FGF-2 Immunoreactivity Research Articles

The distribution and immunolocalization of fibroblast growth factors (FGFs) in the rat oviduct during early pregnancy and the post-partum period.

The mammalian oviduct provides a favourable environment for several reproductive processes, including ovum transport, sperm capacitation, fertilization and pre-implantation embryonic development. This environment is regulated by cyclic ovarian steroids, that is oestrogen, and growth factors. Fibroblast growth factors (FGFs) regulate the differentiation and growth of various cell types in the female genital tract. This study aimed to determine the localization of FGF1, FGF2, FGF receptor 1 (FGFR1) and 2 (FGFR2) in the rat oviduct, by immunohistochemistry, on day 5 of pregnancy and post-partum days 1, 3 and 5, and to demonstrate the possible functions of these proteins during early pregnancy and the post-partum period. On all examination days, cytoplasmic and nuclear FGF1 immunoreactivity was detected in the epithelium lining the infundibulum, ampulla and isthmus of the oviduct. Immunoreactivity was much stronger in the basal bodies of the cilia on the epithelium lining the infundibulum and ampulla. FGF1 immunoreactivity was also detected in stromal cells, myocytes and endothelial cells. Cytoplasmic FGF2 immunoreactivity was observed in the tunica muscularis, vascular myocytes and endothelial cells. While strong cytoplasmic FGF2 immunoreactions were observed in the stromal cells of the lamina propria, the luminal epithelium, some stromal cells and smooth muscle cells displayed a rather weak FGFR1 and FGFR2 immunoreactivity. Immunoreaction intensity did not differ between the periods examined. This study shows that FGF1, FGF2, FGFR1 and FGFR2 are produced by rat oviduct cells during pregnancy and the post-partum period, and reproductive physiology is regulated not only by hormonal mechanisms, but also by growth factors.

The immunoexpression patterns of fibroblast growth factors in the pregnant and postpartum rat ovary.

Fibroblast growth factors (FGFs) are polypeptides involved in the regulation of oogenesis and folliculogenesis by inducing ovarian mitogenic, homeostatic and angiogenic activity. This study was aimed at determining the localisation of FGF ligands (FGF1 and FGF2) and FGF receptor 2 (FGFR2) in the rat ovary by immunohistochemical analyses, at pregnancy and the postpartum period. During pregnancy and the postpartum period, positive FGF1 immunoreactions were observed in the nucleus and cytoplasm of germinative epithelial cells, granulosa cells of follicles in different developmental stages, theca interna cells, interstitial cells, luteal cells and atretic follicles. FGF2 immunoreactivity was strong in the cytoplasm of the endothelial cells and smooth muscle cells of the ovarian blood vessels and in the smooth muscle cells of the ovarian cortex and medulla. Strong FGFR2 immunoreactivity was observed in the stromal cells surrounding the blood vessels and rete ovarii. Immunoreaction intensity of the FGF1, FGF2 and FGFR2 had relatively similar abundances between the periods examined. Considering that FGFs act as local regulators in oogenesis, folliculogenesis, follicular atresia, ovulation, corpus luteum formation and regression and angiogenesis, this study supports the idea that FGFs may also be involved in these physiological functions in rat ovaries during pregnancy and postpartum period.

Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation.

Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1−/− embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1−/− cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1−/− cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1−/− cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1−/− cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1−/− cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway.

