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Y Courtois

Publications and source records attributed to Y Courtois.

At least 73 records · Page 4Linked to original sources
PubMed1996

The neurotrophic activity of fibroblast growth factor 1 (FGF1) depends on endogenous FGF1 expression and is independent of the mitogen-activated protein kinase cascade pathway.

The expression of fibroblast growth factor (FGF) 1, a potent neurotrophic factor, increases during differentiation and remains high in adult neuronal tissues. To examine the importance of this expression on the neuronal phenotype, we have used PC12 cells, a model to study FGF-induced neuronal differentiation. After demonstrating that FGF1 and FGF2 are synthesized by PC12 cells, we investigated if FGF1 expression could be a key element in differentiation. Using the cell signaling pathway to determine the effects of FGF1 alone, FGF1 plus heparin, or a mutated FGF1, we showed an activation to the same extent of mitogen-activated protein (MAP) kinase kinase and MAP kinase (extracellular regulated kinase 1). However, only FGF1 plus heparin could promote PC12 cell differentiation. Thus, the MAP kinase pathway is insufficient to promote differentiation. Analysis of the PC12 cells after the addition of FGF1 plus heparin or FGF2 demonstrated a significant increase in the level of FGF1 expression with the same time course as the appearance of the neuritic extensions. Transfection experiments were performed to enhance constitutivly or after dexamethasone induction the level of FGF1 expression. The degree of differentiation achieved by the cells correlated directly with the amount of FGF1 expressed. The MAP kinase pathway did not appear to be involved. Interestingly, a 5-fold increase in FGF1 in constitutive transfected cells extended dramatically their survival in serum-free medium, suggesting that the rise of FGF1 synthesis during neuronal differentiation is probably linked to their ability to survive in the adult. All of these data demonstrate that, in contrast to the MAP kinase cascade. FGF1 expression is sufficient to induce in PC12 cells both differentiation and survival. It also shows that auto- and trans-activation of FGF1 expression is involved in the differentiation process stimulated by exogenous FGFs through a new pathway which remains to be characterized.

Adult↗
PubMed1996

In vitro changes in plasma membrane heparan sulfate proteoglycans and in perlecan expression participate in the regulation of fibroblast growth factor 2 mitogenic activity.

Fibroblast growth factor 1 (FGF1) and 2 (FGF2) bind to two classes of receptors: the high affinity receptors, a family of four known transmembrane tyrosine kinases (FGF R1-R4), and the low affinity receptors, cell surface and basement membrane heparan sulfate proteoglycan (HSPG). During early (first and second) passages of retinal pigmented epithelial (RPE) cells, both FGF1 and FGF2 exhibited low mitogenic activity, while in later (fifth to ninth) passages the activity of FGF1 remained constant but FGF2 activity increased two- to threefold. We have investigated aspects of FGF receptor interactions and the role of heparin/heparan sulfate which modulates FGF activity on RPE cells during in vitro senescence. Northern blot analysis demonstrated that FGF receptor type 1 (FGF R1) is the major high affinity receptor expressed in RPE cells and that its level of expression did not change during serially passage. Both the FGF R1 and the FGF low affinity receptors' binding characteristics (i.e., Kd and number of sites per cell) for FGF1 were unaffected by passage number, whereas the capacity of FGF2 binding to FGF R1 and to the low affinity receptors increased by two- and fivefold, respectively, in late passages, although the affinities were unchanged. This change in the capacity of FGF2 to bind to FGF R1 and to HSPG was not due to a switch of the IIIc splice form of FGF R1 to the IIIb splice form since the exon IIIc was the most predominant splice form of FGF R1 during RPE cell cultures. Furthermore the ratio of the IIIb to the IIIc splice form was not modified during cell subcultures. In parallel in the older RPE cell passages, expression of perlecan, the major FGF low affinity binding site localized on the extracellular matrix of RPE cells, was much elevated compared to early RPE cell passages. Moreover, the cell surface of late passage RPE cells had 79% more HSPG than early passage cells. Therefore, it is suggested that the increase in the number of FGF low affinity receptors present on the cell surface or basement membrane could account for a part of the greater proliferative response of aged RPE cells to FGF2.

