Expression Of aFGF Research Articles

AFGF, bFGF and flg mRNAs show distinct patterns of induction in the hippocampus following kainate-induced seizures.

We report that kainic acid-induced seizures lead to marked increases in mRNAs encoding basic and acidic fibroblast growth factors (bFGF and aFGF, respectively) and flg, one of their receptors, in the rat hippocampus. Anticonvulsant pretreatment inhibits the up-regulation of these mRNAs. The observed increase in flg mRNA levels involves the pyramidal cells of all hippocampal subfields and the granular cells of the dentate gyrus. The increased expression of aFGF and bFGF mRNAs is limited to neuron populations that are resistant to seizure-induced injury, the granular cells of dentate gyrus and pyramidal cells of CA1 region, respectively. The results suggest that the increase in the FGFs and flg may play pivotal roles in neuron survival and in long-term changes occurring in the hippocampus following seizure activity.

Acidic FGF expression in the surroundings of senile plaques.

Immunohistochemical examination of postmortem brain tissue of Alzheimer's disease revealed that acidic fibroblast growth factor (aFGF) was specifically expressed in a subpopulation of reactive astrocytes which were congregated at the margin of the senile plaque. Double immunostaining indicated that such upregulation of aFGF expression might be related to the presence of reactive microglia rather than beta-amyloid protein deposits. Although, on the other hand, immunohistochemical staining for fibroblast growth factor receptor-1 occurred in some cortical neurons of Alzheimer's disease, the staining pattern did not differ from that in age-matched controls. Possible significance of aFGF-positive astrocytes in the surroundings of the senile plaque will be discussed in relation to receptor mediated or non-mediated mechanisms.

Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. A clue to the mechanism of transformation from the stable to the unstable clinical state.

The mechanisms responsible for the transformation of stable angina to unstable angina, a major cause of morbidity and mortality, are commonly believed to be plaque rupture and thrombosis. We determined whether additional mechanisms are operative by analyzing the histopathology and immuno-histopathology of coronary plaques retrieved by directional atherectomy of patients with unstable angina in whom no intraluminal thrombus was demonstrated by angiography. The histological findings of atherectomy specimens from 34 patients with unstable angina were compared with those of 24 patients with postangioplasty restenosis, whose lesions are known to be composed of smooth muscle cells (SMCs), and 10 patients with stable angina, whose lesions contain relatively few SMCs. We also studied the expression of acidic and basic fibroblast growth factors (aFGF and bFGF), whose role in the vascular response to injury has been established. Specimens from unstable angina resembled those from postangioplasty restenosis in regard to SMC abundance (scale, 0 to 3; 1.4 +/- 0.9 versus 1.7 +/- 0.9; P = NS), and both differed from those of stable angina. Thrombus and/or hemorrhage occurred in only 34% of patients with unstable angina (compared with 8% of restenosis patients and in none of stable angina patients). Active lesions (defined as lesions (defined as lesions containing one or more of the following: thrombus, hemorrhage, abundant and disorganized SMCs in the presence of loose connective tissue, or inflammatory infiltrate) were observed in 56% of the unstable angina patients and in 50% of the restenosis patients but in none of the stable angina patients. The expression of aFGF and bFGF was detected in 80% to 100% of unstable angina (n = 11) and restenosis (n = 10) specimens but in only 1 of 5 stable angina specimens. Microscopic evidence of thrombosis and plaque rupture occurred in only one third of unstable angina patients, selected because they had no angiographic evidence of intracoronary thrombus. Moreover, their lesions resembled those of restenosis patients in regard to SMC abundance, lesion activity, and the expression of aFGF and bFGF. Our findings therefore suggest that an alternative mechanism to plaque rupture and thrombus formation may be operative in the precipitation of unstable angina; namely, in a subset of patients, SMC proliferation may lead to gradual plaque expansion and thereby to lumenal narrowing and unstable angina. Our data also suggest a role for aFGF and bFGF in this process.

Developmental regulation of acidic fibroblast growth factor (aFGF) expression in bovine retina.