FGF-2 in Astroglial Cells During Vertebrate Spinal Cord Recovery

Fibroblast growth factor-2 is a pleiotrophic cytokine with neurotrophic and gliogenic properties. It is known to regulate CNS injury responses, which include transformation of reactive astrocytes, neurogenesis, and promotion of neurotrophic activities. In the brain, it is localized in astrocytes and discrete neuronal populations. Following both central and peripheral nervous system injury, astrocytes become reactive. These activated cells undergo hypertrophy. A key indicator of astrocyte activation is the increased accumulation of intermediate filaments composed of glial fibrillary acidic protein (GFAP). Following physical insult of brain or spinal cord, reactive astrocytes show increased FGF-2 immunoreactivity. Thus, FGF-2 appears to participate in astrocytic differentiation and proliferation and a good candidate for astrocytic function regulation in healthy, injured, or diseased CNS. To further investigate the cellular mechanisms underlying FGF-2 restorative actions and to analyze the changes within astroglial cells, we studied the localization of GFAP and FGF-2 in adult intact and injured Pleurodeles CNS. Our results show that spinal cord injury triggers a significant increase in FGF-2 immunoreactivity in reactive astrocytes at sites of insult. In addition, these results were time-dependent. Increase in FGF-2 immunoreactivity along the CNS axis, starting 1-week post-injury, was long-lasting extending to 6 weeks. This increase was accompanied by an increase in GFAP immunoreactivity in the same spatial pattern except in SC3 where its level was almost similar to sham-operated animals. Therefore, we suggest that FGF-2 may be involved in cell proliferation and/or astroglial cells differentiation after body spinal cord transection, and could thus play an important role in locomotion recovery.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments. In many cases, suitable treatment is problematic as the therapeutic target remains unknown. Basic fibroblast growth factor (bFGF, FGF-2) is involved in neuronal maintenance and wound repair following nervous system lesions. It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances. Peripheral cranial somatic motor neurons, i.e. hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF-2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system. FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life. Moreover, the modulatory effects of astroglial FGF-2 and the Ca+2-binding protein S100β have been postulated in paracrine mechanisms after neuronal lesions. In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72h or 11 days. Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, glial fibrillary acidic protein (GFAP, as a marker of astrocytes), S100β and FGF-2. The number of Nissl-positive neurons of axotomized XII nucleus did not differ from controls. The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection. An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72h and 11 days after the surgery. The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus; however, the nerve transection increased the number of FGF-2 glial profiles by 72h and 11 days. Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF-2 immunoreactivity in axotomized XII neurons by 72h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of glial nuclei by 72h and 11 days after the two lesions. S100β decreased in astrocytes of 11-day-transected XII nucleus. The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus. Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology.

S100ß and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury

The neurotrophic factor fibroblast growth factor-2 (FGF-2, bFGF) and Ca++ binding protein S100beta are expressed by the Schwann cells of the peripheral nerves and by the satellite cells of the dorsal root ganglia (DRG). Recent studies have pointed out the importance of the molecules in the paracrine mechanisms related to neuronal maintenance and plasticity of lesioned motor and sensory peripheral neurons. Moreover, cultured Schwann cells have been employed experimentally in the treatment of central nervous system lesions, in special the spinal cord injury, a procedure that triggers an enhanced sensorymotor function. Those cells have been proposed to repair long gap nerve injury. Here we used double labeling immunohistochemistry and Western blot to better characterize in vitro and in vivo the presence of the proteins in the Schwann cells and in the satellite cells of the DRG as well as their regulation in those cells after a crush of the rat sciatic nerve. FGF-2 and S100beta are present in the Schwann cells of the sciatic nerve and in the satellite cells of the DRG. S100beta positive satellite cells showed increased size of the axotomized DRG and possessed elevated amount of FGF-2 immunoreactivity. Reactive satellite cells with increased FGF-2 labeling formed a ring-like structure surrounding DRG neuronal cell bodies.Reactive S100beta positive Schwann cells of proximal stump of axotomized sciatic nerve also expressed higher amounts of FGF-2. Reactive peripheral glial cells synthesizing FGF-2 and S100beta may be important in wound repair and restorative events in the lesioned peripheral nerves.

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product

BackgroundGFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms.ResultsRT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization.ConclusionPrevious findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.

Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain

The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783–786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891–910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system.

