Basic Fibroblast Growth Factor Messenger Research Articles

Progesterone, but not β-estradiol, enhances Porphyromonas gingivalis lipopolysaccharide-induced vascular endothelial growth factor-A expression in human THP-1 monocytes

Background/purposeProgesterone and estrogen levels are elevated during pregnancy and play a role in maternal immune responses. In addition, unbalanced metabolism of growth factors has been demonstrated in pregnancy tumors. Therefore, we aimed to investigate the effect of progesterone and β-estradiol on vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) expression in THP-1 monocytes in response to Porphyromonas gingivalis lipopolysaccharide (LPS). Materials and methodsTHP-1 monocytes were incubated with progesterone, β-estradiol, or LPS from Escherichia coli and P. gingivalis for up to 24 hours. The expression of VEGF-A and bFGF was investigated using conventional reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Cell growth was assessed using cell proliferation assay. ResultsWe reported herein that progesterone, but not β-estradiol, increased VEGF-A mRNA expression in THP-1 monocytes. Significantly, progesterone enhanced VEGF-A mRNA expression in P. gingivalis LPS-treated monocytes in comparison with a treatment with P. gingivalis LPS alone. However, neither β-estradiol nor progesterone had any effect on bFGF production at mRNA levels. ConclusionThe enhancing effect of progesterone on VEGF-A mRNA expression may have a role in the pathogenesis of pyogenic granuloma in pregnant women.

Evaluation of angiogenic activities of hyaluronan oligosaccharides of defined minimum size

Aims Oligosaccharides of hyaluronan (o-HAs) were proved to have pro-angiogenic activities. The aim of this study was to obtain four hyaluronan oligosaccharides (o-HAs) of defined molecular weight including 4 saccharide residues (o-HA4), 6 saccharide residues (o-HA6), 8 saccharide residues (o-HA8), and 10 saccharide residues (o-HA10) under optimum conditions, and compare their angiogenic activities. Main methods The four o-HAs were prepared by digesting the native high molecular weight HA with hyaluronidase under optimum conditions. The effects of the four o-HAs on human umbilical vein endothelial cell (HUVEC) proliferation were measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenil tetrazolium bromide (MTT) assay. Angiogenic effects of the four o-HAs were evaluated using chicken chorioallantoic membrane (CAM) assay. The effects of the four o-HAs on the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) messenger ribonucleic acid (mRNA) expressions were detected by semi-quantitative polymerase chain reaction (PCR). Key findings O-HA6, o-HA8 and o-HA10 could effectively promote the proliferation of HUVEC, induce angiogenesis in the CAM assay and increase VEGF mRNA levels in HUVEC. No significant effects were found with o-HA4. Significance These results suggested that o-HA6, o-HA8, and o-HA10, but not o-HA4 were effective angiogenic factors.

Adiponectin Deficiency Protects Mice From Chemically Induced Colonic Inflammation

Adiponectin (APN) is an adipokine that regulates insulin sensitivity and is anti-inflammatory in atherosclerosis. The goal of this study was to investigate the role of APN in intestinal inflammation. APN knockout (KO) mice and their wild-type (WT) littermates received dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce intestinal inflammation. Clinical and histologic scores and proliferation of epithelial cells were assessed. Cytokines and APN levels were measured. Expression of APN and heparin binding epidermal growth factor (HB-EGF) was analyzed by immunohistochemistry. Expression of APN and its receptors, HB-EGF, and basic fibroblast growth factor (bFGF) messenger RNA was assessed by reverse-transcription polymerase chain reaction. Association of serum APN with HB-EGF and bFGF was studied by coimmunoprecipitation. APN KO mice are protected from chemically induced colitis; administration of APN restores inflammation. APN is expressed in the colon, luminal APN associates with colonic epithelial cells. In vitro, APN increases production of proinflammatory cytokines from colonic tissue. Expression of colonic APN overlaps with that of bFGF and HB-EGF, which play a protective role in colitis. Circulating APN binds to bFGF and HB-EGF, likely inhibiting their protective activity. Inhibition of EGF receptor signaling, which is required for biologic activity of HB-EGF, restores inflammation in APN KO mice. APN deficiency is associated with protection from chemically induced colitis. APN exerts proinflammatory activities in the colon by inducing production of proinflammatory cytokines and inhibiting bioactivity of protective growth factors. Thus, in colitis, APN exerts an opposite role compared with atherosclerosis.

