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Proliferative vitreoretinopathy (PVR) is a challenging pathological condition, often causing failure of retinal detachment surgery. The purpose of this study was to evaluate the feasibility of a delivery system of bioactive proteins using anionic and cationic gelatin microspheres and to establish a new PVR model in rabbits by intraocular sustained delivery of basic fibroblast growth factor (bFGF) and interferon-beta (IFNβ). Anionic and cationic gelatin microspheres were prepared and immersed in bFGF and IFNβ solution, respectively, to yield a polyion complex between gelatin matrix and a bioactive protein. The bFGF-impregnated microspheres were injected into the subretinal space in rabbit eyes. At week 2, the IFNβ-impregnated microspheres also were injected into the same space. Control eyes received gelatin microspheres without bFGF or IFNß, or both. The eyes then were observed for 8 weeks by ophthalmoscopy, fundus photography, and fluorescein angiography. The eyes also were evaluated histologically. In the group with both bFGF and IFNβ, the number of eyes with more severe PVR increased over time. Histologic examination showed retinal folds. In contrast, no proliferative changes were seen in any control groups. Subretinal implantation of bFGF and IFNβ-impregnated gelatin microspheres induced reproducible PVR in rabbit eyes. This study guaranteed delivery of bioactive proteins with gelatin microspheres., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Basic fibroblast growth factor (bFGF) is a mitogenic cytokine that can stimulate mesoderm-and neuroectoderm-originated cell proliferation. This study was performed to investigate the effects of bFGF on cell differentiation and the expression of specific markers at different embryonic developmental stages. We firstly evaluated the embryotoxic potential of bFGF in vitro using a modified EST protocol. Sequentially, we further investigated how bFGF impact the different tissue-special genes and proteins expressions during the differentiation of murine ES cells in vitro and attempt to reveal the effects of bFGF on differentiation processes. This analysis was focused on key tissue- and stage-specific genes involved in ectodermal, mesodermal, and endodermal differentiation, including ectodermal-specific gene Nestin, Oligo2 and Syn, mesodermal-specific gene MHC and MyoD, and endodermal-specific gene GATA6, TTR and ALB, as well as undifferentiated gene Sox-2 and Oct-4. The results demonstrate that bFGF could promote expression of ectodermal-specific genes and protein, but suppress the expressions of endoderm-specific and some mesoderm-specific gene and protein. A conclusion can be drawn that bFGF exhibits weak embryotoxicity and mainly promotes ES cell differentiation towards the ectodermal lineages but suppress differentiation into endoderm lineages. These opposing effects of bFGF on the embryonic development of the three germ layers may be related to its weak embryotoxic potential. More specifically, inhibition of expression of the endodermal-specific markers transthyretin (TTR), and albumin (ALB) by bFGF may be of more value in detecting the embryotoxic potential of bFGF.

The cystine/glutamate antiporter (system xc-) transports cystine into cell in exchange for glutamate. Fibroblast growth factor-2 (FGF-2) upregulates system xc- selectively on astrocytes, which leads to increased cystine uptake, the substrate for glutathione production, and increased glutamate release. While increased intracellular glutathione can limit oxidative stress, the increased glutamate release can potentially lead to excitotoxicity to neurons. To test this hypothesis, mixed neuronal and glial cortical cultures were treated with FGF-2. Treatment with FGF-2 for 48 h caused a significant neuronal death in these cultures. Cell death was not observed in neuronal-enriched cultures, or astrocyte-enriched cultures, suggesting the toxicity was the result of neuron-glia interaction. Blocking system xc- eliminated the neuronal death as did the AMPA/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), but not the NMDA receptor antagonist memantine. When cultures were exposed directly to glutamate, both NBQX and memantine blocked the neuronal toxicity. The mechanism of this altered profile of glutamate receptor mediated toxicity by FGF-2 is unclear. The selective calcium permeable AMPA receptor antagonist 1-naphthyl acetyl spermine (NASPM) failed to offer protection. The most likely explanation for the results is that 48 h FGF-2 treatment induces AMPA/kainate receptor toxicity through increased system xc- function resulting in increased release of glutamate. At the same time, FGF-2 alters the sensitivity of the neurons to glutamate toxicity in a manner that promotes selective AMPA/kainate receptor mediated toxicity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: Growth factors such as nerve growth factor and glial cell line-derived neurotrophic factor are known to induce pain sensitization. However, a plethora of other growth factors is released during inflammation and tissue regeneration, and many of them are essential for wound healing. Which wound-healing factors also alter the sensitivity of nociceptive neurons is not well known. We studied the wound-healing factor, basic fibroblast growth factor (bFGF), for its role in pain sensitization. Reverse transcription polymerase chain reaction showed that the receptor of bFGF, FGFR1, is expressed in lumbar rat dorsal root ganglia (DRG). We demonstrated presence of FGFR1 protein in DRG neurons by a recently introduced quantitative automated immunofluorescent microscopic technique. FGFR1 was expressed in all lumbar DRG neurons as quantified by mixture modeling. Corroborating the mRNA and protein expression data, bFGF induced Erk1/2 phosphorylation in nociceptive neurons, which could be blocked by inhibition of FGF receptors. Furthermore, bFGF activated Erk1/2 in a dose- and time-dependent manner. Using single-cell electrophysiological recordings, we found that bFGF treatment of DRG neurons increased the current-density of NaV1.8 channels. Erk1/2 inhibitors abrogated this increase. Importantly, intradermal injection of bFGF in rats induced Erk1/2-dependent mechanical hyperalgesia., Perspective: Analyzing intracellular signaling dynamics in nociceptive neurons has proven to be a powerful approach to identify novel modulators of pain. In addition to describing a new sensitizing factor, our findings indicate the potential to investigate wound-healing factors for their role in nociception., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Different growth factors have been shown to influence the development of form-deprivation myopia and lens-induced ametropias. However, growth factors have relatively little effect on the growth of eyes with unrestricted vision. We investigate whether the combination of insulin-like growth factor 1 (IGF1) and fibroblast growth factor 2 (FGF2) influence ocular growth in eyes with unrestricted vision. Different doses of IGF1 and FGF2 were injected into the vitreous chamber of postnatal chicks. Measurements of ocular dimensions and intraocular pressure (IOP) were made during and at the completion of different treatment paradigms. Histological and immunocytochemical analyses were performed to assess cell death, cellular proliferation and integrity of ocular tissues. Treated eyes had significant increases in equatorial diameter and vitreous chamber depth. With significant variability between individuals, IGF1/FGF2-treatment caused hypertrophy of lens and ciliary epithelia, lens thickness was increased, and anterior chamber depth was decreased. Treated eyes developed myopia, in excess of 15 diopters of refractive error. Shortly after treatment, eyes had increased intraocular pressure (IOP), which was increased in a dose-dependent manner. Seven days after treatment with IGF1 and FGF2 changes to anterior chamber depth, lens thickness and elevated IOP were reduced, whereas increases in the vitreous chamber were persistent. Some damage to ganglion cells was detected in peripheral regions of the retina at 7 days after treatment. We conclude that the extreme myopia in IGF1/FGF2-treated eyes results from increased vitreous chamber depth, decreased anterior chamber depth, and changes in the lens. We propose that factor-induced ocular enlargement and myopia result from changes to the sclera, lens and anterior chamber depth.

