Induction of basic fibroblast growth factor mRNA by basic fibroblast growth factor in Müller cells

PURPOSE: To investigate the induction of basic fibroblast growth factor (bFGF) gene expression in cultured rat Müller cells by bFGF and to study the mechanism of induction. METHODS: Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured with Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, carbonic anhydrase II, and glutamine synthetase. Cells of passages 1 through 4 were treated with bFGF, the protein kinase C (PKC) inhibitor, H-7; calphostin C, or the PKC activator, PMA; and protein kinase A (PKA) inhibitor, H-89; as well as the adenylate cylase activator, forskolin; or the adenylate cyclase inhibitor, SQ22536. Northern blot analysis was performed to determine the mRNA expression of bFGF, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF). RESULTS: Addition of bFGF to culture medium induced bFGF gene expression in a dose- and time-dependent manner. Induction of bFCF mRNA started at a bFGF concentration of 0.1 ng/ml. The bFGF mRNA level was elevated by 2-fold at 1 ng/ml of bFGF, 2.8-fold at 5 ng/ml, and reached a peak of 4-fold at 10 ng/ml and 3.7-fold at 50 ng/ml. At 10 ng/ml of bFGF, induction of bFGF mRNA was observed as early as 2 hours (2-fold) after treatment. The bFGF mRNA level continued to increase to 3.7-fold by 4 hours, and reached a maximum of 4.4-fold by 8 hours. A slow decline of the bFGF mRNA level was observed after 8 hours of bFGF treatment (3.5-fold by 12 hours, and 3-fold by 24 hours). This induction of bFGF gene expression was blocked by PKC inhibitors H-7 (30 microM). The PKC activator PMA (0.1 microM) also upregulated bFGF gene expression, but the effects of bFGF and PMA were not additive. An adenylate cyclase inhibitor, SQ22536 (100 microM), did not inhibit bFGF-induced bFGF gene expression. Although forskolin (5 microM), an adenylate cyclase activator, also upregulated the level of bFGF mRNA, the effects of forskolin and bFGF were additive. In addition, no inhibitory effect on bFGF-induced expression of bFGF mRNA was found using H-89 (1 microM). Exogenous bFGF did not alter the mRNA levels of CNTF and BDNF. CONCLUSIONS: These results indicate that bFGF induces bFGF gene expression in cultured rat Müller cells through PKC activation. The authors' findings raise the possibility that Müller cells in vivo also respond to available bFGF (for example, that released from the endogenous reservoirs in the case of injury) or to exogenous bFGF by producing more bFGF, which could in turn promote photoreceptor survival.     

Structure of a heparin-linked biologically active dimer of fibroblast growth factor

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.     

Immunolocalization of basic fibroblast growth factor and fibroblast growth factor receptor-1 and receptor-2 in rat cranial sutures

Craniosynostosis is a common disorder with an unknown etiology. Recent genetic mapping studies have demonstrated a strong linkage between several familial craniosynostotic syndromes and mutations in fibroblast growth factor receptor 1 (FGF-R1) and 2 (FGF-R2). The purpose of this experiment was to investigate by immunohistochemistry the protein production of these receptors as well as of their most prevalent ligand, basic fibroblast growth factor (bFGF), before, during, and after sutural fusion in rat cranial sutures. The posterior frontal (normally fuses between postnatal days 12 and 22) and sagittal (remains patent) sutures of embryonic day 20 and neonatal days 6, 12, 17, 22, and 62 (n = 3 per group) were harvested, fixed, and decalcified. Five-micrometer sections were stained with polyclonal antibodies against bFGF, FGF-R1, and FGF-R2, and patterns of immunohistochemical staining were assessed by independent reviewers. Our results indicate that increased bFGF production correlates temporally with suture fusion, with increased staining of the dura underneath the fusing suture prior to fusion followed by increased staining within osteoblasts and sutural cells during fusion. FGF-R1 and, to a lesser extent FGF-R2 immunostaining revealed a different pattern of localization with increased immunostaining within the patent sagittal suture at these time points. These results implicate bFGF in the regulation of sutural fusion and may imply autoregulatory mechanisms in fibroblast growth factor receptor expression.     

