Recombinant Acidic Fibroblast Growth Factor Research Articles

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N.benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N.benthamiana. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N.benthamiana-derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli-derived recombinant human acidic fibroblast growth factor. Additionally, N.benthamiana-derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N.benthamiana-derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging.

Acidic fibroblast growth factor promotes the growth of skeletal muscle satellite cells: experiment with rabbits

To explore if human recombinant acidic fibroblast growth factor (rhaFGF) can promote the proliferation of skeletal muscle satellite cells (MSCs). MSCs were obtained from rabbits and cultured, and divided into 2 groups: rhaFGF group, treated with rhaFGF of the concentrations of 0, 5, 10, 20, 40, 60, and 80 microg/L, and rhaFGF + low molecular weight heparin (LMWH) group, treated with rhaFGF of different concentrations and LMWH of the terminal concentration of 10 mg/L. MTT method was used to observe the proliferation of the MSCs so as to determine the appropriate concentration to be used in the next experiment. Other MSCs were cultured and treated with the rhaFGF of the appropriate concentration and then LMWH of the terminal concentration of 10 mg/L was added to be co-cultured for 3 days. Flow cytometry was used to observe the cell cycle of the MSCs. rhaFGF of the concentrations 20 approximately 60 ng/ml promoted the proliferation of MSCs, and 40 ng/ml was selected as the best concentration to be used in the next experiment. Treated with the rhaFGF of the concentration of 40 ng/ml and LMWH for 3 days, the proportion of MSCs at the stage G(0)/G(1) was significantly lower and those at the stage S significantly higher in comparison with the control group (both P < 0.01), however, not significantly different from those of the rhaFGF group. rhaFGF promotes the proliferation of MSCs.

VEGF gene therapy for chronic critical limb ischemia

Therapeutic neovascularization is a new area in cardiovascular medicine that received a lot of attention during the past 4 years. Starting with Schumacher et al.'s trial (1998) using recombinant acidic fibroblast growth factor in a coronary setting of incompletely bypassed patients, and Baumgartner et al.'s gene therapy trial (1998) on a series of patients with critical limb ischemia unsuitable for conventional revascularization, there are presently several ongoing clinical trials. This review article focuses on the rationale of intramuscular gene therapy compared to recombinant protein administration, and gives an overview of the development of vascular endothelial growth factor (VEGF) gene therapy for peripheral arterial occlusive disease. An enormous effort has already been done; much is left to be done. The purpose must be to improve the life of patients.

Size exclusion HPLC method for the determination of acidic fibroblast growth factor in viscous formulations

A size exclusion HPLC method has been developed to determine the protein concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not only contain low levels of protein mass (50 μg ml−1), but also include buffer ions, polysaccharide polyanions to conformationally stabilize aFGF and 1% hydroxyethylcellulose to increase the solution's viscosity. A cesium chloride mobile phase is utilized during SEC-HPLC to dissociate aFGF from the pharmaceutical excipients and to minimize nonspecific interaction of the protein with the column matrix. The protein content of a viscous aFGF formulation is determined by comparison of aFGF peak areas to standards of known concentration. Fluorescence spectroscopy was utilized to directly demonstrate that the protein remains in its native conformation during sample preparation and analysis.

Method of analysis of recombinant acidic fibroblast growth factor by capillary electrophoresis.

Fibroblast growth factors are a series of well characterized proteins that have intriguing pharmacological properties. Acidic fibroblast growth factor (aFGF) recently appeared in the literature for its efficacy in spinal cord repair in rats. The protein has proven difficult to analyze by capillary electrophoresis, because it has a tendency to unfold, aggregate and precipitate, especially near and above physiological temperatures. By studying the turbidity of capillary electrophoresis running buffers and aFGF at 50 degrees C, conditions were found that stabilize the aFGF solution, thereby allowing the capillary electrophoretic separation of the protein from its recombinant production impurities. The buffer system employs 50 mM phosphate buffer at pH 2.5 with 0.25% hydroxypropylmethylcellulose (HPMC) additive. This system provided the best efficiency and selectivity of the systems studied and was developed for pharmaceutical purity analysis.