Purpose: To determine the effect of vascular endothelial growth factor (VEGF) TrapR1R2 on bFGF-induced experimental corneal neovascularization (NV)., Methods: Control pellets or pellets containing 80 ng bFGF were surgically implanted into wild-type C57BL/6 and VEGF-LacZ mouse corneas. The corneas were photographed, harvested, and the percentage of corneal NV was calculated. The harvested corneas were evaluated for VEGF expression. VEGF-LacZ mice received tail vein injections of an endothelial-specific lectin after pellet implantation to determine the temporal and spatial relationship between VEGF expression and corneal NV. Intraperitoneal injections of VEGF TrapR1R2 or a human IgG Fc domain control protein were administered, and bFGF pellet-induced corneal NV was evaluated., Results: NV of the corneal stroma began on day 4 and was sustained through day 21 following bFGF pellet implantation. Progression of vascular endothelial cells correlated with increased VEGF-LacZ expression. Western blot analysis showed increased VEGF expression in the corneal NV zone. Following bFGF pellet implantation, the area of corneal NV in untreated controls was 1.05+/-0.12 mm2 and 1.53+/-0.27 mm2 at days 4 and 7, respectively. This was significantly greater than that of mice treated with VEGF Trap (0.24+/-0.11 mm2 and 0.35+/-0.16 mm2 at days 4 and 7, respectively; p<0.05)., Conclusions: Corneal keratocytes express VEGF after bFGF stimulation and bFGF-induced corneal NV is blocked by intraperitoneal VEGF TrapR1R2 administration. Systemic administration of VEGF TrapR1R2 may have potential therapeutic applications in the management of corneal NV.

The objective of this study was to evaluate the effect of vector architecture on DNA condensation, particle stability, and gene transfer efficiency. Two recombinant non-viral vectors with the same amino acid compositions but different architectures, composed of lysine-histidine (KH) repeating units fused to fibroblast growth factor, were genetically engineered. In one vector lysine residues were dispersed (KHKHKHKHKK)(6)-FGF2, whereas in the other they were in clusters (KKKHHHHKKK)(6)-FGF2. Organization of lysine residues in this manner was inspired by the sequence of DNA condensing motifs that exist in nature (e.g., histones) where lysine residues are organized in clusters. These two constructs were compared in terms of DNA condensation and gene transfer efficiency. It was observed that the construct with KH units in clusters was able to condense pDNA into more stable particles with sizes <150 nm making them suitable for cellular uptake via receptor mediated endocytosis. This in turn resulted in five times higher transfection efficiency for the cKH-FGF2. This study demonstrates that in targeted non-viral gene transfer, the vector architecture plays as significant a role as its amino acid sequence. Thus, in the design of the non-viral vectors (synthetic or recombinant) this factor should be considered of paramount importance.

Purpose: To evaluate the influence of some widely used antiglaucoma agents on angiogenesis in a novel rat cornea model., Methods: Angiogenesis was induced in 32 rats by slow-release polymer pellets containing basic fibroblast growth factor (bFGF) placed in a corneal micropocket. Angiogenesis was later measured and compared in groups of rats given one of four antiglaucoma drug therapies and one control group. The drugs were commercially available preparations of prostaglandins, beta-blockers, alpha-2 agonists, and carbonic anhydrase inhibitors given for 7 days in a manner similar to that used in humans. Growth was measured by calculating the maximum linear vessel growth divided by pellet-limbus distance., Results: Biomicroscopic observation disclosed that all tested animals showed an induction of neovascular reactions in their corneal stroma. The growth index results for the control, latanoprost, dorzolamide, brimonidine, and timolol malate groups were 1.65 +/- 0.16, 1.98 +/- 0.18, 1.85 +/- 0.19, 2.03 +/- 0.38, and 1.65 +/- 0.14, respectively, confirming the hypothesis that topically delivered antiglaucoma drugs modify the normal angiogenic response. Of them, the prostaglandins showed the most prominent angiogenic stimulatory effect (P = 0.03)., Conclusions: This modified micropocket assay of corneal angiogenesis in rats demonstrated the stimulatory effect of several widely used topically delivered antiglaucoma medications on the angiogenic process. The results indicate that the selection of drugs for treating different ophthalmic diseases should take into account their influence on angiogenic processes.

Purpose: To evaluate the angiogenic inhibitory effects of an alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist, EMD478761, released from a polymeric implant in a chick chorioallantoic membrane (CAM) assay and laser-induced experimental choroidal neovascularization (CNV) in rats., Methods: Polyvinyl alcohol-based reservoir implants releasing EMD478761 were designed for placement onto a CAM or intravitreally in rats. In vitro release rates of the implants were measured using HPLC. Angiogenesis was induced on 10-day-old chick embryos by basic fibroblast growth factor (bFGF), and areas of neovascularization were measured. Experimental CNV was induced in the Brown-Norway rat with a diode laser. EMD478761 or sham microimplants were placed within the vitreous chamber of Brown-Norway rats. Two weeks later, areas of CNV were determined by FITC-dextran staining of choroidal flatmounts., Results: Sustained delivery of EMD478761 significantly inhibited bFGF-induced angiogenesis in CAM, as determined by a reduction in angiogenesis areas, without drug toxicity to the normal CAM vasculature. In an experimental rat model, intravitreal EMD478761 implants significantly suppressed laser-induced CNV compared with intravitreal sham implants, with the mean area reduced by 63% (P < 0.05)., Conclusions: Sustained delivery of EMD478761demonstrates potent antiangiogenic properties in vivo. These results suggest that an EMD478761 implant may be beneficial in the treatment of neovascular ocular diseases.

Corneal neovascularization is a vision-threatening condition caused by various ocular pathological conditions. The aim of this study was to evaluate the function of the ephrin ligands and Eph receptors in vitro and in vivo in corneal angiogenesis in a mouse model. The Eph tyrosine kinase receptors and their ligands, ephrins, are expressed on the cell surface. The functions of Eph and ephrins have been shown to regulate axonal guidance, segmentation, cell migration, and angiogenesis. Understanding the roles of Eph and ephrin in corneal angiogenesis may provide a therapeutic intervention for the treatment of angiogenesis-related disorders. Immunohistochemical studies demonstrated that ephrinB1 and EphB1 were expressed in basic fibroblast growth factor (bFGF)-induced vascularized corneas. EphB1 was specifically colocalized with vascular endothelial marker CD31 surrounded by type IV collagen. EphrinB1 was expressed in corneal-resident keratocytes and neutrophils. Recombinant ephrinB1-Fc, which induces EphB receptor activation, enhanced bFGF-induced tube formation in an in vitro aortic ring assay and promoted bFGF-induced corneal angiogenesis in vivo in a corneal pocket assay. Synergistically enhanced and sustained activation of extracellular signal-regulated kinase was noted in vascular endothelial cell lines after stimulation with ephrin B1 and bFGF combinations. These results suggest that ephrinB1 plays a synergistic role in corneal neovascularization.

Purpose: Bone-marrow (BM)-derived hematopoietic precursor cells are thought to participate in the growth of blood vessels during postnatal vasculogenesis. In this investigation, multichannel laser scanning confocal microscopy and quantitative image analysis were used to study the fate of BM-derived hematopoietic precursor cells in corneal neovascularization., Methods: A BM-reconstituted mouse model was used in which the BM from enhanced green fluorescent protein (GFP)-positive mice was transplanted into C57BL/6 mice. Basic fibroblast growth factor (bFGF) was used to induce corneal neovascularization in mice. The vasculogenic potential of adult BM-derived cells and their progeny were tested in this in vivo model. Seventy-two histologic sections selected by systematic random sampling from four mice were immunostained and imaged with a confocal microscope and analyzed with image-analysis software., Results: BM-derived endothelial cells did not contribute to bFGF-induced neovascularization in the cornea. BM-derived periendothelial vascular mural cells (pericytes) were detected at sites of neovascularization, whereas endothelial cells of blood vessels originated from preexisting blood vessels in limbal capillaries. Fifty three percent of all neovascular pericytes originated from BM, and 47% of them originated from preexisting corneoscleral limbus capillaries. Ninety-six percent and 92% of BM-derived pericytes also expressed CD45 and CD11b, respectively, suggesting their hematopoietic origin from the BM., Conclusions: Pericytes of new corneal vessels have a dual source: BM and preexisting limbal capillaries. These findings establish BM as a significant effector organ in corneal disorders associated with neovascularization.

