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Biomedical subjects

D Gospodarowicz

Publications and source records attributed to D Gospodarowicz.

At least 73 records · Page 4Linked to original sources
PubMed1987

Basic fibroblast growth factor is present in cultured human retinoblastoma cells.

Cultured human retinoblastoma cells express the basic fibroblast growth factor (bFGF) gene and they produce material similar, if not identical, to bFGF. The retinoblastoma-derived bFGF can stimulate the proliferation of capillary endothelial cells and this process is inhibited by anti-bFGF antibodies. It is conceivable that the retinoblastoma-derived bFGF contributes to the neovascularization of retinoblastomas.

Cell Line↗
PubMed1986

Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor.

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.

Angiogenesis Inducing Agents↗
PubMed1986

Basic and acidic fibroblast growth factors interact with the same cell surface receptors.

Despite quantitative differences, the activity of basic and acidic fibroblast growth factors (FGF) on a wide variety of normal diploid cells derived from neuroectoderm and mesoderm is intrinsically similar. This suggests that they bind to the same cell surface receptors. This was investigated using a baby hamster kidney cell line (BHK-21) as a model. BHK-21 cell membrane components that interact with basic and acidic FGF have been identified by covalent cross-linking to their respective 125I-labeled ligands. Under appropriate conditions, basic and acidic 125I-FGF were cross-linked, using disuccinimidyl suberate, to two receptor species with apparent molecular masses of 145,000 and 125,000 daltons, respectively. The labeling of those receptors is inhibited when either native basic or acidic FGF are present in excess during incubation of cells with either acidic or basic 125I-FGF. Competition of basic 125I-FGF with increasing concentrations of native acidic FGF results in a preferential decrease in the labeling of the 125,000-dalton species, whereas competition of acidic 125I-FGF with increasing concentrations of native basic FGF leads to a preferential decrease in the labeling of the 145,000-dalton species. The data suggest that qualitatively both mitogens interact with the same 145,000- and 125,000-dalton receptor species. The different affinities displayed by acidic and basic FGF toward their common receptor molecules could explain why acidic FGF, depending on the cell type considered, is 20-100-fold less potent than basic FGF.

Affinity Labels↗
PubMed1986

Human basic fibroblast growth factor: nucleotide sequence and genomic organization.

Clones encoding the angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), have been isolated from human cDNA libraries made from kidney, fetal heart, fetal liver, term placenta, and a breast carcinoma. Basic FGF cDNA clones are present in these libraries at very low levels when compared to the quantity of the growth factor in the tissues. This observation, combined with the fact that several of the clones represent unspliced transcripts, suggests that cytoplasmic basic FGF mRNA is unstable and that the protein is stored in tissues. The amino acid sequence of human basic FGF, deduced from the sequence of these cDNAs and from genomic clones, is 99% homologous to that of bovine basic FGF, implying a strong selection pressure for maintenance of function and structure. As with the bovine factor, human basic FGF does not appear to have a signal peptide sequence. Southern blot analysis of human genomic DNA and mapping of the cloned gene shows that there is only one basic FGF gene. All of the basic, heparin-binding endothelial cell mitogens of similar amino acid composition that have been described must therefore be products of this single gene.

Amino Acid Sequence↗
PubMed1986

Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries.

Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.

Adrenal Cortex↗
PubMed1986

Heparin protects basic and acidic FGF from inactivation.

The ability of heparin or that of hexuronyl hexosaminoglycan sulfate (HHS-4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the growth of baby hamster kidney (BHK-21) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at -80 degrees C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65 degrees C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS-4, basic and acidic FGF retained their potency. The effect of heparin and HHS-4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS-4 concentrations. This indicates that heparin and HHS-4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.

Animals↗
PubMed1986

Effect of lipoproteins and growth factors on the proliferation of BHK-21 cells in serum free culture.

Baby hamster kidney-derived cells (BHK-21 cell line), seeded at low density on gelatin coated dishes and exposed to a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, and basic or acidic fibroblast growth factor (FGF). This serum free medium combination supported cell multiplication at a rate equal to that of serum supplemented medium, and at low cell input (10(3) cells/35-mm dish). Epidermal growth factor (EGF), although mitogenic for BHK-21 cells, was less efficient than either basic or acidic FGF in supporting cell growth. When the potency of basic and acidic FGF were compared, acidic FGF was 10-fold less potent than basic FGF. The requirement of BHK-21 cells for transferrin appears to be minimal since cells exposed to HDL and basic FGF could be serially transferred for at least 50 cumulative population doublings in the absence of transferrin.

Animals↗
PubMed1986

Effect of retina-derived basic and acidic fibroblast growth factor and lipoproteins on the proliferation of retina-derived capillary endothelial cells.