Osteopromotion for cranioplasty: An experimental study in rats using acidic fibroblast growth factor

Many growth factors influence the bone healing cascade. Furthermore, the occasional failure of bone repair may in part be due to perturbation in the activation of local growth factors. Local activation of fibroblast growth factors (FGFs) at the fracture site may serve to increase neovascularization, and induce early granulation formation that can affect bone healing. We have performed a rat parietal (6 x 3 mm) critical size defect (CSD). Human recombinant acidic fibroblast growth factor (hraFGF) imbided in agarose was topically administered at the craniectomy site. Control animals received agarose alone in the same manner. Three weeks after surgery, osteopromotion was histologically evaluated. hraFGF-treated animals show a continuous bridge of regenerated bone extending from one edge of the defect to the other. None of the parietal defects that had been treated with agarose contained new bone in the central portion. Our results suggest an important role of FGFs to promote large cranioplasty repair and support the use of these proteins as an alternative choice for bone grafts and bone substitutes.

5401832 Brain derived and recombinant acidic fibroblast growth factor

5401832 Brain derived and recombinant acidic fibroblast growth factor

Dopamine differentiation factors increase striatal dopaminergic function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice

We have previously shown that muscle-derived differentiation factors (MDF) and human recombinant acidic fibroblast growth factor (aFGF) have beneficial behavioral and neurochemical effects on the nigrostriatal dopaminergic neurons of 6-hydroxy-dopamine (6-OHDA)-lesioned rats (Jin and Iacovitti: Neurobiol Dis 2:1-12, 1995). In the present study, we determined the effects of similar treatments on mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Five days after unilateral striatal infusion of MDF or aFGF into MPTP-lesioned mice, striatal tyrosine hydroxylase (TH) activity and dihydroxyphenylacetic acid (DOPAC) levels were bilaterally increased (20-35%) compared to untreated (lesion only) or control (phosphate buffered saline + bovine serum albumin) mice. These increases, however, were not accompanied by change in dopamine (DA) levels, indicating an elevation of DA synthesis (TH/DA) and turnover (DOPAC/DA). The present findings that MDF and aFGF may have neurochemical effects in vivo on the lesioned nigrostriatal dopaminergic system suggest their potential pharmacological role in the treatment of Parkinson's disease.

Acidic FGF induces NGF and its mRNA in the injured neocortex of adult animals.

Recently we reported that human recombinant acidic fibroblast growth factor (aFGF) is capable of preventing degeneration of nucleus basalis magnocellularis neurons in vivo and inducing growth of astrocytes in vitro. In the present study, the effects of aFGF on the concentration of nerve growth factor (NGF) and its messenger RNA were investigated in the rat cerebral cortex following unilateral cortical infarction. Lesioned animals exhibited a significant increase of NGF in the remaining cortex ipsilateral to the lesion. After combining cortical lesion with intracerebroventricular application of aFGF (12 micrograms/day for 7 days), we observed an 8-fold increase in the NGF concentration and a marked increase in the level of steady state NGF mRNA relative to controls ipsilaterally, and a less pronounced aFGF effect in the contralateral cerebral cortex. These results support the hypothesis that the neurotrophic effects previously shown for aFGF and basic FGF (bFGF) in neurotrophin-sensitive neurons is mediated by inducing increased production of NGF within the injured central nervous system (CNS) of adult animals.

