Taliglucerase alfa leads to favorable bone marrow responses in patients with type I Gaucher disease.

Taliglucerase alfa (Protalix Biotherapeutics, Israel) is a carrot-cell-expressed recombinant human beta-glucocerebrosidase recently approved in the United States for the treatment of type 1 Gaucher disease (GD). As bone disease is one of the most debilitating features of GD, quantification of bone marrow involvement is important for monitoring the response to treatment. Therefore, bone marrow fat fraction (Ff) measured by quantitative chemical shift imaging (QCSI) was included as exploratory parameter to evaluate bone marrow response in treatment naïve GD patients participating in a double-blind, randomized phase III study. Eight GD patients with intact spleens were treated with 30 or 60U/kg biweekly. Ff results were compared to outcomes in 15 untreated Dutch GD patients with a follow-up interval of 1year. Five taliglucerase alfa treated patients had a Ff below the threshold that relates to complication risk (<0.23) at baseline (median (n=8) 0.19, range 0.11-0.35). Ff significantly increased compared to baseline (p=0.012) and compared to untreated patients (p=0.005), already after 1year of follow-up with further improvement up to 36months. In four patients with the lowest Ff, the higher dose resulted in increases above 0.23 within 1year. All patients had sustained improvements in all other parameters. There was no influence of antibodies on response parameters. Treatment with taliglucerase alfa results in significant increases in lumbar spine fat fractions, which indicates clearance of Gaucher cells from the bone marrow.

Mechanical resistance of biological repair cartilage: comparative in vivo tests of different surgical repair procedures.

The former common knowledge that cartilage lesions do not heal has been modified over the last few years due to new technologies. For repair of deeper circumscribed lesions osteochondral press-fit grafting and tissue engineering are used in clinical application. The histological data of the hyaline-like tissue obtained by engineering are just as satisfactory as the surviving grafted hyaline cartilage on top of osteochondral cylinders. But comparative studies are still lacking. To fill the gap and with a view to repairing larger osteoarthritic defects we have performed an in vivo study on 16 goats. Three months after the creation of a full thickness wide cartilage defect on the femoral condyle with harvesting of cartilage samples for tissue cultures we performed secondary cartilage repair procedures on the installed osteoarthritis areas: 1) grafting with autogenic osteochondral press-fit cylinders from the opposite knee, 2) autologous engineered chondrocyte grafting under periosteal flaps, 3) both in combination. The harvesting defects were either left as controls or filled with a hyaluronate fleece. After eight months the repaired areas and the harvesting defects were examined for cartilage stiffness as a novel comparative parameter. Compared to normal the cartilage on top of osteochondral grafts is considerably stiffer. Engineered cartilage is weaker than normal. Spontaneously ingrown fibrous cartilage is much weaker even with a carrier fleece. A combination of osteochondral press-fit grafts with engineered autologous cells restores biomechanical qualities to repaired larger degenerative cartilage defects.

Development of a high throughput screening assay for inhibitors of fibroblast growth factor-receptor-heparin interactions.

High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.

Heparin-like inhibitory activity to fibroblast growth factor-2 in wound fluids of patients with chronic skin ulcers and its modulation during wound healing.

Fibroblast growth factors are potent mitogens and angiogenic factors which play a critical role in wound healing. Fibroblast growth factors require heparan sulfates as cofactors in order to activate their cognate receptors and exert their cellular and biological effects. Heparan sulfates were extracted from wound fluids of 5 patients with chronic diabetic foot ulcers or chronic venous stasis ulcers and tested for their capacity to modulate fibroblast growth factor-receptor binding, during the course of the ulcers' resolution, until complete healing (3-8 months). Total heparan sulfates concentration measured as iduronic acid equivalents, decreased in wound fluids from 1.1 +/-0.3 microg/ml to 0.26 +/-0.1 microg/ml as wound healing progressed. These heparan sulfates exhibited a predominant inhibitory effect on fibroblast growth factor-2 binding to fibroblast growth factor receptor-1, when tested in cells deficient in cell surface heparan sulfates. During wound healing, there was a marked decrease in the relative inhibitory activity of the extracted heparan sulfates on fibroblast growth factor-2-receptor binding. Heparan sulfates extracted from chronic skin ulcers of different etiologies such as diabetic foot or chronic venous stasis ulcers showed the same pattern of alternating balance in heparan sulfates mediated activity. The presence of fibroblast growth factor inhibitory factors which possess heparin-like activity in fluids of chronic skin ulcers and their ability to modulate fibroblast growth factor-receptor activity throughout the process of wound healing, may significantly contribute to the mechanism of chronicity. Treatments to counter this inhibition may offer new possibilities for healing chronic wounds.

