Sint-Pieters-Voeren, Belgium
Sint-Pieters-Voeren, Belgium

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Libert S.,Laboratory of Pathophysiological and Nutritional Biochemistry | Willermain F.,I.R.I.B.H.M | Weber C.,Laboratory of Pathophysiological and Nutritional Biochemistry | Bryla A.,Laboratory of Pathophysiological and Nutritional Biochemistry | And 6 more authors.
Molecular Vision | Year: 2016

Purpose: Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation. Methods: ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites. Results: In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abol­ished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase’s isoforms (p38α and p38β) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation. Conclusions: Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema. © 2016 Molecular Vision.


PubMed | Laboratory of Pathophysiological and Nutritional Biochemistry, CHU Saint Pierre and Brugmann and I.R.I.B.H.M.
Type: | Journal: Molecular vision | Year: 2016

Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation.ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites.In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinases isoforms (p38 and p38) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation.Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.


Motulsky E.,Free University of Colombia | Koch P.,CHU Saint Pierre and Brugmann | Koch P.,Free University of Colombia | Janssens S.,Free University of Colombia | And 10 more authors.
Molecular Vision | Year: 2010

Purpose: Blood-retinal barrier (BRB) breakdown and retinal edema are major complications of autoimmune uveitis and could be related to deregulation of aquaporin (AQP) expression. We have therefore evaluated the expression of AQP1 and AQP4 on BRB cells during experimental autoimmune uveitis (EAU) in mice. Methods: C57Bl6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 1-16. The disease was graded clinically, and double immunolabeling using glial fibrillary acidic protein (GFAP; a marker of disease activity) and AQP1 or AQP4 antibodies was performed at day 28. AQP1 expression was also investigated in mouse retinal pigment epithelium (RPE) cells (B6-RPE07 cell line) by reverse transcriptase PCR and western blot under basal and tumor necrosis factor α (TNF-α)-stimulated conditions. Results: In both normal and EAU retina, AQP1 and AQP4 expression were restricted to the photoreceptor layer and to the Müller cells, respectively. Retinal endothelial cells never expressed AQP1. In vasculitis and intraretinal inflammatory infiltrates, decreased AQP1 expression was observed due to the loss of photoreceptors and the characteristic radial labeling of AQP4 was lost. On the other hand, no AQP4 expression was detected in RPE cells. AQP1 was strongly expressed by choroidal endothelial cells, rendering difficult the evaluation of AQP1 expression by RPE cells in vivo. No major differences were found between EAU and controls at this level. Interestingly, B6-RPE07 cells expressed AQP1 in vitro, and TNF-α downregulated AQP1 protein expression in those cells. Conclusions: Changes in retinal expression of AQP1 and AQP4 during EAU were primarily due to inflammatory lesions, contrasting with major modulation of AQP expression in BRB detected in other models of BRB breakdown. However, our data showed that TNF-α treatment strongly modulates AQP1 expression in B6-RPE07 cells in vitro. © 2010 Molecular Vision.


Dewispelaere R.,M.R.Research | Lipski D.,M.R.Research | Foucart V.,M.R.Research | Bruyns C.,M.R.Research | And 3 more authors.
Experimental Eye Research | Year: 2015

Adhesion molecules play a central role in leukocyte adhesion to the blood-retinal barrier (BRB) during uveitis. VCAM-1 expression on the BRB has been already described but although structurally similar, ICAM-1 has shown in various autoimmunity models to have distinct role and expression. Here, we induced uveitis in C57Bl/6 mice by adoptive transfer of semi-purified T cells from IRBP1-20-immunized mice. Using Flow cytometry analysis on transferred cells and immunofluorescence staining on retina we have studied the comparative ocular expression of both ICAM-1 and VCAM-1 and their ligands LFA-1 and VLA-4 at the surface of uveitogenic cells. Our results showed that LFA-1 and VLA-4 are expressed on both T and non T cells, VLA-4 sparsely and LFA-1 ubiquitously. Considering retinal expression, ICAM-1 is faintly present and VCAM-1 is absent in naive eyes. Only ICAM-1 is present on infiltrating cells in the retina and vitreous, while only VCAM-1 extends to perivascular glial cells and all along the internal limiting membrane. Finally, ICAM-1 is strongly expressed on the RPE, where VCAM-1 expression is much weaker. VCAM-1 seems most strongly expressed on the internal BRB while ICAM-1 predominates on the external BRB. Those major differences in the expression pattern could represent differential entry pathways for inflammatory cells to penetrate the eye. © 2015 Elsevier Ltd.


