Evaluation of biomimetic hyaluronic-based hydrogels with enhanced endogenous cell recruitment and cartilage matrix formation.

Biomaterials play a pivotal role in cell-free cartilage repair approaches, where cells must migrate through the scaffold, fill the defect, and then proliferate and differentiate facilitating tissue remodeling. Here we used multiple assays to test the influence of chemokines and growth factors on cell migration and cartilage repair in two different hyaluronan (HA)-based hydrogels. We first investigated bone marrow Mesenchymal Stromal Cells (BMSC) migration in vitro, in response to different concentrations of platelet-derived growth factor-BB (PDGF-BB), chemokine ligand 5 (CCL5/RANTES) and stromal cell-derived factor 1 (SDF-1), using a 3D spheroid-based assay. PDGF-BB was selected as most favourable chemotactic agent, and MSC migration was assessed in the context of physical impediment to cell recruitment by testing Fibrin-HA and HA-Tyramine hydrogels of different cross-linking densities. Supplementation of PDGF-BB stimulated progressive migration of MSC through the gels over time. We then investigated in situ cell migration into the hydrogels with and without PDGF-BB, using a cartilage-bone explant model implanted subcutaneously in athymic mice. In vivo studies show that when placed into an osteochondral defect, both hydrogels supported endogenous cell infiltration and provided an amenable microenvironment for cartilage production. These processes were best supported in Fibrin-HA hydrogel in the absence of PDGF-BB. This study used an advanced preclinical testing platform to select an appropriate microenvironment provided by implanted hydrogels, demonstrating that HA-based hydrogels can promote the initial and critical step of endogenous cell recruitment and circumvent some of the clinical challenges in cartilage tissue repair. STATEMENT OF SIGNIFICANCE: The challenge of articular cartilage repair arises from its complex structure and architecture, which confers the unique mechanical behavior of the extracellular matrix. The aim of our research is to identify biomaterials for implants that can support migration of endogenous stem and progenitor cell populations from cartilage and bone tissue, in order to permanently replace damaged cartilage with the original hyaline structure. Here, we present an in vitro 3D spheroid-based migration assay and an osteochondral defect model, which provide the opportunity to assess biomaterials and biomolecules, and to get stronger experimental evidence of the not well-characterized dynamic process of endogenous cells colonization in an osteochondral defect. Furthermore, the delicate step of early cell migration into biomaterials towards functional tissue engineering is reproduced. These tests can be used for pre-clinical testing of newly developed material designs in the field of scaffold engineering.

Sequential exposure to fibroblast growth factors (FGF) 2, 9 and 18 enhances hMSC chondrogenic differentiation.

OBJECTIVE: To test the effects of sequential exposure to FGF2, 9 and 18 on human Mesenchymal Stem Cells (hMSC) differentiation during in vitro chondrogenesis. DESIGN: Control and FGF2-expanded hMSC were cultured in aggregates in the presence of rhFGF9, rhFGF18 or rhFGFR3-specific signaling FGF variants, starting at different times during the chondroinductive program. Quantitative real time polymerase chain reaction (qRT-PCR) and immunocytochemistry were performed at different stages. The aggregate cultures were switched to a hypertrophy-inducing medium along with rhFGFs and neutralizing antibodies against FGFR1 and FGFR3. Histological/immunohistochemical/biochemical analyses were performed. RESULTS: FGF2-exposed hMSC during expansion up-regulated Sox9 suggesting an early activation of the chondrogenic machinery. FGF2, FGF9 and 18 modulated the expression profile of FGFR1 and FGFR3 in hMSC during expansion and chondrogenesis. In combination with transforming growth factor-beta (TGF-ß), FGF9 and FGF18 inhibited chondrogenesis when added at the beginning of the program (≤ d7), while exhibiting an anabolic effect when added later (≥d14), an effect mediated by FGFR3. Finally, FGFR3 signaling induced by either FGF9 or FGF18 delayed the appearance of spontaneous and induced hypertrophy-related changes. CONCLUSIONS: The stage of hMSC-dependent chondrogenesis at which the growth factors are added impacts the progression of the differentiation program: increased cell proliferation and priming (FGF2); stimulated early chondrogenic differentiation (TGF-ß, FGF9/FGF18) by shifting the chondrogenic program earlier; augmented extracellular matrix (ECM) production (FGF9/FGF18); and delayed terminal hypertrophy (FGF9/FGF18). Collectively, these factors could be used to optimize pre-implantation conditions of hMSC when used to engineer cartilage grafts.

Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors.

Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering.

T2 mapping and dGEMRIC after autologous chondrocyte implantation with a fibrin-based scaffold in the knee: preliminary results.