Alternative Splicing↗
PubMed1996

Cloning of multiple chicken FGF1 mRNAs and their differential expression during development of whole embryo and of the lens.

Five different 5' untranslated regions (5' UTRs) of FGF1 mRNAs were cloned in chicken. The structure of these transcripts suggests that, as in mammals, distinct 5' untranslated exons are spliced to the first coding exon via alternative splicing and alternative promoter usage. In an attempt to correlate the expression of specific transcripts to distinct biological activities, the distribution of these transcripts in different tissues and during the development of both the whole embryo and the lens was studied. In tissues, we have shown a differential, but not exclusive, expression of these transcripts. In the whole embryo, the expression of one transcript correlates with later developmental processes. In the lens, only two transcripts were detected that are both differently expressed and distributed. These results suggest that the biological properties of FGF1 depend on the expression of specific FGF1 mRNAs. Because these transcripts only differ in their 5' UTRs, they could be involved in distinct translational controls.

Animals↗
PubMed1996

Accumulation of mitochondrial DNA deletions in human retina during aging.

PURPOSE: The authors investigated the presence of mitochondrial DNA (mtDNA) mutations in aging human retina. METHODS: A quantitative polymerase chain reaction technique for studying the common mtDNA 4977-deletion (delta mtDNA4977) in retinal pigment epithelium (RPE) and neural retinal (NR) was developed. RESULTS: Although no deletion was detected in the fetus, every adult RPE and NR had this common deletion. The ratio of the deleted delta mtDNA4977 to the total mtDNA increased significantly in elderly persons 60 to 110 years of age and was greater in peripheral than in central RPE. CONCLUSIONS: These results suggest that at least one type of mutation accumulates in the mtDNA in the retina during aging, reflecting a general phenomenon of genomic instability that could influence its function.

Aged↗
PubMed1996

Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats.

PURPOSE: Inducible nitric oxide (NO) synthase (iNOS) has been implicated in the pathogenesis of endotoxin-induced uveitis (EIU). This study was undertaken to localize the cells, in the eye, which express iNOS during EIU in the rat. METHODS: EIU was induced in Lewis rats by a single foot pad injection of 150 micrograms lipopolysaccharide (LPS) from Salmonella typhimurium. At different time intervals after LPS injection, the authors evaluated ocular inflammation (slit lamp observation), iNOS localization by in situ hybridization, and comparison of OX-42- and ED1-positive cell appearance and of glial response by specific immunohistochemistry. RESULTS: iNOS mRNA was not detected in the iris-ciliary body nor in the retina of control rats. It was detected strongly in the epithelial cells of the iris-ciliary body at 6 hours and also in stromal cells of the ciliary processes at 16 hours after LPS injection. In the neuroretina, iNOS mRNA was observed in the inner layers 16 hours after LPS injection. iNOS-positive cells were also present on the vitreous at this time. At 6 and approximately 16 hours after LPS injection, immunohistochemistry experiments revealed a large number of OX-42- and ED1-positive cells (microglia, macrophages, or polymorphonuclear leukocytes) colocalized in part with some iNOS-positive cells in the ciliary body and in the retina. Furthermore, expression of iNOS in Müller cells cannot be excluded. CONCLUSIONS: These observations confirm that subcutaneous injection of endotoxin dramatically induces NOS mRNA expression in the eye, and they demonstrate that epithelial cells of the iris-ciliary body and cells infiltrating the anterior segment of the eye and the retina are the major source of NO. These results support the hypothesis that both inflammatory and resident ocular cells are involved in iNOS expression during EIU.

Animals↗
PubMed1996

Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye.

PURPOSE: To evaluate the effect of intraocular administration of nitric oxide (NO) donors in the rabbit eye on intraocular pressure (IOP), inflammation, and toxicity. METHODS: Intravitreal and intracameral injections of two NO donors, SIN-1 and SNAP, and SIN-1C and BSS were performed. Clinical examination, IOP measurements, protein evaluation in aqueous humor, and histologic analysis of the ocular globes were realized. Nitric oxide release was demonstrated by nitrite production in the aqueous humor and in the vitreous using the Griess reaction. RESULTS: The drastic decrease of IOP, observed after a single NO donor injection, was correlated directly with nitrite production and, thus, to NO release. Injection of inactive metabolite of SIN-1, SIN-1C, which is not able to release NO, did not modulate IOP. When administered in the aqueous humor or in the vitreous, NO did not diffuse from one segment of the eye to another. No inflammation or histologic damage was observed as a result of a single NO donor administration. CONCLUSIONS: Nitric oxide is implicated directly in the regulation of IOP and its acute, and massive release into the rabbit eye did not induce inflammation or other growth toxic effects on the ocular tissues.

