Humanization of fibroblast growth factor 1 single-chain antibody and validation for its antitumorigenic efficacy in breast cancer and glioma cells.

Single-chain variable fragment (scFv) antibodies are the smallest immunoglobulins with high antigen-binding affinity. We have previously reported that fibroblast growth factor 1 played pivotal roles in cancer development and generated a mouse scFv (mscFv1C9) could effectively prohibit cancer cell proliferation in vitro and in vivo. Here, we further humanized this scFv (hscFv1C9) using a structure-guided complementarity determining region grafting strategy. The purified hscFv1C9 maintained similar antigen-binding affinity and specificity as mscFv1C9, and it was capable of inhibiting growth of different tumours in vitro and in vivo. These data strongly suggested that hscFv1C9 has antitumour potentials.

The FGF-1-specific single-chain antibody scFv1C9 effectively inhibits breast cancer tumour growth and metastasis.

Immunotherapy mediated by recombinant antibodies is an effective therapeutic strategy for a variety of cancers. In a previous study, we demonstrated that the fibroblast growth factor 1 (FGF-1)-specific recombinant antibody scFv1C9 arrests the cell cycle at the G0/G1 transition by blocking the intracrine FGF-1 pathway in breast cancer cells. Here, we further show that the overexpression of scFv1C9 in MCF-7 and MDA-MB-231 breast cancer cells by lentiviral infection resulted in decreased tumourigenicity, tumour growth and lung metastasis through FGF-1 neutralization. We found that scFv1C9 resulted in the up-regulation of p21, which in turn inhibited the expression of CDK2 and blocked cell cycle progression. To explore the potential role of scFv1C9 in vivo, we delivered the gene into solid tumours by electroporation, which resulted in significant inhibition of tumour growth. In tumour tissue sections, immunohistochemical staining of the cellular proliferation marker Ki-67 and the microvessel marker CD31 showed a reduction in the proliferative index and microvessel density, respectively, upon expression of scFv1C9 compared with the appropriate controls. Thus, our data indicate a central role for scFv1C9 in blocking the intracrine pathway of FGF-1, therefore, scFv1C9 could be developed in an effective therapeutic for breast cancer.

Fibroblast growth factor-1 is a mesenchymal stromal cell-secreted factor stimulating proliferation of osteoarthritic chondrocytes in co-culture.

Previously, we showed that mesenchymal stromal cells (MSCs) in co-culture with primary chondrocytes secrete soluble factors that increase chondrocyte proliferation. The objective of this study is to identify these factors. Human primary chondrocytes (hPCs) isolated from late-stage osteoarthritis patients were co-cultured with human bone marrow-derived MSCs (hMSCs) in pellets. Genome-wide mRNA expression analysis and quantitative polymerase chain reactions (qPCR) were used to identify soluble factors that were specifically induced in co-cultures. Immunofluorescent staining combined with cell tracking and enzyme-linked immunosorbent assay (ELISA) were performed to validate up-regulation at the protein level and to identify the cellular origin of the increased proteins. Chemical blockers and neutralizing antibodies were used to elucidate the role of the identified candidate genes in co-cultures. A number of candidate factors were differentially regulated in co-cultures at the mRNA level. Of these, fibroblast growth factor-1 (FGF-1) mRNA and protein expression were markedly increased in co-cultures predominantly due to up-regulated expression in MSCs. Blocking of FGF signaling in co-culture pellets by specific FGF receptor inhibitors or FGF-1 neutralizing antibodies completely blocked hPCs proliferation. We demonstrate that MSCs increase FGF-1 secretion on co-culture with hPCs, which, in turn, is responsible for increased hPCs proliferation in pellet co-cultures.

Fibroblast growth factor-1 within the ventral tegmental area participates in motor sensitizing effects of morphine.