Vascular endothelial growth factor and basic fibroblast growth factor expression in esophageal adenocarcinoma and Barrett esophagus

Objective: This study was undertaken to investigate the role of the angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor in the development and progression of Barrett esophagus and adenocarcinomas of the esophagus and gastroesophageal junction. Methods: Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels, relative to the control gene encoding β-actin, were measured by using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector system) in specimens of Barrett intestinal metaplasia (n = 16), dysplasia (n = 11), adenocarcinoma (n = l 5), and matching normal squamous esophageal tissues (n = 35). Vascular endothelial growth factor and basic fibroblast growth factor protein expression and CD31+ microvessel density were assessed by means of immunohistochemistry in 25 tissue sections that included representative areas for each of these Barrett stages. Results: Expression levels were significantly increased in adenocarcinoma compared with in either normal squamous mucosa (P <.0001 for both genes) or intestinal metaplasia (vascular endothelial growth factor, P =.002; basic fibroblast growth factor, P <.0001). Vascular endothelial growth factor levels were also significantly higher in cancer tissues compared with dysplasia tissues (P =.024, Mann-Whitney U test). Basic fibroblast growth factor expression was also significantly increased in Barrett dysplastic mucosa compared with in intestinal metaplasia or normal esophageal mucosa. Microvessel density was generally higher in adenocarcinoma compared with in preneoplastic Barrett tissues. The pattern of vascular endothelial growth factor and basic fibroblast growth factor protein expression was similar to the messenger RNA expression pattern, with the exception that mucin-containing goblet cells stained intensely for vascular endothelial growth factor and only weak vascular endothelial growth factor staining was present in some adenocarcinomas. Conclusions: Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels are significantly upregulated in esophageal and gastroesophageal junction adenocarcinomas, suggesting a role for these angiogenic factors in the development of these cancers. Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels are also increased in some Barrett esophagus tissues, with this increase occurring at an earlier stage for basic fibroblast growth factor than for vascular endothelial growth factor. Basic fibroblast growth factor protein expression pattern is similar to the messenger RNA expression pattern, but unlike the messenger RNA findings, vascular endothelial growth factor protein expression is strongest in goblet cells.J Thorac Cardiovasc Surg 2003;125:246-53

Inhibition of human colon cancer cell growth by antisense oligodeoxynucleotides targeted at basic fibroblast growth factor.

Basic fibroblast growth factor has been shown to be mitogenic in colon cancer cell lines. In human malignant melanoma cells, antisense oligodeoxynucleotides targeted against basic fibroblast growth factor messenger RNA significantly inhibit cell growth. However, the efficacy of such an antisense oligodeoxynucleotide strategy has not been evaluated for colon cancer cells. To investigate whether basic fibroblast growth factor can stimulate the growth of HT-29 human colon cancer cells and whether antisense oligodeoxynucleotides can inhibit growth of these cells at baseline. Western blotting analyses were used to confirm the presence of basic fibroblast growth factor protein in this cell line. Cell growth was assessed after 2, 4 and 6 days of treatment by cell counting using the trypan blue exclusion method. Phosphorothioate-modified oligodeoxynucleotides (10 microM) were used, complementary to codon 60 of the basic fibroblast growth factor messenger RNA. Cationic liposomes (DOTAP) were used to enhance the cellular uptake of the oligodeoxynucleotides. Western blotting demonstrated the presence of basic fibroblast growth factor protein in this cell line. Basic fibroblast growth factor (1-40 ng/mL) dose-dependently stimulated cell growth and peak values were obtained at a dose of 20 ng/mL. By contrast, antisense oligodeoxynucleotide treatment significantly inhibited cell growth compared with the sense oligodeoxynucleotide-treated cells (P=0.007). This inhibition was reversed by the addition of basic fibroblast growth factor, 20 ng/mL. Treatment targeted against basic fibroblast growth factor messenger RNA inhibits growth of HT-29 human colon cancer cells. This finding may provide a rationale for the therapeutic use of antisense oligodeoxynucleotides targeted at basic fibroblast growth factor for the treatment of colon cancer.

