Energetics of heparin binding to human acidic fibroblast growth factor

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.     

Importance of size, sulfation, and anticoagulant activity in the potentiation of acidic fibroblast growth factor by heparin

Heparin was previously reported to potentiate the mitogenic activity of endothelial cell mitogens in a crude extract of bovine hypothalami (Thornton, S. C., Mueller, S. N., and Levine, E. M. (1983) Science 222, 623-625). We and others (Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484) have reported that the growth stimulatory effects of acidic fibroblast growth factor (aFGF) are potentiated in a similar manner. We have used these observations as the basis of an assay to characterize the importance of size, sulfation, and anticoagulant activity of heparin in mediating this effect. Partial nitrous acid depolymerization of heparin from porcine intestinal mucosa resulted in a mixture of heparin fragments, containing oligosaccharides ranging from disaccharides to polysaccharides of about 40 monosaccharides in length. This mixture was fractionated by ion exchange chromatography and gel permeation chromatography to obtain size-homogeneous oligosaccharides with different degrees of sulfation. Assay of these heparin-derived saccharides in the presence of a suboptimal concentration of aFGF revealed that a minimum chain length and a certain degree of sulfation is required in order to potentiate the action of aFGF. Low sulfate oligosaccharides (4-16 units) were unable to potentiate aFGF, whereas medium sulfate fractions of octadecasaccharides and larger were able to moderately potentiate aFGF. The potentiation of aFGF by the high sulfate fraction correlated with the saccharide size: 12 or more monosaccharide units were necessary to achieve potentiation equivalent to whole heparin, octa- and decasaccharides were mildly stimulatory, and hexasaccharides were without effect. In the absence of aFGF, intact heparin as well as all the oligosaccharides examined, inhibited the proliferation of capillary endothelial cells to approximately the same degree, between 20 and 50% inhibition. When a tetradecasaccharide was separated into a binding and a nonbinding fraction on matrix-bound antithrombin III, no difference was seen for these fractions in the endothelial cell proliferation assay. These results indicate that both size and sulfation of a heparin-derived oligosaccharide contribute to its ability to interact with aFGF and/or endothelial cells and that this interaction is independent of anticoagulant activity. In addition, our findings suggest that the inhibitory and potentiating effects of heparin on capillary endothelial cells have different structural requirements.     

Effect of extension of the heparin binding pocket on the structure,stability, and cell proliferation activity of the human acidic fibroblast growth factor

Acidic human fibroblast growth factor (hFGF1) plays a key role in cell growth and proliferation. Activation of the cell surface FGF receptor is believed to involve the glycosaminoglycan, heparin. However, the exact role of heparin is a subject of considerable debate. In this context, in this study, the correlation between heparin binding affinity and cell proliferation activity of hFGF1 is examined by extending the heparin binding pocket through selective engineering via charge reversal mutations (D82R, D84R and D82R/D84R). Results of biophysical experiments such as intrinsic tryptophan fluorescence and far UV circular dichroism spectroscopy suggest that the gross native structure of hFGF1 is not significantly perturbed by the engineered mutations. However, results of limited trypsin digestion and ANS binding experiments show that the backbone structure of the D82R variant is more flexible than that of the wild type hFGF1. Results of the temperature and urea-induced equilibrium unfolding experiments suggest that the stability of the charge-reversal mutations increases in the presence of heparin. Isothermal titration calorimetry (ITC) data reveal that the heparin binding affinity is significantly increased when the charge on D82 is reversed but not when the negative charge is reversed at both positions D82 and D84 (D82R/D84R). However, despite the increased affinity of D82R for heparin, the cell proliferation activity of the D82R variant is observed to be reduced compared to the wild type hFGF1. The results of this study clearly demonstrate that heparin binding affinity of hFGF1 is not strongly correlated to its cell proliferation activity.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.     

Conversion of bovine growth hormone cysteine residues to serine affects secretion by cultured cells and growth rates in transgenic mice.

GHs have been found to possess two disulfide bonds. We set out to determine the importance of bovine (b) GH's disulfide bonds relative to the ability of the hormone to be secreted by cultured cells in vitro and to promote growth in transgenic mice. We have generated six mutated bGH genes that encode serine (Ser) substitutions for cysteines (Cys). These mutated genes were used to generate bGH analogs in which either one or both disulfide bonds are destroyed. When the small loop of bGH was destroyed (Cys181-Ser or Cys189-Ser), the bGH analogs were found to be secreted by mouse L-cells at levels comparable to those of wild-type bGH. However, secretion was drastically reduced when the large loop was abolished (Cys53-Ser or Cys164-Ser). An immunofluorescence study of these bGH analogs revealed two distinct patterns of subcellular localization. Bovine GH analogs with mutations in the small loop demonstrated a perinuclear distribution similar to that of wild-type bGH, but analogs containing a disrupted large loop revealed a uniform cytoplasmic distribution pattern. When these mutated bGH genes were individually introduced into transgenic mice, only those animals that expressed bGH analogs with the large loop intact demonstrated a growth-enhanced phenotype. Transgenic mice that expressed bGH analogs lacking the large loop showed growth rates similar to those of nontransgenic mice. These results suggest that the integrity of the large loop, but not that of the small loop, is essential for the growth-enhancing activity of bGH in transgenic mice.     