Purpose: To investigate the effects of culture medium of human amniotic membrane (AM) on corneal neovascularization (CNV) induced by basic fibroblast growth factor (bFGF) in mice., Methods: Culture medium of amniotic membrane was prepared by cultivating AM (with epithelium side up) in EGM basic medium for 3 days, and was collected separately to three groups, e.g. control (EGM only), AM with epithelium (AM) and AM without epithelium (De-AM). Corneal neovascularization was induced in mice by using micropocket assay with Hydron polymer pellets containing 100 ng bFGF. Migration and proliferation of human umbilical cord vein endothelial cells (HUVEC) were performed in Boyden chambers and by using the CyQUANT fluorescence binding assay respectively. The levels of tissue inhibitors of metalloproteinase 1 and 2 (TIMP1, TIMP2) in culture medium were determined by ELISA assay., Results: CNV induced by bFGF was significantly suppressed by culture medium of amniotic membrane. When the medium was applied as an eyedrop 4 times a day for 7 days, the area of CNV was (2.48+/-0.76) mm(2),(0.64+/-0.52) mm(2) and (1.96+/-0.65) mm(2) in control, AM and De-AM group respectively. The migration and proliferation of HUVEC were strongly inhibited by culture medium of AM with epithelium, while the De-AM had no effect on the migration of HUVEC cells. The high level of TIMP2 was found in AM group, but not in De-AM group, while there was no difference in the amount of TIMP1 in medium among three groups., Conclusion: Culture medium of amniotic membrane significantly suppresses the corneal nevovascularization induced by bFGF. The mechanism of which at least in part is that high level of TIMP2 protein secreted or released into the culture medium of AM and inhibition of migration and growth of vascular endothelial cells.

Purpose: This study was designed to investigate the effects of the locally supplied endogenous angiogenesis inhibitor vasostatin (VS) on corneal angiogenesis., Methods: Recombinant VS was expressed and purified. The effects of VS on the proliferation of endothelial cells were investigated using the methyl thiazolyl tetrazolium (MTT) assay in the absence or presence of angiogenic factors such as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). Corneal neovascularization was induced by implantation of hydron pellets containing bFGF in rat corneal micropockets. The potency of VS to inhibit corneal angiogenesis was investigated by incorporation of VS with bFGF in hydron pellets or topical application of VS containing eye drops to rat eyes implanted with bFGF pellets. The extent of corneal neovascularization was evaluated by microscopic and histological analyses., Results: VS potently inhibited the growth of endothelial cells in the absence or presence of angiogenic factors such as bFGF or VEGF. In the rat corneal micropocket assay, concurrent incorporation of VS abolished the bFGF induced neovascularization. When formulated in a methylcellulose eye drop, VS remained intact and functional in a 4 degrees C solution for more than 7 days. Topical application of VS eye drops potently inhibited bFGF induced neovascularization in rat corneas., Conclusions: The present study effectively demonstrated the potential feasibility of local application of VS for treatment of corneal angiogenesis.

Conjugated linoleic acid (CLA) is a potent inhibitor of mammary carcinogenesis. Cancer cells produce various angiogenic factors which stimulate host vascular endothelial cell mitogenesis and chemotaxis for their growth and metastasis. Basic fibroblast growth factor (bFGF) is a potent angiogenic factor that is expressed in many tumors. In this study, we found that CLA decreased bFGF-induced endothelial cell proliferation and DNA synthesis in a dose-dependent manner. However, CLA did not inhibit endothelial cell migration. Furthermore, CLA showed a potent inhibitory effect on embryonic vasculogenesis and bFGF-induced angiogenesis in vivo. Collectively, these results suggest that CLA selectively inhibits the active proliferating endothelial cells induced by bFGF, which may explain its anti-carcinogenic properties in vivo.

A novel naturally occurring antiangiogenic agent isolated from cartilage, referred to as Neovastat (AE-941), was examined for its efficacy against tumor neovascularization and progression. Exposure to Neovastat results in ex ovo antiangiogenic properties in the chorioallantoid membrane of chicken embryo (71% decrease in the angiogenic index as compared to the basic fibroblast growth factor (bFGF) treated control embryos, P < 0.0001). Oral administration of Neovastat inhibits bFGF-induced angiogenesis in the Matrigel mouse model (87.5% decrease in hemoglobin as compared to the bFGF-treated control implants, P < 0.0001). Neovastat also induces a dose response decrease of lung metastases in the Lewis lung carcinoma model (oral administration; 69.1% of inhibition obtained at the maximal dose of 0.5 ml/day, P < 0.0001). Combined with a sub-optimal dose of cisplatinum (2 mg/kg, i.p.), Neovastat (0.5 ml/day) improved the therapeutic index by increasing the antimetastatic efficacy and by exerting a protective activity against cisplatinum-induced body weight loss and myelosuppression. In summary, our experimental data provide evidence of antiangiogenic and antimetastatic properties of Neovastat, following oral administration.

Purpose: To examine the applicability of culture supernatant of human amniotic cells on basic fibroblast growth factor (bFGF)-induced corneal neovascularization., Methods: Human amniotic epithelial and mesenchymal cells (AC) were obtained from human amniotic membranes by digesting with collagenase and maintained in serum-containing medium. The AC preparations predominantly contained cytokeratin-positive cells (91.2 +/- 3.1%, n = 4). The culture supernatant was prepared by cultivating AC in serum-free medium for 24 hours. Neovascularization was obtained by a micropocket assay with Hydron pellets containing bFGF. Migration assay was carried out by a double-chamber method using human umbilical vein endothelial cells (HUVEC). Cell growth assay was done by MTT assay using HUVEC., Results: Basic fibroblast growth factor-induced corneal neovascularization was significantly reduced by administration of AC culture supernatant. When either control or AC culture supernatant was administered three times per day for 10 days, the area with neovascularization was 13.2 +/- 3.2 mm2 and 4.0 +/- 1.4 mm2 for the control and AC culture supernatant-treated eyes, respectively (n = 7, p = 0.021). AC culture supernatant strongly inhibited bFGF-induced migration and cell growth of HUVEC., Conclusions: Amniotic cell culture supernatant contains potent inhibitors of neovascularization. This effect is explained in part by inhibition of migration and cell growth of vascular endothelial cells. AC culture supernatant may be applicable for treatment of corneal diseases with neovascularization.

Purpose: Vascular endothelial growth factor (VEGF) causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization (NV). Basic fibroblast growth factor (bFGF) also has been implicated in the production of ocular NV. This study was performed to investigate the ability of simultaneous sustained intravitreal release of both VEGF and bFGF to induce robust retinal NV in the rabbit., Methods: Intravitreal implantation of sustained-release Hydron polymeric pellets containing both 20 microg of VEGF and 20 microg of bFGF was performed on adult male Dutch belted rabbits. In other animals either 20 microg or 50 microg bFGF-containing pellets was implanted intravitreally; also, either 20 microg VEGF or 50 microg VEGF-containing pellets was implanted. Control rabbits received either blank polymeric pellets or a pellet containing 30 microg bovine serum albumin. Eyes were examined by indirect ophthalmoscopy after surgery at 24 hrs, 48 hrs, 4 days, 7 days, 14 days, 21 days, and 28 days. Findings were documented by color fundus photography and fluorescein angiography (FA). Eyes were enucleated and prepared for histologic analysis at 28 days following intravitreal implantation of the VEGF/bFGF-containing pellets., Results: In all eyes implanted with VEGF/bFGF pellets, dilation and tortuosity of existing blood vessels were observed within 48 hrs after pellet implantation. The progression of retinal vascular changes was rapid and occurred over the entire optic disk and medullary rays between 4 and 7 days. Hemorrhage occurred as early as 14 days after VEGF/bFGF pellet implantation. In eyes with massive hemorrhage, total traction retinal detachment developed after the second week. The presence of abnormal tissues at the vitreo-retinal interface within 28 days was demonstrated by light microscopy while FA showed profuse leakage of dye from anomalous vessels within the first week. Neither bFGF-exposed eyes nor control eyes showed any vascular changes. Eyes that received only VEGF-containing pellets exhibited tortuosity of existing vessels, but neither hemorrhaging nor retinal detachment occurred., Conclusions: These results demonstrate that retinal vascular changes leading to hemorrhaging is produced rapidly in the rabbit by simultaneous intravitreal release of both VEGF and bFGF. Understanding how these growth factors induce retinal NV may suggest novel therapeutic treatment strategies.

