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Biomedical subjects

D Moscatelli

Publications and source records attributed to D Moscatelli.

At least 37 records · Page 2Linked to original sources
PubMed1990

Growth factor control of extracellular proteolysis.

The involvement of proteases and growth factors in angiogenesis is complex. The angiogenic factor basic fibroblast growth factor (bFGF) induces increased synthesis of both plasminogen activator and collagenase in endothelial cells. In addition, bFGF increases the number of plasminogen activator receptors on the cell surface. Increased production of plasmin may be responsible for the release of soluble complexes of heparan sulfate-bFGF which may be the active form of bFGF. The activity of a negative regulator of angiogenesis, transforming growth factor beta (TGF-beta), is also regulated by proteases since the released latent form of TGF-beta is activated by a surface proteolytic assembly plasminogen activator and plasmin. Since TGF-beta induces an inhibitor of plasminogen activator, the activation reaction is self-regulatory.

Animals↗
PubMed1990

A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF.

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.

Amino Acid Sequence↗
PubMed1990

Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor.

The radius of diffusion of basic FGF (bFGF) in the presence and in the absence of the glycosaminoglycans heparin and heparan sulfate was measured. Iodinated 125I-bFGF diffuses further in agarose, fibrin, and on a monolayer of bovine aortic endothelial (BAE) cells in the presence of heparin than in its absence. Heparan sulfates affected the diffusion of 125I-bFGF in a manner similar to, though less pronounced than, heparin. When applied at the center of a monolayer of BAE cells, bFGF plus heparin stimulated morphological changes at a 10-fold greater radius than bFGF alone. These results suggest that bFGF-heparin and/or heparan sulfate complexes may be more effective than bFGF alone in stimulating cells located away from the bFGF source because the bFGF-glycosaminoglycan complex partitions into the soluble phase rather than binding to insoluble glycosaminoglycans in the extracellular matrix. Thus, the complex of bFGF and glycosaminoglycan may represent one of the active forms of bFGF in vivo.

Animals↗
PubMed1990

Turnover of functional basic fibroblast growth factor receptors on the surface of BHK and NIH 3T3 cells.

The recovery of functional cell-surface bFGF receptors after trypsin treatment was studied in BHK cells and NIH 3T3 cells. Restoration of functional bFGF receptors occurred at an approximately linear rate with 50% of the high-affinity binding capacity restored after 4 hr. Restoration of functional receptors required protein synthesis but not RNA synthesis. Upon exposure of BHK cells to bFGF, cell-surface receptors were rapidly lost, with only 25% remaining after 1 hr. When the bFGF was removed, down-regulated BHK and NIH 3T3 cells recovered cell-surface receptors at about the same rate observed in trypsin-treated cells. The recovery of receptors after down-regulation was inhibited by protein synthesis inhibitors. Addition of the protein synthesis inhibitor cycloheximide to unperturbed cultures of BHK or NIH 3T3 cells resulted in a time-dependent loss of cell-surface bFGF receptors, demonstrating that the receptors turn over constantly in the absence of ligand. These results suggest that bFGF receptors do not recycle and must be continuously synthesized.

Animals↗
PubMed1989

An amino-terminally extended and post-translationally modified form of a 25kD basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. bFGF proteins characteristically have a molecular weight of 18,000 which is consistent with the predicted primary translation product of 155 amino acids from the cDNA. More recently, higher molecular weight forms of bFGF have been identified but their structural relationship to the commonly known 18kD bFGFs has not been established. We now show that a 25kD bFGF purified from guinea pig brain tissue is an N-terminally extended and post-translationally modified form of the growth factor. Although the exact nature of the post-translational modifications has not been determined, circumstantial evidence suggests that they may be methylated arginines.

Amino Acid Sequence↗
PubMed1989

Role of extracellular matrix in the action of basic fibroblast growth factor: matrix as a source of growth factor for long-term stimulation of plasminogen activator production and DNA synthesis.

When bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37 degrees C, washed extensively with phosphate-buffered saline (PBS) and incubated in bFGF-free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin-like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long-term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCl and incubated in bFGF-free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF-free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long-term (24-48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF-containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4 degrees C, incubated in bFGF-free medium for 24 hours at 37 degrees C, and assayed for 3H-thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4 degrees C was carried out in the presence of heparin. Thus, long-term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrix may act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.

