No description available.

No description available.

No description available.

No description available.

Swelling-activated Cl- current (ICl,swell) is an outwardly rectifying Cl- current that influences cardiac electric activities and acts as a potential effector of mechanoelectrical feedback that antagonizes the effects of stretch-activated cation channels. Persistent activation of ICl,swell has been observed in multiple models of cardiovascular diseases. Previously we showed that angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are involved in the activation of ICl, swell by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ETA receptor blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell and the relationship between AngII and ET-1 signaling. Under isosmotic conditions, ET-1 elicited an outwardly rectifying Cl- current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB and by osmotic shrinkage. Selective ETA blockade (BQ123), but not ETB blockade (BQ788), fully suppressed the ET-1-induced current. ET-1-induced ICl,swell was abolished by blockade of EGFR kinase (AG1478) and PI-3K inhibitors (LY294002 and wortmannin), which also suppress beta1-integrin stretch- and swelling-induced ICl,swell. ET-1-induced ICl,swell was abrogated by ebselen, a membrane-permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O, suggesting that ROS were required intermediates in ET-1-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin or mitochondrial complex I with rotenone both completely suppressed ET-1-induced ROS generation and activation of ICl,swell, indicating that ROS from both NOX and mitochondria were required to activate ICl,swell, and complete block by inhibitors of either ROS source suggests mitochondrial and NOX must act in series rather than in parallel. ICl,swell elicited by antimycin A, which stimulates superoxide production by mitochondrial complex III, was insensitive to NOX inhibitor apocynin and the NOX fusion peptide inhibitor gp91ds-tat. Activation of ICl,swell induced by diazoxide, which stimulates mitochondrial ROS production by opening mitochondrial KATP channels, was not affected by gp91ds-tat. These data suggests that mitochondrial ROS is downstream from NOX in the regulation of ICl,swell. Mitochondrial ROS production that is enhanced by NOX ROS is likely to be responsible for the activation of ICl,swell by ET-1. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors (PD 98059 and U0216) on the activation of ICl,swell elicited by ET-1, EGF, and H2O2. ERK inhibitors partially blocked ET-1-induced ICl,swell but fully inhibited activation of ICl,swell in response to EGF. However, ERK inhibitors did not affect ICl,swell elicited by exogenous H2O2. We also established the the relationship of ET-1 to AngII and osmotic swelling in the regulation of ET-1 ICl,swell. ETA blockade abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade did not effect ET-1-induced ICl,swell, suggesting that ET-1 signaling is downstream from AngII and osmotic swelling. HL-1 cell is a murine atrial cell line that retain phenotypic characteristics of adult cardiomyocytes. We showed that osmotic swelling and ET-1 turned on DCPIB-sensitive outwardly rectifying Cl- current in HL-1 cells with both physiological and symmetrical Cl- gradients. The swelling-induced current was suppressed by gp91ds-tat and rotenone but insensitive to apocynin. Blockade of ETA receptor (BQ123) and NOX (gp91ds-tat) completely inhibited ET-1-induced ICl,swell in HL-1 cells. These data indicate that ICl,swell is present in HL-1 cell and regulated by similar mechanisms as in native cells. Finally, we confirmed the production of ROS by ET-1 signaling by flow cytometry of HL-1 cells using the nominally H2O2-selective fluorescent probe C H2DCFDA-AM. Exposure to ET-1 increased ROS production, as did H2O2, a positive control. ET-1-induced ROS production was fully suppressed by both gp91ds-tat and rotenone. HL-1 cell ROS production also was stimulated by the mitochondrial complex III inhibitor antimycin A, and antimycin A-induced ROS production was blocked by rotenone but not by gp91ds-tat. These data suggest that ET-1 ETA receptor signaling elicits ICl,swell by sequentially stimulating ROS production by NOX and mitochondria. ETA receptor signaling is down stream from AngII in the osmotic swelling-induced activation of ICl,swell and is upstream from EGFR kinase and PI-3K. Endothelin signaling is likely to be an important means of activating ROS production and ICl,swell in a variety of cardiovascular diseases.