Acidic fibroblast growth factor (aFGF) is a signalling molecule implicated in a wide variety of biological processes such as cell growth, differentiation and survival. It has been purified from bovine retina. The present study was carried out to detect which cells in the bovine retina expressed aFGF at the different stages of embryonic and post-natal development. The specific aFGF mRNA and protein were detected by in situ hybridization employing riboprobes and immunocytochemistry using affinity purified polyclonal human recombinant aFGF antibodies respectively. No signal was detected by either technique until 4-5 months and then there was progressive expression of aFGF with terminal morphogenesis of the retina. By 8-9 months of embryonic development, nuclei of the 3 neuronal layers (ganglion cell layer, inner and outer nuclear layers) were all uniformly and intensely labeled. A slight labeling of the pigmented epithelium of the retina was also visible throughout development and maturation. These results showed a good correlation between message and protein expression in these cell types. In contrast, glial cells in the nerve fiber layer and vascular endothelial cells displayed a nuclear immunostaining for the protein in the absence of message. These data suggest that aFGF plays a role in the late steps of retinal differentiation by autocrine and paracrine mechanisms.

Acidic and basic fibroblast growth factor mRNAs are increased in striatum following MPTP-induced dopamine neurofiber lesion: assay by quantitative PCR

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF), the two best characterized members of a growing family of heparin-binding growth factors, have been shown to affect both survival of cultured neurons and regeneration of nerve terminals when applied exogenously. The endogenous expression of these growth factors in response to brain injury is not well understood. We have utilized the Swiss-Webster mouse, treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and a quantitative polymerase chain reaction assay to examine changes in endogenous synthesis of mRNA for both aFGF and bFGF in the striatum and substantia nigra. We have found that MPTP treatment results in a loss of 95% of dopaminergic function and is accompanied by an increase in expression of both aFGF and bFGF in the striatum at 1 week post-lesion. After 5 weeks, the terminals appear to be regenerating and FGF mRNA expression has returned to control levels. These results suggest that cellular reaction to chemical lesion in the brain may involve changes in growth factor expression, including both aFGF and bFGF.

Altered Acidic and Basic Fibroblast Growth Factor Expression Following Spinal Cord Injury

In normal spinal cord, acidic fibroblast growth factor (aFGF) immunoreactivity was localized in the cytoplasm of ventral motor neurons and sensory fibers in the dorsal columns. Basic FGF (bFGF) immunoreactivity was restricted to astrocyte nuclei and the cytoplasm of a few neurons in the intermediate gray matter. Spinal cord lesions resulted in complete destruction of the dorsal columns at T8. Two days postlesion, aFGF immunoreactivity was increased in ventral motor neurons and was now seen in intermediate gray matter neurons. Acidic FGP was not detected in the lesioned fasciculus gracilis at T4-5, but markedly increased in the fasciculus cuneatus. At L1-2, aFGF-immunoreactive fibers in the fasciculus gracilis also increased. This aFGF immunostaining was maintained 5 and 12 days postlesion. A lesion-induced loss of aFGF immunoreactivity in the nucleus gracilis suggests that aFGF is anterogradely transported in ascending sensory fibers. Two days postlesion, glial fibrillary acidic protein immunoreactivity increased at the lesion site, as well as at T4-5 and L1-2, with no change in bFGF staining. Five days postlesion, increased bFGF immunoreactivity appeared at the edge of the cystic cavity and the dorsal columns at T4-5 in both the nucleus and the cytoplasm of reactive astrocytes, and was increased at 12 days postlesion. The differential cellular, temporal, and spatial expression of aFGF and bFGF following spinal cord lesion suggest they subserve distinct roles in the response to CNS injury.

Acidic fibroblast growth factor and heart development. Role in myocyte proliferation and capillary angiogenesis.