Complexation of basic fibroblast growth factor with gelatin

Polyion complexation between basic fibroblast growth factor (bFGF) and gelatin was studied by the turbidity change of mixed solution, heparin high performance liquid affinity chromatography (HPLAC), and isoelectric electrophoresis. When an aqueous solution of acidic gelatin with an isoelectric point (IEP) of 5.0 was mixed with that of bFGF, the turbidity of the mixed solution increased with time, whereas basic gelatin with and IEP of 9.0 did not cause any solution turbidity. A maximum turbidity of the mixed bFGF and acidic gelatin solution was observed around a bFGF/gelatin molar ratio of 1.0, irrespective of the gelatin concentration and solution temperature. The solution turbidity decreased with an increase in the ionic strength of the mixed solution. Complexation of bFGF with acidic gelatin was slower than that with poly(acrylic acid) probably because of the lower density of gelatin negative charge than that of poly(acrylic acid). HPLAC study revealed that complexation of bFGF with the acidic gelatin reduced the affinity of bFGF for heparin, in contrast to the basic gelatin, although the extent became smaller with the increasing ionic strength of the solution. An electrophoretic experiment showed that the IEP of bFGF shifted to a lower value after its gelatin complexation. These findings indicate that an electrostatic interaction is the main driving force for the complexation between acidic gelatin and basic bFGF.     

Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment

OBJECTIVE: To determine if insulinlike growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), individually or in combination, support the growth and viability of human septal chondrocytes in a serum-free medium (SFM) and a serum-enhanced culture medium. DESIGN: Chondrocytes were recovered from enzymatically digested human septal cartilage and were plated for monolayer culture in a newly developed medium. The medium included Dulbecco modified Eagle medium mixed 1:1 with Ham F12 medium and a supplement of known amounts of 2 growth factors-bFGF (100 ng/mL) and IGF-I (100 ng/mL)-used in combination and separately. RESULTS: The combination of IGF-I and bFGF enhanced chondrocyte growth and maintained a high degree of viability in SFM and 10% fetal calf serum. After an initial lag, the SFM, augmented with both growth factors, produced a comparable number of viable cells (4.25+/-0.31 x 10(4)) to that of the medium with 10% fetal calf serum (4.64+/-0.35 x 10(4)) by the seventh day of the experiment. Combined with the 2 growth factors, 10% fetal calf serum provided the greatest proliferation by the end of the experiment. However, the overall mean cell counts for the IGF-I- and bFGF-enhanced SFM were not statistically different. CONCLUSIONS: The combination of IGF-I and bFGF in a serum-free and a serum-supplemented environment supports the growth and viability of human septal chondrocytes in short-term culture. In an SFM, the results obtained approximate those produced in a medium enhanced with 10% fetal calf serum.     

Multivalent ligand-receptor binding interactions in the fibroblast growth factor system produce a cooperative growth factor and heparin mechanism for receptor dimerization

The binding interactions for the three primary reactants of the fibroblast growth factor (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the cofactor heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<==>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<==>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<==>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed growth factor and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatocyte growth factor and fibroblast growth factor 2 are overexpressed after cerulein-induced acute pancreatitis

Competency assessments are a growing function of the consultation-liaison (C-L) psychiatrist. Such consultation requests often mask a variety of psychosocial issues that are a source of frustration to the referring physician responding to the pressures of the changing health care delivery system in the acute care setting. This study identifies the issues and the outcome of psychiatric consultation in these patients. The implications of this burgeoning role for the C-L psychiatrist are also explored.     

Studies on the expression of fibroblast growth factors and fibroblast growth factor receptors in human prostate cancer

PURPOSE: We tried to clarify the role of fibroblast growth factors (FGFs) and those receptors (FGF-Rs) in cell proliferation of human prostate cancer. METHODS: The mRNA expression of FGF1, FGF2, FGF7, FGF-R1, FGF-R2 (IIIb), and FGF-R2 (IIIc) was investigated by RT-PCR in androgen sensitive cells (LNCaP), androgen-independent cells (PC3) and primary cultured stromal (PS) and epithelial cells (PE) from benign prostatic hyperplasia (BPH). Expression of the mRNA of FGF-R1, FGF-R2 (IIIb) and FGF-R2 (IIIc) in human prostate cancer tissue was similarly analyzed. Furthermore, the level of FGF-R1 expression in human prostate cancer was measured by semi-quantitative RT-PCR. RESULTS: FGF-R1 mRNA was detected in LNCaP, PC3 and the primary cultured stromal cells of BPH. FGF-R2 (IIIb) was seen in LNCaP cells and the primary cultured epithelial cells of BPH, while FGF-R2 (IIIc) was only observed in PC3. FGF1 mRNA was expressed in LNCaP and PC3, while FGF2 mRNA was in PC3 alone. The expression of FGF7 mRNA was detected only in the primary cultured stromal cells. Of 17 patients with human prostate cancer, FGF-R2 (IIIb) was detected in 2 and FGF-R2 (IIIc) in 15. Histological type of two cases having FGF-R2 (IIIb) were well differentiated adenocarcinoma. The mRNA levels of FGF-R1 in poorly and moderately differentiated types were significantly higher than those in well differentiated ones (p < 0.05). CONCLUSION: These findings suggest that several changes of expression in FGFs and FGF-Rs may correlate with malignant progression of human prostate cancer.     