Diabetic nephropathy is the main cause for initiation of renal replacement therapy and early symptoms in patients include increased glomerular filtration rate (GFR), decreased oxygen tension and albuminuria, followed by a progressive decline in GFR and loss of kidney function. Experimental models of diabetes display increased GFR, decreased tissue oxygenation and nitric oxide bioavailability. These findings are likely to be intertwined in a mechanistic pathway to kidney damage and this thesis investigated their roles in the development of diabetic nephropathy. In vivo, diabetes-induced oxidative stress stimulates renal tubular Na+ transport and in vitro, proximal tubular cells from diabetic rats display increased transport-dependent oxygen consumption, demonstrating mechanisms contributing to decreased kidney oxygenation. In control animals, endogenous adenosine reduces vascular resistance of the efferent arteriole via adenosine A2-receptors resulting in reduced filtration fraction. However, in diabetes, adenosine A2-signalling is dysfunctional resulting in increased GFR via increased filtration fraction. This is caused by reduced adenosine A2a receptor-mediated vasodilation of efferent arterioles. The lack of adenosine-signaling in diabetes is likely due to reduced local adenosine concentration since adenosine A2a receptor activation reduced GFR only in diabetic animals by efferent arteriolar vasodilation. Furthermore, sub-optimal insulin treatment also alleviates increased filtration pressure in diabetes. However, this does not affect GFR due to a simultaneously induction of renal-blood flow dependent regulation of GFR by increasing the filtration coefficient. In diabetes, there is decreased bioavailability of nitric oxide, resulting in alterations that may contribute to diabetes-induced hyperfiltration and decreased oxygenation. Interestingly, increased plasma concentration of l-arginine, the substrate for nitric oxide production, prevents the development of increased GFR and proteinuria, but not increased oxygen consumption leading to sustained intra-renal hypoxia in diabetes. This thesis concludes that antioxidant treatment directed towards the NADPH oxidase as well maneuvers to promote nitric oxide production is beneficial in diabetic kidneys but is targeting different pathways i.e. transport-dependent oxygen consumption in the proximal tubule by NADPH oxidase inhibition and intra-renal hemodynamics after increased plasma l-arginine. Also, the involvement and importance of efferent arteriolar resistance in the development of diabetes-induced hyperfiltration via reduced adenosine A2a signaling is highlighted.

No description available.

