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D B Rifkin

Publications and source records attributed to D B Rifkin.

At least 109 records · Page 6Linked to original sources
PubMed1990

Immunohistochemical localization of basic fibroblast growth factor in astrocytomas.

Because of the prominent neovascularization observed in the growth of brain tumors, we studied the occurrence of basic fibroblast growth factor (bFGF), a potent angiogenic factor in astrocytomas, the most aggressive of which often have marked vascular hyperplasia. Using immunohistochemical methods, we examined 21 examples of such tumors, 7 glioblastomas multiforme, 7 anaplastic astrocytomas, and 7 low grade astrocytomas. Using polyclonal and affinity-purified rabbit antisera to human bFGF, we detected immunoreactive bFGF in all cases of glioblastoma multiforme. bFGF was present in both endothelial cells and neoplastic astrocytes. In 4 of 7 anaplastic astrocytomas, the tumor astrocytes had bFGF immunoreactivity and, in 5 of 7 cases, endothelial cells were also immunopositive. In glioblastomas multiforme and anaplastic astrocytomas, capillaries adjacent to tumor showed bFGF immunoreactivity, whereas capillaries distant from the tumors were not immunostained. In low grade astrocytomas, astrocytic cells were weakly immunoreactive in 2 of 7 cases, and in only 1 of the 7 cases capillaries were immunostained. In each grade, reactive astroglial cells showed variable bFGF immunoreactivity. The immunostaining was not seen with the flow-through fraction obtained after affinity purification of the bFGF antiserum with pure recombinant bFGF. These results suggest a possible role for bFGF in tumor growth and in angiogenesis in astrocytomas.

Astrocytes↗
PubMed1990

Bimodal relationship between invasion of the amniotic membrane and plasminogen activator activity.

Three human tumor cell lines, Bowes' melanoma, HT1080 and Osmond cells, were characterized for their ability to invade the amniotic membrane and their production of plasminogen activator. Bowes' melanoma cells, which release large amounts of tissue plasminogen activator (tPA), were poorly invasive on the amniotic membrane. The addition of plasmin inhibitors, anti-tPA antibody or tissue inhibitor of metalloproteinase (TIMP) to the amnion assay enhanced invasiveness. The depletion of plasminogen from the growth medium also enhanced the degree of invasiveness. Similarly, HT1080 cells, which produce high levels of urokinase-type plasminogen activator (uPA), were poorly invasive under standard conditions but invasion was enhanced by plasmin inhibitors or anti-uPA antibodies. Conversely, Osmond cells, which produce low levels of uPA, were very invasive on the amniotic membrane. Invasion by these cells was blocked by the addition of plasmin inhibitors or anti-uPA antibodies to the amnion assay. These results suggest that invasion requires only a minimum level of PA activity and that, as PA production exceeds this optimal level, the degree of invasion decreases. We propose that high levels of plasmin, generated by the tPA or uPA secreted by the cells, may cause uncontrolled matrix degradation and interrupt the interaction of cells and matrix in the early stages of invasion. The inhibition of excessive plasmin activity may stabilize and increase cell matrix contacts and result in an enhancement of invasion.

Amnion↗
PubMed1990

Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice.

The stimulatory effect of recombinant basic fibroblast growth factor (bFGF) on wound healing was assessed using healing-impaired (db/db) mice. Full-thickness wounds were made in female diabetic C57BL/KsJ db/db mice, and their normal (db/+) littermates with a punch biopsy instrument. Recombinant bFGF was applied locally to the open wound once a day. The mice were later killed and histological sections of the wounds were prepared. The degree of wound healing was evaluated using several histological parameters such as degree of reepithelialization, granulation tissue thickness, matrix density, number of infiltrated cells, and number of capillaries. Wounds from normal mice displayed good reepithelialization rates and granulation tissue formation, while wounds from db/db mice had poor responses, especially in the dermal parameters. Although the application of bFGF to wounds in the normal (db/+) mice had little effect, application of bFGF to wounds in db/db mice induced significant responses in all of the dermal parameters compared with nontreated db/db mice (p less than 0.001). In the presence of bFGF, these parameters approximated those observed in nontreated littermates. A minimum of 0.5 microgram bFGF in either single or multiple applications was required for a significant effect. bFGF that was either boiled or pretreated with neutralizing antibody had little stimulatory effect. Time-course experiments indicated that the granulation response in bFGF-treated mice peaked between 8 and 12 d, and decreased after 12 d, while matrix density continued to increase until the 18th day (p less than 0.05). The breaking strength of healed linear wounds in db/db mice was also decreased when compared with heterozygous littermates. This parameter was also improved by the administration of bFGF to the wounds (p less than 0.05).