Aortic coarctation hypertension induces fibroblast growth factor-2 immunoreactivity in the stimulated nucleus tractus solitarii

The actions of neurotrophic factors i.e. basic fibroblast growth factor (bFGF, FGF-2) to neurons are related not only to neuronal development and maintenance but also to synaptic plasticity regarding neurotransmission. We analyzed here the levels of FGF-2 immunoreactivity in the nucleus tractus solitarii (NTS) of Wistar Kyoto rats in response to alterations of neuronal activity promoted by the stimulation of the baroreceptor reflex following an aortic coarctation-induced-hypertension. The FGF-2 immunoreactivity (IR) was found in the cytoplasm of the neurons and in the nuclei of the glial cells in the NTS. A large number of NTS neurons expressed FOS immunoreactivity 4 h after coarctation, as an indication of neuronal activity. Stereological methods showed an increased number of FGF-2 immunoreactive (ir) neuronal profiles (90%) and glial profiles (149%) in the NTS of the 72 h aortic coarctated rats. 1-week later, FGF-2 ir neurons were still increased (54%) but no change was found in the number of FGF-2 ir glial profiles. The double immunoperoxidase method revealed that the majority of the FGF-2 ir glial cells was glial fibrillary acidic protein (GFAP) positive astrocytes. GFAP immunohistochemistry showed an astroglial reaction at 72 h time-interval (55%) but not 1 week after stimulation. The number of the cresyl violet positive neurons and OX42 ir profiles (marker of activated microglia) in the NTS of coarctated rats were not different from control by 1 week and 1 month after the surgery, indicating a lack of NTS injury in this period following coarctation hypertension. FGF-2 may be an important neurotrophic factor in areas involved in the control of blood pressure. The increased FGF-2 IR in the NTS cells following neuronal stimulation may represent trophic and plastic adaptive responses in this nucleus in an autocrine/paracrine fashion.

CELLULAR ANALYSIS OF S100ß AND FIBROBLAST GROWTH FACTOR-2 IN THE DORSAL ROOT GANGLIA AND SCIATIC NERVE OF RODENTS. FOCUS ON PARACRINE ACTIONS OF ACTIVATED SATELLITE CELLS AFTER AXOTOMY

The role of satellite cells, a type of peripheral glia, in the paracrine mechanisms related to neuronal maintenance and plasticity in the dorsal root ganglia (DRG) needs to be further investigated. This study employed immunohistochemistry and image analysis to investigate basic fibroblast growth factor (bFGF, FGF-2) and S100ß immunoreactivities in the DRG and sciatic nerve of the rat and mouse. Well-characterized antibodies against bovine (residues 1–24) and rat (residues 1–23) FGF-2 were employed. Furthermore, the state of satellite cell reaction and changes in the FGF-2/S100ß immunoreactivity were analyzed after axotomy of rat sciatic nerve. Scattered neurons and the majority of the satellite cells of the rat DRG and also Schwann cells of the rat sciatic nerve stained for S100ß. In the mouse, strong S100ß was encountered in the majority of sensory neurons and Schwann cells. Moderate FGF-2 (residues 1–24) immunoreactivity was found in scattered small size neurons of the rat DRG. A strong FGF-2 (residues 1–23) immunoreactivity was achieved in the satellite cells of rat DRG. Both FGF-2 antisera showed strong labeling in the mouse DRG sensory neurons. Activated satellite cells of the axotomized DRG possessed increased amount of FGF-2 and S100ß immunoreactivity as demonstrated by quantitative image analysis. The proximal stump of the lesioned rat sciatic nerve showed increased FGF-2 (residues 1–24 and 1–23) in the Schwann cells, myelin sheaths, and neuronal fibers, without changes in the level of S100ß immunoreactivity. Results suggested a possible interaction between FGF-2 and S100ß in activated satellite cells of the DRG, which might trigger paracrine actions in the axotomized sensory neurons.

Mast cells and eosinophils: A novel link between inflammation and angiogenesis in allergic diseases

Mast cells and eosinophils are the key cells in the early and late stages of allergic inflammation. There is increasing evidence that angiogenesis plays an important role both in the development of inflammation and in the pathophysiology of tissue remodeling during allergic disorders. In this review we provide recent data showing a link between allergy and angiogenesis and some possible mechanisms through which vascular endothelial growth factor and the immune system can interact. We discuss the multifaceted roles of mast cells and eosinophils in tissue remodeling and angiogenesis during allergic diseases and whether these cells can be both source and target cells for pro-angiogenic mediators.