Retina-derived capillary endothelial (RCE) cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM)-coated dishes in the presence of serum-supplemented medium. This cell type formed at confluence a monolayer of small, tightly packed, contact inhibited cells which expressed Factor VIII-related antigen. Both retina-derived basic and acidic fibroblast growth factors (FGF) were mitogenic when RCE cells were maintained on gelatin-coated dishes and exposed to serum-supplemented medium. Half-maximal stimulation of cell proliferation was observed with concentrations of 13 pg ml-1 for basic FGF and 2.5 ng ml-1 for acidic FGF. Maintaining RCE cells on extracellular matrix (ECM)-coated dishes greatly reduced their requirement for EGF in order to proliferate actively. Heparin strongly reduced the proliferative response of RCE cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on gelatin or on ECM-coated dishes. When RCE cells maintained on ECM-coated dishes were exposed to defined medium, high density lipoproteins, transferrin, and FGF were required in order for these cells to proliferate actively.

Animals↗
PubMed1986

Isolation of fibroblast growth factor from bovine adrenal gland: physicochemical and biological characterization.

The angiogenic growth factors present in the bovine adrenal gland have been purified by a combination of differential salt precipitation, ion exchange chromatography, and heparin-Sepharose chromatography. They consist of 2 single chain polypeptides with apparent mol wt of 16,000 and 15,000. Sequence analysis of the first 14 residues of both peptides identified the sequences as Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro for 1 of the peptides and His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-x-Lys-Asn-Gly-Gly for the other. The first sequence is identical to residues 1-14 of bovine pituitary and brain fibroblast growth factor (FGF), while the second is identical to residues 1-14 of the corpus luteum (CL) FGF, which is an amino-terminally truncated form of FGF and is otherwise similar, if not identical, to FGF. The biological activity of adrenal FGF is indistinguishable from that of pituitary or brain FGF and CL FGF. They are highly active in triggering the proliferation of culture bovine vascular endothelial cells derived from either large vessels (aortic arch) or CL and adrenal cortex capillaries (half-maximal stimulation at 20-40 pg/ml and saturation at 400-600 pg/ml). In vivo implants containing 50 ng to 1 microgram adrenal-derived growth factors stimulate neovascularization in the chorioallantoic membrane of the chick embryo. In addition to being mitogenic for vascular endothelial cells, adrenal FGFs stimulate the proliferation of a wide variety of mesoderm- and neuroectoderm-derived cells, including vascular smooth muscle cells, granulosa and adrenal cortex cells, rabbit costal chondrocytes, and corneal endothelial cells.

Adrenal Cortex↗
PubMed1985

Retina- and eye-derived endothelial cell growth factors: partial molecular characterization and identity with acidic and basic fibroblast growth factors.

Two retina-derived growth factors have been isolated on the basis of their ability to stimulate the proliferation of capillary endothelial cells in vitro. Gas-phase sequence analysis identified the amino-terminal sequence of the major form of the mitogen as being identical with residues 1-35 of bovine basic fibroblast growth factor (FGF). Amino-terminal sequence analysis of the second form identified 28 residues that are indistinguishable from those of brain acidic FGF (residues 1-28). The possibility that these retina-derived endothelial cell growth factors are related to, if not identical with, basic and acidic FGF is supported by observations that they have similar molecular weights (15000-16000), similar retention behavior on all steps of chromatography (ion-exchange, heparin-Sepharose), and similar amino acid compositions and that they cross-react with antibodies to basic and acidic FGF. The eye-derived growth factors, like FGF, are potent stimulators of capillary endothelial cell growth in vitro. The results identify the major retina-derived endothelial cell growth factor as indistinguishable from basic FGF and demonstrate the presence of an acidic FGF in the eye. They suggest that at least some of the mitogenic, angiogenic, and neovascularizing activities described as being present in the retina are due to the existence of FGF in this tissue. The implications of this finding on the etiology and pathophysiology of vasoproliferative diseases of the eye are discussed.

Amino Acid Sequence↗
PubMed1985

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.

Amino Acid Sequence↗
PubMed1985

Isolation and partial characterization of an endothelial cell growth factor from the bovine kidney: homology with basic fibroblast growth factor.

Two kidney-derived mitogens have been isolated by ion exchange, heparin-Sepharose and reverse-phase high-performance liquid chromatography on the basis of their capacity to stimulate the proliferation of bovine vascular endothelial cells in vitro. Gas phase sequence analysis identified the amino terminal sequences His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-Phe-Phe-Leu and His-Phe-Lys-Asp-Pro-Lys-Arg-Leu, respectively. The sequences are identical to residues 16-32 and 16-23 of bovine basic pituitary Fibroblast Growth Factor (FGF). The possibility that these kidney-derived mitogens are related, if not identical, to pituitary basic FGF is supported by the observations that they have similar molecular weights (15-16 kDa), similar retention behavior on all steps of chromatography and similar amino acid compositions, and they share at least some structural homology. Moreover, the kidney-derived growth factors, like basic FGF, are potent stimulators of capillary endothelial cells, granulosa cells, adrenocortical cells and vascular smooth-muscle cells (ED50 = 50 pg). The results demonstrate the existence of a kidney-derived FGF and suggest that at least some of the mitogenic, angiogenic and neovascularising activities described to be present in the kidney are due to the presence of an FGF-like molecule in this tissue.