Dexamethasone selectively suppresses microglial trophic responses to hippocampal deafferentation

Hippocampal deafferentation increases the expression of insulin-like growth factor-1 by microglia, and of ciliary neurotrophic factor and basic fibroblast growth factor by astroglia in fields and periods of reactive axonal growth. Glucocorticoids attenuate lesion-induced hippocampal sprouting, possibly by reducing trophic signals that stimulate growth. With an interest in this hypothesis, the present studies evaluated the influence of systemic treatment with the synthetic glucocorticoid dexamethasone on entorhinal lesion-induced increases in neurotrophic factor expression in young adult rat hippocampus. Daily dexamethasone injections almost completely blocked increases in insulin-like growth factor-1 messenger RNA content, but did not perturb increases in ciliary neurotrophic factor or basic fibroblast growth factor messenger RNA content, in the deafferented dentate gyrus molecular layer. To determine if the suppression of insulin-like growth factor-1 expression was secondary to a general inhibition of microglial responses, and to identify the time period of glucocorticoid sensitivity, additional rats were prepared to evaluate the effects of semi-chronic (i.e. daily) and single dexamethasone injections on microglial proliferation, ED-1 immunoreactivity (a marker of microglial reactivity) and insulin-like growth factor-1 messenger RNA expression. Semi-chronic dexamethasone treatment attenuated all three measures of deafferentation-induced microglial reactivity. However, a single dexamethasone injection given two (but not one or three) days postlesion inhibited deafferentation-induced increases in insulin-like growth factor-1 messenger RNA content, without having significant effects on other measures. These results demonstrate that dexamethasone treatment preferentially suppresses microglial, as opposed to astroglial, trophic responses to deafferentation, and suggest that glucocorticoids attenuate reactive axonal sprouting by inhibiting the microglial production of insulin-like growth factor-1.

Spatial learning and physical activity contribute to the induction of fibroblast growth factor: neural substrates for increased cognition associated with exercise

New evidence indicates that neural activity regulates the expression of trophic factors in the brain but regulation of these molecules by select aspects of behaviour remains solely a fascinating possibility. We report that following training in the Morris water maze, a spatial memory task, the hippocampus and cerebellum of learning rats exhibited an increase in basic fibroblast growth factor messenger RNA. Basic fibroblast growth factor messenger RNA levels were higher during the learning of the task and decreased once asymptotic performance was reached, suggesting an involvement of basic fibroblast growth factor in learning/memory. An active control group, which exercised for the same time as the learning group but the spatial learning component of the task was minimized, exhibited a minor increase in basic fibroblast growth factor messenger RNA. The intensification of the physical activity component of the task by massed or intensive training resulted in greater increases in basic fibroblast growth factor messenger RNA for both learning and yoked groups, but levels of basic fibroblast growth factor messenger RNA in the learning group remained higher than yoked only in the cerebellum. Changes in basic fibroblast growth factor were accompanied by an increase in astrocyte density in the hippocampus in agreement with described roles of basic fibroblast growth factor in astrocyte proliferation/reactivity. Results suggest that learning potentiates the effects of physical activity on trophic factor induction in select brain regions. Trophic factor involvement in behaviour may provide a molecular basis for the enhanced cognitive function associated with active lifestyles, and guide development of strategies to improve rehabilitation and successful ageing.

Angiotensin-converting enzyme inhibitor cilazapril suppresses expression of basic fibroblast growth factor messenger ribonucleic acid and protein in endothelial and intimal smooth muscle cells in a vascular injury model of spontaneous hypertensive rats.