Drug addiction is viewed as a form of neural plasticity, and neurotrophic factors have been implicated in many forms of plasticity in the adult nervous system. Here we show that the fibroblast growth factor-1 (FGF-1), that is expressed on dopamine and GABA neurons of the ventral tegmental area (VTA), is involved in the sensitizing effects of morphine. The receptor FGFR-1 is expressed on VTA astrocytes, as well as dopamine and GABA neurons. FGF-1 or anti-FGF-1 infusions into the VTA during the induction (not expression) phase of sensitization advanced or blocked morphine's activating motor effects respectively, in a dose-dependent manner. Infusions into the adjacent substantia nigra, whose neurons also express FGF-1 and FGFR-1, did not modify normal morphine-induced sensitization. Biochemical traits related to morphine's sensitizing effects were altered by intra-VTA anti-FGF-1 because morphine-induced upregulation of both tyrosine hydroxylase (TH) and N-methyl d-aspartate glutamate receptor 1 (NMDAR1) in the VTA was blocked after anti-FGF-1. Changes in the activation state of VTA calcium/calmodulin-dependent kinase type II seem to participate in FGF-1-induced effects as well. We conclude that the FGF-1 system of the ventral tegmental area is required for biochemical and behavioral sensitization to this drug.

Assessment of the VEGF, bFGF, aFGF and IL8 angiogenic activity in urinary bladder carcinoma, using the mice cutaneous angiogenesis test.

Development of new blood vessels in solid tumors depends on changes in equilibrium between angiogenic stimulators and inhibitors. Overexpression of angiogenic growth factors has been shown in bladder carcinoma. The 'mice cutaneous angiogenesis test' is a good method for assessment of the total angiogenic potential of bladder cancer tissue. The analysis of the levels of proangiogenic factors could be useful for the choice of properly directed angiogenesis inhibitors. The aim of our study was to investigate the influence of blocking some angiogenic factors on the angiogenic activity of bladder cancer tissue. Tumor tissue obtained from 12 patients with invasive bladder carcinoma was used. Cancer tissue homogenates were incubated in the presence of specific antibodies against VEGF, bFGF, Il-8 and aFGF. Cytokine levels were determined using the ELISA test. Cutaneous angiogenesis assay in Balb/c mice was performed to detect the angiogenic activity of the tumor tissue. VEGF, bFGF and Il-8 were present in all examined cancer tissues (aFGF level was not estimated). Cytokine concentration and angiogenic activity of bladder cancer tissue showed individual variation. There was no correlation between the cytokines content in tumor tissue and the ability of this tissue to induce angiogenesis. Absorption caused significant reduction in cytokines level. The reduction of angiogenic activity was observed in the cancer tissue of 1 patient after VEGF absorption, in 3 patients' tissue homogenates after incubation with anti-aFGF and in 2 patients' homogenates after bFGF absorption. There was no reduction of angiogenic activity after Il-8 absorption.

Development of enzyme-linked immunosorbent assay for acidic fibroblast growth factor and its clinical application.

We have developed, for the first time, an enzyme-linked immunosorbent assay (ELISA) system for the measurement of human acidic fibroblast growth factor (aFGF). Anti-bovine aFGF rabbit IgG was conjugated with N-hydroxysuccimidobiotin, and the resulting IgG-biotin conjugate was used as the second antibody. This assay was highly specific and reproducible, enabling us to detect aFGF at a concentration as low as 1 microg/l without any prior processing of samples. With this method, it was possible to determine human aFGF up to 833 x 10(3) ng/l, with the use of anti-bovine aFGF IgG as the first and second antibody. There was no significant cross-reactivity of the antibody with other growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The aFGF concentration in pericardial fluid was significantly higher in patients with unstable angina than in those with other heart diseases, suggesting that the aFGF plays an important role(s) in the course of collateral growth in coronary artery disease. Therefore, our ELISA system may be useful in determining unknown biological function(s) or pathological role(s) of aFGF in various disease entities.

A single pre-training glucose injection induces memory facilitation in rodents performing various tasks: contribution of acidic fibroblast growth factor.