Fibroblast growth factor‐2 immunoreactivity is present in the central and peripheral auditory pathways of adult rats

Recent findings have pointed out the role of neurotrophic factors in the survival and maintenance of neurons of the auditory system. Basic fibroblast growth factor (bFGF, FGF-2) is a potent neurotrophic molecule whose actions can be seen in the central and peripheral nervous systems. In the present study, FGF-2 immunoreactivity was analyzed in the auditory pathways of the adult rat, employing a well-characterized polyclonal antibody against FGF-2. In the cochlea, FGF-2 immunoreactivity was observed in the inner and outer hair cells of the organ of Corti, spiral ganglion neurons, spiral limbus, and stria vascularis. Stereological methods employing optical fractionator revealed the presence of 84.5, 15, and 0.5% of spiral ganglion neurons possessing FGF-2 immunoreactivity of strong, moderate, and weak intensity, respectively. In the central auditory pathways, FGF-2 immunoreactivity was found in the cytoplasm of the neurons of the cochlear nuclei, trapezoid body nuclei, medial geniculate nucleus, and inferior colliculus. The two-color immunoperoxidase method showed FGF-2 immunoreactivity in the nuclei of astrocytes throughout the central auditory pathway. Computer-assisted microdensitometric image analysis revealed higher levels of specific mean gray values of FGF-2 immunoreactivity in the trapezoid body and ventral cochlear nucleus and also in the spiral ganglion and inner hair cells. Sections incubated with FGF-2 antibody preabsorbed with human recombinant FGF-2 showed no immunoreaction in the majority of the studied regions, exhibiting only a slight immunoreactive product in the hair cells of the organ of Corti. Furthermore, no changes in immunoreactivity were observed in sections incubated with FGF-2 antiserum preincubated with human recombinant acidic FGF (FGF-1). The findings suggest that FGF-2 may exert paracrine and autocrine actions on neurons of the central and peripheral auditory systems and may be of importance in the mechanism of hearing diseases.

Time-dependent increase in basic fibroblast growth factor protein in limbic regions following electroshock seizures

Brief experimentally induced seizures have been shown to increase the expression of mRNA encoding basic fibroblast growth factor (FGF-2) in specific brain regions. However, the extent to which this change in mRNA affects the expression of FGF-2 protein in these brain regions has not been examined. In the present study, we exposed rats to brief non-injurious seizures to determine whether this treatment would lead to an increase in FGF-2 protein expression in selected brain regions. Because initial results indicated that the elevation of FGF-2 protein was not significant following acute seizure exposure, we examined both acute and chronic seizure treatment to determine whether FGF-2 protein expression could be increased under conditions of repeated seizures. Brief limbic seizures were induced by minimal electroconvulsive shock (ECS) given as daily treatments for 1 (acute) or 7 (chronic) days. FGF-2 protein was measured in hippocampus, rhinal cortex, frontal cortex, and olfactory bulb at 20, 48, and 72 h following the last seizure. No significant increases in FGF-2 protein were observed in any region following acute ECS. In the chronic ECS-treated groups, significantly elevated FGF-2-like immunoreactivity was found in the frontal and rhinal cortex as compared with the same regions from both control and acute ECS animals. Increases after chronic ECS were maximal at 20 h, and remained significantly elevated as long as 72 h. These increases were predominantly observed for the 24-kDa and 22/22.5-kDa FGF-2 isoforms. Because chronic ECS, which has been shown to be protective against neuronal cell death, induced significantly more FGF-2 immunoreactivity than did acute ECS, we suggest that FGF-2 expression may be an important substrate for the neuroprotective action of non-injurious seizures. A prolonged induction of the high molecular weight isoforms of FGF-2, as occurs after chronic ECS, may selectively reduce the vulnerability of certain brain regions to a variety of neurodegenerative insults.