The use of a novel immune complex to isolate neutralizing antibodies to basic fibroblast growth factor

This paper describes the use of a novel immune complex (IC) to generate neutralizing monoclonal antibodies to basic fibroblast growth factor. The IC uses a non-neutralizing monoclonal antibody bound to Protein A, itself coated on a solid support, to capture the antigen. Presumably, the capture antibody binds to a region of the antigen distal from the neutralizing site. Animals, immunized with the IC, develop a neutralizing titer and hybridomas producing neutralizing antibodies to basic FGF were obtained. This method can be used to generate neutralizing monoclonals to highly conserved growth factors whenever the antigen can be captured at a site distal to the neutralizing eptiopes.     

Identification and characterization of a novel member of the fibroblast growth factor family

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.     

Alanine screening mutagenesis establishes tyrosine 60 of bovine insulin-like growth factor binding protein-2 as a determinant of insulin-like growth factor binding

The determinants of insulin-like growth factor (IGF) binding to its binding proteins (IGFBPs) are poorly characterized in terms of important residues in the IGFBP molecule. We have previously used tyrosine iodination to implicate Tyr-60 in the IGF-binding site of bovine IGFBP-2 (Hobba, G. D., Forbes, B. E., Parkinson, E. J., Francis, G. L., and Wallace, J. C. (1996) J. Biol. Chem. 271, 30529-30536). In this report, we show that the mutagenic replacement of Tyr-60 with either Ala or Phe reduced the affinity of bIGFBP-2 for IGF-I (4.0- and 8.4-fold, respectively) and for IGF-II (3.5- and 4.0-fold, respectively). Although adjacent residues Val-59, Thr-61, Pro-62, and Arg-63 are well conserved in IGFBP family members, Ala substitution for these residues did not reduce the IGF affinity of bIGFBP-2. Kinetic analysis of the bIGFBP-2 mutants on IGF biosensor chips in the BIAcore instrument revealed that Tyr-60 --> Phe bIGFBP-2 bound to the IGF-I surface 3.0-fold more slowly than bIGFBP-2 and was released 2.6-fold more rapidly than bIGFBP-2. We therefore propose that the hydroxyl group of Tyr-60 participates in a hydrogen bond that is important for the initial complex formation with IGF-I and the stabilization of this complex. In contrast, Tyr-60 --> Ala bIGFBP-2 associated with the IGF-I surface 5.0-fold more rapidly than bIGFBP-2 but exhibited an 18.4-fold more rapid release from this surface compared with bIGFBP-2. Thus both the aromatic nature and the hydrogen bonding potential of the tyrosyl side chain of Tyr-60 are important structural determinants of the IGF-binding site of bIGFBP-2.     

Sinusoidal capillarization of human hepatocellular carcinoma: possible promotion by fibroblast growth factor

Expression of acidic and basic fibroblast growth factors (aFGF and bFGF) and von Willebrand factor (vWF) was immunohistochemically investigated in 55 nodules of human hepatocellular carcinoma (HCC), 15 nodules of adenomatous hyperplasia (AH) of the liver and 10 cirrhotic livers (LC). AH, a putative preneoplastic lesion in the cirrhotic liver, was subdivided into ordinary and atypical types: the former was characterized by little cellular and structural atypia and the latter had some atypia equivocal as to benignity and malignancy. The positive rates of FGF (aFGF and/or bFGF) were as follows: 0% in LC, 20% in AH (ordinary and atypical) and 42% in HCC, whereas the positivity of vWF was 10% in LC, 20% in ordinary AH, 30% in atypical AH and 40% in HCC. There was no correlation between the expression of FGF or vWF and the size of HCC. No correlation was also found between the positivity of FGF and that of vWF in HCC and atypical AH. While vWF was not constantly expressed in the vicinity of FGF-positive HCC cells, capillarized sinusoids were significantly more numerous in FGF-positive cases than in FGF-negative cases (p < 0.01). These data indicate that FGF may be pathogenetically linked to the multistep development of HCC in relation to sinusoidal capillarization.     