PubMed | CHU Saint Pierre and Brugmann and M.R.Research
Type: | Journal: Experimental eye research | Year: 2015

Adhesion molecules play a central role in leukocyte adhesion to the blood-retinal barrier (BRB) during uveitis. VCAM-1 expression on the BRB has been already described but although structurally similar, ICAM-1 has shown in various autoimmunity models to have distinct role and expression. Here, we induced uveitis in C57Bl/6 mice by adoptive transfer of semi-purified T cells from IRBP1-20-immunized mice. Using Flow cytometry analysis on transferred cells and immunofluorescence staining on retina we have studied the comparative ocular expression of both ICAM-1 and VCAM-1 and their ligands LFA-1 and VLA-4 at the surface of uveitogenic cells. Our results showed that LFA-1 and VLA-4 are expressed on both T and non T cells, VLA-4 sparsely and LFA-1 ubiquitously. Considering retinal expression, ICAM-1 is faintly present and VCAM-1 is absent in naive eyes. Only ICAM-1 is present on infiltrating cells in the retina and vitreous, while only VCAM-1 extends to perivascular glial cells and all along the internal limiting membrane. Finally, ICAM-1 is strongly expressed on the RPE, where VCAM-1 expression is much weaker. VCAM-1 seems most strongly expressed on the internal BRB while ICAM-1 predominates on the external BRB. Those major differences in the expression pattern could represent differential entry pathways for inflammatory cells to penetrate the eye.


Salik D.,Free University of Colombia | Motulsky E.,Free University of Colombia | Gregoire F.,Free University of Colombia | Delforge V.,Free University of Colombia | And 5 more authors.
Acta Ophthalmologica | Year: 2016

Purpose The goal of this study was to investigate the modifications of aquaporin (AQP) expression in ARPE-19 cells in response to fenretinide-induced transdifferentiation into neuronal-like cells Methods ARPE-19 cells were treated daily for 7 days with 3 μm fenretinide or dimethyl sulphoxide as control. mRNA and protein expression were evaluated by real-time quantitative PCR, Western blot analysis and immunofluorescence. Results Control ARPE-19 cells expressed AQP1, AQP4, AQP6 and AQP11 at the mRNA level, but only AQP4, AQP6 and AQP11 at the protein level. Fenretinide induced the transdifferentiation of ARPE-19 cells into neuronal-like cells. Indeed, fenretinide induced morphological changes similar to neurons characterized by elongated cell body and the formation of neurite branching. Moreover, ARPE-19 cells transdifferentiated to neuron-like cells were characterized by significant decrease in retinal pigmented epithelium markers, for example cytokeratin 8 and cellular retinaldehyde-binding protein, as well as an increase in neuronal markers such as synaptophysin and calretinin. AQP4 expression, at both mRNA and protein levels, and AQP6 expression, only at protein level, were significantly decreased in ARPE-19 cells transdifferentiated into neuronal-like cells. Conclusions The expression of AQP4 and AQP6 is downregulated during fenretinide-induced transdifferentiation. © 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.


Willermain F.,CHU Saint Pierre and Brugmann | Willermain F.,Free University of Colombia | Libert S.,CHU Saint Pierre and Brugmann | Libert S.,Free University of Colombia | And 7 more authors.
Frontiers in Physiology | Year: 2014

The retinal pigmented epithelium (RPE) is composed of retinal pigmented epithelial cells joined by tight junctions and represents the outer blood-retinal barrier (BRB). The inner BRB is made of endothelial cells joined by tight junctions and glial extensions surrounding all the retinal blood vessels. One of the functions of the RPE is to maintain an osmotic transepithelial gradient created by ionic pumps and channels, avoiding paracellular flux. Under such physiological conditions, transcellular water movement follows the osmotic gradient and flows normally from the retina to the choroid through the RPE. Several diseases, such as diabetic retinopathy, are characterized by the BRB breakdown leading to leakage of solutes, proteins, and fluid from the retina and the choroid. The prevailing hypothesis explaining macular edema formation during diabetic retinopathy incriminates the inner BRB breakdown resulting in increased osmotic pressure leading in turn to massive water accumulation that can affect vision. Under these conditions, it has been hypothesized that RPE is likely to be exposed to hyperosmolar stress at its apical side. This review summarizes the origins and consequences of osmotic stress in the RPE. Ongoing and further research advances will clarify the mechanisms, at the molecular level, involved in the response of the RPE to osmotic stress and delineate potential novel therapeutic targets and tools. © 2014 Willermain, Libert, Motulsky, Salik, Caspers, Perret and Delporte.