Antidepressant treatments induce the expression of basic fibroblast growth factor in cortical and hippocampal neurons.

New experimental evidence suggests that the mechanism of action of antidepressants includes the induction of neurotrophic factor synthesis in selected brain areas. The present study is aimed at establishing whether prolonged antidepressant treatments increase the expression of basic fibroblast growth factor (FGF2), a polypeptide growth factor that has a broad neurotrophic activity in the adult central nervous system. Rats received a single dose or long-term (3 weeks) administration of desipramine (DMI), fluoxetine (FLU), and mianserin (MIA), then were sacrificed at 5 and 24 h after the last injection. RNase protection assay and Western blot analysis revealed that all antidepressant drugs elicited an anatomically specific increase in FGF2 mRNA and protein. The increase in FGF2 mRNA after a single injection was seen only at 5 h after the injection and was restricted to the entorhinal cortex, whereas the effect of the long-term treatments lasted up to 24 h and occurred in the entire cortex and hippocampus. Immunohistochemical analysis of FGF2 immunoreactivity was carried out to investigate which cell types responded to the antidepressant treatments. DMI and MIA increased FGF2 proteins predominantly in neurons of layer V throughout the cerebral cortex and in some neurofilament-positive cells of the hippocampus. FLU increased FGF2 immunoreactivity mainly in neurofilament-positive cells of the hippocampus. These findings may explain the therapeutic efficacy of antidepressants in affective disorders.

Basic fibroblast growth factor and fibroblast growth factor receptors in adult olfactory epithelium

Basic fibroblast growth factor (FGF2) stimulates proliferation of the globose basal cells, the neuronal precursor in the olfactory epithelium. The present study investigates the expression of basic fibroblast growth factor and fibroblast growth factor receptors in the adult olfactory epithelium. FGF2 immunoreactivity was expressed widely in the olfactory epithelium, with the highest density of immunoreactivity in the supporting cells. In contrast, most cells in the epithelium expressed FGF2 mRNA. Fibroblast growth factor receptor-1 (FGFr1) immunoreactivity was densest in the basal cell and neuronal layers of the olfactory epithelium and on the apical surface of supporting cells. In the lamina propria FGF2 immunoreactivity and mRNA were densest in cells close to the olfactory nerve bundles. FGFr1 immunoreactivity was heaviest on the olfactory ensheathing cells. Using reverse transcriptase-polymerase chain reaction analysis, the olfactory epithelium was shown to express only three receptor splice variants, including one (FGFr1c) with which basic fibroblast growth factor has high affinity. Other receptor splice variants were present in the lamina propria. Taken together, these observations indicate endogenous sources of FGF2 within the olfactory epithelium and lamina propria and suggest autocrine and paracrine pathways via which FGF2 might regulate olfactory neurogenesis. The observation of only three receptor splice variants in the olfactory epithelium limits the members of the fibroblast growth factor family which could act in the olfactory epithelium. The widespread distribution of receptors suggests that fibroblast growth factors may have roles other than proliferation of globose basal cells.

Survival and Differentiation of Dopaminergic Mesencephalic Neurons Are Promoted by Dopamine-Mediated Induction of FGF-2 in Striatal Astroglial Cells