Basic fibroblast growth factor impregnated hydrogel microspheres for embolization of cerebral arteriovenous malformations

To assess the prevention of recanalization at embolized sites in cerebral arteriovenous malformations, the authors devised a novel embolic material, hydrogel microspheres prepared from poly(ethylene glycol) diacrylate impregnated with basic fibroblast growth factor. In this article, preparation of the microspheres, and preliminary study of in vitro and in vivo performance are discussed. Poly(ethylene glycol) diacrylate, prepared from end capping of poly(ethylene glycol) (molecular weights, 1,000, 2,000, and 4,000) with acryloyl chloride and benzophenone derived poly(ethylene glycol), prepared from poly(ethylene glycol) (molecular weight, 2,000) with benzoyl benzoic acid chloride as a photoinitiator, were dissolved in a buffer solution with or without basic fibroblast growth factor. The mixed solution was dropped stepwise into liquid paraffin with stirring. Ultraviolet light irradiation resulted in the formation of relatively rigid hydrogel microspheres (diameter, 100-400 microm). The in vitro study showed that the higher the molecular weight of poly(ethylene glycol) diacrylate used, the faster the release rate of immobilized protein. Canine kidneys were embolized with these microspheres via the femoral artery using a microcatheter. Histologic examination showed that microspheres occluded arterioles. The degree of accumulation of fibroblasts and extracellular matrix were larger for basic fibroblast growth factor impregnated microspheres than for nonimpregnated ones. Basic fibroblast growth factor released from microspheres may help regenerate tissues at arteriovenous malformation sites, and recanalization is expected to be prevented.     

Effects of modulation of basic fibroblast growth factor on tumor growth in vivo

BACKGROUND: Recombinant human basic fibroblast growth factor (rHu-bFGF) is known to stimulate proliferation in some tumor cells and to modulate tumor vascularization. PURPOSE: The purpose of this study was to examine the possible role of this agent in the development of tumors. The study was designed to determine the effects of modulating bFGF activity in vivo in tumor models from cell lines with different responses to bFGF and with different content and receptor levels of bFGF. METHODS: Two tumor cell lines (human DLD-2 colon carcinoma and rat C6 glioma) were characterized for bFGF content and bFGF receptor levels by Western blot analysis in cultured cells and by studies of [125I]rHu-bFGF binding to sections from xenografts grown in nude mice. Tumor cell proliferation was monitored after treatment with rHu-bFGF or the DG2 or DE6 IgG monoclonal antibody to rHu-bFGF in culture and in vivo. RESULTS: C6 cells exhibited 7800 high-affinity receptors for rHu-bFGF per cell (dissociation constant [Kd] = 46 pM), while DLD-2 cells lacked high-affinity receptors. rHu-bFGF stimulated [3H]thymidine uptake by C6 cells, but the addition of DG2 IgG prevented this stimulation; rHu-bFGF had no effect on [3H]thymidine incorporation by DLD-2 cells. C6 cells had higher levels of immunoreactive bFGF than did DLD-2 cells. The xenografts from both cell lines exhibited high-affinity [125I]rHu-bFGF binding that was concentrated on vascular-like structures. rHu-bFGF at a dosage of 0.25 mg/kg given intraperitoneally daily for 18 days caused a twofold increase in DLD-2 tumor weight but had little effect on the growth of C6 xenografts. In contrast, daily intravenous injections of DG2 IgG given to mice had no effect on DLD-2 tumor growth but reduced growth of C6 tumors by approximately 30%--a statistically significant difference. CONCLUSIONS: The addition of exogenous rHu-bFGF or of a neutralizing antibody resulted in significant alterations in tumor growth in vivo, which were specific for tumor type and bFGF characteristics. While some of these effects may be mediated by the bFGF-responsive endothelial cells of the tumor vasculature (DLD-2 colon carcinoma), others may result from inhibition of bFGF-dependent tumor cell proliferation (C6 glioma). IMPLICATIONS: Studies that measure tumor blood flow are necessary to confirm that these effects are mediated by changes in tumor vasculature.     

A natural kinase-deficient variant of fibroblast growth factor receptor 1

A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation.     

