Exogenous bFGF Research Articles

Basic fibroblast growth factor (bFGF) enhances tissue sparing and functional recovery following moderate spinal cord injury.

The rapid increase in basic fibroblast growth factor (bFGF) production following spinal cord injury (SCI) in rats is thought to serve a role in the cellular processes responsible for the functional recovery often observed. In this study, bFGF was intrathecally administered continuously for 1 week beginning 30 min after a moderate (12.5 mm) spinal cord contusion in adult rats using the New York University impactor device. Osmotic minipumps were implanted into the lateral ventricle and lumbar thecal sac to deliver bFGF at a rate of 3 microg or 6 microg per day versus control vehicle. Animals were behaviorally tested for 6 weeks using the Basso, Beattie, Bresnahan locomotor rating scale and histologically assessed for both tissue sparing and glial reactivity rostral and caudal to the lesion. Rats treated with bFGF regained coordinated hindlimb movements earlier than controls and demonstrated consistent coordination from 4 to 6 weeks. Vehicle-treated rats showed only modest improvements in hindlimb function. The amount of spared tissue was significantly higher in bFGF-treated rats than in controls. Astrocyte and microglial reactivity was more pronounced in bFGF-treated animals versus controls. In summary, intrathecal infusion of exogenous bFGF following SCI significantly reduces tissue damage and enhances functional recovery. Early pharmacological intervention with bFGF following SCI may serve a neuroprotective role and/or create a proregenerative environment, possibly by modulating the neuroglial response.

Possible mechanism for lead inhibition of vascular endothelial cell proliferation: a lower response to basic fibroblast growth factor through inhibition of heparan sulfate synthesis

Although lead inhibits the proliferation of vascular endothelial cells, the mechanism has been incompletely understood. A lower response to basic fibroblast growth factor (bFGF) of growing bovine aortic endothelial cells after exposure to lead was investigated using a cell culture system in the present study. It was shown that lead significantly decreased the incorporation of [ 3H]thymidine into the acid-insoluble fraction of the cells but the inhibition disappeared in the presence of bFGF neutralizing antibody. Pretreatment with lead resulted in a reduction of the stimulation by exogenous bFGF on the [ 3H]thymidine incorporation. Lead decreased endogenous bFGF bound to cell surface heparan sulfate proteoglycans in a concentration-dependent manner but not the high affinity FGF receptor without a change of the accumulation within the cells. In spite of such a change in the endogenous bFGF distribution, the total amount of the growth factor synthesized was not significantly changed by lead. Although the binding of [ 125I]bFGF to heparan sulfate proteoglycans can be directly inhibited by lead, the inhibition was not so marked. On the other hand, lead markedly suppressed the incorporation of [ 35S]sulfate into heparan sulfate accumulated in the cell layer and the conditioned medium, suggesting that the metal inhibited the synthesis of the glycosaminoglycan in growing endothelial cells. Inhibition of the [ 3H]thymidine incorporation by lead was significantly restored by heparin. Since the binding of bFGF to its receptor is strongly promoted by heparan sulfate, the present data suggest that lead inhibits vascular endothelial cell proliferation by induction of a lower response to endogenous bFGF through a suppression of heparan sulfate synthesis.

Effects of Basic Fibroblast Growth Factor (bFGF)-Neutralizing Antibody and Platelet Factor 4 on Facial Nerve Regeneration

Exogenous basic fibroblast growth factor (bFGF) has been shown to prevent death of injured cholinergic neurons and stimulate neurite outgrowth from the proximal stump of the transected sciatic nerve. The present study was designed to examine the role of endogenous bFGF, rather than exogenous bFGF in the regenerative process of the transected facial nerve of guinea pig, by using the so-called silicone tubulization model which enabled us to bridge the transected facial nerve with a silicone tube and to inject into the tube bFGF-neutralizing antibody, normal IgG, saline, or platelet factor 4 (an antagonist for bFGF receptor). Under light microscopy, treatment with bFGF-neutralizing antibody caused significant decreases in vascular number, vascular area, and regenerating axons in the middle point of regeneration chambers at the third week after facial nerve transection, even though electron microscopy revealed that the bFGF-neutralizing antibody increased the number of thin axons with caliber smaller than 1 μm. Treatment with platelet factor 4 exhibited similar but more conspicuous effects on facial nerve regeneration. These findings suggest that endogenous bFGF not only facilitates angiogenesis within the transected facial nerve but also acts as a neurotrophic agent during facial nerve regeneration; it appears that endogenous bFGF contributes to the enlargement of axon caliber and increases the number of relatively large caliber axons.

Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.

Basic fibroblast growth factor (bFGF, FGF-2) is progressively lost from mammary epithelial cells as they become malignant. To investigate the effects of restoring the expression of bFGF in breast cancer cells, we constructed MCF-7 cells that permanently overexpress 18-kD cytoplasm-localizing bFGF (MCF-7/deltaA(FGF)(18) cells) and cells that express both the 18-kD along with the 22- and 24-kD nucleus-localizing bFGF peptides (MCF-7/NCF(FGF)(18,22,24) cells), using retroviral transduction. These stable cell constructs grew more slowly and had a larger fraction of their populations in the G0/G1 phase of the cell cycle than control cells. All forms of bFGF were eluted from MCF-7/NCF(FGF)(18,22,24) cell monolayers with 2 M NaCl, in contrast to fibroblasts that were demonstrated to secrete only the 18-kD bFGF isoform. High-affinity binding of 18-kD 125I-bFGF to these cells was significantly decreased, probably because of competitive binding by the autocrine-secreted bFGF. Recombinant 18-kD bFGF that was previously demonstrated in our laboratory to inhibit proliferation, activate MAP kinase, and induce the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in MCF-7 cells, further inhibited MCF-7/deltaA(FGF)(18) cells but had no effect on MCF-7/NCF(FGF)(18,22,24) cells. The total cellular content of the high-affinity FGF receptors 1-3 was unchanged, but FGF receptor 4 was decreased in MCF-7/NCF(FGF)(18,22,24) cells. Both cell types overexpressing bFGF isoforms had elevated levels of the cyclin-dependent kinase inhibitor p27Kip1 but not that of p21WAF1/CIP1. In MCF-7/deltaA(FGF)(18) cells, FGFR1 and MAP kinase were constitutively phosphorylated. Exogenous recombinant 18-kD bFGF did not accentuate these effects but did induce an increase in the levels of p21WAF1/CIP1 corresponding to the further inhibition induced by exogenous bFGF in these cells. In MCF-7/NCF(FGF)(18,22,24) cells, FGFR1 and MAP kinase were not phosphorylated at baseline nor upon stimulation with recombinant bFGF, and exogenous bFGF only had a minimal effect on low steady-state p21WAF1/CIP1 levels. However, stimulation of these cells with phorbol ester or insulin did result in MAP kinase phosphorylation. While growth-inhibited in the G1 phase of the cell cycle, MCF-7/NCF(FGF)(18,22,24) cells retained active isoforms of cdk2 and the hyperphosphorylated form of Rb. These data suggest that high molecular weight forms of bFGF overexpressed in MCF-7 cells do not activate the receptor-mediated MAP kinase pathway, and do not induce p21WAF1/CIP1 in an autocrine manner, but inhibit proliferation through other, possibly direct nuclear signalling mechanisms.