Background: This laboratory previously demonstrated that placement of fibroblast growth factor-2 (FGF-2)-soaked beads adjacent to the developing ventricle at stage 24 caused cardiovascular anomalies by embryonic day 15. We sought to characterize early cellular changes that may suggest mechanisms for the abnormalities observed at day 15. Because levels of both myocyte proliferation and immunohistochemically detectable endogenous FGF-2 begin to decline before stage 24 in untreated embryos, it was of interest to determine whether exogenous FGF-2 might maintain cardiac myocyte proliferation at or near peak levels., Methods: Chick embryos were incubated to stage 18 (2.8 days), at which time beads soaked in phosphate-buffered saline (PBS) or 100 microg/ml FGF-2 were placed adjacent to the developing ventricle and development was allowed to continue. After 3 days (stage 29), bromodeoxyuridine (BrdU) was applied to mark dividing cells, followed by double fluorescent assessments to detect relative numbers of dividing and nondividing cells., Results: Quantitative image analysis, using Metamorph software, showed that exogenous FGF-2 caused a 62% increase in the overall number of dividing cells (P < 0.01), concomitant with a 25% increase in total cell number (cell density: P < 0.05). Expressed in relative terms, these changes corresponded to a 25% increase in the proliferation labeling index: 30% of all cells were proliferating in FGF-treated hearts, in contrast with only 24% in control hearts., Conclusions: Taken together, these data suggest that an FGF-induced imbalance in myocardial cell proliferation at early developmental stages of heart development causes cardiovascular anomalies during late embryogenesis., (Copyright 2000 Wiley-Liss, Inc.)

A 1-mo toxicity study followed by a 1-mo recovery period of recombinant human basic fibroblast growth factor (bFGF) was performed using Beagle dogs at doses of 30, 120 or 480 mg/kg/d to estimate the no observed adverse effect level (NOAEL). Subcutaneous thickening was seen and its incidence, as well as that of stiffness of the injection sites, increased with dose. There were neither dead animals nor significant changes of body weight during the experimental period. In addition, no significant bFGF-related changes were found in ophthalmologic and histopathological examination, urinalysis and hematological, biochemical and organ weight parameters. At necropsy, red-brownish spots and/or nodule formations were recognized in a dose-dependent manner. Splenomegaly was noted in the 480 mg/kg group, but these findings had a low incidence in all dose groups. The findings in the dosing period disappeared or were ameliorated during the recovery period. The above data suggests the NOAEL of bFGF in Beagle dogs is >480 mg/kg/d.

Overexpression and activation of matrix metalloprotease (MMP) have been implicated in angiogenesis. However, the involvement of cysteine proteases, such as calpains (EC 34.22.17), is obscure. Thus, the purpose of this experiment was to study the involvement of cysteine proteases in angiogenesis induced by basic fibroblast growth factor (bFGF) in guinea pig and rat corneas using cysteine protease inhibitors. Sustained-release polymers containing bFGF were implanted into guinea pig and rat corneas to induce angiogenesis. For treatment of corneal angiogenesis, polymers containing cysteine protease inhibitors, leupeptin or SJA6017, were also implanted into corneas. Using the slit lamp, the corneas were observed for nine days after polymer implantation. Soluble proteins and albumin levels were used as markers of corneal injury by angiogenesis. bFGF induced angiogenesis in guinea pig and rat corneas. In guinea pig cornea, wet weight, the amount of soluble protein and albumin was highest at four days after bFGF-containing pellet implantation. In rat cornea, the amount of soluble protein and albumin was highest at six days, and wet weight increased within four days. One hundred nmole of leupeptin showed a tendency to reduce bFGF-induced angiogenesis in guinea pig cornea, and 10 nmole of SJA6017 was effective in reducing bFGF-induced angiogenesis in rat cornea, although SJA6017 showed a stronger effect than leupeptin. Ten nmole of SJA6017 significantly reduced the number of new blood vessels. These data suggested involvement of cysteine proteases in angiogenesis in guinea pig and rat cornea.

Background: The proliferation of vascular endothelial cells and ultimately angiogenesis is inhibited by blocking integrin-mediated cell-matrix interaction. To asses the therapeutic potential of alpha(v)-integrin antagonists LM609 and cRGDfV in neovascularization of the anterior segment, their inhibitory effect on angiogenesis was studied in two rat models for corneal neovascularization., Methods: Corneal neovascularization was induced in Wistar rats (n=51) either by silver nitrate burns or intrastromal implantation of polymer pellets containing 400 ng of fibroblast growth factor (bFGF). Animals were treated with subcutaneous injections of a cyclic alpha(v)-integrin antagonist (cRGDfV, 15 mg/kg body wt) or saline twice daily. Additional animals received intrastromal implants containing 400 ng bFGF together with either Lm609 (mAb, anti-alpha(v)beta(3)) or control antibody. Four days later, the animals were killed and the percentage of the surface area covered with vessels determined using digital image analysis., Results: Systemic treatment with cRGDfV resulted in a significant reduction of corneal vessel growth in animals with bFGF-induced corneal vascularization. In corneas with silver nitrate burns, systemic cRGDfV treatment showed no significant reduction of vascularization compared with controls. Pellets containing bFGF and LM609 mAb induced significantly less neovascularization than pellets containing bFGF and control mAb., Conclusion: Our results suggest that in the rat cornea, alpha(v)beta(3) ligation does inhibit bFGF-induced neovascularization. A chemical burn of the cornea induces angiogenisis which is not inhibited by blocking alpha(v)-integrins. This suggests an angiogenic pathway independent of alpha(v)-integrins.

Background and Objective: We investigated the effect of photodynamic therapy (PDT) using topically administered ATX-S10(Na) on corneal neovascularization (CoNV)., Material and Methods: Rabbit eyes with induced CoNV were treated with ATX-S10(Na) eye drops (10 mg/mL) every 5 minutes, 5 to 25 times. Five to ninety minutes after topical administration, the CoNV were irradiated with a diode laser using a wavelength of 670 nm., Results: The CoNV were occluded fluorescein angiographically in 7 of 16 treated eyes. The eyes having occluded, CoNV were irradiated using fluence of 510-1019 J/cm2 within 20 minutes of eye-drop administration. However, the effect was more variable than what we found using systemic administration in our previous investigation., Conclusions: Experimental CoNV was occluded by photodynamic therapy using topically administered ATX-S100(Na), suggesting this modality as a possible treatment for CoNV avoids the side effects found with systemic administration of the dye. Further efforts to improve the eye drops in terms of pH and osmotic pressure are needed to achieve increased dye accumulation.