Repair of chronic tympanic membrane perforations with fibroblast growth factor

A number of angiogenic growth factors have been shown to accelerate wound healing. Previous work has demonstrated that topical application of epidermal growth factor is effective in healing chronic tympanic membrane perforations in an animal model. Theoretically, fibroblast growth factor may result in a superior healed membrane through preferential stimulation of the fibroblasts within the middle layer of the tympanic membrane. To test this hypothesis, the effects of exogenously applied fibroblast growth factor on the chronically perforated tympanic membrane were evaluated. A buffered solution of fibroblast growth factor (25 microliters of fibroblast growth factor, 0.2 mg/ml) was administered to a Gelfoam pledget placed over chronic tympanic membrane perforations in chinchillas. Control ears were treated with Gelfoam and the buffer solution only. Complete closure of the tympanic membrane perforation was observed in 81% (13 of 16) of the fibroblast growth factor-treated ears, but in only 41% (7 of 17) of the controls (p = 0.05). Heading took place gradually, requiring an average of 4 weeks for the fibroblast growth factor-treated and 6.5 weeks for the control ears that healed. The relatively high healing rate for the control group does not imply that the pretreatment perforations were not chronic, rather there appears to be some efficacy to the control protocol of repeated applications of Gelfoam and buffer. A histologic analysis of the fibroblast growth factor-healed eardrums immediately after closure demonstrated hypertrophy of the squamous and fibrous layers of the tympanic membrane. Over time, the eardrum thinned to reach proportions similar to those of the normal tympanic membrane, including the presence of a substantial middle fibrous layer. A screening ototoxicity study revealed no structural damage to the organ of Corti after growth factor treatment. To assess the potential for systemic toxicity, blood and peripheral tissues were analyzed for radioactivity at time points during a 48-hour period after application of 25 microliters of 125I-fibroblast growth factor to the perforated tympanic membrane. More than 78% of the radioactivity remained at the application site. Given the tiny original dosage, the small fraction absorbed systemically is minuscule and highly unlikely to induce adverse effects in light of published toxicity data. On the basis of these promising safety and efficacy data in the chinchilla model, clinical trials of fibroblast growth factor in repair of chronic tympanic membrane perforations in human beings are being initiated.     

Expression of receptors for basic fibroblast growth factor on human periodontal ligament cells

Basic fibroblast growth factor (FGF-2; bFGF) is a major mitogen for connective tissue cells, and participates in the healing process. It has already been reported that FGF-2 could be applicable to enhance periodontal regeneration. In the present study, we examined FGF receptor (FGFR) expression on human periodontal ligament (PDL) cells. The binding of [125I]-labeled FGF-2 to human PDL cells was studied by radioreceptor assay. The binding of [125I]-FGF-2 to PDL cells reached a plateau after 2.5 h incubation at 4 degrees C and was inhibited by the addition of unlabeled FGF-2 and acidic FGF (FGF-1; aFGF), but not insulin-like growth factor-I, platelet-derived growth factor and transforming growth factor-beta 1. Scatchard analysis revealed the presence of approximately 1.0 x 10(5) FGF-2 binding sites per cell with an apparent Kd of 1.2 x 10(-10) M. Interestingly, the binding of [125I]-FGF-2 on PDL cells reached its maximum at d 6 of the culture and then gradually decreased. Scatchard analysis also demonstrated that the number of FGFRs on a PDL cell was altered during the course of the culture, while the affinity between FGF-2 and its receptor was not. The responsiveness of PDL cells to FGF-2, which was monitored by the inhibitory effect on alkaline phosphatase activity, was reduced in proportion to the decrease in the number of FGFRs on the PDL cells. The present study suggests that PDL cells alter the responsiveness to FGF-2 during the course of the culture by changing the density of its receptor, and that the density of FGFR expression might be a marker of the cytodifferentiation of PDL cells into mineralized tissue forming cells.     

Binding of basic fibroblast growth factor to fibrinogen and fibrin

Fibrin is formed at sites of tissue injury and provides the temporary matrix needed to support the initial endothelial cell responses needed for vessel repair. Basic fibroblast growth factor (bFGF) also acts at sites of injury and stimulates similar vascular cell responses. We have, therefore, investigated whether there are specific interactions between bFGF and fibrinogen and fibrin that could play a role in coordinating these actions. Binding studies were performed using bFGF immobilized on Sepharose beads and soluble 125I-labeled fibrinogen and also using Sepharose-immobilized fibrinogen and soluble 125I-bFGF. Both systems demonstrated specific and saturable binding. Scatchard analysis indicated two classes of binding sites for each with Kd values of 1.3 and 260 nM using immobilized bFGF; and Kd values of 0.9 and 70 nM using immobilized fibrinogen. After conversion of Sepharose-immobilized fibrinogen to fibrin by treatment with thrombin, bFGF also demonstrated specific and saturable binding with two classes of binding sites having Kd values of 0.13 and 83 nM. Fibrin binding was also investigated by clotting a solution of bFGF and fibrinogen, and two classes of binding sites were demonstrated using this system with Kd values of 0.8 and 261 nM. The maximum molar binding ratios of bFGF to fibrinogen were between 2.0 and 4.0 with the four binding systems. We conclude that bFGF binds specifically and saturably to fibrinogen and fibrin with high affinity, and this may have implications regarding the localization of its effect at sites of tissue injury.