Effects of a pre-training intraperitoneal glucose injection on learning and memory were tested using two tasks: passive avoidance and Morris water maze. In the former task, mice that had received glucose 2 h prior (but not 1, 3, or 5 h prior) to a trial that combined acquisition with passive avoidance of foot shock showed a significantly increased retention latency when tested 24 h later. Thus, this effect was time-dependent, and it was also found to be dose-dependent by further experiment. In contrast, 2-deoxy-D-glucose and fructose had no such effect. In the Morris water maze task, glucose injection 2 or 3 h before a block of trials enhanced the spatial memory performance of mice. These glucose-induced memory-facilitation effects were abolished by an intracerebroventricular injection of anti-acidic fibroblast growth factor antibody 30 min before the glucose injection, suggesting a critical role for endogenous acidic fibroblast growth factor in this facilitatory effect. Furthermore, continuous intracerebroventricular infusion of acidic fibroblast growth factor in rats significantly increased retention latency (when tested repeatedly on successive days using a passive avoidance task). Our earlier studies demonstrated that brain acidic fibroblast growth factor is produced in the ependymal cells of the cerebroventricular system, and is released into the cerebrospinal fluid following either a meal or a (intraperitoneal or intracerebroventricular) glucose injection. This released acidic fibroblast growth factor also diffuses into the brain parenchyma, and is taken up by neurons in the hippocampus, hypothalamus, and elsewhere in the brain some 2 h after the meal or glucose injection. These and the present findings indicate (i) that pre-training glucose injection improves memory performance, and (ii) that acidic fibroblast growth factor, especially by its action within the hippocampus, is involved in this enhancement process.

Amphibian FGF-1 is structurally and functionally similar to but antigenically distinguishable from its mammalian counterpart.

Recent studies have shown that fibroblast growth factors (FGF) play an important role in the diverse cellular mechanisms involved with vertebrate development. One system which has received a great deal of attention is the developing limb in part because of the extensive epithelial-mesenchymal interactions that take place during this process. Because it closely parallels the developmental process of the limb and is a model for wound repair, the phenomenon of amphibian limb regeneration has been used to investigate the role of FGF in these processes. We have recently reported on the cloning and functional characterization of an FGF receptor (FGFR) isolated from amphibian regenerative tissue. In this report, we describe the isolation and characterization of an FGF-1 molecule from the newt, Notophthalmus viridescens. Amino acid sequence comparisons indicate that the newt FGF-1 exhibits between 79 to 83% identity with FGF-1 from mammalian and avian species. The full length cDNA of the newt FGF-1 was cloned into a prokaryotic expression vector and purified from E. coli. Although the newt FGF-1 shares a high degree of primary amino acid sequence similarity with other FGF-1 molecules, the recombinant protein was not detected in a Western blot analysis using a polyclonal antibody directed against mammalian FGF-1. Despite the antigenic divergence, the newt FGF-1 was capable of binding to NIH/3T3 and Chinese hamster ovary cells overexpressing mammalian and amphibian FGFRs with dissociation constants comparable to those reported for mammalian FGF-1. Newt FGF-1 could also be cross-linked to receptors on the surface of NIH/3T3 cells. In addition, it elicits a mitogenic response in NIH/3T3 cells indistinguishable from human recombinant FGF-1.

Immunolocalization of FGF-1 and receptors in glomerular lesions associated with chronic human renal allograft rejection.

Glomerular lesions are considered one of the more detrimental pathologic changes associated with chronic rejection of renal allografts. To elucidate potential pathophysiologic mechanisms associated with transplant glomerulopathy, we examined the expression of acidic fibroblast growth factor (FGF-1) and its high-affinity receptors (FGFR) in both relevant renal transplant controls (n=5) and tissue from patients (n=19) who underwent nephrectomy following graft loss secondary to chronic rejection. In situ immunohistochemical analyses demonstrated minimal staining and distribution of FGFR and FGF-1, which was localized to the mesangial matrix in glomeruli from normal human kidneys. In situ hybridization failed to detect the presence of FGF-1 mRNA in control tissue. In contrast, each stage of the developing glomerular lesion associated with chronic rejection demonstrated the exaggerated appearance of FGF-1 protein in visceral and parietal epithelial cells. Intense staining for FGF-1 protein did not correlate with the increased appearance of FGF-1 mRNA, which was restricted to circulating inflammatory cells. Glomeruli in kidneys with findings of chronic rejection also exhibited increased immunodetection of both FGFR and PCNA in mesangial and epithelial cells. Immunogold labeling of chronically rejected visceral epithelial cells revealed both cytoplasmic and nuclear/localization of FGF-1, thereby establishing mitogenic potential of the growth factor. The enhanced appearance of both biologically active FGF-1 and FGFR suggests that this polypeptide may serve as an important mediator of growth responses associated with glomerular lesion development during chronic rejection.

