Anne Vejux
Université de Bourgogne, UFR SVTE, Faculty Member
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ABSTRACT But Des mécanismes inflammatoires locaux sont mis en cause dans la genèse des lésions de dégénérescence maculaire liée à l’âge (DMLA). Les capacités pro inflammatoires des oxystérols ont ainsi été testées sur des cellules... more
ABSTRACT But Des mécanismes inflammatoires locaux sont mis en cause dans la genèse des lésions de dégénérescence maculaire liée à l’âge (DMLA). Les capacités pro inflammatoires des oxystérols ont ainsi été testées sur des cellules épithéliales pigmentaires en culture. La sécrétion d’IL-8, chémokine connue pour ses pouvoirs chimioattractif surles cellules de l’inflammation et pour son rôle pro-angiogénique a été particulièrement étudiée ainsi que les protéines impliquées dans le contrôle de sa sécrétion. Matériels et Méthodes Des cellules épithéliales pigmentaires en lignée (ARPE-19) ont été cultivées et traitées par différents oxystérols : le 7-cétocholestérol, le 7- betahydroxycholestérol et le 25-hydroxycholestérol (25-OH). Après 24 h et 48 h de traitement à différentes concentrations les surnageants étaient récupérés et un dosage de plusieurs cytokines a été réalisé d’une part avec une technique d’analyse multiplexée et d’autre part par méthode ELISA. L’implication de la voie des MAP-kinases dans la sécrétion d’IL-8 a été évaluée en utilisant des inhibiteurs spécifiques de cette voie U0126 et PD98059. Résultats Le dosage a été réalisé par cytométrie en flux avec analyse simultanée de la sécrétion de 6 cytokines (IL-8, IL-1ß, IL-6, IL-10, TNF- αet IL-12). Seule la sécrétion d’IL-8 a été identifiée sous l’influence du 25-OH. Le 7-céto et le 7- βn’induisent pas la sécrétion d’IL-8. Ceci a été confirmé par les dosages avec la méthode ELISA qui montrent que seul le 25-OH stimule la sécrétion d’IL-8. L’utilisation des inhibiteurs spécifiques de la voie des MAP-kinases diminue la sécrétion d’IL-8 induite par le 25-OH. Les valeurs mesurées sont alors comparables à celles obtenues sur le surnageant de cellules non traitées. Discussion L’IL-8 est la seule cytokine dont la sécrétion par les cellules ARPE-19 est stimulée et ce uniquement par le 25-OH. Cet oxystérol qui n’a par ailleurs aucun effet cytotoxique peut donc être considéré comme un promoteur de l’inflammation. La voie des MAP-kinases semble être une des voies de signalisation majeure impliquée dans le contrôle de la sécrétion d’IL-8 par les cellules ARPE-19. Conclusion Le 25-OH semble être un agent pro-inflammatoire qui favorise au travers de la sécrétion d’IL-8 le recrutement des cellules inflammatoires contribuant auxlésions observées dans la DMLA. Par ailleurs l’IL-8 possède aussi des propriétés pro-angiogèniques qui pourraient favoriser le développement des lésions néo vasculaires observées au cours de la DMLA.
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Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation... more
Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, referred to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7alpha-, 7beta-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O2*-) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O2*- overproduction quantified with hydroethidine was identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, with cholesterol-5alpha, 6alpha-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7beta-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.
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Ageing is accompanied by increasing vulnerability to major pathologies (atherosclerosis, Alzheimer's disease, age-related macular degeneration, cataract, and osteoporosis) which can have similar underlying pathoetiologies. All of... more
Ageing is accompanied by increasing vulnerability to major pathologies (atherosclerosis, Alzheimer's disease, age-related macular degeneration, cataract, and osteoporosis) which can have similar underlying pathoetiologies. All of these diseases involve oxidative stress, inflammation and/or cell death processes, which are triggered by cholesterol oxide derivatives, also named oxysterols. These oxidized lipids result either from spontaneous and/or enzymatic oxidation of cholesterol on the steroid nucleus or on the side chain. The ability of oxysterols to induce severe dysfunctions in organelles (especially mitochondria) plays key roles in RedOx homeostasis, inflammatory status, lipid metabolism, and in the control of cell death induction, which may at least in part contribute to explain the potential participation of these molecules in ageing processes and in age related diseases. As no efficient treatments are currently available for most of these diseases, which are predicted to...
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The development of cataract is associated with some lipid changes in human lens fibers, especially with increased accumulation and redistribution of cholesterol inside these cells. Some direct and indirect lines of evidence, also suggest... more
The development of cataract is associated with some lipid changes in human lens fibers, especially with increased accumulation and redistribution of cholesterol inside these cells. Some direct and indirect lines of evidence, also suggest an involvement of cholesterol oxide derivatives (also named oxysterols) in the development of cataract. Oxysterol formation can result either from nonenzymatic or enzymatic processes, and some oxysterols can induce a wide range of cytotoxic effects (overproduction of reactive oxygen species (ROS); phospholipidosis) which might contribute to the initiation and progression of cataract. Thus, the conception of molecules capable of regulating cholesterol homeostasia and oxysterol levels in human lens fibers can have some interests and constitute an alternative to surgery at least at early stages of the disease.
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In demyelinating or non-demyelinating neurodegenerative diseases, increased levels of 7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC) and 24(S)-hydroxycholesterol (24S-OHC) can be observed in brain lesions. In 158N murine... more
In demyelinating or non-demyelinating neurodegenerative diseases, increased levels of 7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC) and 24(S)-hydroxycholesterol (24S-OHC) can be observed in brain lesions. In 158N murine oligodendrocytes, 7KC triggers a complex mode of cell death defined as oxiapoptophagy, involving simultaneous oxidative stress, apoptosis and autophagy. In these cells, 7KC as well as 7β-OHC and 24S-OHC induce a decrease of cell proliferation evaluated by phase contrast microscopy, an alteration of mitochondrial activity quantified with the MTT test, an overproduction of reactive oxygen species revealed by staining with dihydroethidium and dihydrorhodamine 123, caspase-3 activation, PARP degradation, reduced expression of Bcl-2, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC, 7β-OHC and 24S-OHC promote conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II...
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Research Interests: Multiple sclerosis, Atherosclerosis, Phospholipids, Inflammation, Oxidative Stress, and 15 moreApoptosis, Cardiovascular disease, Macular Degeneration, Biological Sciences, Humans, Intact polar lipids, Cell Death, TNF, Age related macular degeneration, Neoplasms, LC, MAPK, Biological activity, Human Disease, and PARP
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Research Interests: Flow Cytometry, Enzyme Inhibitors, Transmission Electron Microscopy, Mitochondria, Fluorescent Dyes and Reagents, and 12 moreApoptosis, Multidisciplinary, Fluorescence Resonance Energy Transfer, Humans, Permeability, Cholesterol, Cell Fractionation, Lipid metabolism, Cell nucleus, Benzimidazoles, Subcellular Fractions, and Spectral Imaging
Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also... more
Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C-reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol), and various cytokines. Different flow cytometric bead-based assays were used to quantify some cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, IFN-gamma, MCP-1, MIP-1beta, or TNF-alpha) in the culture media of oxysterol-treated U937 and THP-1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods we...
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Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable,... more
Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, "adaptive" resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.