Neovascular diseases are the leading causes of blindness in humans. Although several promising compounds have been isolated, pharmacological treatment remains difficult. Thalidomide has inhibitory effects on angiogenesis in the corneal micropocket assay. However, the results vary considerably depending on the administration route and animal model. The aim of this study, therefore, was to investigate thalidomide and two of its derivatives, supidimide and EM12, in the rabbit corneal micropocket assay. Using both basic fibroblast growth factor and vascular endothelial growth factor for initiation of the neovascular response, we were able to show a significant inhibition of neovascularisation with all three substances. EM12, the most teratogenic derivative analysed, was demonstrated to be the most potent inhibitor of angiogenesis in this model. Thalidomide and supidimide did not show systemic side effects in the applied dosage. An equal dosage of EM12, however, resulted in significant weight loss of the animals, but did not increase angiogenic activity compared with lower doses. Together with earlier findings, these data support a strong correlation between the antiangiogenic potential and the teratogenic activity of thalidomide and structurally related compounds.

Background: Suramin, which binds to and blocks autocrine and paracrine growth factors required for the proliferation of neoplastic cells, is a clinically effective antitumor agent against some human tumors; however, efficacy often is limited by toxicity. In this study, suramin treatment was combined with a fibroblast growth factor (FGF) receptor-directed toxin chimera, basic FGF-saporin (bFGF-SAP), based on the authors' previous observations that autocrine-mediated resistance to bFGF-SAP in melanoma in vitro is abrogated by suramin treatment., Methods: Severe-combined immunodeficient-Beige mice bearing SK-Mel-5 human melanoma xenografts received weekly treatments of suramin (200 or 75 mg/kg intraperitoneally) beginning on Day 5 after tumor implantation followed 18 hours later by a treatment with bFGF-SAP (0.5-5 microg/kg intravenously) for 4 weeks. The optimal interlude between the administration of suramin and bFGF-SAP was determined by tumor excision assays. The efficacy of combination therapy as a function of alternative dosing regimens was determined by tumor growth inhibition (TGI) studies., Results: Fifty days after implantation, a 79-82% TGI was observed in animals receiving the suramin (200 mg/kg) plus bFGF-SAP combination regimens compared with median tumor volumes from vehicle-treated controls (3070+/-440 mm(3)). TGI observed for combination therapies varied significantly (P<0.05-0.001) from TGI observed in treatment groups receiving suramin alone (57%) or bFGF-SAP alone (34-38%). Combining bFGF-SAP (5 microg/kg) with a low, therapeutically ineffective dose of suramin (75 mg/kg) produced a 68% rate of TGI compared with controls, thus lowering the therapeutic effective dose of suramin and eliminating the suramin-related lethal toxicity (12% mortality rate) observed in animals treated with high dose suramin., Conclusions: The results of the current study suggest that combining suramin with receptor-directed therapies offers a more effective regimen for the treatment of malignant melanoma., (Copyright 1999 American Cancer Society.)

Background: Basic fibroblast growth factor receptors on DU145 human prostatic carcinoma xenografts serve as targets for the delivery of a growth factor-toxin chimera, basic fibroblast growth factor-saporin (bFGF-SAP), which produces significant antitumor activity in a nude mouse model. However, DU145 tumors often become resistant to prolonged treatment., Methods: Nude mice bearing DU145 xenografts were intravenously administered bFGF-SAP (0.05 microg/kg weekly for 4 weeks), and a panel of eight tumors was isolated from the treated animals and established in monolayer culture., Results: In cell-survival assays, sensitivity of the treated tumor-derived cell lines to bFGF-SAP (IC50 = 12-100 nM) varied widely from cells derived from a vehicle-treated control tumor (IC50 = 10 nM). A significant inverse correlation was observed between increased IC50 values in vitro and increased tumor growth delay in vivo. Pretreatment of tumor cells with suramin or neutralizing antibodies to bFGF or keratinocyte growth factor (KGF) circumvented resistance in one of the tumor lines, confirming autocrine-mediated resistance. In another tumor subline, a 3-fold decrease in bFGF high-affinity receptor sites, which concurred with a 4-fold decrease in ability to internalize the bFGF ligand, was consistent with a decrease in total cellular expression of the FGF2 receptor (Bek). Resistance was circumvented by alternatively targeting FGF1 receptor (Flg) on these cells with a saporin immunotoxin., Conclusions: These studies identify alterations in the ligand-targeted receptor as a frequent contributor to resistance arising in DU145 tumors to in vivo treatment with a bFGF receptor-directed-toxin chimera, and provide the basis for designing methods to circumvent resistance for the purpose of enhancing efficacy of receptor-directed therapies in the treatment of prostate cancer., (Copyright 1999 Wiley-Liss, Inc.)

Recombinant FGF-2-SAP is a mitotoxin consisting of the plant-derived ribosome-inactivating toxin saporin (SAP) fused to basic fibroblast growth factor (FGF-2). FGF-2-SAP targets and kills cells bearing upregulated FGF receptors. In vivo, FGF-2-SAP inhibits smooth muscle cell hyperplasia in models of restenosis. The present study examined the potential for a differential effect of FGF-2-SAP on canine vascular endothelial cells (EC) and smooth muscle cells (SMC) separately as well as in a novel co-culture model. Canine vascular SMC and EC cultures were established separately and made quiescent once cells reached 80% confluence. Following the release from growth arrest, both cell types were treated with FGF-2-SAP, or FGF-2, or SAP alone for 48 h. [3H]TdR incorporation was used to determine the growth response of SMC and EC. The co-culture system was created by plating canine vascular SMC and EC on either side of a microporous 13 microm thick polyester membrane insert. Both cell types were grown to 80% confluence and independently made quiescent. Following the release from growth arrest, cells were treated with FGF-2-SAP, or FGF-2, or SAP alone. Negative and positive control groups were untreated wells containing phosphate buffered saline and complete growth media, respectively. After 48 h, both [3H]TdR incorporation and total DNA content, by fluorometric measurement, were quantitated in SMC and EC independently. FGF-2-SAP showed a concentration-dependent cytotoxicity in both canine SMC and EC but cytotoxicity for EC required substantially higher concentrations. In co-cultured SMC, FGF-2-SAP significantly decreased both [3H]TdR uptake and total DNA content at 0.5, 5, 50, and 500 ng/ml (0.01-10 nM) compared to positive controls. In co-cultured EC, FGF-2-SAP decreased [3H]TdR uptake at 50 and 500 ng/ml and total DNA content at 500 ng/ml compared to positive controls. Neither SAP alone nor FGF-2 alone showed a significant effect on [3H]TdR uptake or DNA content of either SMC or EC. In this unique co-culture model, which better replicates the relationship between SMC and EC in vivo, we demonstrated a dose-response range of FGF-2-SAP at which both the proliferation and total cell number of SMC, but not EC, is significantly reduced. These data suggest that FGF-2-SAP may have therapeutic utility in inhibiting myointimal hyperplasia in the absence of a deleterious effect on regenerating endothelium following vascular reconstructions.

The presence and distribution of functional, high-affinity receptors for fibroblast growth factors (FGFs) in the neonatal organ of Corti were probed using the intracellular toxin saporin conjugated to basic FGF (FGF-2). FGFs that bind to high-affinity FGF receptors are internalized as part of the normal process of receptor inactivation. The receptor can thus be used for the targeted delivery of molecules conjugated to FGF into the cytoplasm. Incubation of postnatal day 5 (P5) rat organ of Corti cultures with FGF-saporin caused a dose dependent destruction of outer hair cells, Deiters cells and outer pillar cells. Inner hair cells and other cells were unaffected. Organ of Corti cultures at P0 and P10 showed much less damage than at P5. The results suggest that outer hair cells and adjacent supporting cells in the organ of Corti transiently express high-affinity FGF receptors, and that these receptors can mediate the intracellular delivery of bioactive molecules.