The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea.

Here we show that the mature cochlear neurons are a rich source of acidic fibroblast growth factor (aFGF), which is expressed in the neuronal circuitry consisting of afferent and efferent innervation. The site of action of neuronal aFGF is likely to reside in the organ of Corti, where one of the four known FGF receptor (FGFR) tyrosine kinases--namely, FGFR-3 mRNA--is expressed. Following acoustic overstimulation, known to cause damage to the organ of Corti, a rapid up-regulation of FGFR-3 is evident in this sensory epithelium, at both mRNA and protein levels. The present results provide in vivo evidence for aFGF being a sensory neuron-derived, anterogradely transported factor that may exert trophic effects on a peripheral target tissue. In this sensory system, aFGF, rather than being a neurotrophic factor, seems to promote maintenance of the integrity of the organ of Corti. In addition, aFGF, released from the traumatized nerve endings, may be one of the first signals initiating protective recovery and repair processes following damaging auditory stimuli.

Developmental time course of acidic and basic fibroblast growth factors' expression in distinct cellular populations of the rat central nervous system.

Acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) are expressed in high levels in adult central nervous system (CNS). We report the time course of developmental appearance and distribution of these factors and of two FGF receptors, FGFR-1 and FGFR-2, in the CNS of rats ranging in age from embryonic day 16 to adult. Immunohistochemical analysis showed that sensory neurons in the midbrain were the first cells to contain detectable aFGF immunoreactivity at embryonic day 18. The next cell group to contain aFGF were motor neurons, which were found to be aFGF-positive at the day of birth. A number of other subcortical neuronal populations were observed to contain aFGF immunoreactivity after postnatal day 7. Adult levels and distribution patterns of aFGF were reached in all CNS areas by postnatal day 28. Basic FGF immunoreactivity was observed at postnatal day 0 in neurons in the CA2 subfield of hippocampus. Astrocytes contained detectable bFGF immunoreactivity, starting at postnatal day 7. Adult levels and patterns of distribution of bFGF were reached in all CNS areas by postnatal day 28. These immunohistochemical observations were confirmed by using bioassay and Western blot techniques. FGFR-1 and FGFR-2 mRNA were expressed in significant levels in all CNS areas at all time points analyzed. The observation that aFGF and bFGF appear in specific and distinct cellular populations at relatively late developmental times suggests that these FGFs may be involved in specific mechanisms of CNS maturation, maintenance, and repair.

Branching morphogenesis of embryonic mouse lung epithelium in mesenchyme-free culture.

Embryonic mouse lung epithelium was separated from its mesenchyme and cultured under mesenchyme-free conditions. When covered with Matrigel, the cultured epithelium underwent branching morphogenesis in medium containing acidic fibroblast growth factor (aFGF), in which the epithelial cells constructed a simple columnar cell layer forming a lumen, as seen in normal development. The epithelial growth and branching morphogenesis induced by aFGF was completely inhibited by an antibody against aFGF. Heparin caused extra epithelial growth in cooperation with aFGF, but its use resulted in luminal expansion instead of enhanced branching. Basic FGF induced abnormal morphogenesis of the epithelium, though the lumen formed was lined by a simple columnar cell layer. Epidermal growth factor could not maintain epithelial cell growth, and the epithelium became a smaller and smoother ball than that at the start of cultivation. When covered with a collagen gel instead of Matrigel, the epithelium remained in its initial form, neither newly branching nor becoming a smooth ball, in the presence of aFGF. These results show that the epithelium of lung rudiments was able to branch under mesenchyme-free culture conditions in which a basement membrane matrix and aFGF were substitutes for the mesenchyme.

Detection of T cells responsive to a vascular growth factor in rheumatoid arthritis.