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.     

Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.     

Structure and stability of an acidic fibroblast growth factor from Notophthalmus viridescens

The three-dimensional solution structure of an acidic fibroblast growth factor (nFGF-1) from the newt (Notophthalmus viridescens) is determined using multidimensional NMR techniques. Complete assignment of all the atoms ((1)H, (15)N, and (13)C) has been achieved using a variety of triple resonance experiments. 50 structures were calculated using hybrid distance geometry-dynamical simulated annealing technique with a total of 1359 constraints. The atomic root mean square distribution for the backbone atoms in the structured region is 0.60 A. The secondary structural elements include 12 beta-strands arranged antiparallely into a beta-barrel structure. The protein (nFGF-1) exists in a monomeric state upon binding to the ligand, sucrose octa sulfate (SOS), in a stoichiometric ratio of 1:1. The SOS binding site consists of a dense cluster of positively charged residues located at the C-terminal end of the molecule. The conformational stabilities of nFGF-1 and its structural and functional homologue from the human source (hFGF-1) are drastically different. The differential stabilities of nFGF-1 and hFGF-1 are attributed to the differences in the number of hydrogen bonds and the presence of solvent inaccessible cavities in the two proteins.     

Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate

The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]₂-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and trisulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluoresence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.Abbreviations aFGF,bFGF acidic and basic fibroblast growth factor - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - U,2S-(14)-GlcNS,6S O--L-ido(ene-pyranosyluronic acid 2-O-sulfate)-(14)-2-sulfoamino-2-deoxy-D-glucose-6-O-sulfate - U-(14)-GlcNS,6S O-(ene-pyranosyluronic acid)-(14)-2-sulfoamino-2-deoxy-D-glucose-6-O-sulfate - IdoUA iduronic acid - GlcUA glucuronic acid - GlyUA uronic acid; GlcNAcN-acetylglycosamine - GlcNS N-sulfated glucosamine - GlcNS,6S N,6-disulfated glucosamine - Gal galactose - Xyl xylose - Ser serine - HS heparan Sulfate     

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.     

Peroxynitrite modulates acidic fibroblast growth factor (FGF-1) activity

To establish peroxynitrite (ONOO(-)) as a mediator of acidic fibroblast growth factor (FGF-1) function, preparations of recombinant human FGF-1 were treated with the pro-oxidant in vitro and identified amino acid modifications were correlated with biologic activity. The sequence of FGF-1 amino acid modifications induced by increasing concentrations of ONOO(-) was from cysteine oxidation to dityrosine formation, and to tyrosine/tryptophan nitration. Low steady-state ONOO(-) concentrations (10-50 microM) induced formation of dityrosine, which involved less than 0.1% of the total tyrosines. Treatment of FGF-1 with ONOO(-) induced a dose-dependent (10-50 microM) loss of sulfhydryl groups that correlated with formation of reducible (dithiothreitol, arsenite) FGF-1 aggregates containing 50% latent biologic activity. Treatment with 0.1-0.5mM ONOO(-) induced increasing formation of non-reducible, inactivated FGF-1 structures. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that six residues (Y29, Y69, Y108, Y111, Y139, and W121) were nitrated by ONOO(-). ONOO(-) treatment (0.1mM) of an active FGF-1 mutant (cysteines converted to serines) induced dose-dependent, non-reversible inhibition of biologic activity that correlated with nitration of Y108 and Y111, both of which reside within a conserved domain encompassing the putative FGF-1 receptor binding site. Collectively, these observations predict a role for low levels of ONOO(-) during secretion of FGF-1 as an extracellular complex containing latent biologic activity. High steady-state levels of ONOO(-) may induce extensive cysteine oxidation, critical tyrosine nitration, and non-reversible inactivation of FGF-1, a potential inhibitory feedback mechanism restoring cellular homeostatis during the resolution of inflammation and repair.     

Enhanced growth of canine bone marrow stromal cell cultures in the presence of acidic fibroblast growth factor and heparin

Summary The ex vivo establishment, expansion, transduction, and reintroduction of autologous bone marrow stromal cells offers a potential efficacious system for somatic cell gene therapy. It is likely that any ex vivo system will require the use of large numbers of cells which express high levels of transgene products. We present a method for routine expansion of canine bone marrow stromal cells, established from initial 10–20 ml marrow aspirates, to greater than 10⁹ cells. This high level expansion of cell cultures uses the stimulatory effect of acidic fibroblast growth factor (aFGF) and heparin. In the absence of these factors, stromal cell cultures grow actively for only 1 to 2 passages, become flattened in morphology, and expand to only 10⁸ cells. In the presence of heparin (5 U/ml), aFGF exerts its effect over a wide range of concentrations (0.1–10 ng/ml) in a dose-dependent manner. The stimulatory effect is dependent on the presence of both aFGF and heparin. Immunocytochemical and cytochemical analyses phenotypically characterize these stromal cells as bone marrow stromal myofibroblasts. Stromal cells grown in the presence of aFGF and heparin grow actively and maintain a fibroblast-like morphology for a number of passages, transduce efficiently with a human growth hormone (hGH) expression vector, and express and secrete high levels of hGH. Human marrow stromal cells were also established and expanded by the same culture method. This culture method should be of great value in somatic cell gene therapy for the delivery of secreted gene products to the plasma of large mammals.     