The temporal roles of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the development of sensory neurons have been studied in a cell culture preparation which models normal embryonic inner ear development (normocytic). Previous studies showed that FGF-2 stimulated migration and differentiation of ganglion cells for the first 2 days in vitro, but after 5 days led to degeneration, implicating other factors in their later development. To see if BDNF could be such a factor, otocysts were explanted from white leghorn embryos at the time when ganglion cell precursors normally start migrating from the otic epithelium. Cultures were grown in a defined medium, either with or without human recombinant FGF-2 for 2 days or with BDNF. On Day 3, FGF-2 was replaced either with BDNF in defined medium or with defined medium only. Measurements of neuroblast migration and neurite outgrowth were made by time-lapse imaging in living cultures. In cultures receiving BDNF on Day 3, cell migration and neurite outgrowth from the explant increased for more than 3 weeks but not in cultures receiving only defined medium from Day 3. Cultures did not survive more than 3-4 days when receiving either BDNF in defined medium or defined medium alone from the first day. A neutralizing antibody to BDNF inhibited neuronal migration and neurite outgrowth, and it also blocked the effects of exogenous BDNF. BDNF did not enhance the effects of FGF-2 by interacting with it. These experiments defined a temporal sequence in which FGF-2 acts early in development, while BDNF affects a later stage.

The neurotrophic effects of basic fibroblast growth factor on hippocampal neurons have been well documented. However, the acute effects of basic fibroblast growth factor on hippocampal electrical activities in vivo have not yet been studied. To assess the impact of basic fibroblast growth factor on hippocampal electroencephalographic (EEG) activity, two doses of basic fibroblast growth factor were injected into the right ventral hippocampus of freely moving animals. Unilateral injection of 50 ng of basic fibroblast growth factor into the dentate region of the hippocampus resulted in immediate EEG ictal discharges and behavioral seizures in 7/9 rats. These seizures lasted about 2 h following the injection. Injection of 25 ng of basic fibroblast growth factor into the same sites induced ictal discharges of approximately 60 min duration in 3/7 rats. Injection with heat-inactivated basic fibroblast growth factor into the same sites did not cause any EEG or behavioral modifications. These results suggest that in addition to growth-promoting effects, basic fibroblast growth factor is able to acutely alter the electrical activity of hippocampal neurons and cause seizures.

Myoblast-mediated gene transfer and its repeated applications were tested for achieving a long-term stable systemic production of human factor IX (hFIX) at a therapeutic level in SCID mice. Primary skeletal myoblasts were stably transfected with a hFIX expression plasmid vector, pdLMe4 betaA-hIXm1, which contains a hFIX minigene under the control of a beta-actin promoter with muscle creatine kinase enhancers. Myotubes derived from the myoblasts produced 1,750 ng hFIX/10(6) cells/24 hours in culture. hFIX secretion by the myoblasts and thereof derived myotubes were equally efficient, and myotubes were shown to have a sufficient secretory capacity to handle a substantially elevated production of hFIX. After intramuscular injection of 5, 10, and 20 x 10(6) myoblasts, SCID mice stably produced hFIX into the systemic circulation proportional to the number of implanted cells, and the expression levels were maintained for at least up to 10 months (end of the experiment). Additional cell injections administered to animals that originally received 10 x 10(6) cells approximately 2 months later elevated the systemic hFIX levels to an average of 182 +/- 21 ng/mL, a therapeutic level, which persisted for at least 8 months (end of the experiment). These results indicate that long-term, stable systemic production of hFIX at therapeutic levels can be achieved by repeated application of myoblast-mediated gene transfer.

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

The plasticity of mature oligodendrocytes was studied in aggregating brain cell cultures at the period of maximal expression of myelin marker proteins. The protein kinase C (PKC)-activating tumor promoters mezerein and phorbol 12-myristate 13-acetate (PMA), but not the inactive phorbol ester analog 4alpha-PMA, caused a pronounced decrease of myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity. In contrast, myelin/oligodendrocyte protein (MOG) content was affected relatively little. Northern blot analyses showed a rapid reduction of MBP and PLP gene expression induced by mezerein, and both morphological and biochemical findings indicate a drastic loss of compact myelin. During the acute phase of demyelination, only a relatively small increase in cell death was perceptible by in situ end labeling and in situ nick translation. Basic fibroblast growth factor (bFGF) also reduced the levels of the oligodendroglial differentiation markers and enhanced the demyelinating effects of the tumor promoters. The present results suggest that PKC activation resulted in severe demyelination and partial loss of the oligodendrocyte-differentiated phenotype.

Basic fibroblast growth factor (bFGF) is a potent mitogen for bone. In this study, we utilized the clonal rat osteoblastic cell line, Py1a, to examine signal transduction by bFGF and to determine the role of mitogen activated protein kinases (MAPK) and induction of c-fos mRNA in the mitogenic response to bFGF. Stimulation of [3H]thymidine incorporation (TDR) into DNA by bFGF was determined in the presence of phorbol myristate acetate of (PMA) to down-regulate the protein kinase C (PKC) pathway, genistein, an inhibitor of tyrosine kinase and H-7, a PKC inhibitor, bFGF 10(-8) M and PMA 10(-7) M increased TDR by 242 and 245%, respectively. Treatment with bFGF or PMA for 5 or 30 minutes increased tyrosine phosphorylation of multiple proteins, and immunoblotting with MAPK-specific antibody revealed that two of these bands were the 42 and 44 kD isoforms of MAPK. PMA and bFGF induced c-fos mRNA expression at 30 minutes. Genistein at 10 micrograms/ml blocked the mitogenic effect of bFGF and partially inhibited the mitogenic effect of PMA. Genistein at 100 micrograms/ml also blocked both bFGF- and PMA-induced increases in c-fos mRNA. A 24 h pretreatment with PMA at 10(-7) M inhibited the mitogenic response, tyrosine phosphorylation of MAPK, and induction of c-fos mRNA subsequent to the addition of PMA, but not bFGF. H-7 at 50 microM blocked bFGF-induced mitogenesis and c-fos induction, but did not inhibit bFGF-induced tyrosine phosphorylation of MAPK. In this study, we show that the signaling pathway of bFGF and PMA are similar in that they both induce tyrosine phosphorylation of MAP kinases and activate c-fos. However, the signaling pathways ultimately diverge in that once the PKC pathway is down-regulated by PMA pretreatment or blocked by the PKC inhibitor H-7, tyrosine phosphorylation of MAP kinase, c-fos induction, and the mitogenic effect of PMA is blocked. In contrast, down-regulation of the PKC pathway inhibits c-fos and the mitogenic response to bFGF, but not bFGF's effects on tyrosine phosphorylation of MAP kinase.

Background: We have shown that the angiogenic peptides basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) enhance canine coronary collateral development when administered for > or = 4 weeks. bFGF, a pluripotent mitogen of mesodermally derived cells, could theoretically exacerbate neointimal smooth muscle cell hyperplasia, a fundamental component of atherosclerosis. VEGF, an endothelial cell-specific mitogen and vascular permeability factor, could have deleterious effects related to vascular hyperpermeability. The present investigation had two aims: (1) to ascertain whether brief (7-day) systemic arterial treatment with bFGF or VEGF would improve myocardial collateral perfusion and (2) to determine whether these peptides induce neointimal accumulation in vivo., Methods and Results: Dogs were subjected to ameroid-induced occlusion of the left circumflex coronary artery and randomized to bFGF 1.74 mg (n = 9), VEGF 0.72 mg (n = 9), or saline (n = 10) as a daily left atrial bolus (days 10 to 16). Additional dogs were randomized to VEGF 0.72 mg (n = 6) or saline (n = 5); however, treatment was delayed by 1 week. Coincident with the institution of treatment, all dogs underwent balloon denudation injury of the iliofemoral artery. bFGF markedly increased maximal collateral flow but did not exacerbate neointimal accumulation. VEGF had no discernible effect on maximal collateral flow, but it exacerbated neointimal thickening after vascular injury., Conclusions: Short-term treatment with bFGF enhanced collateral development without increasing neointimal accumulation at sites of vascular injury. Although VEGF did not increase collateral development as administered in this study, it significantly exacerbated neointimal accumulation. These data provide support for the clinical investigation of bFGF in selected patients with ischemic heart disease.