Regulation of new blood vessel growth into ischemic skeletal muscle

Purpose: In a rabbit model, transposition of a muscle pedicle flap to an ischemic hind limb has been shown to result in the development of new blood vessels that connect the arterial circulation of the flap to the circulation of the limb. The hypothesis that exogenous recombinant basic fibroblast growth factor (bFGF) would enhance the development of this new blood supply was examined and the regulation of bFGF in this process was investigated. Methods: The right common iliac artery was ligated in 12 male New Zealand white rabbits. An abdominal wall muscle flap based on the left inferior epigastric artery was transposed to the right thigh. bFGF in phosphate-buffered saline (PBS) at 3 ng/h (n = 6), or PBS alone (n = 6), was infused for 7 days via mini-osmotic pumps with an infusion catheter positioned at the flap-muscle interface. The flap-muscle interface was immunostained with anti-α–actin antibody to determine blood vessel density (number of vessels/mm) and with anti-bFGF antibody to evaluate bFGF distribution. RNA was isolated from these sections, and polymerase chain reaction (PCR) was used to examine endogenous bFGF messenger RNA (mRNA) expression. Results: Blood vessel density was significantly increased in animals receiving exogenous bFGF (22.0 ± 10.6 vessels/mm vs. 10.7 ± 8.8 vessels/mm, P = .009). In the controls, neovessels were arranged in clusters with endogenous bFGF concentrated around these clusters. In bFGF-treated animals, vessels were diffusely scattered throughout the flap-limb interface, corresponding to the distribution pattern of infused bFGF. There was no difference in bFGF mRNA expression between the control and the bFGF-treated groups. Conclusion: Exogenous bFGF infusion significantly augmented new blood vessel development at the flap-limb interface. Endogenous bFGF was up-regulated around the newly developed microvessels in control animals, and vessel growth correlated with the diffuse distribution of exogenous bFGF, implicating bFGF as an important factor in angiogenesis. Exogenous bFGF did not affect bFGF mRNA expression, suggesting that the regulation of bFGF is not under autocrine control. (J Vasc Surg 1998;28:919-28.)

Basic Fibroblast Growth Factor Stimulates Angiogenesis in the Hindlimb of Hyperglycemic Rats

Angiogenic growth factors including basic fibroblast growth factor (bFGF) have therapeutic value for chronic ischemia in nondiabetic animals. However, angiogenic therapy for chronic ischemia in a background of diabetes remains unexplored. In the present study, we evaluated the effects of exogenous bFGF on angiogenesis in streptozotocin-induced diabetic rats with ischemic and nonischemic limbs. We produced ischemia of the left lower limb by excising the superficial femoral artery. At 2 weeks, the rats received an intramuscular injection of vehicle (group A), 0.3 μg bFGF/day (group B), or 1 μg bFGF/day (group C), daily for 2 weeks. At 4 weeks, we assessed limb angiogenesis by skeletal muscle capillary density (cap/mm2) and capillary per muscle fiber ratio (cap/F) counts. Group C had significantly higher mean levels compared to group A for calf capillary density (P< 0.0024) and capillary per muscle fiber ratio in both thigh (P< 0.0015) and calf (P< 0.0001). There was a trend toward increased mean capillary per muscle fiber ratio with increasing dose. This trend was significant in the calf (P< 0.0015) and just missed statistical significance in the thigh. There was a similar trend in calf capillary density. We conclude that exogenous bFGF enhances angiogenesis and, possibly, collateral circulation in ischemic limbs of diabetic rats.

Expression of the basic fibroblast growth factor gene in mild and more severe head injury in the rat.

The goal of this study was to investigate the relationship between basic fibroblast growth factor (bFGF) gene expression and neuropathological changes in the hippocampus after varying degrees of brain injury. Mild and severe brain injury in rats was produced by using Marmarou's method. There were 25 animals in each brain injury group and 25 additional animals served as controls. Basic fibroblast growth factor gene expression was investigated by means of RNA hybridization, in situ hybridization, immunohistochemical analysis, and histological analysis using hematoxylin and eosin staining. A 3.7-kb bFGF messenger (m)RNA was detected in the rat hippocampus in both control and injured rats. In the mild injury group its expression was increased at 12 hours after injury and peaked on the 3rd day. Neuronal degeneration in the hippocampal CA2 and CA3 sectors was maximum on that day. In the severe injury group, the expression of the bFGF gene was the same as that in the mild injury group at corresponding times, but the number of surviving neurons in the CA2 and CA3 sectors was much lower than in the mild injury group. In situ hybridization showed that the main cells that expressed bFGF mRNA were pyramidal and granulocytic neurons in all three experimental groups. The number of neurons expressing bFGF mRNA in the severe injury group was less than that in the mild injury group, but the intensity of expression was greater. Immunohistochemical staining showed that the number of neurons expressing the bFGF protein was less in the severe injury group than in the mild injury group. It is concluded that after mild injury there is a close relationship between the expression of the bFGF gene and the degree of histological change in the hippocampus; this indicates that as one of the growth factors, bFGF may participate in the protection and repair processes of neurons following brain injury. In severe injury there is a reduced expression of bFGF. The reason for this appears to be that more of the cells that have the potential to express bFGF have died, reducing the ability to express the bFGF gene. Conversely, it is possible that there may be an intrinsic insufficiency of expression of the gene, compatible with the known vulnerability of the hippocampus to many pathological conditions. Consideration should be given to supplying exogenous bFGF to protect the brain, particularly the hippocampus, after injury.