Acetonitriles↗
PubMed1996

[Effects of human recombinant basic Fibroblast Growth factor on endothelial wound healing in organ culture of human cornea].

PURPOSE: The effects of human recombinant bFGF has been evaluated on 9 paired human donor corneas (age 75 +/- 8 years), preserved in organ culture medium. MATERIAL AND METHODS: The endothelium of corneas were mechanically wounded (area of 7.53 +/- 2.05 mm2) and placed in culture medium during 14 days. For each pair, one cornea was tested with bFGF (50 ng/ml), delivered in two times (day 0, day 7), according to the stockage of the bFGF on the basal membranes (low affinity receptor), while the ipsilateral cornea served as control. Endothelium was assessed by trypan staining at day 0, day 7, and day 14. At this term of fourteenth day, alizarine red and trypan blue staining permitted morphometric data. RESULTS: The bFGF factor increases significantly cell density in the wound area (p < 0.05), and in the transitional area (p < 0.01), compared to the control group. In the transitional area, cells depletion was only 15% (392 +/- 55 cells/mm2) in the treated group compared to the 28% (716 +/- 0.1 cells/mm2) in the untreated group. In the wound area, the mean cell area was averaged 2581 microns2 in the control group and 2161 microns2 in the bFGF treated group (p < 0.05); in the transitional area the mean cell size was 549 microns2, and 479 microns2 in the control and the bFGF treated group (p < 0.05) respectively. The bFGF group do not affect the shape factor. CONCLUSION: This assay demonstrates that human bFGF greatly facilitates wound closure in endothelium of human cornea. The cellular migration from the transitional zone seems the dominant healing mechanism.

Cell Count↗
PubMed1995

Involvement of DNase II in nuclear degeneration during lens cell differentiation.

The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation. The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract. In fibers, both types of nuclease activities are found and appear higher than in epithelial cells. Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms. DNase II like-nuclease, present in fibers, is represented by three major bands (60,23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells. DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells. Immunocytochemistry studies show both nucleases present in lens cell sections. DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers. DNase I is always concentrated in nuclei of epithelial and fiber cells. DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer. In addition, DNase II antibody is able to prevent this degradation. These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.

Animals↗
PubMed1995

Fibroblast growth factors decrease inducible nitric oxide synthase mRNA accumulation in bovine retinal pigmented epithelial cells.

Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon gamma (IFN gamma) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF-1 (acidic FGF) and FGF-2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co-stimulated with FGF-2 and LPS/IFN gamma accumulate lower levels of NO synthase than in the absence of FGF-2. Northern blot analysis by cross-species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4-kb mRNA accumulates when RPE cells are activated with LPS/IFN gamma. The level of inducible NO synthase mRNA in LPS/IFN gamma-activated RPE cells is markedly reduced by FGF-1 or FGF-2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFN gamma receptors was excluded, since IFN gamma binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine-mediated tissue damage.

Amino Acid Oxidoreductases↗
PubMed1995

Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase.

Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory diseases. In the present study, we investigated the potential role of NO in an ocular model of inflammation, endotoxin-induced uveitis (EIU), in Lewis rats. Injection of LPS in one footpad induces severe uveitis after 16 h, which is accompanied by an increase of NO in the aqueous and vitreous humors, as evaluated by nitrite assay. Reverse transcriptase-PCR experiments reveal a large increase of inducible NO synthase (iNOS) mRNA in the iris/ciliary body, from 2 to 24 h after LPS treatment. In the retina, maximal increase of iNOS mRNA was detected 16 h after LPS treatment. Two i.p. injections of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), which inhibits nitrite release in the aqueous and vitreous humors, profoundly reduce clinical and histologic inflammation in EIU rats. These results implicate the NO pathway in the pathogenesis of EIU and demonstrate the possibility of modulating this inflammatory disease by injection of a NOS inhibitor.

