Effect of heparin on the stimulation of non-vascular cells by human acidic and basic FGF

We have purified acidic and basic fibroblast growth factors from human brain (h-aFGF, h-bFGF) and studied the effect of heparin on the growth stimulation by these factors of hamster fibroblast CC139 cells and bovine epithelial lens (BEL) cells. In both the presence and the absence of foetal calf serum (FCS) heparin cooperates with h-aFGF in a dose dependent manner to stimulate both types of cells. The cooperation with h-bFGF is much less. An unpurified human brain fraction containing both factors behaves differently: in the absence of FCS, heparin enhances the activity of the crude fraction on BEL cells, while in the presence of FCS, it decreases this activity. These results indicate that heparin cooperates strongly with h-aFGF to stimulate non-vascular cell proliferation while in a partially purified extract and in the presence of serum it can induce the opposite effect.     

Modulation of growth of human carcinoma SW-13 cells by heparin and growth factors

This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type-e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW-13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin-Sepharose column with 2 M NaCl and 1.6 M NaCl, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin-Sepharose column with 0.5 M NaCl. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW-13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW-13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW-13 cells. A dose-dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) were unable to overcome heparin-induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR-2B and partially inhibited AKR-2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW-13 cells and possibly of other epithelial cells in complex ways and that heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth.     

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.     

Neuronal-derived factors regulating glial cell proliferation and maturation

We purified to homogeneity two active factors, named astroglial growth factors (AGFs: AGF2 and AGF1), from bovine brain after two and three chromatographic steps, respectively. We found that AGFs have a strong affinity for heparin. Therefore, heparin affinity chromatography was used to purify rapidly and efficiently these growth factors. The purified AGF1 is an acidic protein (pI: 5.5) with an apparent molecular weight of about 17,500 daltons; the AGF2 is a basic protein (pI: 9.5) of 18,500 daltons. The comparison of the physico-chemical properties, the aminoacid composition and the amino-terminal sequence of the AGFs with that of other growth factors isolated from the brain and affecting the proliferation of other cell types has indicated that AGF1 and AGF2 are identical to the acidic and basic fibroblast growth factor (FGF), respectively. Both factors stimulate the proliferation as well as the morphological and biochemical maturation of the astroglial cells. Both factors enhance also the multiplication of oligodendroglial cells. Polyclonal and monoclonal antibodies against AGFs have been prepared and used for immunocytochemical localization of these molecules in the rat brain and cerebellum. AGFs are found exclusively in neuronal cells.     

Differential Effects of Acidic and Basic Fibroblast Growth Factors on Spinal Cord Cholinergic, GABAergic, and Glutamatergic Neurons

When spinal cord cultures from embryonic day 12 rats were cultured at low density, both acidic and basic fibroblast growth factors significantly increased neuronal survival and neurite outgrowth in a dose-dependent manner. The effects of acidic fibroblast growth factor were independent of heparin, in contrast to its mitogenic effects on both NIH3T3 cells and cerebral cortical astrocytes. In high-density cultures, acidic fibroblast growth factor increased choline acetyltransferase activity by 57%, glutamic acid decarboxylase activity by 58%, and aspartate aminotransferase activity by 65%. Basic fibroblast growth factor increased choline acetyltransferase activity by 73% and glutamic acid decarboxylase activity by 200% but decreased aspartate aminotransferase activity by 40%. Growing these cultures in the presence of a mitotic inhibitor did not significantly alter the effect of acidic or basic fibroblast growth factor on these enzyme activities. These results demonstrate that acidic and basic fibroblast growth factors differentially affect neurotransmitter enzyme levels of multiple classes of neurons, rather than having effects on a single neuronal population.     

Heparin-binding growth factors and their receptors

Heparin-binding growth factors modulate diverse biological activities including cellular proliferation, cellular differentiation, morphogenesis, and angiogenesis. Biochemical characterization for two members of the heparin-binding growth factor family, acidic and basic fibroblast growth factors, is extensive, while characterization of the remaining five members is forthcoming. Cell surface receptors have been identified for acidic and basic fibroblast growth factors, but little is known concerning their sites of action in vivo or the mechanisms involved in transducing the energy of growth factor binding to a biological response. An understanding of the biological basis for the diversity of the heparin binding growth factor family and the in vivo actions of these factors will prove a major challenge to future research efforts.     

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.     