Animals↗
PubMed1990

Studies on the role of basic fibroblast growth factor in vivo: inability of neutralizing antibodies to block tumor growth.

Affinity-purified polyclonal rabbit antibodies prepared against recombinant basic fibroblast growth factor (bFGF) neutralized the ability of bFGF to stimulate plasminogen activator (PA) production and endothelial cell migration in vitro. After iodination and intraperitoneal injection of the antibodies in mice, approximately 76% of the maximum circulating level of 125I-anti-bFGF antibodies (AF) was found as intact IgG after 24 hr. Furthermore, the circulating 125I-AF retained the ability to bind bFGF. Studies were performed to determine whether the growth of three different murine tumors (CT26, EHS, or B16/BL6) could be inhibited with affinity-purified neutralizing antibodies against bFGF. Tumors were injected subcutaneously in syngeneic mice, and neutralizing antibodies against bFGF were injected daily into the peritoneum. All studies, which varied in tumor burden, antibody dose, and study length, indicated that neutralizing antibodies against bFGF had no effect on tumor size, tumor growth, or tumor histology.

Animals↗
PubMed1990

Nuclear and cytoplasmic localization of different basic fibroblast growth factor species.

The subcellular distribution of basic fibroblastic growth factor (bFGF) was analyzed by subcellular fractionation and immunofluorescence to gain insight into potential mechanisms for its release from cells. Subcellular fractionation of either SK-Hep-1 cells or NIH 3T3 cells transfected with a bFGF cDNA revealed that the 18 kd form of bFGF was found primarily in the cytosolic fraction, whereas the 22 and 24 kd forms of bFGF were found preferentially in ribosomal and nuclear fractions. Analysis of bFGF distribution by immunofluorescence using an antibody that recognized all forms of bFGF indicated both cytoplasmic and nuclear localization but failed to reveal any growth factor in structures representing secretory vesicles. Therefore, bFGF has a distribution inconsistent with that of a secretory protein.

Animals↗
PubMed1990

Expression of tissue factor procoagulant activity: regulation by cytosolic calcium.

Intact bovine fibroblasts, pericytes, and kidney cells manifested significantly less tissue factor procoagulant activity than their disrupted counterparts. Addition of calcium ionophore A23187 rapidly and reversibly enhanced the cell-surface expression of tissue factor in intact cells up to the level achieved by disruption. Inhibitors of calmodulin blocked the ionophore-dependent enhancement of procoagulant activity. Similar kinetic parameters were obtained for factor X hydrolysis by tissue factor-factor VIIa on unperturbed pericytes and phosphatidylcholine vesicles. Increase in Vmax and decrease in apparent Km for this reaction were seen after either disruption or ionophore stimulation of the pericytes. Addition of phosphatidylserine to the reconstituted phospholipid vesicles also increased the Vmax and decreased the apparent Km for factor X hydrolysis. These data agree with the hypothesis that the expression of tissue factor procoagulant activity on cell surfaces is modulated by calcium-mediated changes in the asymmetric distribution of phosphatidylserine in plasma membrane.

Animals↗
PubMed1990

Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells.

The levels of endogenous basic fibroblast growth factor (bFGF) in seven clones of cultured bovine capillary endothelial (BCE) cells were assayed, and their relation to cell morphology, bFGF receptor number, cell migration, amniotic membrane invasivity, and proteinase levels were studied. Immunoblotting experiments with anti-bFGF IgG demonstrated that cells from these clones contained different amounts of bFGF. The cells containing high levels of bFGF had a spindle or elongated appearance at confluence and a low number of high affinity receptors for bFGF. The cells containing low levels of bFGF had a cobblestone-like appearance and a higher number of high affinity receptors. When exposed to 10 ng/ml bFGF, cells containing a low level of bFGF took on an elongated appearance with a crisscross pattern similar to that seen with the high producer bFGF cells. The endogenous bFGF levels of the BCE cell clones correlated with the extent of cell migration after wounding of a monolayer and the degree of invasion of the human amniotic membrane. Cells from the clone with the highest endogenous bFGF level migrated well, invaded the amnion membrane without the addition of exogenous bFGF, and were relatively unaffected by the addition of bFGF. Cells from the clone containing the lowest level of bFGF did not migrate or invade under normal conditions. However, the addition of bFGF to the culture medium strongly enhanced both of these processes. The inclusion of anti-bFGF IgG in the media suppressed cell migration and invasion. The plasminogen activator (PA) activities of cell lysates of the clones, assayed by the 125I-fibrin plate technique, indicated that the PA levels did not correlate with the bFGF levels. Metalloproteinase activities in the conditioned medium, assayed by gelatin zymography, correlated with the endogenous bFGF levels, suggesting that the degree of expression of metalloproteinases might be critical for cell migration and invasion. These data suggest that endogenous bFGF may have an important role for migration and invasion of BCE cells during neovascularization via the induction and/or activation of specific metalloproteinases.