Amino Acid Oxidoreductases↗
PubMed1995

Fibroblast growth factors alter light responses and dark voltage in retinal rods of the frog (Rana temporaria).

Fibroblast growth factors (FGF-1 and FGF-2) were applied intracellularly via whole-cell patch-clamp electrodes while the membrane voltage was recorded simultaneously. During recording the exchange of substances by diffusion between cytosol and pipette medium affects the cell's function. Under control conditions, the loss of nucleotides is reflected by a slow hyperpolarization of the dark voltage and prolongated light responses. Addition of FGF-1 and FGF-2 to the pipette medium accelerated the time course of the hyperpolarization and intensified the prolongation of the light responses. The depolarization of photoreceptor cells after intracellular application of the nitric oxide (NO)-synthase cofactor nicotinamide adenine dinucleotide phosphate (NADPH) and the stabilization of light response recovery by L-arginine is abolished by FGF-2. FGF-2 was ineffective when it was applied together with the calcium chelator ethylene glycol-bis(2-aminoethylether)tetraacetate (EGTA). The results indicate a possible role of FGF in the regulation of NO and calcium in photoreceptor cells and may explain protective effects of FGF in degenerative processes of photoreceptor cells.

Amino Acid Oxidoreductases↗
PubMed1995

The role of exogenous/endogenous basic fibroblast growth factor (FGF2) and transforming growth factor beta (TGF beta-1) on human corneal endothelial cells proliferation in vitro.

Adult human corneal endothelial cells (HCEC) have extremely low turnover rates but undergo rapid division in vitro when stimulated with soluble growth factors. We have investigated the role played by FGF2 and TGF beta-1 in the regulation of HCEC growth stimulation. HCEC from donors who were over 30 years old were cultured and experiments performed on cultures between the 2nd and the 6th passage in the presence of 5% NCS. Cell counts revealed a maximal stimulation of 2.1x for FGF2 and 1.9x for TGF beta-1 compared to control cultures. When both factors were added, a synergistic effect was noticed with a maximal stimulation of the proliferation rate of 4.5x over controls. In addition, endogenous FGF2 produced by HCEC was quantitated in a sensitive EIA assay. After 5 days in culture, 10(6) cells contained 150 ng FGF2 and 35 ng was extracted from trypsin-digested ECM. Two molar NaCl washes of ECM released 15.6 ng FGF2, which induced a slight mitogenic activity (1.5x over control) in HCEC cultures, which was partially inhibited by an anti-FGF2 antibody. Northern blot analysis of HCEC extracts revealed the presence of FGF receptors R1 and R2 mRNA. The bioactive FGFRs were demonstrated by the toxic effect of a mitotoxin FGF2-SAP. These results suggest that FGF2 could participate in the autocrine regulation of HCEC proliferation and survival. The synergy between exogenously added FGF2 and TGF beta demonstrates that a combination of different growth factors may be important to stimulate proliferation of these cells in vivo.

Adult↗
PubMed1995

Corneal wound healing modulation using basic fibroblast growth factor after excimer laser photorefractive keratectomy.

Photorefractive keratectomy with the 193-nm excimer laser is one of the most promising innovations in refractive surgery. However, routine clinical application is hindered by two main obstacles, i.e., postoperative subepithelial opacity and possible regression of the refractive result. Because we have previously demonstrated beneficial effects of basic fibroblast growth factor (bFGF) on corneal epithelial healing in different in vivo models, we investigated the influence on both epithelial and stromal healing of topical bFGF after excimer laser keratomileusis in a rabbit model. After 7-mm circular deepithelialization, the eyes of 24 New Zealand White rabbits received identical deep stromal laser ablations (depth 50 microns, 6 D, diameter 5 mm) and were randomly assigned to one of four treatment groups [control (phosphate-buffered saline, PBS), bFGF 10 micrograms/application, dexamethasone 0.1%, bFGF + dexamethasone, n = 12 eyes/group]. All treatments were administered four times daily--bFGF until complete epithelial healing, dexamethasone and PBS until 3 months postsurgery. Wound surface regression on time was determined by means of computer-assisted image analysis of fluorescein-stained corneas. Corneal opacity was observed biomicroscopically and graded using a previously established scoring system. After bFGF application for 2-3 days only, a highly significant acceleration in epithelial wound healing speed was found compared with the rate of the other three treatment groups (p < 0.001). A combined therapy (bFGF + steroid) had no effect on the healing rate. Compared with control values, mean scores for subepithelial haze in the other groups were found to be nearly 50% lower during the first 2 postoperative months.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Characterisation of eye-lens DNases: long term persistence of activity in post apoptotic lens fibre cells.