PubMed | CHU Saint Pierre and Brugmann and Free University of Colombia
Type: Journal Article | Journal: Acta ophthalmologica | Year: 2016

The goal of this study was to investigate the modifications of aquaporin (AQP) expression in ARPE-19 cells in response to fenretinide-induced transdifferentiation into neuronal-like cellsARPE-19 cells were treated daily for 7 days with 3 m fenretinide or dimethyl sulphoxide as control. mRNA and protein expression were evaluated by real-time quantitative PCR, Western blot analysis and immunofluorescence.Control ARPE-19 cells expressed AQP1, AQP4, AQP6 and AQP11 at the mRNA level, but only AQP4, AQP6 and AQP11 at the protein level. Fenretinide induced the transdifferentiation of ARPE-19 cells into neuronal-like cells. Indeed, fenretinide induced morphological changes similar to neurons characterized by elongated cell body and the formation of neurite branching. Moreover, ARPE-19 cells transdifferentiated to neuron-like cells were characterized by significant decrease in retinal pigmented epithelium markers, for example cytokeratin 8 and cellular retinaldehyde-binding protein, as well as an increase in neuronal markers such as synaptophysin and calretinin. AQP4 expression, at both mRNA and protein levels, and AQP6 expression, only at protein level, were significantly decreased in ARPE-19 cells transdifferentiated into neuronal-like cells.The expression of AQP4 and AQP6 is downregulated during fenretinide-induced transdifferentiation.


Arsenijevic T.,Free University of Colombia | Vujovic A.,Free University of Colombia | Libert F.,Free University of Colombia | De Beeck A.O.,Free University of Colombia | And 9 more authors.
Cell Death and Disease | Year: 2013

Osmotic changes occur in many tissues and profoundly influence cell function. Herein, we investigated the effect of hyperosmotic stress on retinal pigmented epithelial (RPE) cells using a microarray approach. Upon 4-h exposure to 100mM NaCl or 200mM sucrose, 79 genes were downregulated and 72 upregulated. Three gene ontology categories were significantly modulated: cell proliferation, transcription from RNA polymerase II promoter and response to abiotic stimulus. Fluorescentactivated cell sorting analysis further demonstrated that owing to hyperosmotic stimulation for 24 h, cell count and cell proliferation, as well as the percentage of cells in G0/G1 and S phases were significantly decreased, whereas the percentage of cells in G2/M phases increased, and apoptosis and necrosis remained unaffected. Accordingly, hyperosmotic conditions induced a decrease of cyclin B1 and D1 expression, and an activation of the p38 mitogen-activated protein kinase. In conclusion, our results demonstrate that hypertonic conditions profoundly affect RPE cell gene transcription regulating cell proliferation by downregulation cyclin D1 and cyclin B1 protein expression. © 2013 Macmillan Publishers Limited All rights reserved.


Makhoul D.,CHU Saint Pierre and Brugmann | Kolyvras N.,CHU Saint Pierre and Brugmann | Benchekroun S.,CHU Saint Pierre and Brugmann | Willermain F.,CHU Saint Pierre and Brugmann | Caspers L.,CHU Saint Pierre and Brugmann
Ocular Immunology and Inflammation | Year: 2010

Objective: To report the case of a young boy, homozygous for the hemoglobin S, who presented a pseudouveitis in the setting of severe sickle cell retinopathy complicated by macular infarction. Methods: Case report. Results: A 15 year-old boy with a history of hypertensive uveitis of two months duration was reffered to our institution. He was treated with topical prednisolone acetate, beta-blockers and acetazolamide.The visual acuity was 20/200 RE and 20/25 LE. Anterior inflammation included fine inferior keratic precipitates with 2+ cells RE and 1+ cells LE. Vitreous haze was 2+ preventing clear vieuw of subretinal infiltrates scattered around the posterior pole and midperipheral retina, some of them having a salmon patch appearence. Fluorescein angiograms revealed multiple preretinal haemorrahge and some areas of retinal ischemia. Fundus examination of the left eye was normal. A diagnosis of panuveitis was done and a sickle cell retinopathy was suspected. Systemic workup showed an hemoglobin at 8,2 mg/dl and sickle cells on direct examination. Two days later he developped sudden loss of vision in the right eye. Funduscopy an angiogram revealed macular infarction with occlusion of the retinal arterioles surrounding the foveal avascular zone. The clinical picture was not improved by erythrocyte transfusion. Intraocular pressure raised again after few days, and was finally controlled by anterior chamber paracentesis. The patient was later found to be homozygous for HbS. Conclusions: Sickle cell retinopathy can rarely masquerade as panuveitis, and can lead to severe ocular complications as an irreversible macular ischemia. © 2010 Informa Healthcare USA, Inc.

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