Micropattern-immobilization of heparin to regulate cell growth with fibroblast growth factor

Heparin was immobilized on a polystyrene plate in a specificpattern by photolithography. Heparin was coupled with azidoaniline. Thederivatized heparin was cast on the polystyrene plate from aqueoussolution. After drying, the plate was photo-irradiated in the presence of aphotomask. The micropatterning was confirmed by staining with a dye,ethydium bromide. Since heparin has negative charges, the cationic dyewas adsorbed on the regions where heparin was immobilized. In thepresence fibroblast growth factor (FGF), the growth of mouse fibroblastSTO cells was enhanced only on the heparin-immobilized regions. Thisresult indicated that micropattern-immobilized heparin activated FGF forcell growth activity.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.     

Similarities and differences between the effects of heparin and glypican-1 on the bioactivity of acidic fibroblast growth factor and the keratinocyte growth factor

The keratinocyte growth factor (KGF or FGF-7) is unique among its family members both in its target cell specificity and its inhibition by the addition of heparin and the native heparan-sulfate proteoglycan (HSPG), glypican-1 in cells expressing endogenous HSPGs. FGF-1, which binds the FGF-7 receptor with a similar affinity as FGF-7, is stimulated by both molecules. In the present study, we investigated the modulation of FGF-7 activities by heparin and glypican-1 in HS-free background utilizing either HS-deficient cells expressing the FGF-7 receptor (designated BaF/KGFR cells) or soluble extracellular domain of the receptor. At physiological concentrations of FGF-7, heparin was required for high affinity receptor binding and for signaling in BaF/KGFR cells. In contrast, binding of FGF-7 to the soluble form of the receptor did not require heparin. However, high concentrations of heparin inhibited the binding of FGF-7 to both the cell surface and the soluble receptor, similar to the reported effect of heparin in cells expressing endogenous HSPGs. The difference in heparin dependence for high affinity interaction between the cell surface and soluble receptor may be due to other molecule(s) present on cell surfaces. Glypican-1 differed from heparin in that it stimulated FGF-1 but not FGF-7 activities in BaF/KGFR cells. Glypican-1 abrogated the stimulatory effect of heparin, and heparin reversed the inhibitory effect of glypican-1, indicating that this HSPG inhibits FGF-7 activities by acting, most likely, as a competitive inhibitor of stimulatory HSPG species for FGF-7. The regulatory effect of glypican-1 is mediated at the level of interaction with the growth factor as glypican-1 did not bind the KGFR. The effect of heparin and glypican-1 on FGF-1 and FGF-7 oligomerization was studied employing high and physiological concentrations of growth factors. We did not find a correlation between the effects of these glycosaminoglycans on FGFs biological activity and oligomerization. Altogether, our findings argue against the heparin-linked dimer presentation model as key in FGFR activation, and support the notion that HSPGs primarily affect high affinity interaction of FGFs with their receptors.     

Stabilization by heparin of acidic fibroblast growth factor mitogenicity for human endothelial cells in vitro

The effects of heparin and other glycosaminoglycans (GAGs) on the mitogenicity and stability of acidic fibroblast growth factor (aFGF) were studied. The mitogenic activity of aFGF was assayed utilizing cultured adult human endothelial cells (AHECs) isolated from iliac arteries and veins as target cells. In most experiments, aFGF purified from bovine brain was employed; in some experiments recombinant bovine aFGF was used and qualitatively similar results were obtained. In the presence of heparin, bovine aFGF at doses between 0.5 and 1.0 ng/ml (30-60 pM) elicited half the maximum AHEC growth over a 4-day period depending on the cell line tested; in the absence of heparin, significant growth was not observed at aFGF concentrations less than 10-20 ng/ml. This effect of heparin was dose-dependent over the range 0.1-10 micrograms/ml (half-maximum dose, 2 micrograms/ml). The mitogenic activity of bovine aFGF for AHECs decreased by 50% after preincubation in culture medium without cells at 37 degrees C for 2 1/2 to 3 hours. In contrast, the mitogenic activity of bovine aFGF preincubated in the presence of heparin-containing culture medium without cells was dramatically stabilized (half-life 24-29 hours). These effects also were observed in serum-free medium. Several GAGs structurally related to heparin such as chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and hyaluronic acid neither potentiated nor stabilized aFGF mitogenic activity. However, heparan sulfate from bovine lung was found to be nearly as active as heparin in both these effects. These data suggest that the binding and stabilization of mitogens by extracellular and tissue-associated heparan sulfates might play important roles in the regulation of AHEC growth.