Proliferative growth of the ventricular myocyte (cardiomyocyte) is primarily limited to fetal and early neonatal periods of development. In concert with the neonatal "transition" from proliferative to hypertrophic growth, ventricular remodeling of the nonmyocyte compartment is characterized by increased extracellular matrix synthesis/deposition and capillary angiogenesis. A role for locally generated and bioactive ventricular acidic fibroblast growth factor (aFGF) in these processes is proposed and substantiated by the following: 1) colocalization of aFGF peptide and fibroblast growth factor receptor (flg) transcripts to the developing fetal cardiomyocyte by immunohistochemistry, immunoelectron microscopy, and in situ hybridization, 2) continued localization of aFGF peptide and transcripts to the neonatal/mature cardiomyocyte, and 3) localization of flg immunoreactivity and transcripts to specific neonatal ventricular nonmuscle cell types. Specific ventricular cell types at distinct developmental stages appear to be responsive to ventricular myocyte-derived aFGF (myocytes in the fetal heart and nonmyocytes/endothelial cells in the neonatal heart). These data indicate that expression of aFGF and one of its receptors (flg) are most pronounced in the fetal to early neonatal ventricle, the presence of both suggesting an autocrine/paracrine growth regulatory function. As the animal matures, ventricular capillary angiogenesis may be facilitated by "release" of cardiomyocyte-derived fibroblast growth factors into the surrounding extracellular space/matrix functioning as a "paracrine" angiogenic stimuli. Therefore, the results of our study suggest that myocyte-derived aFGF may function to increase the fetal ventricular cardiomyocyte population in absolute number as well as to facilitate the subsequent increase in capillary angiogenesis that occurs during cardiomyocyte maturation and ventricular remodeling.

Increasing cell density down-regulates the expression of acidic FGF by human RPE cells in vitro.

Previous studies have reported the expression of acidic fibroblast growth factor (aFGF) by rat, bovine, and human retinal pigment epithelium (RPE) in vivo. To critically examine the expression of aFGF by RPE cells, we studied the density dependence of steady-state levels of mRNA and protein expression in vitro. Northern blot analysis demonstrated 5 transcripts ranging from 4.5 kB to 1 kB. Steady-state levels of all the transcripts decreased as a function of culture density. A polyclonal antibody was raised against recombinant human aFGF and affinity purified on aFGF coupled to AffiGel-10. The resulting antibody crossreacted with bFGF but not FGF-5, but this crossreactivity could be eliminated by absorption of the antibody on bFGF coupled to AffiGel-10. The final antibody preparation recognized only a single band at approximately 18.5 kD in lysates of RPE. Immunohistochemical staining with this antibody preparation demonstrated a marked dependence on cell density after 3 days in culture. Low culture density yielded cells staining moderately for aFGF, while confluent cells exhibited little or no staining. The reduction of aFGF from RPE cells in culture in a density-dependent fashion could also be demonstrated by Western blot analysis.

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.

Differential expression of acidic and basic FGF in the rat substantia nigra during development.

Both acidic (aFGF) and basic (bFGF) fibroblast growth factors have been shown to be present in the adult rat ventral mesencephalon and to exert effects on cultured mesencephalic cells. In the present study we have examined the expression of aFGF and bFGF in the rat ventral mesencephalon at various stages of development. bFGF was present at all ages examined [embryonic day 16 (E16) to postnatal day 90 (P90)]. In contrast, aFGF was not detectable at embryonic and early postnatal ages, but was observed at later (P20, P60, P90) postnatal stages. These data suggest that aFGF and bFGF may have functions in mesencephalic dopamine neurones in different stages of development.

Endogenous aFGF expression and cellular changes after a demyelinating lesion in the spinal cord of adult normal mice: Immunohistochemical study

Fibroblast growth factors (FGFs) are known to act on glial cells in vitro. At the present time, their involvement in the remyelinating process of the adult central nervous system (CNS) is still unknown. In the present study, using immunohistochemistry (IHC), we investigated the evolution in time and space of acidic FGF (aFGF) expression and CNS cell changes occurring after a chemically induced demyelinating lesion. In a first early period, aFGF immunostaining was shown to decrease around the demyelinated area. A dramatic increase was then observed and was accompanied by an increase of cell density around and inside the lesion. This was correlated with the beginning of remyelination. Late after demyelination, while remyelination was still in progress, aFGF immunostaining of the lesion and unlesioned spinal cord were comparable. A role of aFGF in remyelination is proposed.