Characteristics of cartilage biopsies used for autologous chondrocytes transplantation.

Biopsies removed from 57 patients considered for cartilage transplantation were grown at CTI Ltd. (47 biopsies) and at Tel Aviv University (10 biopsies). Tissue processing took place in dedicated laboratories. Explant cultures allowed cell number expansion. Fifty-four out of 57 biopsies grew cells. Fanning out of the cells began after 5-15 days in culture. Two passages later, cell numbers in the 10(7) range were achieved. Cells from all cultures expressed mRNA of aggrecan and link protein but not of alkaline phosphatase. Histochemical stains such as alcian blue pH 1 were negative in sparse monolayer cultures, but positive in pellet cultures. Immunohistochemistry demonstrated expression of collagen type I in monolayer cultures, switching to collagen type II in micromass cultures. Fibroblast growth factor receptor 3, a recently described characteristic receptor of precartilaginous cells, was expressed in monolayers and disappeared in micromass cultures. In conclusion, explants of articular chondrocytes cultured in vitro consistently yield monolayer cultures. The cells appear to revert to dedifferentiated chondrocytes, expressing a mesenchymal stem cell protein profile. Simultaneously, these cells regained their capacity to proliferate. Cultures held as micromass allowed reexpression of the differentiated phenotype traits.

[Autologous chondrocyte transplantation--from science fiction to routine clinical practice].

Adult articular cartilage lacks the capacity for self-repair. The limiting factor appears to be the inability of chondrocytes to proliferate while embedded in the extracellular matrix typical of hyaline cartilage. Cartilage defects larger than 1 cm2 change articular biomechanics and lead to eventual osteoarthritis and joint destruction. During the past decade, several competing techniques have evolved to stimulate articular cartilage repair. Small lesions can be successfully treated by either micro-fracture or osteochondral cylinder grafting. The latter technique allows immediate weight bearing but leads to damage of previously uninvolved areas of articular cartilage, which limits its application to lesions of less than 2 cm2. When the damaged area is more extensive, grafting of autologous chondrocytes should be considered. First a diagnostic arthroscopy is performed to assess the damaged area and a small cartilage biopsy is taken. 6 weeks later, arthrotomy and chondrocyte transplantation are performed. In the interval, the antologous chondrocytes have expanded by 2 to 3 orders of magnitude. Our experience to date includes 10 cases with follow-up of 6 months to 5 years. Preoperative complaints of crepitation and locking disappear. There is functional improvement and pain reduction of approximately 50%. This procedure, currently limited to patients under 55 years of age with limited damage to an articular surface, for the first time allows reconstruction of damaged articular areas without resorting to allografts.

Porphyrin analogues as novel antagonists of fibroblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis.

Fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) play a pivotal role in the multistep pathway of tumor progression, metastasis, and angiogenesis. We have identified a porphyrin analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra -p-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding and activation. TMPP demonstrated potent inhibition of binding of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at submicromolar concentrations. TMPP inhibits binding of radiolabeled FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an in vitro angiogenic assay measuring the extent of endothelial cell growth, tube formation, and sprouting, TMPP dramatically reduced the extent of the FGF2-induced endothelial cell outgrowth and differentiation. In a Lewis lung carcinoma model, mice receiving TMPP showed a marked inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon alternate daily injections of TMPP at 25 microg/g of body mass. Finally, novel meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel corrole-based derivative, with >50-fold increase in activity in vitro, had a significantly improved efficacy in blocking tumor progression and metastasis in vivo.

Autologous chondrocyte transplantation for reconstruction of isolated joint defects: the Assaf Harofeh experience.