Dopamine differentiation factors produce partial motor recovery in 6-hydroxydopamine lesioned rats

Two-week infusion of muscle-derived differentiation factor (MDF), or human recombinant acidic fibroblast growth factor (aFGF) and/or its muscle-derived activating substance into the striatum of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats caused a significant and long lasting (40 days) reduction (48–100%) in amphetamine-induced rotational asymmetry. In parallel with behavioural recovery, striatal tyrosine hydroxylase (TH) activity, dopamine (DA) and dihydroxy-phenyl-acetic acid (DOPAC) levels recovered in a dose-dependent manner in all treated rats when compared to controls. The greatest increments were observed in rats infused with aFGF and its activator. Increases in biochemical indices were not reflected in trophic changes of the dopamine system; thus, the number of TH-immunoreactive neurones and their striatal innervation were unmodified by treatment with MDF. In contrast with the lesioned brain, infusion of these agents into the intact brain produced no change in nigrostriatal dopamine biochemistry. Our results suggest that dopamine differentiation factors may be important in regulating the production of dopamine in the injured brain and, therefore, may be useful in the treatment of DA imbalances associated with certain neurological disorders such as Parkinson's disease.

5401832 Brain derived and recombinant acidic fibroblast growth factor : Linemeyer David; Kelly Linda; Gimenez-Gallego Guillermo; Thomas Kenneth A Westfield, NJ, United States Assigned to Merck & Co Inc

5401832 Brain derived and recombinant acidic fibroblast growth factor : Linemeyer David; Kelly Linda; Gimenez-Gallego Guillermo; Thomas Kenneth A Westfield, NJ, United States Assigned to Merck & Co Inc

Reversed-phase high-performance liquid chromatography assay for recombinant acidic fibroblast growth factor in E. coli cell suspensions and lysate samples

A reversed-phase HPLC assay for acidic Fibroblast Growth Factor (aFGF) expressed in E. coli is described. The assay was developed on a Polymer Labs. PLRP-S macroporous poly(styrene-benzene) column. A sample preparation procedure was developed to permit quantitation of aFGF in crude samples, such as cell suspensions and cell lysates. The assay was linear over the range of 25–140 μg/ml with an accuracy of 5%. By using a POROS column containing a stationary phase with “through-pores” and a superficial mobile phase velocity of 1440 cm/h, the analysis could be performed in 3 min. The polymeric supports used for this assay were durable; periodic washing with sodium hydroxide maintained column performance for extended periods.

Partially structured self-associating states of acidic fibroblast growth factor

A combination of near- and far-UV circular dichroism, Fourier-transform infrared spectroscopy, tryptophan fluorescence, size-exclusion chromatography, and a fluorescent extrinsic hydrophobic probe has been employed to characterize partially structured states of human recombinant acidic fibroblast growth factor (aFGF). At low pH, the addition of specific polyanionic ligands or moderate amounts of salts induces states with high secondary but low tertiary structure content. At neutral pH, intermediate amounts of chaotropic agents impose similar partially structured conformational states which also display noncooperative unfolding transitions. Kinetic evidence indicates that similar forms of the protein exist in the first few hundred milliseconds in the refolding pathway of aFGF. The kinetics of their formation appear to be temperature-independent, implying lack of an energy barrier, which is characteristic for further slow folding into the native state. Unlike the native and fully unfolded states, these partially structured conformations exhibit very low solubility, resulting in irreversible aggregation. Potential physiological implications of the existence of such "molten globule" states with regard to the growth factor's transport and biological activity are considered.

Effect of in vivo administration of recombinant acidic fibroblast growth factor on thyroid function in the rat: induction of colloid goiter.