Expression of aFGF in porcine myocardium depends on severity of ischemia: Stefan Sack, Jutta Schaper, Masahiro Mohri, Renate Möhren, Sabine Bernotat-Danielowski, Margarete Arras, Ernst Rüdiger Schwarz, Wolfgang Schaper, Max-Planck-Institute, Department of Experimental Cardiology, Bad Nauheim, F. R. Germany

Expression of aFGF in porcine myocardium depends on severity of ischemia: Stefan Sack, Jutta Schaper, Masahiro Mohri, Renate Möhren, Sabine Bernotat-Danielowski, Margarete Arras, Ernst Rüdiger Schwarz, Wolfgang Schaper, Max-Planck-Institute, Department of Experimental Cardiology, Bad Nauheim, F. R. Germany

Immunohistochemical detection of acidic fibroblast growth factor in bladder transitional cell carcinoma.

Acidic fibroblast growth factor (aFGF) is a regulatory peptide which, on account of its structural homologies with the products of oncogenes, is involved in cell proliferation, differentiation, and motility. We previously reported the presence of aFGF in the urine of patients with transitional cell carcinoma (TCC). aFGF can also induce the motility of a rat-derived bladder carcinoma cell line (NBTII). This immunohistochemical study used polyclonal rabbit antibodies against acidic and basic FGF and peroxidase detection. Native NBTII nude mice xenografts and aFGF transfected NBTII (NFS14) nude mice xenografts were used as tissue controls for antibody specificity. The samples included 4 normal urothelia and 12 TCC. In addition, cytospins of 4 different tumoral cell lines of human bladder and normal bladder cells were stained. The results showed strong immunostaining in all tumoral urothelium samples using anti-aFGF and a very low amount of staining or none at all in healthy tissues. A primary analysis suggested that the strongest reaction was obtained in high-grade tumors (3 + vs + for lower-grade tumors). Using bFGF antibody, strong immunohistochemical staining was detected on basal membranes and stromal vessels and none in urothelium. These data confirm aFGF expression in the epithelial cell compartment of bladder cancer and the likely involvement of this regulatory peptide in the biology of TCC.

Heterogeneity of 3′untranslated region of bovine acidic FGF transcripts

A bovine aFGF genomic clone (14.2Kb) has been isolated and characterized. This clone contains exons 2 and 3 interrupted by 6.7 Kb long intron. Exon 3 contains part of the coding region and the 3′ untraslated region (3′UTR). Two overlapping cDNA clones specific for this 3′UTR have been isolated from bovine retina cDNA libraries or after amplification of RNA by the RACE technique. Analysis of these clones and RNAse protection assay demonstrate alternative termination of aFGF transcripts giving rise to differently sized 3′UTR of 2.5 Kb and at least 3.9 Kb. The sequence of these long 3′UTR is highly conserved among species (70% identity between human and rat) which suggests an important role for aFGF expression.

Acidic fibroblast growth factor (aFGF) in developing normal and dystrophic (mdx) mouse muscles. Distribution in degenerating and regenerating mdx myofibres.

Affinity purified polyclonal antibodies directed against human recombinant acidic FGF (aFGF), were used in immunofluorescence studies to localize this growth factor in several normal and dystrophic (mdx) mouse skeletal muscles. The expression of aFGF was detected throughout the life of both the control and mdx mice. In striated muscles, examined up to 3 weeks postnatal, aFGF was localized around the myofibres and this pattern was consistent in both mdx and the normal counterpart strain. However, the intensity of the signal was much stronger in the mdx strain. In mdx mouse skeletal muscles, examined during the acute phase of degeneration and regeneration (3-14 weeks) aFGF was localized around the myofibres, in approximately 60% of the nuclei of newly formed or regenerated myofibres and also in the pockets of necrosis which represented actively degenerating myofibres. In normal mouse skeletal muscles, studied over the same period, the antibodies localized aFGF mainly to the periphery of the muscle fibres. The augmentation of aFGF observed by immunofluorescence in mdx mouse muscles was confirmed by enzyme immunoassay (EIA) analysis of the same muscles over the same period of time. The data from the EIA indicated a 3.5-fold increase in aFGF in mdx as compared to normal muscles at 3 weeks, and an approximate 26-fold increase during the period of active degeneration-regeneration. This increased concentration of aFGF noted in the mdx muscles suggests that this endogenous aFGF may participate in the high level of regenerative activity observed in mdx mouse.