Animals↗
PubMed1990

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity.

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.

Adrenal Cortex↗
PubMed1990

Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system.

The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system.

Animals↗
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

Long-term culture of human bone marrow stromal cells in the presence of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells. Normally, large numbers of human bone marrow stromal cells are difficult to obtain. However, nanogram/ml concentrations of bFGF stimulate the growth of passaged bone marrow stromal cells both in media formulated for optimal growth of stromal cells and in a simple mixture of RPMI-1640 and 10% fetal calf serum facilitating the successive expansion of stromal cells through multiple passages. bFGF also greatly accelerates the formation of a primary stromal cell layer following inoculation of newly harvested bone marrow cells into dishes. In the presence of bFGF, the stromal cells attain high densities, lose their contact inhibition and grow in multilayered sheets. Heparin greatly potentiates the stimulatory effect of low concentrations of bFGF. The effects of bFGF are fully reversible: cells cultured in the presence of this factor for multiple passages revert to normal growth rates following trypsinization and subculture. A short (4 h) exposure of the cells to bFGF elicits profound growth stimulation. This supports the hypothesis that this factor binds to glycosaminoglycans in the cell matrix which act as a storage reservoir for this cytokine.

Bone Marrow Cells↗
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

Alpha 2-macroglobulin is a binding protein for basic fibroblast growth factor.

After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.

Animals↗
PubMed1989

Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan.

Fibroblast growth factors (FGF) are a family of heparin-binding angiogenic polypeptide mitogens. In the presence of heparin, recombinant human basic fibroblast growth factor (bFGF) is fully protected from tryptic digestion and partially protected from chymotryptic digestion. Complete protection of bFGF by heparin is achieved at ratios of growth factor:heparin of approximately 10 or less (w/w). The protection requires bioactive bFGF because inactivated bFGF is rapidly degraded by trypsin or chymotrypsin in the presence of heparin. The bFGF-heparin interaction forms hydrophobic complexes which become insoluble in aqueous buffers at bFGF:heparin ratios of 8 to 10 (w/w). The heparin was found to bind up to a tenfold excess of bFGF on a weight basis. bFGF in the presence of heparin is as active as bFGF alone in inducing 3H-thymidine incorporation into Swiss 3T3 fibroblast DNA.

Animals↗
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

In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases.

The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under normal conditions. Addition of human bFGF to either the BM or to the stromal aspect of the amnion induced BCE cell invasion with a dose-dependent response. This effect was maximal in the presence of 70 ng/ml bFGF, and was inhibited by anti-FGF antibody. Transforming growth factor beta, as well as plasmin inhibitors and anti-tissue type plasminogen activator antibody inhibited BCE cell invasion. The tissue inhibitor of metalloproteinases, 1-10 phenanthroline, anti-type IV and anti-interstitial collagenase antibodies had the same effect. On the contrary, anti-stromelysin antibody and Eglin, an inhibitor of elastase, were ineffective. The results obtained show that both the plasminogen activator-plasmin system and specific collagenases are involved in the invasive process occurring during angiogenesis.

Amnion↗
PubMed1989

Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture.

When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded with a razor blade, endothelial cells (ECs) spontaneously move into the denuded area. If bovine pericytes or smooth muscle cells (SMCs) are plated into the denuded area at low density, they block the movement of the ECs. This effect is dependent upon the number of cells plated into the wound area and contact between ECs and the plated cells. Antibodies to transforming growth factor-beta 1 (TGF-beta 1) abrogate the inhibition of BAE cell movement by pericytes or SMCs. TGF-beta 1, if added to wounded BAE cell monolayers, also inhibits cell movement. When cultured separately, BAE cells, pericytes, and SMCs each produce an inactive TGF-beta 1-like molecule which is activated in BAE cell-pericyte or BAE cell-SMC co-cultures. The activation appears to be mediated by plasmin as the inhibitory effect on cell movement in co-cultures of BAE cells and pericytes is blocked by the inclusion of inhibitors of plasmin in the culture medium.

Animals↗