Le TRALI représente un œdème pulmonaire lésionnel aigu survenant au cours d’une transfusion. Son mécanisme étiopathologique encore très imprécis conduit aujourd’hui à une sous-estimation de son incidence. Des études clinico-anatomiques ont souligné le rôle central des polynucléaires neutrophiles (PMN) en montrant que le TRALI résulte de l’accumulation de PMN au contact de l’endothélium lésé des capillaires pulmonaires. De nombreux investigateurs ont tenté de définir le facteur déclenchant présent dans le produit sanguin transfusé et évoquèrent l’existence d’un conflit immunologique par infusion d’anticorps anti-HLA. En appui avec les données de la littérature, ce travail a pour but d’apporter une meilleure connaissance du mécanisme du TRALI afin d’en améliorer son diagnostic, sa prévention et la prise en charge du patient. Tout d’abord, nous confirmons l’implication des anticorps anti-HLA dans la survenue du TRALI en validant pour la première fois l’hypothèse du modèle en deux étapes: une première étape préalable est requise chez le patient présentant une situation clinique ou thérapeutique prédisposante qui aboutit à une pré-stimulation des PMN, puis une seconde étape, dépendante de l’apport d’anticorps anti-HLA lors de la transfusion, entraîne l’activation de la NADPH oxydase phagocytaire. Cela conduit à l’activation des PMN et la libération de dérivés réactifs de l’oxygène qui sont directement responsables de la lésion endothéliale pulmonaire et provoque une augmentation de la perméabilité endothéliale. Nous démontrons en plus l’existence d’un seuil d’anticorps anti-HLA nécessaire pour déclencher une forte activation des PMN. Enfin, nous avons mis en évidence un mécanisme d’activation des PMN par les anticorps anti-HLA faisant intervenir la formation de complexes immuns antigène – anticorps à la surface des PMN. Ces complexes immuns sont reconnus avec une affinité plus grande que les anticorps seuls par les récepteurs Fc des PMN. Cette double interaction au sein d’un même PMN pourrait favoriser la formation de cluster de récepteurs Fc activés au niveau de radeaux lipidiques, ce qui induirait une activation optimisée de ces récepteurs, entraînant une cascade de signalisation aboutissant à l’activation de la NADPH oxydase des PMN. Nos résultats constituent un rationnel scientifique solide pour accéder à une meilleure connaissance du TRALI. / TRALI represents an acute non-cardiogenic pulmonary oedema following blood transfusion. The unknown etiopathological mechanism of TRALI leads to an underestimation of the incidence. Clinical and anatomical studies highlighted the major role of neutrophils (PMN) and showed that TRALI results from an increased number of neutrophils within the pulmonary capillary endothelium. Many evidence suggest that antibodies recognizing human leukocyte antigens (HLA) present in the blood transfusion are the predominant trigger leading to TRALI. Towards theses findings, we investigated the precise mechanism in TRALI in order to get a better knowledge of its diagnosis, its prevention and the patient care. We confirm the major role of anti-HLA antibodies and validate for the first time the two-hit model: the first-hit related to the patient clinical condition leads to their PMN stimulation, followed in the second-hit by the infusion of blood products containing anti-HLA antibodies that activate the phagocytic NADPH oxidase. This event induces PMN activation and the release of reactive oxygen species that are directly responsible for the pulmonary endothelial damage and cause the endothelial permeability increase. We also demonstrate the cut-off of anti-HLA antibodies that raises PMN activation. Finally, we showed that both the antigen-binding and the Fc-binding systems to antibodies are needed to induce a major PMN activation. We found that the binding of anti-HLA antibodies to HLA antigens promote the formation of cluster of Fc receptors within lipid rafts. The translocation of Fc receptors into lipid rafts improve Fc receptors activation, leading to intracellular signal transduction and activation of effector functions, such as NADPH oxidase activation and release of reactive oxygen species involved in tissue damage.