Purpose: To develop a non-inflammatory model of both acute and chronic angiogenesis in the rabbit cornea using a known directly angiogenic cytokine., Methods: Pellets made of the slow-release polymer Hydron (polyhydroxyethylmethacrylate) and containing sucralfate and/or basic fibroblast growth factor (basic-FGF) were implanted into rabbit corneas. The neovascular response to the implantation of pellets containing basic-FGF alone, sucralfate alone or a titration of basic-FGF in the presence of a constant amount of sucralfate was measured. The role of inflammation in the neovascular response was also investigated., Results: The addition of sucralfate to the pellets led to the sustained release of basic-FGF resulting in a predictable and aggressive neovascular response with a low dose of basic-FGF that by itself was unable to elicit neovascularisation. At a dose of 500 ng per pellet, approximately one-third of the surface area of the cornea was vascularised within eight days of implantation. Minimal or no vascularisation occurred with the same dose of basic-FGF without sucralfate. While this dose of basic-FGF induced corneal oedema, only minimal inflammation was observed and the response was unaffected by ionising radiation. A less aggressive though still robust neovascular response with no or only minimal oedema was observed when the dose was lowered to 50 ng of basic-FGF per pellet. Some induced vessels persisted for more than three months., Conclusion: This is an inexpensive in vivo model of angiogenesis with the advantages of the neovascularisation being aggressive, predictable, persistent, unassociated with an obvious inflammatory response and induced by the sustained release of an agent known to have a direct stimulatory action on endothelial cells.

The effects of basic fibroblast growth factor (FGF-2) on presumptive auditory and vestibular neurons from the medulla were studied in primary cell cultures. The part of the rhombic lip that forms nucleus magnocellularis (homologue of the mammalian anteroventral cochlear nucleus) was explanted from white leghorn chicken embryos at Hamburger-Hamilton stage 28 (E5.5), the time when precursors of the magnocellularis bushy cells migrate and begin to differentiate in situ. In vitro the neuroblasts migrated onto 2-D substrates of purified collagen, differentiated, and expressed neuronal markers. One-half of the cultures were supplemented with human recombinant FGF-2 (10 ng/ml daily) for 5-7 days; the others, with fetal bovine serum. FGF-2 more than doubled the length of neurite outgrowth during the first 3 day treatment compared to serum, but the number of migrating neuroblasts was unaffected. Although neurites attained greater lengths in FGF-2, they usually degenerated after 4-5 days; in serum their growth continued for several weeks. Differentiation of neuronal structure, including axons and dendrites, began within 1-2 days in bFGF but required at least 5-7 days in serum. Histochemical observations in vitro and in situ with antibodies to FGF receptor demonstrated immunopositive patches on acoustico-vestibular neuroblasts at stage 28, when they are migrating and first forming their axons. The findings suggest that FGF-2 stimulates neurite outgrowth in the cochlear and vestibular nuclei. FGF-2 may accelerate cell death by overstimulating neuroblasts, but other factors are needed to sustain their further development.

Podocyte injury is believed to contribute to glomerulosclerosis in membranous nephropathy. To identify the factors involved, we investigated the effects of basic fibroblast growth factor (bFGF), a cytokine produced by podocytes, on rats with membranous nephropathy (passive Heymann nephritis [PHN]). All rats received a daily i.v. bolus of 10 microg bFGF or vehicle from days 3-8 after PHN induction. In proteinuric PHN rats on day 8, bFGF injections further increased proteinuria. Podocytes of bFGF-injected PHN rats showed dramatic increases in mitoses, pseudocyst formation, foot process retraction, focal detachment from the glomerular basement membrane, and desmin expression. bFGF injections in PHN rats did not alter antibody or complement deposition or glomerular leukocyte influx. bFGF-injected PHN rats developed increased glomerulosclerosis when compared with control PHN rats. Also, bFGF induced proteinuria and podocyte damage in rats injected with 10% of the regular PHN-serum dose. None of these changes occurred in bFGF-injected normal rats, complement-depleted PHN rats or rats injected with 5% of the regular PHN serum dose. These divergent bFGF effects were explained in part by upregulated glomerular bFGF receptor expression, induced by PHN serum. Thus, bFGF can augment podocyte damage, resulting in increased glomerular protein permeability and accelerated glomerulosclerosis. This bFGF action is confined to previously injured podocytes. Release of bFGF from glomerular sources (including podocytes themselves) during injury may represent an important mechanism by which podocyte damage is enhanced or becomes self sustained.

Purpose: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs)., Method: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis., Results: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL., Conclusions: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

Human interferon-inducible protein 10 (IP-10), a member of the alpha chemokine family, inhibits bone marrow colony formation, has antitumor activity in vivo, is chemoattractant for human monocytes and T cells, and promotes T cell adhesion to endothelial cells. Here we report that IP-10 is a potent inhibitor of angiogenesis in vivo. IP-10 profoundly inhibited basic fibroblast growth factor-induced neovascularization of Matrigel (prepared by H. K. Kleinman) injected subcutaneously into athymic mice. In addition, IP-10, in a dose-dependent fashion, suppressed endothelial cell differentiation into tubular capillary structures in vitro. IP-10 had no effect on endothelial cell growth, attachment, and migration as assayed in vitro. These results document an important biological property of IP-10 and raise the possibility that IP-10 may participate in the regulation of angiogenesis during inflammation and tumorigenesis.

Basic fibroblast growth factor (bFGF), a soluble mitogen, has been isolated and purified from various organs, including the retina. In vivo angiogenic activity of bFGF has been demonstrated with several assays. An experimental model of choroidal neovascularization was developed in the mini pig by perfusion of recombinant human bFGF through an osmotic minipump. Endogenous bFGF and bFGF receptors were localized in the normal pig retina by immunohistochemistry and autoradiography after binding. The perfusion of exogenous bFGF induced well-organized new vessels along the last 3 mm of the catheter in the suprachoroidal space. This neovascularization did not penetrate the normal Bruch's membrane. Vascular cells (identified by von Willebrand factor antibody staining) increased in number and in surface from the proximal part to the end of the intraocular catheter in all bFGF perfused eyes. In eyes perfused with phosphate buffered saline (controls), but not in the bFGF perfused eyes, an inflammatory response occurred (identified by a macrophage specific antibody). These results demonstrate that choroidal angiogenesis can be achieved without an inflammatory response by perfusing an excess of bFGF in the suprachoroidal space.

A novel method for quantitating angiogenesis and its inhibition has been developed for the chick embryo. The method is based on the vertical growth of new capillary blood vessels into a collagen gel through two parallel nylon meshes which align the capillaries for counting. Angiogenesis is induced by basic fibroblast growth factor contained within the gel and slowly released by aluminum sucrose octasulfate (sucralfate) or by tumor cells implanted on the gel. The potency of four different angiogenesis inhibitors was compared at concentrations of 0.6 to 600 nmole. This technique may facilitate the discovery and development of angiogenesis inhibitors for clinical application.