Mesoderm induction and blood island formation by angiogenic growth factors and embryonic inducing factors

Factors which induce mesoderm, including endothelium lined cavities and primitive blood cells in omnipotent amphibian ectoderm, have been isolated from different sources. Recently it was shown that angiogenic factors, which belong to the protein families of the heparin binding growth factors (acidic and basic fibroblast growth factor) and the transforming growth factors (TGF-beta 1 and -beta 2), also induce mesodermal tissues in amphibian ectoderm. In triturus ectoderm, capillary like endothelial networks are induced preferentially by the transforming growth factors. The relationship between growth factors and inducing factors is discussed.     

Enhancement of DNA synthesis of human epithelial carcinoma cells by acidic fibroblast growth factor

Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

The analysis of heparin-protein interactions using evanescent wave biosensor with regioselectively desulfated heparins as the ligands

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin-protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.     

SR 25989 inhibits healing of a mechanical wound of confluent human saphenous vein endothelial cells which is modulated by standard heparin and growth factors

The thienopyridine, ticlopidine, a potent platelet antiaggregating agent and SR 25989, an esterified derivative of ticlopidine, devoid of antiplatelet activity, were tested in an in vitro model of healing of a mechanical wound in confluent endothelium. This model allows exploration of substances involved in wound healing and angiogenesis. These two compounds inhibited both cell proliferation and cell migration during lesion repair in a dose-dependent manner (18–150 μM), SR 25989 being twice as active as ticlopidine. Its effect was not inhibited by acidic or basic fibroblast growth factor or by platelet derived growth factor. In contrast, it exerted a conjugated inhibition with standard heparin and was able to totally reverse the healing increase induced by a mixture of acidic fibroblast growth factor and heparin. The mechanism of action of SR 25989 is not yet elucidated, but it does not seem to involve competition with fibroblast growth factors since these substances were not able to alter their binding to receptors on the endothelial cell surface. SR 25989 therefore appears as a promising new candidate for inhibition of angiogenesis. © 1994 Wiley-Liss, Inc.     

Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 micrograms/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.     

The genes for basic and acidic fibroblast growth factors are on different human chromosomes

Basic and acidic fibroblast growth factor (FGF) are related both structurally and functionally. A bovine basic FGF cDNA and a human acidic FGF genomic fragment were used as hybridization probes in Southern blot analysis of DNAs isolated from a panel of 30 mouse-human cell hybrids. The gene encoding basic FGF was assigned to human chromosome 4, and the gene for acidic FGF to human chromosome 5. The two growth factors which are presumed to have a common evolutionary ancestor are therefore not linked. A HindIII restriction fragment length polymorphism was detected for human basic FGF.     

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.     

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.     

Stimulation of cell growth and inhibition of prostacyclin production by heparin in human umbilical vein endothelial cells

We characterized human umbilical vein (HUV) endothelial cells as to cell growth and prostacyclin production to get a better understanding of the properties of endothelial cells. Endothelial cell growth supplement (ECGS) and basic fibroblast growth factor (FGF) stimulated HUV endothelial cell growth. Heparin further enhanced the cell growth stimulated by ECGS, but not the cell growth stimulated by FGF or in the absence of these growth factors. In the presence of ECGS, the prostacyclin-producing capacity of the cells was inhibited by heparin. However, in the presence of FGF of in the absence of growth factors, heparin did not inhibit prostacyclin production. Therefore, it is likely that there is a specific correlation between heparin and growth factors for endothelial cells in the blood vessel to maintain nonthrombogenicity properly. Heparin-treated cultures may not be suitable for some examinations of prostacyclin production by vascular endothelial cells.     

Endothelial cell mitogens derived from retina and hypothalamus: biochemical and biological similarities

Bovine retina and hypothalamus contain anionic endothelial cell mitogens that display unusual affinities for the negatively charged glycosaminoglycan heparin. Both growth factor activities are acidic polypeptides (pl's of 5.0) as determined by isoelectric focusing and DEAE-affinity chromatography. In spite of their anionic nature, the factors bound to heparin-Sepharose columns with high affinity and could be eluted only at high salt concentrations (0.9-1.1 M NaCl). The affinity of the retina-derived growth factor (RDGF) for heparin permitted a 15,000-fold purification of the mitogen in two steps: heparin-affinity chromatography and size exclusion high-performance liquid chromatography. RDGF and the anionic hypothalamus-derived factor (aHDGF) exhibit three major biochemical similarities including isoelectric point, (pl's of 5.0), heparin affinity (elution at 0.9-1.1 M NaCl) and molecular weight (18,000). Additionally, the two factors display similar biological activities, stimulating the proliferation of capillary and human umbilical vein endothelial and 3T3 cells but not vascular smooth muscle cells. We suggest that RDGF and aHDGF are related if not identical growth factor molecules.