Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.     

Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells.

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.     

Expression and functional role of bTRPC1 channels in native endothelial cells

We have analyzed the expression and localization of bovine transient receptor potential-C1 (bTRPC1) in bovine aortic endothelial cells, and its possible involvement in the store-independent calcium influx induced by basic fibroblast growth factor (bFGF). RT-PCR experiments confirmed the existence of two btrpc1 mRNA isoforms; conversely, the btrpc3 gene was not transcribed. Anti-TRPC1 antibody revealed the presence of the protein in the membrane-rich compartment only. Application of anti-TRPC1 during the response to bFGF caused a partial but significant reduction of calcium entry. This is the first evidence of TRP channel involvement in a non-capacitative calcium influx induced by a biologically relevant agonist such as the angiogenic factor bFGF in native endothelial cells.     

The disulfide structure of bovine pituitary basic fibroblast growth factor.

Basic fibroblast growth factor has 4 cysteine residues in its amino acid sequence, two of which are perfectly conserved within the fibroblast growth factor family of proteins suggesting a disulfide bond at this position. Furthermore, thiol titration of bovine pituitary basic fibroblast growth factor (bFGF) indicates the presence of two free thiols, which is consistent with an intramolecular disulfide. Direct analysis of natural and recombinant fibroblast growth factor proteins have not confirmed the existence of such a disulfide. Instead, the two nonconserved cysteines of bFGF purified from bovine pituitaries are S-thiolated with glutathione. Inclusion of 75 mM N-ethylmaleimide during the homogenization of the pituitaries effectively blocks the S-thiolation, demonstrating that this modification is an artifact of the purification procedure. Analysis of the N-ethylmaleimide purified bovine pituitary bFGF suggests that the natural protein is in the correct redox state when all 4 cysteines are in the reduced form.     

Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine

We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector. This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88. All three constructs were cloned and expressed in Escherichia coli and the proteins purified. The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF. The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays. Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups. This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond. Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration. Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation. These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.     

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.     

Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells

Bovine embryonic stem-like cells (ES-like cells) appear to maintain a normal diploid karyotype indefinitely during culture in vitro and to express marker proteins that are characteristic of ES cells from mice, namely, alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1), STAT-3, and Oct 4. After proliferation of undifferentiated ES-like cells in vitro, some bovine ES-like cells differentiated to neural precursor cells, which were cultured in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). In addition, calves were successfully cloned using ES-like cells and the frequency of term pregnancies for blastocysts derived from ES-like cells was higher than those of early pregnancies and maintained pregnancies after nuclear transplantation (NT) with bovine somatic cells. Successful cloning from bovine ES-like cells should allow the introduction into cattle of specific genetic characteristics of biomedical and/or agricultural importance.     

The stimulatory effect of PDGF on vascular smooth muscle cell migration is mediated by the induction of endogenous basic FGF

The migration of arterial smooth muscle cells from the media to the intima is a crucial event for the development of the atherosclerotic lesion, and platelet derived growth factor (PDGF) is thought to play an important role in this process. Here we report that the spontaneous migration of bovine smooth muscle (BSM) cells is dependent on endogenously produced basic fibroblast growth factor (bFGF). PDGF stimulates the migration of BSM cells and its effect is abolished by affinity purified anti-bFGF antibody. PDGF induces bFGF mRNA in BSM cells. These results indicate that the effect of PDGF on the migration of BSM cells may be mediated by the induction of endogenous bFGF.     

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.     

Biologically active synthetic fragments of human basic fibroblast growth factor (bFGF): identification of two Asp-Gly-Arg-containing domains involved in the mitogenic activity of bFGF in endothelial cells.