BACKGROUND: Articular cartilage is incapable of undergoing self-repair since chondrocytes lose their mitotic ability as early as the first year of life. Defects in articular cartilage, especially in weight-bearing joints, will predictably deteriorate toward osteoarthritis. No method has been found to prevent this deterioration. Drilling of the subchondral bone can lead to fibrocartilage formation and temporary repair that slowly degrades. Animal experiments indicate that introducing proliferating chondrocytes such as cultured articular chondrocytes can reliably reconstruct joint defects. OBJECTIVES: To describe our clinical experience in culturing and transplanting autologous chondrocytes. METHODS: Biopsies were obtained from 10 patients, aged 18-45, undergoing a routine arthroscopy in which a cartilage defect was identified with indications for cartilage transplantation. The biopsies were further processed to establish chondrocyte cultures. ACT was performed in 8 of the 10 patients because of persistent symptoms for at least 2 months post-arthroscopy. All patients (6 men and 2 women) had a grade IV cartilage defect in the medial or lateral femoral condyle, and three had a defect in the trochlear region as well. Biopsies were removed from the lateral rim of the superior aspect of the femur, and cells were cultured in a clean room. Following a 2 order of magnitude expansion, cells were implanted under a periosteal flap. RESULTS: The eight patients implanted with autologous cells were followed for 6 months to 5 years (average 1 year). Complaints of giving-way, effusion and joint locking resolved in all patients, and pain as assessed by the visual analogue score was reduced by an average of 50%. Follow-up magnetic resonance imaging studies in all patients revealed that the defects were filled with tissue having similar signal characteristics to cartilage. CONCLUSIONS: Chondrocyte implantation is a procedure capable of restoring normal articular cartilage in cases with isolated joint defects. Pain can be predictably reduced, while joint locking and effusion are eliminated. The effect on osteoarthritis progression in humans has not yet been elucidated.

Oligomerization reduces heparin affinity but enhances receptor binding of fibroblast growth factor 2.

The biological response of cells to fibroblast growth factors (FGFs) depends on heparan sulphate glycosaminoglycans sharing particular structural motifs. Heparin induced FGF dimerization has been suggested to mediate receptor dimerization and activation. Here we demonstrate that heparin-derived oligosaccharides that promote receptor binding and activation specifically induce the dimerization of basic FGF (FGF2). These heparin-induced dimers of FGF2 acquire high affinity for receptor binding and are biologically active. Using biotinylated FGF2 bound to immobilized streptavidin gradually saturated with biotin, enabled a quantitative analysis of heparin-dependent and heparin-independent FGF2 monomers and oligomers. Streptavidin induced FGF2 dimers bind and activate FGF receptors only in the presence of heparin. An excess of streptavidin, forcing biotin-FGF2 into monomers, reduces receptor binding and blocks FGF-dependent cell proliferation. All these suggest predominant receptor binding and activation by heparin associated FGF2 oligomers. Unexpectedly, heparin induced dimers and higher order oligomers lose most of their affinity towards heparin. Direct binding of soluble FGF receptors (FGFRs) to either monomers or dimers of FGF2, immobilized on heparin, confirm the preferred association of FGFRs with dimers of FGF2. Computerized molecular docking predicts a cis-oriented FGF2 dimer, stabilized by heparin, which complies with all the experimental data.

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.

Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.

Properly oriented heparin-decasaccharide-induced dimers are the biologically active form of basic fibroblast growth factor.

Interaction of basic fibroblast growth factor (FGF-2) with heparin or heparan sulfate proteoglycans (HSPGs) is required for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the FGF receptor by FGF-2 and heparin, we have used NMR, dynamic light scattering, and HSPG-deficient cells and cell-free systems. The first 28 N-terminal residues in FGF-2 were found to be highly mobile and flexible, consistent with the disorder found in both the NMR and X-ray structures. The structure of an FGF-2-heparin-decasaccharide complex that binds to and activates the FGF receptor was compared to a heparin-tetrasaccharide-induced complex that does not promote an interaction with the receptor. The major change observed upon the addition of the tetrasaccharide to FGF-2 was an increase in the correlation time consistent with the formation of an FGF-2 dimer. The NMR line widths of FGF-2 in the presence of the decasaccharide are severely broadened relative to the tetrasaccharide, consistent with dynamic light scattering results which indicate FGF-2 is a tetramer. The interaction of these heparin species with FGF-2 does not induce a significant conformational change in the overall structure of FGF-2, but small chemical shift changes are observed in both heparin and receptor binding sites. A trans-oriented symmetric dimer of FGF-2 is formed in the presence of the tetrasaccharide whereas two cis-oriented dimers in a symmetric tetramer are formed in the presence of the decasaccharide. This suggests that the cis-oriented FGF-2 dimer is the minimal biologically active structural unit of FGF-2. These data allow us to propose a novel mechanism to explain the functional interaction of FGF-2 with heparin and its transmembrane receptor.