The relationship between the expression of basic fibroblast growth factor (bFGF) messenger ribonucleic acid (mRNA) and protein, a potent mitogen for vascular smooth muscle cells in vivo, and administration of the angiotensin-converting enzyme inhibitor cilazapril, which suppresses smooth muscle cells proliferation in denuded arteries, was studied in spontaneously hypertensive rats using the in situ hybridization technique and immunohistochemical study. The effect of cilazapril on neointimal formation through modification of bFGF expression was evaluated using the increased tissue expression of the renin-angiotensin system in spontaneously hypertensive rats. Arterial injury was produced by using balloon catheter denudation in the left carotid artery of rats. The effects were evaluated 2 weeks later. bFGF mRNA and protein were observed only in the endothelial cells of sham-operated rats. bFGF mRNA and protein were observed in both endothelial cells and intimal smooth muscle cells in operated rats receiving only vehicle. Expression of bFGF mRNA and protein was suppressed in both endothelial cells and intimal smooth muscle cells of operated rats receiving cilazapril. These data suggest that cilazapril suppresses smooth muscle cell proliferation through modification of the expression of bFGF mRNA and bFGF protein in addition to other genes.

Acute intermittent nicotine treatment produces regional increases of basic fibroblast growth factor messenger RNA and protein in the tel- and diencephalon of the rat

Several findings show a neuroprotective effect of nicotine treatment in different experimental models, and a negative correlation has been observed between cigarette smoking and the incidence of Parkinson's disease. It seems possible that nicotine may in part exert its neuroprotective actions by favouring the synthesis of neurotrophic factors. The aim of this study was to determine whether the nicotine treatment could be associated with the induction of a neurotrophic factor in brain regions with nicotinic receptors. Thus, we analysed by in situ hybridization and RNAse protection assay the effects of (−)nicotine on basic fibroblast growth factor messenger RNA and by immunocytochemistry fibroblast growth factor-2 protein in the tel- and diencephalon of rats following single or acute intermittent (−)nicotine treatment. The present results showed that acute intermittent (−)nicotine treatment (four i.p. injections at intervals of 30 min), but not single injections, lead to a substantial and dose-related (0.1–2 mg/kg) up-regulation of fibroblast growth factor-2 messenger RNA levels in the cerebral cortex, in the hippocampus, in the striatum and ventral midbrain. This induction of fibroblast growth factor-2 expression peaked 4 h after the first injection and returned to normal levels within 24 h. The change of fibroblast growth factor-2 messenger RNA levels was associated with increased fibroblast growth factor-2 immunoreactivity mainly localized to nerve cells. The treatment was effective also when repeated in the same animals three or five days after the first injection. The pre-treatment with the non-competitive (−)nicotine receptor antagonist mecamylamine blocked the (−)nicotine effects on fibroblast growth factor-2 messenger RNA levels. In the above areas, no changes were observed in the fibroblast growth factor-1, 2 and 3 receptor messenger RNA levels nor in brain-derived neurotrophic factor messenger RNA levels. The present data indicate an ability of intermittent (−)nicotine to increase fibroblast growth factor-2 in many tel- and diencephalic areas. In view of the trophic function of fibroblast growth factor-2, the previously observed neuroprotective effects of (−)nicotine may at least in part involve an activation of the neuronal fibroblast growth factor-2 signalling, and open up new avenues for treatment of Parkinson's disease and Alzheimer's disease based on the existence of nicotinic receptor subtypes enhancing fibroblast growth factor-2 signalling in many regions of the tel- and diencephalon.

Basic fibroblast growth factor messenger ribonucleic acid levels in human placentas from normal and pathological pregnancies

This study was designed to determine whether basic fibroblast growth factor (bFGF) gene expression in human placenta varies as a function of gestational age and to evaluate whether bFGF synthesis might be altered in pathological pregnancies. Moreover, we also investigated whether human placental cells express the bFGF receptor gene. The presence of mRNA for bFGF and its receptor was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on total RNA derived from human placental cells at different times of culture. Levels of bFGF mRNA were determined by competitive RT-PCR in human placental tissues collected at the beginning and at the end of pregnancy. Competitive RT-PCR was also employed to evaluate bFGF synthesis in term placentas derived from pregnancies complicated by diabetes. mRNA for both bFGF and its receptor were demonstrated in human placenta starting as early as 8 weeks of pregnancy. Values of bFGF mRNA were significantly higher in first trimester compared with term placentas. Term placentas derived from pregnancies associated with type I diabetes expressed levels of bFGF mRNA higher than those present in normal term placentas. These data demonstrate that bFGF and its receptor are synthesized in human placental cells throughout gestation. Moreover, bFGF gene expression is developmentally regulated. Finally, bFGF might also be partially responsible for the placental alterations observed in pregnancies complicated by diabetes.