Electron Microscopic Immunolocalization of Basic Fibroblast Growth Factor-Like Molecules in Capillary Endothelial Cells

Abstract Basic fibroblast growth factor (bFGF) is a potent angiogenic polypeptide. It promotes angiogenesis in vivo and in vitro by stimulating migration, proliferation and proteolytic activity of endothelial cells. Whereas several effects of exogenous bFGF on endothelial cells have been described, it has remained unclear how endogenous bFGF produced by vascular endothelial cells regulate angiogenesis. To further investigate functional implications of the distribution of bFGF, we undertook the present study. Our aims were (i) to identify the specific location of bFGF in endothelial cells using electron microscopy immunogold labeling technique (ii) to determine the distribution of bFGF in capillaries of different types of tissues. Tissue samples from sciatic nerve, hippocampus, adrenal gland and kidney of normal adult rats were fixed in 4% paraformaldehyde/1 to 5% glutaraldehyde and embedded in Spurr's resin. Ultrathin sections were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics) specific for bFGF using a two-step immunogold labeling method.

Exercise training enhances basic fibroblast growth factor-induced collateral blood flow.

This study evaluated whether daily exercise would enhance the peripheral collateral vessel development found in response to exogenous basic fibroblast growth factor (bFGF) infusion. After bilateral femoral occlusion, male Sprague-Dawley rats (approximately 325 g) received intra-arterial infusions of either bFGF (1 microg/day; n = 15) or carrier solution (n = 13) via osmotic pumps for 2 wk. Subgroups of each treatment were kept sedentary (cage activity) or trained by walking at 20 m/min at 15% grade, two times a day, 5 days/wk for 4 wk. Training markedly increased citrate synthase activity in the active muscle (P < 0.001). Muscle function and blood flows (85Sr microsphere) were evaluated using an isolated hindquarter perfused at 100 mmHg via the abdominal aorta. The significant increase in blood flow to the entire hindlimb in the sedentary animals, caused by bFGF infusion (P < 0.05), was further increased (P < 0.01) in the bFGF-trained group. The quantitatively largest increases in blood flows were observed in the collateral-dependent tissues of the distal hindlimb. Blood flows to the entire calf muscle group increased approximately 140% in carrier-trained (P < 0.001), approximately 180% in bFGF sedentary (P < 0.001), and approximately 240% in the bFGF-trained (P < 0.001) groups compared with the carrier sedentary group. The increases in collateral blood flow were functionally important, as improvements in calf muscle performance correlated with measured blood flows. Our results demonstrate that exogenous bFGF administration in combination with a moderate-intensity exercise program greatly increases collateral-dependent blood flow and improves muscle performance. That physical activity enriched the bFGF response is consistent with the hypothesis that hemodynamic factors are important contributors to collateral vessel enlargement.

Normal Human Melanocytes That Express a bFGF Transgene Still Require Exogenous bFGF for Growth In Vitro

The expression of basic fibroblast growth factor (bFGF) has been implicated as an important factor in the development of malignant melanoma. The timing of this expression suggests that bFGF plays a role early in melanoma tumor progression. Benign nevi produce bFGF, and cells cultured from these lesions show a loss of dependence on exogenous bFGF for growth. We have examined the effects of constitutive bFGF expression on the in vitro growth requirements of normal human melanocytes. bFGF was overexpressed in normal human epidermal melanocytes through genomic insertion of a human bFGF cDNA in a retroviral vector. These melanocytes produced the 18 kDa bFGF isoform as well as the higher molecular weight isoforms. The bFGF was not released into the culture medium, but it was present in the cell nucleus. The bFGF produced by these cells was mitogenic for 3T3 fibroblasts and therefore possessed functional activity; however, melanocytes producing bFGF had the same appearance and growth patterns as those infected with control virus or uninfected melanocytes. Expression of bFGF did not confer independence from the exogenous mitogen, nor would these cells form colonies in a soft-agar medium. These results indicate that expression of bFGF alone is not enough to cause aberrant growth of normal human melanocytes.