We have recently demonstrated that the iv administration of 0.6-60 micrograms/kg.day of acidic fibroblast growth factor (acidic FGF) increases thyroid weight in male and female rats. Interestingly, measurement of serum TSH and thyroid hormones in rats treated with 6 micrograms/kg.day acidic FGF for 30 days revealed only a slight increase in serum T4 and reverse T3 concentrations. Since thyroid function was only examined 24 h after the 30th daily treatment, we performed a series of experiments to evaluate the effects of acidic FGF on thyroid function following single and 6 multiple injections of acidic FGF. There was a small increase in the serum TSH concentrations at 2, 4, 8, and 24 h after a single high dose iv injection of acidic FGF (60 micrograms/kg). In contrast, serum T3 concentrations were slightly decreased at 2, 4, and 8 h after acidic FGF administration. There was no effect of a single injection of acidic FGF on serum T4, reverse T3, or thyroglobulin concentrations. After 6 days of treatment, there was a 34% increase in the thyroid weights of rats treated with acidic FGF. Analysis of serum hormones revealed a slight increase in serum TSH, T3, and T4 concentrations in acidic FGF-treated rats, but no change in serum reverse T3 or thyroglobulin concentrations. There was no effect of acidic FGF administration on thyroid radioiodine uptake, the intrathyroidal metabolism of radioiodine, or the relative amounts of thyroidal thyroglobulin or peroxidase messenger RNAs, or on liver 5'-deiodinase activity. In hypophysectomized rats, with no detectable levels of serum TSH, acidic FGF failed to increase thyroid weight. These data suggest that FGFs may participate with TSH in the regulation of thyroid weight and colloid accumulation, and that autocrine or paracrine growth factors may be involved in the pathogenesis of colloid goiter.

Effect of in vivo administration of recombinant acidic fibroblast growth factor on thyroid function in the rat: induction of colloid goiter

Effect of in vivo administration of recombinant acidic fibroblast growth factor on thyroid function in the rat: induction of colloid goiter

Induction of 5‐Deiodinase Activity in Astroglial Cells by 12‐O‐Tetradecanoylphorbol 13‐Acetate and Fibroblast Growth Factors

In the brain, 5'-deiodinase (5'-D) is responsible for the metabolic activation of thyroxine (T4) into 3,5,3'-triiodothyronine (T3) and 5-deiodinase (5-D) deiodinates T4 and T3 into inactive metabolites. This study examines the effects of factors known to induce astroglial 5'-D activity on the 5-D activity in cultured rat astroglial cells. The potencies of these factors were compared after 8 h of incubation, when stimulations by these factors near their maximal effects. 12-O-Tetradecanoylphorbol 13-acetate (TPA) at 10(-7) M was a potent inducer of 5-D activity, producing a 30- to 80-fold increase after 8 h. The maximal effect of TPA was observed after about 14 h. The TPA stimulation of 5-D activity was not dependent on glucocorticoids, unlike 5'-D activity. In comparison with TPA, 8-bromo-cyclic AMP (10(-3) M) was a poor inducer of 5-D activity whereas it is an excellent inducer of 5'-D activity. It produced a 2- to 20-fold increase in 5-D activity after 8 h. Natural acidic fibroblast growth factor (20 ng/ml) produced a degree of stimulation similar to that of TPA after 8 h. The maximal effect of acidic fibroblast growth factor was observed after about 16 h (until a 120-fold increase). Recombinant acidic fibroblast growth factor also induced 5-D activity. Basic fibroblast growth factor was less potent than acidic fibroblast growth factor for increasing 5-D activity (maximal increase by 40- to 50-fold after 8 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Human Acidic Fibroblast Growth Factor Has Trophic Effects on Cultured Neurons from Multiple Regions of Brain and Retina

Neurotrophic activities of human recombinant acidic fibroblast growth factor (haFGF) were evaluated on primary cultured neurons from various brain regions and compared with those of CS23, modified human basic fibroblast growth factor. Survival of cultured neurons from embryonic day 16 (E16) rat cortex and substantia nigra was significantly increased by the addition of more than 10 ng/ml haFGF and that from the hippocampus was increased by 100 and 1000 ng/ml. However, enhancement of viability by haFGF was observed only in 1000 ng/ml-treated neurons from the striatum, thalamus, colliculus and cerebellum; and it was not observed in septal neurons. Survival of cultured neurons from postnatal day 15 rat on glial feeder layer was significantly increased by the addition of 1000ng/ml haFGF, except for neurons from the septum. CS23 (lOng/ml) increased the survival of cultured neurons from all regions mentioned above, and its effects were stronger than those of 1000 ng/ml haFGF. Addition of more than 10 ng/ml haFGF significantly increased the survival of cultured neurons from neonatal day 2 rat retina. Addition of 1000 ng/ml haFGF increased the choline acetyltransferase activity of E16 septal neurons slightly but significantly, but didn’t increase the dopamine uptake activity of embryonic day E15 ventral midbrain. These results show that haFGF is effective on limited regions and ages of brain and retina, while CS23 is effective on all regions tested.