Ontogeny of epidermal growth factor (EGF), EGF receptor (EGFR) and basic fibroblast growth factor (bFGF) mRNA levels in pancreas, liver, kidney, and skeletal muscle of pig

Epidermal growth factor (EGF), EGF receptor (EGFR), and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) levels were examined by Northern blot analysis in four tissues (pancreas, liver, kidney, and skeletal muscle) of pig from fetal 90 d to postnatal 180 d of age. The present study shows for the first time that EGF mRNA increased with advancing age in the kidney and skeletal muscle of pig. A high level of EGF mRNA was observed in the kidney compared with the liver and skeletal muscle. In the pancreas, high levels of EGF mRNA were found in fetuses and newborns and were low in older pigs. Pancreatic EGFR mRNA level parallelled its EGF mRNA, whereas in the kidney and skeletal muscle, patterns of EGFR mRNA were reversed to their EGF mRNA levels. In the liver, EGFR mRNA was abundant but EGF mRNA was undetected. In the pancreas and skeletal muscle, the highest levels of bFGF mRNA were found in fetuses of 90 d of age and then decreased with advancing age. In the liver and kidney, there were no major changes in bFGF mRNA levels during the examined developmental periods. These results show that EGF, EGFR, and bFGF mRNA levels are developmentally and tissue specifically regulated in pig. In the pancreas, mRNA levels of EGF, EGFR and bFGF were high in fetal and neonatal life and low thereafter. In the kidney and skeletal muscle, EGF mRNA increased with advancing age. EGF may play a role in muscle growth and maintenance in growing pigs during the later stage of development.

Acidic and basic fibroblast growth factor messenger RNA and protein show increased expression in adult compared to developing normal and dystrophic rat retina.

To further elucidate the possible roles of fibroblast growth factors (FGFs) in retinal pathophysiology, messenger RNA levels of acidic and basic FGF (aFGF and bFGF, respectively) were measured throughout embryonic and postnatal development until adulthood in normal and dystrophic (Royal College of Surgeons, RCS) rat retinas using sensitive reverse transcription-coupled polymerase chain reaction (PCR) techniques. In normal rats, both aFGF and bFGF transcript levels remained steadily low throughout embryogenesis and up until 7 d of postnatal age. By 13 d bFGF mRNA had increased 30-fold, and by adulthood (4 mo) levels were 150 times greater than in newborn retina. Dystrophic RCS retinas followed the same basic pattern, except that bFGF expression levels were increased relative to normal rats: By 4 d postnatal RCS retinas contained three times more bFGF mRNA than normal, by 7 d they contained six times more, and by 10 d they contained eight times more. In contrast, aFGF mRNA levels rose only threefold between embryonic and adult stages, and did not show any differences between normal and RCS rats. In parallel, staining of lightly fixed frozen sections of young (< 20 d) normal rat retina with antibodies to bFGF revealed only faint labeling of neural cells, whereas adult retinal sections were labeled strongly, especially within the photoreceptor layer. Twenty-day RCS rat retina showed detectable bFGF-like immunoreactivity. Hence, these data indicate that major aFGF and bFGF expression occurs only late in retinal maturation, suggesting these factors act principally as survival factors, especially for photoreceptors. In addition, the increased expression in a degenerative mutant strain may indicate the early onset of general cellular stress.

Elevated basic fibroblast growth factor levels in stroke-prone spontaneously hypertensive rats.