Animals↗
PubMed1989

Transformation of NIH 3T3 cells with basic fibroblast growth factor or the hst/K-fgf oncogene causes downregulation of the fibroblast growth factor receptor: reversal of morphological transformation and restoration of receptor number by suramin.

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.

Animals↗
PubMed1988

Modulation of plasminogen activator activity in human endometrial adenocarcinoma cells by basic fibroblast growth factor and transforming growth factor beta.

Human endometrial adenocarcinoma HEC-1-A and HEC-1-B cells produce and secrete the urokinase-type plasminogen activator and trace amounts of the tissue-type plasminogen activator. The two clones, which originate from the same tumor, possess high affinity binding sites for basic fibroblast growth factor (bFGF) and they respond to the addition of human recombinant bFGF with an increase of the synthesis and secretion of urokinase-type and tissue-type plasminogen activators and with an increase in cell proliferation. Transforming growth factor beta, (TGF beta), when added alone or 6 h before bFGF, induces an increase of plasminogen activator synthesis in both cell lines and stimulates the production of the endothelial cell-type plasminogen activator inhibitor in HEC-1-A cells, but not in HEC-1-B cells. Moreover, TGF beta inhibits basal proliferation of both cell lines and suppresses the mitogenic activity of bFGF. We have previously demonstrated that HEC-1-A and HEC-1-B cells produce significant amounts of bFGF. On the basis of the well-established coordinate modulation of solid tumor growth and plasminogen activator production, our data suggest that bFGF may contribute, in an autocrine fashion, to the development of endometrial carcinomas. Moreover, endometrial tumor cells appear also to be a target for TGF beta. Our results demonstrate also that significant qualitative and quantitative differences in the response to growth factors exist in clones derived from the same tumor, and support the view that the properties of in vivo tumors cannot be extrapolated from results obtained with a single isolate of tumor cells.

Adenocarcinoma↗
PubMed1988

The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF. The identification of bFGF in adherent elicited murine peritoneal macrophages.

Polyclonal antibodies which have the capacity to neutralize the biological activity of basic fibriblast growth factor (bFGF) in vitro, have been raised in rabbits against the 157 amino acid form of bFGF purified from human placenta. In a dot blot assay the anti-bFGF antibodies do not recognize the acidic form of FGF (aFGF) with which the basic form shares significant amino acid sequence homology. As determined by immunoblotting, bFGF antibodies recognized only bFGF in a mixture of placentally derived heparin-binding proteins, demonstrating the specificity of these antibodies. Using the anti-human bFGF antibodies, we have developed a solid-phase competitive radioimmunoassay sensitive to 7.8 ng/ml (0.4 pmol/ml) for bFGF. aFGF does not compete with bFGF for binding to the antibodies in the radioimmunoassay even at 2.04 micrograms/ml. The specificity of the assay was further demonstrated by a lack of competition of cytochrome C, myoglobin, epidermal growth factor or bovine serum albumin with bFGF for binding to the antibodies. We have identified bFGF in extracts of adherent thioglycollate-stimulated mouse peritoneal macrophages by immunological criteria including the ability of the extract to compete with 125I-bFGF for binding to affinity-purified anti-human bFGF antibodies in the RIA and the ability of these antibodies in inhibit the bFGF-like biological activity of the macrophage extract.

Amino Acids↗
PubMed1988

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor.

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.

Animals↗
PubMed1988

Multiple forms of an angiogenesis factor: basic fibroblast growth factor.

An angiogenesis factor has been isolated from human placenta and human hepatoma cells on the basis of its ability to stimulate protease production in cultured capillary endothelial cells. The purified angiogenesis factor also stimulated DNA synthesis and motility in capillary endothelial cells and induced angiogenesis in vivo. Amino acid sequence data revealed that the angiogenesis factor was human basic fibroblast growth factor (bFGF). Other angiogenesis factors isolated on the basis of their ability to stimulate endothelial cell proliferation have also been identified as bFGFs. The bFGFs that have been sequenced show variability in their N-termini. These different forms of bFGF may be naturally occurring processed forms or may be generated by proteases released during the isolation procedure. Recently a bFGF with a large N-terminal extension has been identified. This Mr 25,000 bFGF has the same biological activity and the same affinity for the bFGF receptor as the typical Mr 18,000 bFGFs. The Mr 25,000 bFGF can be converted into an Mr 18,000 form by treatment with low concentrations of trypsin, suggesting that it may be a precursor to the Mr 18,000 bFGF.

Amino Acid Sequence↗
PubMed1988

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation.

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

Animals↗