OBJECTIVE: To assess repair tissue (RT) after the implantation of BioCartII, an autologous chondrocyte implantation (ACI) technique with a fibrin-hyaluronan polymer as scaffold. T2 mapping and delayed Gadolinium Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) were used to gain first data on the biochemical properties of BioCartII RT in vivo. METHODS: T2 mapping and dGEMRIC were performed at 3T in five patients (six knee joints) who had undergone ACI 15-27 months before. T2 maps were obtained using a pixel wise, mono-exponential non-negative least squares fit analysis. For quantitative T1 mapping a dual flip angle 3D GRE sequence was used and T1 maps were calculated pre- and post-contrast using IDL software. Subsequent region of interest analysis was carried out in comparison with morphologic MRI. RESULTS: A spatial variation of T2 values in both hyaline, normal cartilage (NC) and RT was found. Mean RT T2 values and mean NC T2 values did not differ significantly. Relative T2 values were calculated from global RT and NC T2 and showed a small range (0.84-1.07). The relative delta relaxation rates (rDeltaR1) obtained from the T1 maps had a wider range (0.77-4.91). CONCLUSION: T2 mapping and dGEMRIC provided complementary information on the biochemical properties of the repair tissue. BioCartII apparently can provide RT similar to hyaline articular cartilage and may become a less-invasive alternative to ACI with a periosteal flap.

Results of chondrocyte implantation with a fibrin-hyaluronan matrix: a preliminary study.

UNLABELLED: Fibrin, a homologous polymer, is the natural scaffold of wound healing and therefore a candidate as a carrier for cell transplantation. We explored a novel matrix-based implant cartilage repair composed of both fibrin and hyaluronan in a defined ratio that takes advantage of the biological and mechanical properties of these two elements. The matrix was seeded with autologous chondrocytes expanded in the presence of a proprietary growth factor variant designed to preserve their chondrogenic potential. We prospectively followed eight patients with symptomatic-chronic cartilage defects treated with this carrier. Patients had arthroscopy to harvest autologous chondrocytes then grown in autologous serum. Chondrocytes were cultured in the presence of the FGF variant and then seeded on the fibrin-hyaluronan matrix. About 4 weeks following biopsy, the patients underwent implantation of the constructs by miniarthrotomy. Three of the eight patients had transient effusion. Clinical performance was measured by Lysholm and IKDC scores, MRI, and the need for secondary surgery. The clinical outcome of a 1-year followup demonstrated increase of clinical scores. The MRI followup showed good filling of the defect with tissue having the imaging appearance of cartilage in all patients. Apart from the transient effusion in three patients we observed no other adverse events during the followup. LEVEL OF EVIDENCE: Level IV, therapeutic study.

FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: implications for endochondral ossification.

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.

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.

FGF receptors ubiquitylation: dependence on tyrosine kinase activity and role in downregulation.

A crucial aspect of ligand-mediated receptor activation and shut-down is receptor internalization and degradation. Here we compared the ubiquitylation of either wild type or a K508A 'kinase-dead' mutant of fibroblast growth factor receptor 3 (FGFR3) with that of its naturally occurring overactive mutants, G380R as in achondroplasia, or K650E involved in thanatophoric dysplasia. Fibroblast growth factor receptors ubiquitylation was found to be directly proportional to their intrinsic tyrosine kinase activity, both of which could be blocked using kinase inhibitors. Despite excessive ubiquitylation, both overactive mutants failed to be efficiently degraded, even when challenged with ligand or overexpression of c-Cbl, a putative E3 ligase. We conclude that phosphorylation is essential for FGFR3 ubiquitylation, but is not sufficient to induce downregulation of its internalization resistant mutants.

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.

Role of FGF9 and FGF receptor 3 in osteochondroma formation.

Osteochondromas are chondro-osseous protuberances that occur in metaphyses of long bones. The cartilaginous cap is assumed to be responsible for the growth of the lesions during childhood and adolescence, but mitotic figures are rarely seen in the cap. Therefore, another cell population, probably mesenchymal cells, is responsible for proliferation and growth. Residual mesenchymal cells capable of rapid proliferation are difficult to detect due to lack of specific histologic features. Two specific markers for mesenchymal cells, FGF receptor 3 (FGFR3) and collagen type IIa, have been described. Osteochondroma mesenchymal cells are found in the soft tissues overlying the cartilage cap. The surrounding areas of typical cartilage are negative for both mesenchymal cell associated antigens. The soft tissues overlying the cartilage do not have cartilaginous features. The undifferentiated cells overlying the exostosis yield in culture a rapidly proliferating homogenous population of fibroblast-like cells. Expression at the mRNA level of FGF9, FGFR3, and collagen type IIa is found in these cells, but not in skin fibroblasts from afflicted or healthy individuals. Exogenous administration of TGFbeta1 to cultures of hereditary multiple exostosis eliminates FGF9 expression. These results indicate fibrous regions contain the mesenchymal cells responsible for osteochondroma growth.