Engineering of fibroblast growth factor: alteration of receptor binding specificity.

A five-residue loop structure in basic fibroblast growth factor (FGF-2) which extends from amino acid residue 118 to residue 122 was replaced, by cassette mutagenesis, with the corresponding seven-residue loop structure from the structural homologue acidic fibroblast growth factor (FGF-1) or the corresponding five-residue loop from interleukin-1 beta to give FGF-2LA and FGF-2LI, respectively. The mutants were expressed in Escherichia coli and purified to homogeneity, and their heparin and receptor binding and biological properties were examined. The ability of FGF-2LA to induce endothelial cell proliferation was the same as that of FGF-2. Affinities of the mutants to heparin and to cells that express FGF receptor-1 (FGFR-1) were identical to those of the wild-type protein. The role of the loop structure in FGF-1 and FGF-2 was elucidated by using soluble FGF receptor systems, which display distinct ligand binding specificities. Thus, FGF-2LA bound, with the same affinity as FGF-1 and FGF-2, to FGFR-1 and FGFR-2, whereas only FGF-1 and the FGF-1 loop-containing mutant, FGF-2LA, bound to the keratinocyte growth factor receptor. A change in receptor binding specificity was not observed with the FGF-2LI engineered mutant. That the binding specificity of FGF-2 was dramatically altered by transfer of a loop structure from FGF-1 to resemble the binding profile of the donor protein provides strong evidence that this motif is a receptor binding specificity determinant of fibroblast growth factors.

Heparin-dependent binding and autophosphorylation of epidermal growth factor (EGF) receptor by heparin-binding EGF-like growth factor but not by EGF.

Heparin-binding EGF-like growth factor (HB-EGF) is a recently identified member of the EGF family of growth factors and a potent mitogen for smooth muscle cells and fibroblasts. Chinese hamster ovary (CHO) cells genetically engineered to express the human EGF receptor bind with high affinity both EGF and HB-EGF. CHO mutant cells lacking heparan sulfate proteoglycans (HSPG) bind EGF equally well to wild-type cells and EGF binding is not affected by exogenous heparin. However, HSPG-deficient EGF receptor-expressing cells do not bind significant levels of HB-EGF unless heparin is present in the binding medium. Moreover, binding of radiolabeled EGF to HSPG-deficient EGF receptor-expressing cells is efficiently displaced by nonlabeled HB-EGF only in the presence of heparin. Signal transduction by the EGF receptor tyrosine kinase as evidenced by receptor autophosphorylation is induced by HB-EGF only in the presence of heparin, in contrast to EGF-induced receptor autophosphorylation, which is independent of heparin. These results directly demonstrate that HB-EGF but not EGF requires heparin or cell surface HSPG for binding and activation of the EGF receptor and that HB-EGF receptor interactions can be tightly regulated by the available local concentration of heparin-like molecules.

Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis.

A survey of defined species of cell surface and extracellular matrix heparan sulfate proteoglycans (HSPG) was performed in a search for cellular proteoglycans that can promote bFGF receptor binding and biological activity. Of the various affinity-purified HSPGs tested, perlecan, the large basement membrane HSPG, is found to induce high affinity binding of bFGF both to cells deficient in HS and to soluble FGF receptors. Heparin-dependent mitogenic activity of bFGF is strongly augmented by perlecan. Monoclonal antibodies to perlecan extract the receptor binding promoting activity from active HSPG preparations. In a rabbit ear model for in vivo angiogenesis, perlecan is a potent inducer of bFGF-mediated neovascularization. These results identify perlecan as a major candidate for a bFGF low affinity, accessory receptor and an angiogenic modulator.

Diminished heparin binding of a basic fibroblast growth factor mutant is associated with reduced receptor binding, mitogenesis, plasminogen activator induction, and in vitro angiogenesis.

