneity from bovine brain and its complete primary structure established by gas-phase sequence analysis. The 140 amino acid (Mr 16,000) protein has been previously shown to be a potent growth factor for many diverse cell types of mesodermal origin, in vitro, and an angiogenic agent, in vivo. The amino acid sequence of bovine brain acidic FGF has a 53% absolute homology with that of bovine pituitary basic FGF suggesting that these endothelial cell mitogens are derived from a single ancestral gene.

Extracellular Matrix and Control of Proliferation of Vascular Endothelial Cells

Bovine vascular endothelial cells plated at low cell density in the presence of high (10%) concentrations of serum and maintained on plastic tissue culture dishes proliferate slowly. If the cultures were exposed to fibroblast growth factors (FGF), the cells proliferated actively and, after a week, a monolayer composed of closely apposed and highly contact-inhibited mononucleated cells formed. In contrast to cultures maintained on plastic, cultures maintained on dishes coated with an extracellular matrix produced by corneal endothelial cells proliferated rapidly and no longer required FGF to reach confluence. Addition of FGF to such cultures did not decrease the mean doubling time, which was already at a minimum (18 h), nor did it result in a higher final cell density, which was already at a maximum (700-1,000 cells/mm(2)). Likewise, although human umbilical vein endothelial cells plated at low density on plastic did not proliferate, they proliferated rapidly when plated on dishes coated with an extracellular matrix. However, unlike bovine vascular endothelial cells, they still required FGF if the cultures were to become confluent. The ability of plasma vs. serum to sustain cell proliferation was analyzed using low density bovine-vascular endothelial cell cultures maintained either on plastic or on dishes coated with an extracellular matrix. Cells plated on plastic had a lower growth rate when exposed to plasma than to serum. In both cases, FGF was required for the cultures to become confluent. In contrast, when cells were plated on an extracellular matrix, they proliferated equally well, regardless of whether they were exposed to plasma or serum, and no longer required FGF to become confluent. Because the growth rate of the cultures maintained on an extracellular matrix was a direct function of the serum or plasma concentrations to which they were exposed, it is likely that the extracellular matrix had a permissive rather than a direct mitogenic effect on the cells. Therefore, one can conclude that the simple change of substrate from plastic to extracellular matrix will restore the sensitivity of vascular endothelial cells to physiological agents present in plasma or serum.

Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells

Cultured cells derived from bovine retinal pigment epithelium (RPE) express the basic fibroblast growth factor (bFGF) gene and they contain 3.7 and 7.0 kb bFGF gene transcripts which are translated into immunoreactive bFGF of Mr 17,500 and 18,000, respectively. The RPE cell-derived bFGF is bioactive, i.e., it can stimulate the proliferation of capillary endothelial cells and the stimulation of cell proliferation is blocked by anti-bFGF antibodies. We suggest that RPE cell-derived bFGF may be involved in the repair mechanisms following retinal injury and in the intravitreal pseudoneoplastic proliferation of the injured RPE.

Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin.

Follistatin, a hormone which acts to suppress the release of follicle-stimulating hormone (FSH) by pituitary-derived gonadotrophs, has previously been identified only in the liquor folliculi of ovarian follicles. By microsequencing of fractions derived from conditioned medium, we show here that bovine pituitary-derived folliculo stellate cells are also capable of producing and secreting this hormone. These results suggest that folliculo stellate cells may serve as a source of follistatin within the pituitary itself and that the regulation of FSH release from the pituitary could therefore involve a paracrine mechanism.