We have constructed growth factor-toxin chimeric molecules composed of basic fibroblast growth factor (bFGF) and two different binding mutant forms of Pseudomonas exotoxin termed bFGF-PE40 and bFGF-PE4E KDEL. The chimeric molecules were expressed in Escherichia coli and localized to both inclusion bodies and the spheroplast cytoplasm. The bFGF-toxin fusion protein that was isolated and purified from inclusion bodies was 3-fold more active in inhibiting protein synthesis than that purified from spheroplast cytoplasm. Immunoreactivity of purified bFGF-toxin fusion protein to anti-bFGF antibodies was similar to that of native bFGF, as determined by ELISA analysis. A variety of carcinoma cell lines were sensitive to bFGF-PE40 and bFGF-PE4E KDEL, including H3396 (breast), Hep G2 (hepatocellular), and A431 (epidermoid). The concentration of chimeric toxin that inhibited protein synthesis by 50% (EC50) was 110, 70, and 18 ng/mL for bFGF-PE40 and 15, 1, and 18 ng/mL for bFGF-PE4E KDEL. In comparison with fusion-toxins composed of acidic fibroblast growth factor (aFGF) and either PE40 or PE4EKDEL, bFGF-PE40 and bFGF-PE4E KDEL were similarly cytotoxic on most cell lines tested. Human aortic smooth muscle cells were sensitive to both bFGF and aFGF toxin fusion proteins. However, human aortic endothelial cells were sensitive to the bFGF-toxins but were resistant to both aFGF-toxin forms. Time course studies showed that bFGF-PE40 needed a 4-6-h exposure to target cells for peak inhibition of protein synthesis on both MCF-7 and A431 cells, while aFGF-PE40 was almost fully active within a 2-h incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravenous administration of human basic fibroblast growth factor up to 100 micrograms/kg/day to Sprague-Dawley rats caused changes in the kidneys that included enlargement, vacuolation, and karyomegaly of podocytes in glomeruli, dilatation and cast formation in tubules, thickening of the media in the lobular arteries, and hyperplasia of the epithelium of the papilla and collecting ducts. In cynomolgus monkeys there was hyperplasia of the parietal epithelium of Bowman's capsule in the glomeruli, tubular dilatation, and minimal arteriopathy. These changes were only seen at 100 micrograms/kg/day. The development and eventual recovery over time were investigated in a sequence of sacrifices. In monkeys the first changes were seen after 7 days of treatment, but in rats only after 16 days. In both species the changes had partially resolved after 30 days of recovery and were considered to return to normal after 60 days without treatment. The morphological changes were accompanied by functional alterations that included proteinuria and raised blood urea. Changes that occurred in other tissues including bone, red blood cells, adrenals, ovaries, liver, gall bladder, spleen, mesenteric lymph nodes, thymus, aorta, salivary glands, and injection site are not described in this paper.

Basic fibroblast growth factor-saporin (FGF-SAP) is an extremely potent cytotoxic agent for cells bearing the FGF receptor. For its synthesis, the two free cysteines on the surface of basic FGF are available for linking by disulfide bridge to SAP which has been derivatized with a reactive sulfhydryl. Because of the heterogeneous nature of the synthesis, the resulting conjugate is heterogeneous as judged by gel electrophoresis. We have removed by site-directed mutagenesis one of the reactive cysteines of basic FGF and have purified monoderivatized SAP to use as the reactants. The resulting chemical conjugate shows a single band containing only 1 mol of basic FGF and 1 mol of SAP per molecule. This homogeneous conjugate is a potent cytocidal agent to cells bearing the FGF receptor.

Basic fibroblast growth factor (b-FGF) is one of the multifunctional growth factors with important therapeutic potential in the field of ophthalmology. It is also implicated in pathogenesis of vitreoretinal proliferative diseases. In the present study, we evaluated its vitreoretinal toxicity by means of clinical observation, electroretinography (ERG), and histopathology after injection of different doses of b-FGF into the vitreous of rabbit eyes. Doses of b-FGF up to 2 micrograms per eye caused no toxicity; however, injection of 4 micrograms or more resulted in sight-threatening vitreoretinal proliferative changes. This information is important for studies aimed at evaluating the therapeutic potential of b-FGF in retinal diseases. Despite some degree of vitreous organization and opacification, retinal folds, and small areas of traction retinal detachment, the amplitudes of ERGs were normal or even increased (hyperpolarization) in eyes which received 8 micrograms of b-FGF.

Fibroblast growth factors (FGFs) have been implicated in a number of proliferative lesions, including malignant tumor growth and vascularization. As a result, cytotoxic agents that target cell surface FGF receptors are currently under investigation. Previous reports have shown that conjugation of basic FGF with the ribosome inactivator, saporin, results in a potent cytotoxin specific for cells bearing high-affinity FGF receptors. In this report, we have used this FGF receptor-dependent cytotoxin to study receptor interactions at the surface of embryonal carcinoma cells, which express low numbers of high-affinity FGF receptors. The growth of three embryonal carcinoma cell lines and one embryonic stem cell line was shown to be inhibited by bFGF-saporin, suggesting that these cells are able to bind and internalize FGF through high-affinity FGF receptors. In addition, we determined that the responses of these cells to bFGF-saporin are qualitatively different than the responses of CHO-KI cells, which also exhibit low numbers of high-affinity FGF receptors. Specifically, pretreatment with bFGF-saporin reduces the cloning efficiency of CHO-KI cells 8- to 10-fold, whereas bFGF-saporin has little or no effect on the cloning efficiency of embryonal carcinoma cells. This finding suggests that bFGF-saporin is cytotoxic for CHO-KI cells, but not for embryonal carcinoma cells. Thus, our findings argue strongly that other factors, in addition to high-affinity FGF receptor number, are important in determining sensitivity of cells to bFGF-saporin.

Repeated intravenous administrations were carried out in cynomolgus monkeys and rats (S.D.) for a maximum of 4 weeks at doses of 1, 10 and 100 micrograms/kg/day in stable formulation. Three main target organs were identified: red blood cells (RBC), kidney glomeruli (KG) and bone at the top dose level. RBC: Normochromic normocytic anaemia started in rats and monkeys during the second week of treatment (decrease in red blood cell production). The kinetics of this anaemia, as well as its recovery, will be discussed. Bone: Dramatic hyperostosis in rats was present by day 10 in long or spongious bone. This became marked on day 29 and regressed after treatment was stopped. KG: In the rat glomerular lesions were present starting from day 16. They consisted of enlargement and vacuolation of podocytes with loss of foot processes and adhesions between glomerular tuft and Bowman's capsule. Proteinuria was a striking feature. In the monkey the lesions were hyperplasia of the parietal epithelium of Bowman's capsule which involved replacement of normally flattened epithelium by cuboidal cells, with some pseudostratification. Proteinuria also occurred in monkeys, accompanied by a lowering of serum protein (albumin). In two animals, death (by day 15) was preceded by high levels of urea and blood creatinine. The above lesions (KG) disappeared almost completely over a recovery period. It is suggested that these phenomena are not the expression of direct toxicity in the form of lethal insults, but rather a manifestation of a change in cell activity.

To evaluate whether or not the corneal micropocket implantation is effective for determining the toxicity of polymeric materials, currently used biodegradable polymers such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), LA-GA copolymers, and three kinds of poly(2-cyano-acrylate)s (PCA) were implanted in a rabbit corneal pouch and the tissue responses were observed macroscopically and microscopically. It was found that PLA induced no vascularization, whereas a residual solvent and ethylene oxide gas remaining in the PLA matrix invoked vascularization. Vascularization clearly took place when PGA was implanted in the cornea, which became opaque, probably because of cellular infiltration. In the case of PCA implantation, severe inflammation as well as vascular invasion occurred in the initial stage. It is likely that these tissue reactions were caused by the leachables from the implanted materials, the extent being dependent on the leaching rate and the toxicity. It was concluded that the corneal micropocket assay is a good means to detect trace amounts of leachables from implanted materials without sacrificing the animals with the implanted materials.