Survival of dopaminergic (DAergic) midbrain neurons during development and after lesioning depends, in part, on the presence of astroglia-derived growth factors, as, e.g., fibroblast growth factor (FGF)-2. Astrocytes express DA receptors in a brain-region-specific manner. We show here that DA (10−3 to 10−6 mol/liter) applied continuously for 12 h or as a 10-min pulse significantly upregulates FGF-2 immunoreactivity quantified by Western blot and densitometry in astrocytes cultured from two target areas of DAergic neurons, striatum and cortex, but not in mesencephalic astroglia. Semiquantitative competitive RT-PCR confirmed the increase in FGF-2 on the mRNA level. The effects were specific in that glutamate, which can also activate receptors on astroglial cells, did not influence FGF-2 synthesis. In addition to the DA-mediated increase in FGF-2 synthesis the capability of conditioned medium (CM) from DA-stimulated striatal and cortical astrocytes to promote survival and process formation of cultured rat DAergic neurons was significantly enhanced. These effects could be fully blocked by preincubation of the CM with an FGF-2-specific polyclonal antiserum. Our results suggest that DA released from DAergic axon terminals in target regions of DAergic neurons and astroglial FGF-2 production are interdependent in that DA triggers synthesis of FGF-2, which, in turn enhances survival and differentiation of DAergic neurons.

A subpopulation of fibroblast growth factor-2-binding heparan sulfate is lost in human papillary thyroid carcinomas.

We hypothesized that a change in composition of proteoglycans can regulate the bioactivity of fibroblast growth factor (FGF)-2 in the thyroid. In order to test this hypothesis, we established a simple and sensitive method for detecting FGF-2-binding heparan sulfates and characterized them in papillary thyroid carcinomas and normal thyroids. The thyroid extracts were applied to a Q-Sepharose anion exchange column. After the column was washed with 10 mM of phosphate buffer, 1 microgram of human recombinant FGF-2 was added onto the column. The column was eluted with a gradient of NaCl (0.3-1.5 M). Each fraction was blotted onto nitrocellulose membrane. Immunoreactivity of heparan sulfate and FGF-2 was revealed by the incubation of membranes with the specific antibodies, and quantitatively estimated by measuring the density of the color product. In normal thyroids, immunoreactivity of heparan sulfate was detected as two peaks at 0.7 and 0.9 M of NaCl. Heparan sulfate-containing fractions also showed FGF-2 immunoreactivity, indicating the complex formation of FGF-2 and heparan sulfate. In papillary thyroid carcinomas, immunoreactivity of heparan sulfate showed various elution profiles on Q-Sepharose chromatography, including single peak at 0.7 M of NaCl and the one similar to that of the normal thyroids. However, FGF-2 immunoreactivity was detected only in the fractions eluting at 0.7 M of NaCl. This loss of a subpopulation of FGF-2-binding heparan sulfate in human papillary thyroid carcinomas may lead to the increase of free FGF-2 bioavailable in extracellular matrix.

Ontogeny of fibroblast growth factors in the early development of the rat endocrine pancreas.

Pancreatic islet ontogeny involves endocrine cell neogenesis from ductal epithelium and islet expansion by cell replication, balanced by apoptotic deletion of endocrine cells which, in rat, is pronounced in the neonate. Fibroblast growth factors (FGF) are involved in tissue morphogenesis, and we examined the distribution and ontogeny of several FGF within rat pancreas from late fetal life until weaning. Islet cell replication (immunohistochemistry for proliferating cell nuclear antigen) did not change, but a transient increase in ductal epithelial cell replication existed between postnatal days (pnd) 10 and 14. Immunoreactive FGF-1 was found mainly in alpha cells of islets, and FGF-2 immunoreactivity and mRNA throughout the islets, their distribution increasing with age. Both FGF-1 and -2 were also located in ductal epithelium, being maximally distributed at pnd 10-14, coincident with increased cell replication, and when mRNA transcripts encoding FGF-1 (4.4 kb) and FGF-2 (7 kb) were relatively increased in pancreata. FGF-4 and -6 immunoreactivities were localized strongly within islets and ductal cells. In contrast, immunoreactive FGF-7 was associated with pancreatic mesenchyme and intra-and extraislet endothelial cells, and mRNA abundance was transiently increased between pnd 4 and 12, suggesting a role in the initiation of endocrine cell neogenesis. Exogenous FGF-7 was fivefold more potent than FGF-1 or -2 in stimulating DNA synthesis within isolated rat islets. Multiple FGF are expressed within defined compartments of developing pancreas and may contribute to endocrine cell neogenesis and islet function.