Basic fibroblast growth factor is a biologically active polypeptide with mitogenic, angiogenic and neurotrophic properties. In the present study, the temporal and spatial expressions of basic fibroblast growth factor in stroke-prone spontaneously hypertensive rats were compared to two related strains of rat: spontaneously hypertensive rats and normotensive Wistar Kyoto rats. Higher levels of total RNA concentration were found in cerebral cortex of four-week-old stroke-prone rats compared to spontaneously hypertensive rats and Wistar Kyoto rats. Northern blot analysis showed no changes in levels of basic fibroblast growth factor messenger RNA with increasing age in cerebral cortex of Wistar Kyoto and spontaneously hypertensive rats. However, significant increases were found in 26- and 38-week-old stroke-prone rats compared to four-week-old stroke-prone rats. Although messenger RNA increases were also found in subcortical and cerebellar regions, a significant difference in levels of basic fibroblast growth factor messenger RNA was observed only in cerebral cortices among these three strains. This age-related increase in basic fibroblast growth factor messenger RNA correlated with the increase incidence of stroke in stroke-prone rats. Immunohistochemical study further revealed a dramatic increase in levels of basic fibroblast growth factor immunoreactivity in cerebral cortex of 30-week-old stroke-prone rats as compared to young stroke-prone rats, as well as age-matched Wistar Kyoto and spontaneously hypertensive rats. This increase in basic fibroblast growth factor immunoreactivity therefore appears very specific to aged stroke-prone rats. However, immunoreactivity decreased once severe tissue damages were observed in the cerebral cortex. Basic fibroblast growth factor-positive cells were diffusely expressed in cerebral cortex; double staining with glial fibrillary acidic protein showed the majority of these basic fibroblast growth factor-positive cells to be astrocytes. In summary, although young stroke-prone spontaneously hypertensive rats showed significantly higher RNA concentration, significant increases in levels of basic fibroblast growth factor, including both messenger RNA and protein expression, were observed in aged stroke-prone rats with a high incidence of stroke. These findings suggest the possibility that basic fibroblast growth factor may play a role in the developmental sequelae of cerebral lesions in stroke-prone spontaneously hypertensive rats.

Chronic continuous infusion of (−)nicotine reduces basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat

A negative correlation has been found between smoking and Parkinson's disease. There is evidence to suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells in vivo and in vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the vental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125 mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbrain. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions.

Basic fibroblast growth factor messenger ribonucleic acid levels in eutopic and ectopic human endometrial stromal cells as assessed by competitive polymerase chain reaction amplification

Previous studies have localized basic fibroblast growth factor (bFGF) and its mRNA in normal and neoplastic endometrium but the expression of bGFG mRNA in endometriosis cells is virtually unknown. Our purpose was to investigate the presence of bFGF and its receptor mRNAs in highly purified primary cultures of stromal cells isolated from six eutopic endometrial samples obtained from patients without evidence of endometriosis and five endometriosis cyst linings. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single major DNA bands of the expected sizes for bFGF and its receptor (354 and 661 bp, respectively) were detected in both endometrial and endometriosis samples. A competitive RT-PCR, that uses coprimer extension and amplification of a bovine RNA as an internal standard, was developed for semiquantitative estimation of bFGF gene expression. The target to standard mean ratios ± SEM in six normal endometrial samples and in five endometriosis cultures were 26.7 ± 10.7 and 9.2 ± 3.0, respectively. Furthermore, when data were analyzed according to the cyst diameter, levels of bFGF mRNA resulted statistically higher ( P < 0.05) in bigger cysts when compared to those detected in smaller ones (16 ± 2.7 and 4.7 ± 1.8, respectively). These results demonstrate that the genes coding for bFGF and its receptor are expressed in endometriosis cells, but levels of bFGF mRNA are generally lower than those detected in their eutopic counterpart. Moreover, they indicate that endometriosis cells derived from large cysts have increased bFGF mRNA levels. Thus, bFGF could be one of the factors responsible for a more or less active behavior of the endometnotic lesion.

Fasting increases the expression of basic fibroblast growth factor (FGF-2) messenger ribonucleic acid in rat hypothalamus.

Basic fibroblast growth factor (FGF-2) has been isolated from the brain, but the regulation of FGF-2 synthesis in the brain is not yet fully understood. Since exogenously administered FGF-2 has been reported to suppress food intake as well as the secretion of gastric acid and pepsin in rats, we examined the effect of fasting on FGF-2 mRNA levels in the hypothalamus and the cerebral cortex of male rats, using RNase protection assay. Fasting for 72 h resulted in an approximately 2-fold increase in FGF-2 mRNA level in the hypothalamus but did not affect FGF-2 mRNA level in the cerebral cortex significantly. These findings support the hypothesis that FGF-2 plays a significant role in regulation of hypothalamic function.

Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons

The present study investigates the temporal and spatial changes of the cellular expression of basic fibroblast growth factor messenger RNA and immunoreactivity after a 6-hydroxydopamine-induced lesion in the nigrostriatal dopamine system. In situ hybridization revealed a sustained (from 4 h to two weeks) and strong (300–400% of control, at the peak intervals) increase of basic fibroblast growth factor messenger RNA in the pars compacta of the substantia nigra and the ventral tegmental area ipsilateral to the lesion. A short-lasting increase of basic fibroblast growth factor messenger RNA was observed in the ipsilateral pars reticulata of the substantia nigra (from 4–24 h, 300% of control) and neostriatum (24 h, 180% of control) as well as in the ipsilateral and contralateral hippocampus and neocortex (by 4 h, 200% of control). Brightfield microscopy showed an increased number of putative glial cells expressing the basic fibroblast growth factor messenger RNA signal. Basic fibroblast growth factor immunohistochemistry revealed on control brains the protein in the nuclei of glial cells throughout the forebrain and the midbrain and in the nuclei of neurons of the layer II of the retrosplenial granular cortex, the CA2 region of the hippocampus and the fasciola cinereum as well as in the nuclei of ependymal cells. The injection of 6-hydroxydopamine increased basic fibroblast growth factor immunoreactivity in the nuclei of astrocytes only within the ipsilateral substantia nigra and ventral tegmental area. By 2 h after the drug injection, the density of glial basic fibroblast growth factor-immunoreactive profiles was increased in the pars compacta of the substantia nigra and the ventral tegmental area. The density, size and intensity of the astroglial basic fibroblast growth factor immunoreactive nuclei were increased in the entire substantia nigra and the ventral tegmental area at 72 h, and peaked one week after the 6-hydroxydopamine injection. The saline injection promoted a time-dependent increase in the density of the glial basic fibroblast growth factor immunoreactivity but only in the ipsilateral pars compacta of the substantia nigra. In conclusion, the dopamine cell degeneration may give rise to extracellular signals activating the surrounding astroglia, leading to a sustained increased synthesis of astroglial basic fibroblast growth factor, which may exert neuroprotective action and increase repair on the nigrostriatal dopamine system.

Increase of basic fibroblast growth factor (FGF) and FGF receptor messenger RNA during rat thyroid hyperplasia: temporal changes and cellular distribution

Goitre was induced in adult rats by acute (1 or 2 weeks) or chronic (4 or 10 weeks) administration of methimazole together with a low iodine diet. Involution of thyroid growth was then observed at 16 weeks, 4 weeks after withdrawal of goitrogens and reversion to a normal diet. Experimental animals quickly became hypothyroid compared with controls and exhibited thyroid hyperplasia (control (n = 10): total serum thyroxine (T4) 66 +/- 4 nmol/l, thyroid weight 5 +/- 1 mg/100 g body weight, means +/- S.D.; experimental (n = 10): T4 undetectable, thyroid weight 27 +/- 4 mg/100 g body weight after 2 weeks of treatment). Thyroid growth rate subsequently slowed between 2 and 10 weeks. Messenger RNA for basic fibroblast growth factor (basic FGF) and for the high-affinity FGF receptor, was compared in the thyroids and livers of control and goitrous rats by ribonuclease protection assay. Low levels of mRNA for basic FGF and its receptor were detectable in thyroids from control rats at all times, while none was detected in the livers from any animal. Basic FGF and receptor mRNAs increased, and were detected at greatest abundance in hyperplastic thyroids at 1 and 2 weeks respectively, during goitre formation, but subsequently declined in parallel with thyroid growth rate at 4 and 10 weeks. When quantified by radioimmunoassay, basic FGF extracted from thyroids was fivefold greater than in controls after 1 week of goitrogen treatment (control (n = 4): 24 +/- 9 pmol/micrograms DNA; goitre (n = 4): 100 +/- 16 pmol/micrograms DNA; P < 0.05). Basic FGF and FGF receptor mRNAs localized by in situ hybridization predominantly to the epithelial cell population within follicles. Localization by immunohistochemistry demonstrated that basic FGF was present in the thyroids of control rats, and was largely associated with the basement membrane of follicles. During thyroid hyperplasia, increased basic FGF immunoreactivity appeared over the cytoplasm of follicular epithelial cells and was lost from the extracellular matrix. Thyroid involution following removal of goitrogen/low iodine treatment was associated with a decrease in mRNA for basic FGF or its receptor, and a loss of immunoreactive basic FGF from the cytoplasm of follicular cells. These results suggest that autocrine expression of basic FGF and FGF receptor could contribute to thyroid hyperplasia in rats.