Human Acidic Fibroblast Growth Factor Has Trophic Effects on Cultured Neurons from Multiple Regions of Brain and Retina.

Neurotrophic activities of human recombinant acidic fibroblast growth factor (haFGF) were evaluated on primary cultured neurons from various brain regions and compared with those of CS23, modified human basic fibroblast growth factor. Survival of cultured neurons from embryonic day 16 (E16) rat cortex and substantia nigra was significantly increased by the addition of more than 10 ng/ml haFGF and that from the hippocampus was increased by 100 and 1000 ng/ml. However, enhancement of viability by haFGF was observed only in 1000 ng/ml-treated neurons from the striatum, thalamus, colliculus and cerebellum; and it was not observed in septal neurons. Survival of cultured neurons from postnatal day 15 rat on glial feeder layer was significantly increased by the addition of 1000 ng/ml haFGF, except for neurons from the septum. CS23 (10 ng/ml) increased the survival of cultured neurons from all regions mentioned above, and its effects were stronger than those of 1000 ng/ml haFGF. Addition of more than 10 ng/ml haFGF significantly increased the survival of cultured neurons from neonatal day 2 rat retina. Addition of 1000 ng/ml haFGF increased the choline acetyltransferase activity of E16 septal neurons slightly but significantly, but didn't increase the dopamine uptake activity of embryonic day E15 ventral midbrain. These results show that haFGF is effective on limited regions and ages of brain and retina, while CS23 is effective on all regions tested.

High-level production of human acidic fibroblast growth factor in [formula omitted] cells: Inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor

Recombinant human acidic fibroblast growth factor has been produced in E. coli cells at a level of at least 50 mg/l culture. The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations. The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.

Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo.

We assessed the ability of acidic and basic fibroblast growth factor (FGF) to stimulate tyrosine kinase activity in intact cells. Immunoblot with polyclonal antiphosphotyrosine antibodies detected a 90-kDa phosphotyrosine-bearing protein in lysates of Swiss 3T3 cells exposed to pituitary-derived FGF, recombinant acidic FGF, or recombinant basic FGF, but not from unstimulated cells or cells exposed to epidermal growth factor or platelet-derived growth factor. Phosphotyrosine and its analogue phenyl phosphate, but not phosphoserine, phosphothreonine, or tyrosine itself, blocked recognition of the 90-kDa protein by antiphosphotyrosine antiserum. A monoclonal antiphosphotyrosine antibody also recognized the 90-kDa protein and was used to partially purify the protein by immunoaffinity chromatography. Phosphoamino acid analysis of the 90 kDa band revealed that it contained 20% phosphotyrosine, 35% phosphothreonine, and 45% phosphoserine. Tyrosine phosphorylation of the 90-kDa protein was detectable within 30 s and reached a plateau within 10 min of FGF addition. The addition of suramin, which blocks the interaction of FGF with its receptor, caused rapid disappearance of the 90 kDa band. Cell fractionation experiments were consistent with the 90-kDa protein being membrane-associated, but cross-linking studies revealed that the FGF receptor had an Mr between 145 and 210 kDa in Swiss 3T3 cells, distinct from the 90-kDa major substrate for tyrosine phosphorylation. These data demonstrate that both acidic and basic FGF activate a tyrosine kinase in vivo leading to phosphorylation of a unique 90-kDa substrate, and they suggest that protein modification by phosphorylation at tyrosine is involved in eliciting the mitogenic effect of FGF.