Human dermal fibroblasts express multiple bFGF and aFGF proteins

We investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions.

Prominent expression of acidic fibroblast growth factor in motor and sensory neurons

Several growth factors originally characterized and named for their action on a variety of cells have more recently been suggested to be importantly involved in the development and maintenance of the nervous system. Acidic fibroblast growth factor (aFGF) is a member of a family of seven structurally related polypeptide growth factors. The cells responsible for expression of aFGF in the nervous system of adult rats have been identified using an affinity-purified antibody to aFGF in immunohistochemical studies and synthetic oligonucleotide probes for in situ hybridization studies. High levels of aFGF expression were observed in motoneurons, primary sensory neurons, and retinal ganglion neurons. Glial cells did not express detectable amounts of aFGF. Confocal and electron microscopic analysis suggested that a large portion of aFGF immunoreactivity was associated with the cytoplasmic face of neuronal membranes, consistent with the hypothesis that aFGF is a sequestered growth factor.

Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential.

Addition of exogenous acidic fibroblast growth factor (aFGF) to NBT-II epithelial carcinoma cells results in fibroblastic transformation and cell motility. We have generated aFGF-producing NBT-II cells by transfection with recombinant expression vectors containing human aFGF cDNA, or the human aFGF cDNA coupled to a signal peptide (SP) sequence. The effects of the nonsecreted and the secreted 16-kDa growth factor on the morphology, motility, and cell invasive potential (gelatinase activity) were compared. aFGF coupled to a SP was actively secreted out of the producing cells. The secretion of aFGF was not necessary for induction of gelatinase activity, as this was observed in NBT-II cells producing aFGF with or without SP. Production of aFGF, whether secreted or not secreted, resulted in increased in vitro motility of most isolated clones; however, there was no correlation between aFGF level and motility rate. The data suggest that expression of aFGF in NBT-II cells induces metastatic potential through an autocrine or intracrine mechanism.

Acidic Fibroblast Growth Factor Overexpression in Corneal Epithelial Wound Healing

aFGF expression was studied in normal and regenerating cornea of adult rats. aFGF mRNA and proteins were expressed mainly in corneal epithelium but not in stroma. After burning of the epithelium by iodine vapours, the intact epithelial cells migrated to cover the wounded area during the first 4 days and then divided to reconstitute a normal multilayered epithelium 6 days after injury. aFGF mRNA localized by in situ hybridization on regenerating epithelium showed a peak between 6 hr and 2 days after denudation, decreasing to basal levels 6 days later. This induction of aFGF mRNA preceded the increased amount of aFGF peptides, as assessed by indirect immunofluorescence staining. Thus aFGF overexpression is clearly correlated with active migration in epithelial wound healing.

Reactive Astrocytes Express Acidic Fibroblast Growth Factor in Alzheimer's Disease Brain

Localization of acidic fibroblast growth factor (aFGF) was investigated immunohistochemically in postmortem brain tissue of Alzheimer''s disease (AD), amyotrophic lateral sclerosis (ALS), and age-matched control cases using a specific rabbit antiserum to aFGF. Small numbers of glial cells were faintly stained in white but not gray matter of control cases. Larger numbers of reactive astrocytes were stained intensely for aFGF in severely affected areas of AD cases and in the primary motor cortex of ALS cases. In AD, aFGF-positive astrocytes congregated at the margin of senile plaques. Double immunostaining indicated that such upregulation of aFGF expression might be related to the presence of reactive microglia rather than β-amyloid protein deposits.