Basic fibroblast growth factor: a potential inhibitor of glutamine synthetase expression in injured neural tissue.

Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of glutamine synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Müller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of glutamine synthetase expression. This inhibition appears to be mediated by the c-Jun protein which accumulated, in response to bFGF, exclusively in Müller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c-Jun protein and a decline in glutamine synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down-regulates glutamine synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down-regulation of glutamine synthetase.

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.

Mapping a heparin binding site on ErbB-3 epidermal growth factor receptor.

Signaling via the ErbB-family of receptors plays an important role in mammalian development and oncogenesis. Here we show that the ErbB-3 receptor, but not other members of this receptor family, binds to immobilized heparin and can be dissociated only at a high ionic strength comparable to that required for fibroblast growth factor receptors. Competition-binding analysis suggests that this interaction is specific and requires highly sulfated species of heparan sulfate. Primary sequence analysis of ErbB-3 identified a basic amino acid cluster (466)KHNRPRR(472) localized to the proximal, cysteine-rich extracellular ligand binding domain of the receptor, with charge density and distribution compatible with, but different to, known linear heparin binding motifs. Site-directed mutagenesis, replacing this sequence with the corresponding residues from ErbB-1, resulted in complete loss of heparin binding activity of the chimeric receptor. Finally, antibodies directed to the putative heparin binding peptide, efficiently bind the native receptor suggesting a novel target for blocking heparin mediated ErbB-3 interactions.

Anti-VEGFR-2 scFvs for cell isolation. Single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/flk-1) on the surface of primary endothelial cells and preselected CD34+ cells from cord blood.

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

Soluble and matrix-associated heparan sulfate proteoglycans increase expression of erb-B2 and erb-B3 in colon cancer cell lines.

We investigated the effect of hepatocyte-derived extracellular matrix (ECM) on the expression of erb-B2 and erb-B3 in colon cancer cell lines, as well as the role of erb-B2 and erb-B3 in colon cancer cell proliferation. Colon cancer cell lines plated on hepatocyte-derived ECM had increased protein levels of both erb-B2 and erb-B3. The addition of soluble recombinant proteoglycan syndecan-4 also resulted in higher expression of erb-B2 and erb-B3. We prepared hepatocyte-derived ECM from 1 to 7 days' cultures of hepatocytes, which contained different amounts of sulfated glycosaminoglycans. There was a direct correlation between the amounts of sulfated glycosaminoglycans in the ECM and the levels of erb-B2 and erb-B3 in the colon cell line KM12. The stimulatory effect of hepatocyte-derived ECM was abolished when the colon cancer cells were cultured in the presence of antibodies to erb-B2. These studies show that hepatocyte-derived ECM and the heparan sulfate proteoglycans present in it are responsible for inducing erb-B2 and erb-B3 in colon cancer cells. The growth stimulatory effect of extracellular matrix is mediated, at least in part, by increased expression of erb-B2 and erb-B3.

Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization.

The crystal structure of a dimeric 2:2:2 FGF:FGFR:heparin ternary complex at 3 A resolution has been determined. Within each 1:1 FGF:FGFR complex, heparin makes numerous contacts with both FGF and FGFR, thereby augmenting FGF-FGFR binding. Heparin also interacts with FGFR in the adjoining 1:1 FGF:FGFR complex to promote FGFR dimerization. The 6-O-sulfate group of heparin plays a pivotal role in mediating both interactions. The unexpected stoichiometry of heparin binding in the structure led us to propose a revised model for FGFR dimerization. Biochemical data in support of this model are also presented. This model provides a structural basis for FGFR activation by small molecule heparin analogs and may facilitate the design of heparin mimetics capable of modulating FGF signaling.

Sorting polyclonal antibodies into functionally distinct fractions using peptide phage display: 'a library on top of a library'.

A general approach for sorting antibodies (Abs) to a restricted protein domain was developed using phage-displayed peptide libraries. The method is demonstrated by fractionating polyclonal antibodies (pAbs), raised against a short peptide derived from the extracellular, juxtamembrane region of fibroblast growth factor receptor 1 (FGFR1) into fractions with distinct chemical and biological characteristics. Screening two combinatorial peptide libraries, with the pAb, several sequences, homologous to different regions within the original peptide, were identified. Four of the corresponding peptides were synthesized and used as peptide-conjugated affinity columns for the fractionation of the pAbs. The fractions obtained were unique in their recognition patterns and in their capacity to immunoprecipitate and immunoblot, as well as to modulate the activity of FGFR1. This technique is, therefore, highly sufficient in separating pAbs to monospecific fractions and may also be used for fine mapping of different, even overlapping, sequences within a restricted peptide or protein domain.

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.

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.