Short- and long-term induction of basic fibroblast growth factor gene expression in rat central nervous system following kainate injection

Both RNase protection assay and in situ hybridization were used to investigate the effect of intraperitoneal injection of kainate on the messenger RNA levels for basic fibroblast growth factor in the rat central nervous system. Limbic motor seizures were produced by kainate injection and this event was followed by a significant elevation of basic fibroblast growth factor gene expression in rat hippocampus and striatum 6 h after the convulsant injection. The increase in hippocampus was maximal at 24 h and it was delayed with respect to nerve growth factor induction, which peaked 3 h after kainate injection. Animals that suffered prolonged seizure activity also showed a significant elevation of basic fibroblast growth factor gene expression four and 14 days after kainate, when no changes in nerve growth factor gene expression were observed. We show that, within the hippocampus, the increase of basic fibroblast growth factor messenger RNA was localized in dentate gyrus and the CA1 layer 6 and 24 h after kainate injection. Long-term effects on its gene expression were measurable only in the CA1 hippocampal subfield, where major cell damage and astrocytosis have been reported to occur following kainate-induced seizure activity [Ben-Ari Y. et al. (1981) Neuroscience 7, 1361–1391; Lothman E. W. and Collins R. C. (1981) Brain Res. 218, 299–318; Schwob J. E. et al. (1980) Neuroscience 5, 991–1014]. Indeed, the animals which displayed elevated messenger RNA levels for basic fibroblast growth factor four and 14 days after kainate injection showed a marked induction of messenger RNA expression for the astroglial marker glial fibrillary acidic protein. These results indicate that the glutamate analogue kainate produces short- and long-term increases of basic fibroblast growth factor messenger RNA expression with a specific anatomical pattern. Therefore, the gene expression for this neurotrophic factor is probably regulated by neuronal activity at early points in time, whereas the induction observed at later time points is related to adaptive mechanisms taking place following kainate-induced neuronal degeneration.

Increase of basic fibroblast growth factor (bFGF, FGF-2) messenger RNA and protein following implantation of a microdialysis probe into rat hippocampus.

In vivo microdialysis is an established tool for sampling extracellular fluid compartments. However, microdialysis faces the problem that the implantation of the probe damages the microenvironment from which measurements are derived. In this study, we examined the expression of basic fibroblast growth factor mRNA and protein at the cellular level after implantation of a microdialysis probe into the dorsal hippocampus and found that 8 h after inserting the probe bFGF mRNA was markedly increased in a relatively large area centered around the probe, involving both the dorsal hippocampus and the overlying cerebral cortex, as revealed by radioactive in situ hybridization. Using nonradioactive in situ hybridization with digoxigenin-labelled riboprobes, combined with immunohistochemistry for glial fibrillary acidic protein we demonstrated that bFGF mRNA was exclusively increased in astrocytes at the probe insertion site. Using immunohistochemistry we also found that bFGF-like immunoreactivity was increased after implantation of the probe close to the lesion site, as shown by an increased number of bFGF immunoreactive nuclear glial profiles. These results provide evidence that the implantation of a microdialysis probe into the brain induces activation of bFGF gene expression in astrocytes associated with nuclear bFGF-like immunoreactivity. We conclude that lesion-induced effects have to be considered when evaluating microdialysis data, and that mechanical trauma to the brain will activate astroglial trophism, as seen from the increased density of astroglial profiles demonstrating bFGF mRNA and protein levels.