The primary lesion in rheumatoid arthritis (RA) is a destructive synovitis characterized by proliferation of endothelial cells, fibroblasts, and vascular smooth muscle cells, and with perivascular lymphocyte aggregates. A nonhematopoietic growth factor, acidic fibroblast growth factor (aFGF), may induce many of the biological features found in rheumatoid synovium, including T cell activation. To determine if aFGF-responsive T cells are increased in RA, we developed an assay to measure the frequency of peripheral blood T cells that are costimulated by aFGF. The data indicate that the frequency of aFGF-responsive T cells is increased in RA and may change with disease activity and treatment.

Characterization of recombinant human acidic fibroblast growth factor: comparative studies with bovine acidic fibroblast growth factor.

Recombinant human acidic fibroblast growth factor (haFGF) was purified from E. coli lysate by heparin-sepharose affinity chromatography. The purified haFGF exhibited potent mitogenic activity in stimulating DNA synthesis in 3T3 cells and this activity could be significantly increased by heparin. By analysis of mitogenic activity and immunological properties, a marked difference was found between haFGF and bovine aFGF (baFGF). The main difference was that the heparin-dependence of haFGF was stronger than that of baFGF.

Acidic fibroblast growth factor is expressed abundantly by photoreceptors within the developing and mature rat retina.

In order to further understand the role(s) of fibroblast growth factors (FGFs) in the development, differentiation and function of the central nervous system, we analysed the expression of the mRNA, and the presence and tissue distribution of the translated product, of one member of the FGF family, acidic FGF (aFGF), within the mammalian retina. Firstly, the relative abundance of aFGF mRNA was assayed in embryonic (between 14 and 17 days of gestation), postnatal (between 1 and 17 days after birth) and adult rat retina by quantitative reverse transcription-coupled polymerase chain reaction amplification using specific aFGF oligonucleotides. The level of expression remained uniformly low throughout the embryonic period and until postnatal day 7. Therefore the quantity of aFGF mRNA increased rapidly, reaching 80% of adult levels by eye opening (postnatal day 13). Adult levels were three-fold higher than at early developmental times. In situ hybridization of adult rat retina using specific antisense aFGF riboprobes revealed labelling in all cellular layers. Antisera raised against recombinant human aFGF revealed very little labelling of 4-day postnatal retina, but by postnatal days 8 and 17 immunoreactive aFGF was localized mainly within the photoreceptor cell bodies. Western blots of retinal extracts derived from 17-day embryonic, 4-day postnatal and adult retina probed with the same antibody revealed a single immunoreactive band of the expected molecular weight (18 kDa) in all extracts. Thus aFGF is mostly transcribed and translated within the retina subsequent to the major steps of cell birth, migration and differentiation, and seems to be abundantly expressed by maturing photoreceptor cells.

Increasing cell density down-regulates the expression of acidic FGF by human RPE cells in vitro.

Previous studies have reported the expression of acidic fibroblast growth factor (aFGF) by rat, bovine, and human retinal pigment epithelium (RPE) in vivo. To critically examine the expression of aFGF by RPE cells, we studied the density dependence of steady-state levels of mRNA and protein expression in vitro. Northern blot analysis demonstrated 5 transcripts ranging from 4.5 kB to 1 kB. Steady-state levels of all the transcripts decreased as a function of culture density. A polyclonal antibody was raised against recombinant human aFGF and affinity purified on aFGF coupled to AffiGel-10. The resulting antibody crossreacted with bFGF but not FGF-5, but this crossreactivity could be eliminated by absorption of the antibody on bFGF coupled to AffiGel-10. The final antibody preparation recognized only a single band at approximately 18.5 kD in lysates of RPE. Immunohistochemical staining with this antibody preparation demonstrated a marked dependence on cell density after 3 days in culture. Low culture density yielded cells staining moderately for aFGF, while confluent cells exhibited little or no staining. The reduction of aFGF from RPE cells in culture in a density-dependent fashion could also be demonstrated by Western blot analysis.

A histological processing technique that preserves the integrity of calcified tissues (bone, enamel), yolky amphibian embryos, and growth factor antigens in skeletal tissue.