Synthetic peptides derived from the amino acid sequence of human basic fibroblast growth factor (bFGF) have been assayed for the capacity to exert bFGF agonist and antagonist activities in cultured endothelial cells. bFGF fragments A and C, which correspond to the sequences bFGF (38-61) and bFGF (82-101), induce a limited but statistically significant increase in cell number when administered to cultures of fetal bovine aortic endothelial GM 7373 cells and adult bovine aortic endothelial cells. The two peptides also exert a partial antagonist activity when GM 7373 cells are stimulated to proliferate by bFGF, but they do not affect cell proliferation induced by serum, epidermal growth factor (EGF), phorbol ester (TPA), or 1,2-diacylglycerol (diC8). Moreover, antibodies raised against peptides A and C specifically quench the mitogenic activity of bFGF. Peptides A and C contain the amino acid sequence Asp-Gly-Arg (DGR), which is the inverse of the cell adhesion signal sequence RGD recognized by integrins. DGR- and RGD-containing tetra- and heptapeptides inhibit the mitogenic activity exerted by bFGF and by the two active bFGF fragments. They do not affect cell proliferation induced by acidic FGF, EGF, serum, TPA, and diC8. However, neither peptides A and C, their corresponding antibodies, nor DGR-and RGD-containing peptides inhibit the binding of 125I-bFGF to its low and high affinity binding sites. The data suggest that amino acid residues 38-61 and 82-101, both containing a core DGR sequence, represent two "activation" domains of bFGF. Both domains are involved in the modulation of the mitogenic activity of bFGF without interacting directly with the bFGF receptor.     

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1 -expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resitance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition. © 1995 Wiley-Liss, Inc.     

Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells

We have examined the effect of tumor necrosis factor (TNF) on the proliferation of capillary endothelial cells derived from brain or adrenal cortex. In both cell types, TNF inhibits basal as well as basic fibroblast growth factor (bFGF)-stimulated cell proliferation. TNF induces an additional cytotoxic effect in bFGF-stimulated, but not in unstimulated, capillary endothelial cells. These results suggest that TNF could act as a negative regulator of angiogenesis in vivo and further, that TNF might induce selective cytotoxicity of capillary endothelial cells stimulated by tumor-derived bFGF. These results could explain why TNF induces hemorrhagic necrosis of certain, solid tumors.     

The int-2 gene product acts as a growth factor and substitutes for basic fibroblast growth factor in promoting the differentiation of a normal mouse mammary epithelial cell line.

We have investigated the effect of basic fibroblast growth factor (bFGF) and the related int-2 gene on the growth, transformation, and differentiation of HC11 mouse mammary epithelial cells. We show that in HC11 cells infected with int-2 retroviral expression vectors, the int-2 protein can function as a bFGF-like growth factor in stimulating: (a) HC11 cell proliferation in monolayer, (b) anchorage-independent growth in soft agar, and (c) soft agar growth of the bFGF-responsive SW13 tumor cell line. These effects are observed irrespective of whether the int-2 protein is expressed in its wild-type form or is linked to a signal peptide. A candidate bFGF receptor, which is the product of the flg gene and which may recognize the int-2 protein, is expressed at high levels in HC11 cells. Following epidermal growth factor or bFGF priming and subsequent treatment with lactogenic hormones, all of the int-2 infected and the parental HC11 cells synthesize similar levels of beta-casein. However, the autocrine expression of int-2 in HC11 cells abrogates their requirement for either exogenous epidermal growth factor or bFGF priming. These data suggest that, in HC11 cells, the growth factor activity of the int-2 gene is indistinguishable from that of bFGF and does not interfere with the mammary cell differentiation program associated with lactogenesis.     

Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.

Normal adult bovine aortic endothelial cells were infected with various recombinant retroviruses expressing one, two, or three human basic fibroblast growth factor (bFGF) proteins normally synthesized by an alternative use of translation initiation codons. We show here that the constitutive expression of the AUG-initiated from (18 kDa) leads the transfected cells to form colonies in soft agar. The expression of the high molar weight (HMW) forms (22.5 and 21 kDa) initiated at one of the two CUG initiation codons allows cell immortalization, whereas the tumorigenic potential is reached when the three forms are constitutively expressed. Furthermore, we provide evidence that constitutive expression of (HMW) bFGF forms has a down-regulation effect on bFGF synthesis from the gene naturally active in parental endothelial cells.     

Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells

Transforming growth factor beta-1 (TGF beta-1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGF beta-1, but it is blocked if cells are cultured on extracellular-matrix-coated dishes instead of plastic. TGF beta-1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGF beta-1 might act through regulation of bFGF synthesis in BCE cells.