Using modeling of heparin-fibroblast growth factor interactions, we replaced four basic residues of basic fibroblast growth factor (FGF-2) with neutral glutamine residues by site-specific mutagenesis to give the mutants K128Q, K138Q, K128Q-K138Q, R129Q, K134Q, and R129Q-K134Q. The FGF mutants were characterized for their receptor and heparin binding affinities, mitogenic and cell proliferation activities, and their ability to induce plasminogen activator (PA) production and in vitro angiogenesis by cultured endothelial cells. Heparin binding properties and biological activities of the three mutants involving R129 and K134 remained essentially unchanged; however, significant changes for three mutants involving K128 and K138 were found. The KD values for heparin binding for K128Q and K138Q mutants were increased about 10-fold, and that for the K128Q-K138Q double mutant was increased by about 100-fold. The mutant K128Q-K138Q required a 10-fold higher concentration of heparin to promote binding to heparan sulfate proteoglycan (HSPG)-deficient CHO cells transfected with fibroblast growth factor receptor-1 (FGFR1) or to induce DNA synthesis in HSPG-deficient myeloid cells transfected with FGFR1. Binding affinities of the mutants to cell surface receptors on BHK-21 cells, however, were similar to that of wild-type FGF-2. In endothelial cell proliferation assays the activities of K128Q and K128Q-K138Q were about 10-fold lower than that of the wild-type protein, whereas the K138Q mutant exhibited wild-type activity. In addition, the K128Q-K138Q mutant displayed a markedly lowered capacity to induce PA activity in cultured endothelial cells and to form capillary-like structures in an in vitro angiogenesis model.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor.

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). The capacity of various species of heparin and heparan sulfate (HS) to promote bFGF receptor binding was investigated using both Chinese hamster ovary mutant cells deficient in cell surface HSPG and a soluble bFGF receptor-alkaline phosphatase fusion protein. Highly sulfated oligosaccharides were more effective than medium and low sulfate fractions of the same size oligosaccharide. O-Sulfation in heparin was found to be critical for its capacity to promote binding of bFGF to its receptors. The highest level of bFGF-receptor binding was achieved in the presence of over-sulfated heparin fragments (% sulfur > 14) regardless of whether the N-position was sulfated or acetylated. Unlike receptor binding of bFGF which requires oligosaccharides containing at least 8-10 sugar units, displacement of heparin- or HS-bound bFGF was obtained by oligosaccharides containing as little as four sugar units and by an N-sulfated, O-desulfated heparin fragment (% sulfur = 5.3). A preparation of total cell surface-derived HS induced bFGF receptor binding. A preliminary survey of several defined and affinity purified species of cell surface HSPG, including syndecan, fibroglycan, and glypican failed to identify natural HSPG that promote high affinity receptor binding of bFGF. A similar lack of activity was observed with species of HS isolated from bovine arterial tissue and characterized for their effect on vascular smooth muscle cell proliferation. Moreover, most of these species of HS inhibited in a dose-dependent manner the restoration of bFGF-receptor binding induced by heparin or by total HSPG. These results suggest the involvement of defined heparin-like oligosaccharide sequences and unique species of cell surface and extracellular matrix HS in the regulation of bFGF receptor binding and biological activity.

Isolation of peptides that inhibit binding of basic fibroblast growth factor to its receptor from a random phage-epitope library.

Basic fibroblast growth factor (bFGF) is known to bind to its cell-surface receptors with high affinity and in a heparin-dependent manner. In an attempt to predict the receptor recognition site on bFGF we screened phage-epitope libraries with monoclonal antibodies DG2 and DE6, which inhibit bFGF binding to its receptor. On the affinity-isolated phages, we identified several peptide sequences as the putative antibody-binding epitopes on bFGF. The identified library epitopes shared the consensus sequence Pro-(Pro/Ser)-Gly-His-(Tyr/Phe)-Lys, corresponding to two continuous protein sequences of bFGF: Pro-Pro-Gly-His-Phe-Lys and Arg-Thr-Gly-Gln-Tyr-Lys at amino acids 13-18 and 120-125 of bFGF, respectively. Synthetic peptides of the corresponding phage epitopes or of the above bFGF sequences specifically inhibited binding of the antibodies to bFGF, blocked binding of bFGF to its high-affinity receptor, and inhibited basal and bFGF-induced proliferation of vascular endothelial cells at submicromolar peptide concentrations. The potent inhibition of bFGF binding and biological activity by peptides recognized by the antibodies suggests that these sequences are functionally involved in receptor binding and may constitute part of the receptor-binding determinants on bFGF.