We have devised a processing technique to embed calcified tissues, such as bone and tooth enamel, in paraffin, to preserve the delicate antigenic sites of molecules such as growth factors. The same technique, omitting the decalcification step, allows delicate tissues, such as axolotl embryos (Ambystoma mexicanum) containing large yolk masses, to be easily handled during tissue processing and to be serially sectioned. Specimens were all fixed in periodate-lysine-paraformaldehyde (PLP) fixative at 5 degrees C. Bone and teeth were decalcified in an EDTA-G solution at -4 degrees C. Maintaining a temperature of 5 degrees C, the decalcified samples were then washed (with PBS, pH 7.2, under vacuum) to remove glycerol. Both the decalcified tissues and the yolky embryos were dehydrated through an ascending series of isopropanol and embedded in low melting-point paraffin under vacuum. Acidic fibroblast growth factor (aFGF) was located in cells of the expanded cambial layer in the early fracture calluses of male CD-1 mice, demonstrating retention of antigenic sites. The results reported here have not previously been obtained with existing processing and embedding techniques.

Distribution of acidic and basic fibroblast growth factors in ovine skin during follicle morphogenesis.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been localized by immunochemistry in ovine skin during wool follicle morphogenesis. At 40 days of gestation, prior to the appearance of follicle primordia, bFGF immunoreactivity was detected in the intermediate and periderm layers of the epidermis and at the dermal-epidermal junction. Antibodies to aFGF did not bind to skin at this age. During early follicle formation, at 76 days of gestation, both FGFs were found in the epidermis and associated with the follicle primordia. Antibodies to aFGF, in particular, bound to the basal cells of the epidermis and the follicle cell aggregations. With the development of epidermal plugs, bFGF was confined to the intermediate layers of the epidermis and the dermal-epidermal junction, whereas aFGF staining was associated with the cells of the epidermis and the plugs. At 90 days, when many different stages of follicle development were in evidence, immunoreactivity for both FGFs was associated with the cells of the elongating epidermal column, particularly those adjacent to the dermal-epidermal junction. During follicle maturation, bFGF was found in the suprabasal layer of the epidermis, in the outer root sheath of the follicle and in the basement membrane zone surrounding the bulb matrix. Conversely, strong staining for aFGF was observed in the epidermis and pilary canal contiguous with the epidermis, and in cells of the upper bulb matrix of the follicle in the region of the keratogenous zone. Western blotting of extracts of mature follicles that had been isolated from the skin showed the presence of a major aFGF immunoreactive band with an apparent molecular mass of 27 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation and localisation of monoclonal antibodies against fibroblast growth factors in ischaemic collateralised porcine myocardium.

OBJECTIVE: The collateral circulation plays a critical role in the prognosis of ischaemic heart disease, with a clear correlation between neovascularisation, infarction, and tissue recovery. The aim of the study was to address the question of whether endogenous acidic and basic fibroblast growth factors (FGF) participate in ischaemia induced collateral enlargement and development in the myocardium, and also which cells may represent the source of these growth factors. METHODS: Eight pigs received an ameroid constrictor around the left circumflex coronary artery which, by slow coronary occlusion, induces ischaemia and collateral growth in the left ventricle. The degree of stenosis and development of collaterals were determined angiographically. About 14 d after constrictor implantation pigs were killed and hearts were excised and prepared for western blot analysis and immunohistochemistry. Monoclonal antibodies were raised against acidic and basic FGF, characterised for their specificity, and used for the localisation of growth factors in sections of ischaemic and normal pig heart. RESULTS: The eight pigs included in this study showed a gradual 70-100% left circumflex coronary stenosis about 14 d after implantation of the constrictor. Out of four pigs with a complete occlusion, three developed visible collaterals. Antibody staining to acidic FGF was detected only in hearts from pigs with complete occlusion of the coronary artery. The growth factor was localised in cardiomyocytes close to small necrotic tissue patches. In control tissue no acidic FGF staining was evident. Basic FGF could not be detected in ischaemic or in normal perfused pig hearts. CONCLUSIONS: These data show that (1) complete occlusion of the left circumflex coronary artery in pig hearts induces collateral growth; (2) cardiomyocytes are able to produce acidic FGF in response to ischaemia, thus providing a mitogen for endothelial cells; (3) endogenous basic FGF, which was not detected in normal or ischaemic pig hearts, appears not to play an important role in ischaemia induced collateral growth at the chosen time point of investigation.