Amino Acids↗
PubMed1985

The identification and partial characterization of the fibroblast growth factor receptor of baby hamster kidney cells.

The binding of biologically active, 125I-labeled basic fibroblast growth factor (FGF) to baby hamster kidney-derived cell line cells (BHK-21) was studied at 4 degrees C. Unlabeled FGF displaced cell surface bound 125I-FGF, but platelet-derived growth factor, epidermal growth factor, insulin, or transferrin did not. Binding was saturable both as a function of time and as a function of increasing 125I-FGF concentrations. Scatchard analysis of the binding data revealed the presence of about 1.2 X 10(5) binding sites/cell with an apparent KD of 270 pM. The number of the binding sites was down-regulated following preincubation of the cells with FGF. The density of binding sites/cell also decreased as an inverse function of cell density. When 125I-FGF binding was studied in a BHK-21 cell membrane preparation, it was found that the membranal binding site displayed a lower KD of 21 pM. 125I-FGF was covalently cross-linked to its cell surface receptor on intact BHK-21 cells using the homobifunctional agent disuccinimidyl suberate. Two macromolecular species with an apparent molecular weight of 145,000 and 125,000, respectively, were labeled under both reducing and nonreducing conditions. Unlabeled FGF competed with 125I-FGF for binding to both macromolecular species. The labeling of the macromolecules was also inhibited by heparin. No labeling was observed in the absence of the cross-linkers or when heat-inactivated 125I-FGF was used instead of radiolabeled, biologically active FGF.

Animals↗
PubMed1985

Human brain fibroblast growth factor. Isolation and partial chemical characterization.

Fibroblast growth factor (FGF) has been purified to homogeneity from human brain by a procedure involving salt precipitation, cation-exchange chromatography, Heparin-Sepharose affinity chromatography and reverse-phase HPLC. Isolation was monitored by radioimmunoassay and/or by testing column fractions for their capacity to stimulate the proliferation of vascular endothelial cells in vitro. The amino-terminal sequence of human brain FGF was determined as Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-. This sequence is identical to that of the amino-terminal region of bovine FGF. Additional evidence, including amino acid composition, chromatographic retention behavior, and immunoreactivity suggest that the human and bovine mitogens are very similar in structure.

Amino Acid Sequence↗
PubMed1985

Fibroblast growth factor in the human placenta.

Fibroblast growth factor (FGF) has been purified 333,000-fold from human placenta by a combination of salt precipitation, cation-exchange chromatography, and Heparin-Sepharose affinity chromatography. Molecular weight (15-16 kDaltons), amino acid composition, bioactivity and immunological crossreactivity with bovine pituitary FGF indicate that the mitogens from the two species are closely related molecules.

Amino Acids↗
PubMed1985

Stimulation by glucocorticoid of the synthesis of cartilage-matrix proteoglycans produced by rabbit costal chondrocytes in vitro.

The effect of glucocorticoids on sulfated proteoglycan synthesis by rabbit costal chondrocyte cultures exposed to serum-free conditions has been examined. Low density cultures of rabbit costal chondrocytes were maintained on dishes coated with extracellular matrix produced by bovine corneal endothelial cells and exposed to a 9:1 mixture (v/v) of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with transferrin, high density lipoproteins, fibroblast growth factor, and insulin (Medium A). Chondrocytes maintained in the presence of Medium A supplemented with 10(-7) M hydrocortisone reorganized, at confluence, into a homogeneous cartilage-like tissue composed of round cells surrounded by a refractile matrix in which abundant thin collagen fibrils characteristic of type II collagen were observed. The cell ultrastructure and fibrils of the pericellular matrix were similar to those seen in vivo. In contrast, cells maintained in the presence of Medium A alone, once they reached confluence, formed a fibroblastic multilayer and produced thick collagen bundles. The level of 35SO4(2-) incorporated into large cartilage-specific proteoglycans in glucocorticoid-supplemented cultures was 33-fold higher than that of glucocorticoid-free cultures. The level of 35SO4(2-) incorporated into small ubiquitous proteoglycans was only 4-fold higher than that of glucocorticoid-free cultures. On the other hand, the level of [3H]glucosamine incorporated into hyaluronate in glucocorticoid-supplemented cultures was 4.5-fold lower than that of glucocorticoid-free cultures. Within 24 h of their addition to confluent cultures, hydrocortisone or dexamethasone markedly stimulated proteoglycan synthesis. This effect was not mimicked by androgens, estrogens, progesterone, or an inactive form of glucocorticoids such as deoxycorticosterone. This suggests that glucocorticoids have a direct and specific stimulatory effect on cartilage-specific proteoglycan synthesis and are essential for the maintenance of this synthesis in low density chondrocyte cultures.

Animals↗