Sequence-specific antiproliferative effects of antisense and end-capping-modified antisense oligodeoxynucleotides targeted against the 5'-terminus of basic-fibroblast-growth-factor mRNA in coronary smooth muscle cells.

Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells, has been shown to play a fundamental role in the pathogenesis of arteriosclerosis and restenosis by stimulating the proliferation of vascular smooth muscle cells. We found that partially phosphorothioate-modified 15-residue antisense oligodeoxynucleotides complementary to bFGF mRNA at 0.1-2.0 microM block growth and division of cultured human and bovine coronary smooth muscle cells in a dose-dependent manner. The effect is sequence specific at low (0.1-0.5 microM) nontoxic concentrations. It is associated with inhibition of expression of pericellular and intracellular bFGF, with a decreased de novo synthesis of bFGF and is partly reversible by the addition of exogenous (recombinant) bFGF. The antisense effect lasts 48-72 h and diminishes thereafter. If the antisense oligodeoxynucleotide medium is replaced by an oligonucleotide-free medium after 24 h, the [3H]thymidine incorporation rate returns to control levels. Under the same conditions, the corresponding sense oligodeoxynucleotide exerts negligible nonspecific inhibitory actions. The antiproliferative potency of the 15-residue antisense oligodeoxynucleotide is markedly enhanced by adding 3-4 nonbase-pairing guanosine residues at the 5'- and 3'-termini of the 15-residue antisense oligonucleotide. The data implicate bFGF in the process of smooth muscle cell proliferation and an effective and specific antiproliferative potency of bFGF-specific antisense oligonucleotides. The results point to possible new therapeutic strategies for the use of antisense methodology in the suppression of post-angioplasty restenosis.

Enhanced angiogenesis and unfavorable remodeling in injured porcine coronary artery lesions: effects of local basic fibroblast growth factor delivery.

There is interest in the role of growth factors in the genesis of arterial remodeling. We studied local administration of basic fibroblast growth factor (bFGF) to coronary lesions to determine whether there is a difference in remodeling and whether neovascularization could be induced in such stenoses and distal myocardium. Pigs were randomized to balloon infusion of either saline or bFGF at each thermally injured arterial site. After the animals were killed, their internal elastic lamina, neointima, and lumen areas were measured. Capillaries were counted in the arteries and myocardium. There was a greater loss of lumen and internal elastic in the bFGF group. The neointima, media, and myocardium in the bFGF treated arteries had statistically more capillaries. This study showed that local intracoronary bFGF, at a dose that results in arterial luminal revascularization in injured segments, adversely affects arterial remodeling. Thus, the angiogenic response to exogenous bFGF may be offset by concomitant shrinkage of injured arterial segments.

Photodynamic therapy inactivates extracellular matrix-basic fibroblast growth factor: insights to its effect on the vascular wall.

Photodynamic therapy (PDT), the light activation of photosensitizer dyes for the production of oxygen and other free radical moieties without the generation of heat, has been shown to inhibit the development of experimentally induced intimal hyperplasia. The host response to PDT, a form of vascular injury that results in complete vascular wall cell eradication, is devoid of inflammation and proliferation and promotes favorable vascular wall healing. These effects do not result in intimal hyperplasia and are suggestive of PDT-induced changes in the extracellular matrix (ECM). As a model to better understand the biologic consequences of PDT on the vascular wall matrix proteins, the effect of PDT was studied on the powerful matrix-resident mitogen basic fibroblast factor (bFGF) in vitro. PDT (5 to 200 J/cm2, 100 mW/cm2, 675 nm) was used with the photosensitizer chloroaluminum sulfonated phthalocyanine (5 micrograms/ml) to inactivate bFGF in vitro while 100 J/cm2 of irradiation was administered 24 hours after 5 mg/ml of the photosensitizer was used in vivo. PDT was used on bFGF in solution and on endothelial cell-derived ECM. Enzyme-linked immunosorbent assay was used to quantitate bFGF in solution after PDT treatment or after extraction from the ECM by collagenase and heparin. Functional activity of matrix-associated bFGF was assessed by smooth muscle cell mitogenesis by 3H-thymidine incorporation. To demonstrate the in vivo relevance of these observations, immunohistochemical analysis of PDT-treated rat carotid arteries was undertaken. PDT eliminated detectable levels of bFGF in solution. PDT of ECM significantly reduced matrix-bound bFGF (1.0 +/- 0.6 vs 27.5 +/- 1.3 pg/ml; p < 0.0001). This reduction in bFGF after PDT of the ECM was associated with a decrease in vascular smooth muscle cell mitogenesis (52.4% +/- 4.6%; p < 0.0001) when plated on PDT-treated matrix compared with nontreated matrix. Quantitative replenishment of exogenous bFGF to PDT-treated matrix restored proliferation to baseline levels. PDT of rat carotid arteries demonstrated a loss of bFGF staining compared with control nontreated arteries. PDT inactivation of matrix-resident bFGF and possibly other bioactive molecules can provide a mechanism by which PDT suppresses smooth muscle cell proliferation in the vessel wall. This free radical-mediated alteration of matrix may contribute to favorable vascular healing when PDT is used for the inhibition of injury-induced intimal hyperplasia.