Corneal Insult Affects the Production and Distribution of FGF-2 within the Lacrimal Gland

The purpose of this study was to determine the distribution of FGF-2 within rabbit lacrimal glands and to determine whether corneal insult affects that distribution. The scarified corneas of experimental animals were inoculated either with adenovirus type 5 or buffer. Control animals were either untreated, or animals whose corneas were scarified. Twenty-one days later all animals were killed and the lacrimal glands were studied by immunocytochemistry and Western blotting to detect FGF-2. In untreated control animals, FGF-2 was immunolocalized predominantly within a population of elongated cells in the basal epithelium of ducts, and to a lesser degree in the basal epithelium of the acini. The elongated immunopositive cells appear to be myoepithelial cells known to be present at these sites. Interstitial cells around ducts and acini, and the basement membranes of the ducts and acini, were also immunopositive for FGF-2. Twenty-one days after adenovirus inoculation and scarification of the cornea, immunopositivity for FGF-2 was dramatically decreased in basement membranes, but increased within myoepithelial cells of the duct epithelium. These myoepithelial cells were frequently enlarged, bulging toward the duct lumen. In animals whose corneas were inoculated with buffer and scarified, or animals whose corneas were simply scarified, the changes in the lacrimal gland were similar, but somewhat less pronounced, to those of adenovirus-inoculated animals. Western blots confirmed the presence of FGF-2 immunoreactivity in all groups. The major band in untreated controls was at 24kD, whereas all animals with corneal scarification had major bands at 38kD. Densitometry of Western blots demonstrated that the amount of 24kD FGF-2 present within the lacrimal gland after corneal scarification was at least 50% less than in untreated controls, whereas 38kD FGF-2 was at least ten-fold greater. Our findings indicate that corneal scarification results in an altered distribution of FGF-2 within the lacrimal gland, which involves a decrease in low molecular weight FGF-2 and a dramatic increase in a higher molecular weight isoform of FGF-2. FGF-2 may be released from myoepithelial cells apically (exocrine) into the tear fluid and basally (autocrine/paracrine) into the connective tissue, as well as from extracellular complexes within basal laminae.

Capillary growth in relation to blood flow and performance in overloaded rat skeletal muscle.

Rat extensor digitorum longus muscles were overloaded by stretch after removal of the synergist tibialis anterior muscle to determine the relationship between capillary growth, muscle blood flow, and presence of growth factors. After 2 wk, sarcomere length increased from 2.4 to 2.9 micrometers. Capillary-to-fiber ratio, estimated from alkaline phosphatase-stained frozen sections, was increased by 33% (P < 0.0001) and 60% (P < 0.01), compared with control muscles (1.44 +/- 0.06) after 2 and 8 wk, respectively. At 2 wk, the increased capillary-to-fiber ratio was not associated with any changes in mRNA for basic fibroblast growth factor (FGF-2) or its protein distribution. FGF-2 immunoreactivity was present in nerves and large blood vessels but was negative in capillaries, whereas the activity of low-molecular endothelial-cell-stimulating angiogenic factor (ESAF) was 50% higher in stretched muscles. Muscle blood flows measured by radiolabeled microspheres during contractions were not significantly different after 2 or 8 wk (132 +/- 37 and 177 +/- 22 ml. min-1. 100 g-1, respectively) from weight-matched controls (156 +/- 12 and 150 +/- 10 ml. min-1. 100 g-1, respectively). Resistance to fatigue during 5-min isometric contractions (final/peak tension x 100) was similar in 2-wk overloaded and contralateral muscles (85 vs. 80%) and enhanced after 8 wk to 92%, compared with 77% in contralateral muscles and 67% in controls. We conclude that increased blood flow cannot be responsible for initiating expansion of the capillary bed, nor does it explain the reduced fatigue within overloaded muscles. However, stretch can present a mechanical stimulus to capillary growth, acting either directly on the capillary abluminal surface or by upregulating ESAF, but not FGF-2, in the extracellular matrix.