Fibre cells in the ocular lens exhibit a constitutive apoptotic process of nuclear degradation that includes chromatin breakage, generating a ladder pattern of DNA fragments. This process is intrinsic to the normal terminal differentiation program. Despite the loss of nucleus and cytoplasmic organelles, the terminal differentiated fibre cells remain in the lens during the whole life span of the individual. The lens cells thus provide a unique system in which to determine the presence and fate of endonucleases once the chromatin has been cleaved. We report here on the presence of DNase activity in nucleated and anucleated lens cells. Using a nuclease gel assay and double-stranded DNA as substrate, we found active 30 and 60 kDa DNases. The enzymatic activities were Ca(2+), Mg(2+) dependent, and active at neutral pH. The relative amount of these forms changed during development and aging of the lens fibre cells. Both forms were inhibited by Zn(2+), aurintricarboxylic acid, and G-actin. The proteins were also separated by SDS-PAGE, renatured after removing SDS and incubated in the presence of native DNA adsorbed to a membrane. Therefore it was possible to demonstrate, by means of a nick translation reaction, that the enzymes produced single strand cuts. Based on these findings we propose that these chick lens nucleases are probably related to DNase I.

Journal Article↗
PubMed1995

Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

Animals↗
PubMed1995

In vivo inhibition of lens regrowth by fibroblast growth factor 2-saporin.

PURPOSE: To investigate the ability of fibroblast growth factor (FGF) 2-saporin to prevent lens regrowth in the rabbit. METHODS: Chemically conjugated and genetically fused FGF2-saporin (made in Escherichia coli) were used. Extracapsular extraction of the lens was performed on the rabbit, and the cytotoxin either was injected directly into the capsule bag or was administered by FGF2-saporin-coated, heparin surface-modified (HSM) polymethylmethacrylate intraocular lenses. The potential of the conjugate was checked by slit lamp evaluation of capsular opacification and by measuring crystallin synthesis. Toxin diffusion and sites of toxin binding were assessed by immunohistochemistry. Possible toxicity was determined by histologic analysis of ocular tissues. RESULTS: FGF2-saporin effectively inhibited lens regrowth when it was injected directly into the capsular bag. However, high concentration of the toxin induced transient corneal edema and loss of pigment in the iris. Intraocular lenses coated with FGF2-saporin reduced lens regrowth and crystallin synthesis without any detectable clinical side effect. After implantation, FGF2-saporin was shown to have bound to the capsules and, to a lesser extent, to the iris; no histologic damage was found on ocular tissues as a result of implantation of drug-loaded HSM intraocular lenses. CONCLUSIONS: Chemically conjugated (FGF2-SAP) and genetically fused FGF2-saporin (rFGF2-SAP) bound to HSM intraocular lenses can prevent lens regrowth in the rabbit.

Animals↗
PubMed1994

Multiple deletions in mitochondrial DNA are present in senescent mouse brain.

We report for the first time that multiple deletions occur during ageing of mice brain mitochondrial DNA (mtDNA). Deletions were detected by electrophoresis after amplification using the nested Polymerase Chain Reaction (N-PCR) method. Three mutant mtDNAs with 3726-, 3867- and 4236-bp deletions were directly detected by N-PCR which were undetectable in young brain mice. Each deletion was sequenced: in the region containing the junction fragment three different repeats of 13, 14 and 15 bp were observed. These results support the hypothesis that direct repeats are an initiating factor involved in ageing-associated accumulation of deletion.

Aging↗