Inhibitory effects of hypercholesterolemia and ox-LDL on angiogenesis-like endothelial growth in rabbit aortic explants. Essential role of basic fibroblast growth factor.

Hypercholesterolemic (HC) rabbits exhibit suppressed compensatory vascular growth after restriction of arterial supply. However, neovascularization is commonly found in atheromas containing inflammatory cells. We used an in vitro model to determine the effects of hypercholesterolemia on angiogenesis in the absence or presence of inflammatory cells. HC rabbit aortic explants (1 mm2) with or without (n = 90 each) lesion-forming inflammatory cells were cultured in a collagen matrix with serum-free medium. Explant-derived endothelial cell growth was organized into capillary-like microtubes (CLM) that could be videomicroscopically quantified. CLM growth from lesion-free HC explants was significantly reduced to 13 +/- 4% of the value in explants (n = 90) from normocholesterolemic (NC, n = 15) rabbits (P < .001). In contrast, in lesion-containing HC explants, the matrix was invaded by foam cells, and CLM growth was not inhibited. Immunoassayable basic fibroblast growth factor (bFGF, in pg/mL) in the culture medium was significantly lower in lesion-free HC (< 5) than NC explants (11 +/- 2, P < .01) or HC explants with lesions (14 +/- 3). In addition, CLM growth was reduced in NC explants incubated with oxidized LDL (ox-LDL, 50-100 micrograms/mL). Exogenous bFGF (10 ng/mL) reversed the inhibitory effects of hypercholesterolemia and ox-LDL, whereas bFGF-neutralizing antibody (10 micrograms/mL) abolished CLM growth in all groups. In cultured rabbit aortic endothelial cells, ox-LDL reduced DNA synthesis, but this inhibition was reversed by bFGF. We conclude that hypercholesterolemia and ox-LDL inhibit angiogenesis like endothelial growth because of a suppressed availability of endogenous bFGF. Retained responsiveness to exogenous bFGF suggests that inducing bFGF expression at targeted sites may improve collateral growth in hyperlipidemic arterial disease.

Suppression of autocrine cell proliferation and tumorigenesis of human melanoma cells and fibroblast growth factor transformed fibroblasts by a kinase-deficient FGF receptor 1: evidence for the involvement of Src-family kinases.

Basic Fibroblast Growth Factor (bFGF/FGF2) is thought to play a decisive role in malignant progression. Aberrant expression of bFGF causes constitutive autocrine activation of its cognate receptor and autonomous growth of human melanoma cells or bFGF transformed fibroblasts in culture. It remains to be determined, however, whether the endogenous bFGF confers growth advantage to tumors and what are the downstream targets of the activated FGF receptor critical for its transforming capacity. We therefore transfected metastatic melanoma cells and bFGF transformed mouse fibroblasts with a dominant-negative mutant of the murine FGF receptor 1 (fgfr1/flg), comprising the extracellular and transmembrane domains but lacking the intracellular kinase domain (dnflg). Reverse transcriptase-PCR, 125I-bFGF binding and affinity labeling analyses show that the truncated receptor is targeted to the membrane and is expressed at much higher levels than the endogenous receptor in all of the selected clones. Expression of the dnflg dramatically reduces the basal as well as bFGF induced growth of these cells in vitro and also suppresses their tumorigenic potential in nude mice. The expression of the dnflg does not significantly alter the general level of tyrosyl-phosphorylated proteins in the trunsduced melanoma cells. Rather, a major downstream affected target is a Src-family kinase, whose activity, determined by an in vitro immune kinase assay, is stimulated in normal melanocytes by exogenous bFGF, and is markedly reduced in the dnflg-expressing melanoma cells. The present study demonstrates that direct interference with the activity of FGF receptors has a deleterious effect on cell proliferation and survival in vitro and in vivo leading to the suppression of melanoma tumor progression possibly through the inactivation of a Src-family kinase.

Neurotrophic Factors, Cytokines and Stress Increase Expression of Basic Fibroblast Growth Factor in Retinal Pigmented Epithelial Cells

Basic fibroblast growth factor (bFGF) and FGF receptors have been localized to photoreceptors and retinal pigmented epithelium (RPE), but the function of bFGF in adult retina and RPE is unknown. Exogenous bFGF has a neuroprotective effect in retina and brain and its expression is increased in some neurons in response to cytokines or stress. In this study, we investigated the effect of light, other types of stress, neurotrophic factors, and cytokines on bFGF levels in cultured human RPE.Some agents that protect photoreceptors from the damaging effects of constant light, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, and interleukin-1β, increase bFGF mRNA levels in RPE cells. Intense light and exposure to oxidizing agents also increase bFGF mRNA levels in RPE cells and cycloheximide blocks the increase. An increase in bFGF protein levels was demonstrated by ELISA in RPE cell supernatants after incubation with BDNF or exposure to intense light or oxidizing agents. These data indicate that bFGF is modulated in RPE cells by stress and by agents that provide protection from stress and support the hypothesis that bFGF functions as a survival factor in the outer retina.

Boundary Layer Infusion of Basic Fibroblast Growth Factor Accelerates Intimal Hyperplasia In Endarterectomized Canine Artery

We examined the effects of human recombinant basic fibroblast growth factor (bFGF) on the proliferation and migration of cultured dog smooth muscle cells (SMCs) and endothelial cells (ECs) and the effect of continuous local boundary layer infusion of bFGF on intimal hyperplasia in endarterectomized dog artery.In vitroproliferation and migration of dog SMCs or ECs were performed using direct counting and Boyden's chamber, respectively. At a dose of 10 ng/mL, bFGF significantly promoted both SMC and EC proliferation (7- and 4-fold, respectively) and migration (2.3- and 1.9-fold, respectively). Six dogs underwent bilateral carotid endarterectomies. A newly designed local infusion device with an osmotic pump continuously delivered bFGF to one artery or vehicle solution to the contralateral artery for 14 days. The intimal thickness and area in the bFGF-treated vessels were increased by 72 and 81%, respectively, compared with control arteries (P< 0.05). As assessed by the bromodeoxyuridine index, the proliferative activity was increased by 73% in bFGF-treated arteries (P= 0.03). Furthermore, cell proliferation at the distal anastomoses of local infusion device was significantly increased in the bFGF-infused grafts compared with distal anastomoses in the control grafts (13.24 ± 1.24% versus 5.24 ± 1.01%,P< 0.01). These data demonstrate that human recombinant bFGF has a potent effect on dog SMC and EC proliferation and migration, and that local infusion of exogenous bFGF significantly enhances the intimal hyperplasia formation and cell proliferation to vascular injury. We conclude that the bFGF pathway may contribute to the development of intimal hyperplastic lesions.

Induction of Tubulogenesis of Microvascular Endothelial Cells by Basic Fibroblast Growth Factor from Human SN12C Renal Cancer Cells

To investigate the role of basic fibroblast growth factor (b-FGF) in angiogenesis in renal cell carcinoma. We examined b-FGF expression in 25 patients with renal cell carcinoma (RCC) by reverse transcriptase polymerase chain reaction (RT-PCR). We measured the tube formation by human omental microvascular endothelial (HOME) cells induced by b-FGF in a three-dimensional collagen gel in the absence or presence of SN12C human renal cancer cells by means of computer-image analysis. RT-PCR analysis detected b-FGF m-RNA in 20 (80%) of 25 patients with RCC. The tube formation of HOME cells was increased in the presence of SN12C cells overexpressing b-FGF mRNA, induced by exogenous b-FGF in a dose dependent manner and completely inhibited by anti-b-FGF antibody. Basic-FGF may develop tumor angiogenesis by inducing microvessel tubulogenesis in patients with RCC.

Basic Fibroblast Growth Factor Treatment Delays Age-related Photoreceptor Degeneration in Fischer 344 Rats

This study was undertaken to investigate the potential of basic fibroblast growth factor (bFGF) to delay photoreceptor cell loss in retinas of Fischer 344 rats which exhibit an age-related peripheral retinopathy. Eight male 16-month-old Fischer 344 rats were injected intravitreally with 2.0 μg bFGF in the right eye, while the vehicle was injected into the left eye and all rats were killed at two months post-injection. Eight other Fischer rats were injected with either bFGF or vehicle at 16 and again at 18 months, then killed two months later. After enucleation, eyes were processed for light and electron microscopic and morphometric analyses. The distance from the ora serrata to the point where the outer nuclear layer (ONL) in the superior retina was two cells in thickness, called the die-back zone, that represents the extent of the peripheral retinopathy, was compared in bFGF- and vehicle-injected Fischer rats. These measurements revealed that the die-back zone in retinas of eyes injected twice with bFGF was significantly reduced (P<0.01) when compared to this zone in retinas of age-matched rats that received vehicle injections. However, there was no significant difference (P>0.05) in this degeneration zone in retinas of 18-month-old rats that received a single-bFGF injection when compared to retinas of respective vehicle-injected rats. In addition, the ONL at 2 and 3 mm from the ora serrata in the superior retina of bFGF-injected 20-month-old Fischer rats was significantly thicker than corresponding regions in retinas of vehicle-control rats. Furthermore, the photoreceptor cells in the superior retina of 20-month-old rats which received two injections of bFGF had normal-appearing inner and outer segments, while the few photoreceptors had short inner and outer segments in vehicle-injected retinas of Fisher rats. These findings reveal that injections of bFGF into eyes of Fischer 344 rats significantly delays the progress of photoreceptor cell degeneration suggesting that exogenous bFGF may act as a survival-promoting factor in these aged retinas.

Low Level Expression of Basic FGF Upregulates Bcl-2 and Delays Apoptosis, But High Intracellular Levels are Required to Induce Transformation in NIH 3T3 Cells

We investigated the roles of basic fibroblast growth factor (bFGF) in the transformation and survival of NIH 3T3 cells. We constructed NIH 3T3-derived cell lines expressing human bFGF using retroviral gene transfer with an N2-based vector. Clonally derived cell lines containing a single copy of the vector overexpress bFGF mRNA and produce more immunoreactive protein (0.407 ± 0.010-3.028 ± 0.087 ng bFGF/106 cells) which is biologically active than nontransduced (0.151 ± 0.013 ng bFGF/106 cells) or N2-transduced (0.211 ± 0.029 ng bFGF/106 cells) NIH 3T3 cells. All cells producing excess amounts of bFGF achieve greater density at confluence, show delayed apoptosis and increased survival and have elevated intracellular levels of Bcl-2. However, only cells expressing from 8-15 times background levels of bFGF are phenotypically transformed. The transformed cells form dense foci at confluence, have decreased adherence to tissue culture plates and grow colonies in soft agar. Exogenous bFGF induces higher Bcl-2 levels in a dose dependent manner and recapitulates the antiapoptotic effects of the overexpressed species but fails to induce changes associated with the transformed phenotype. In this study, we demonstrate a dissociation between phenotypic transformation secondary to bFGF overexpression and upregulation of cellular Bcl-2 that correlates with a delay in programmed cell death. Although low level expression of bFGF or exogenous bFGF is sufficient to upregulate Bcl-2 and delay apoptosis, high intracellular levels are required for cellular transformation. These data suggest that overexpression of bFGF modulates cellular transformation and Bcl-2-mediated inhibition of apoptosis through alternate molecular mechanisms.