La Polyarthrite Rhumatoïde est caractérisée par une synovite à l’origine de lésions progressives ostéo-articulaires induites par les formes réactives de l’oxygène (ROS) produites par la NADPH oxydase des polynucléaires neutrophiles (PMN). La NADPH oxydase des phagocytes, est formée d’un centre catalytique membranaire, le cytochrome b558, sur lequel vient s’associer des protéines cytosoliques régulatrices (p67phox, p47phox, p40phox et Rac1/2). Nous avons étudié la spécificité de l’interaction entre la (6-phosphofructokinase 2) et de la 6PGDH (6-phosphogluconate déshydrogénase) et la NADPH oxydase des PMN. D’autre part, nous avons caractérisé les domaines de l’hétérocomplexe S100A8/A9 impliqués dans l’activation de la NADPH oxydase phagocytaire. Par ailleurs, une étude de la signature protéique dans le liquide synovial a été menée afin de rechercher l’empreinte de l’activation du PMN dans la PR.Après stimulation par le PMA, la 6PGDH et la PFK2 co-imunoprécipitent avec les facteurs cytosoliques p67phox, p47phox and p40phox. Les expériences de microscopie confocale suggèrent une co-localisation de ces deux enzymes du métabolisme du glucose avec la NADPH oxydase, dans des micro-domaines membranaires : les radeaux lipidiques. La 6PGDH est impliquée dans l’activation de la NADPH oxydase phagocytaire en élevant la concentration du NADPH cytosolique mais également en augmentant l’affinité de cette enzyme pour son substrat, le NADPH. PFK2 est l’enzyme majeure de la régulation de la glycolyse, voie est essentielle pour la production d’ATP du PMN. L’utilisation du complexe S100A8/A9 et de protéines chimères de fusion nous a permis de révéler que la partie C-terminale de S100A8 est impliquée dans la liaison avec le cytochrome b558 et l’activation de la NADPH oxydase phagocytaire. In vivo, le profil protéique du liquide articulaire de PR a révélé l’empreinte de l’activation du PMN dans cette pathologie avec une surexpression des protéines S100A8 et S100A9. Une production ectopique de S100A8/A9 par les synoviocytes de type fibroblastique a été mise en évidence.En conclusion, la 6PGDH, la PFK2 et l’hétérodimère S100A8/A9 sont de nouveaux partenaires d’activation de la NADPH oxydase des phagocytes. Dans la PR, l’activation des PMNs conduit à la sécrétion de S100A8/A9 qui semblent constituer à la fois des biomarqueurs pertinents, mais également des cibles thérapeutiques potentielles. / Rheumatoid Arthritis (RA) is caused by an inflammation of the synovial membrane leading to progressive joint destruction and deformation, related to the production NADPH oxidase related-reactive oxygen species (ROS) production. The phagocyte NADPH oxidase is a multi-protein complex formed by a catalytic core, i.e. the transmembrane cytochrome b558 and cytosolic regulators (p67phox, p47phox, p40phox and Rac1/2). We aimed at better analyzing the NADPH oxidase activation through the evaluation of the specificity of the interaction with 6PGDH or PFK2 and through the further analysis of the association with the S100A8/A9 heterocomplex. The RA-specific protein profiling was conducted in order to determine whether a PMN activation fingerprint could be revealed among RA specific proteins.Upon PMA stimulation, both 6PGDH and PFK2 co-imunoprecipitated with cytosolic factors p67phox, p47phox and p40phox. At the plasma membrane level, confocal microscopy experiments suggested a co-localization of either 6PGDH or PFK2 with the phagocyte NADPH oxidase in lipid rafts. 6PGDH enhanced the phagocyte NADPH oxidase activity by both improving the availability of cytosolic NADPH content and by increasing the affinity of the NADPH oxidase for its substrate. PFK2 also augmented the NADPH oxidase activity. PFK2 modulated the ATP concentration available for the phosphorylation of the phagocyte NADPH oxidase components and for the NDP Kinase related-Rac activation. The generation of truncated S100A8/S100A9 heterodimer chimera could reveal that the C-terminal region of S100A8 is involved in both the interaction and the activation of the phagocyte NADPH oxidase.In vivo, synovial fluid of RA patients was remarkably labelled with the PMN activation fingerprint. S100A8 and S100A9 proteins clearly distinguished RA synovial fluid from osteoarthritis and non RA-synovial fluids. An ectopic production of S100A8/S100A9 was shown in RA fibroblast like synoviocyte.In conclusion, 6PGDH, PFK2 and S100A8/A9 proteins are surrogate activating partners of the phagocyte NADPH oxidase. In RA, the activation of PMNs leads to the release of S100A8/A9 proteins which may constitute interesting biomarkers and promising therapeutic targets.

La NADPH oxydase des phagocytes (NOX2) est responsable de la production d’anions superoxydes qui sont les précurseurs des autres formes réactives de l’oxygène. NOX2 est une enzyme majeure de la réponse immunitaire. Les dysfonctionnements de NOX2 sont associés à de nombreuses pathologies et donc il convient d’en comprendre les détails de la régulation. Cette oxydase est composée de cinq sous-unités : deux protéines membranaires, gp91phox et p22phox et 3 protéines cytosoliques p47phox, p67phox et p40phox. D’après les études in vitro avec des protéines purifiées, les protéines cytosoliques sont supposées former un complexe ternaire qui se déplace à la membrane avec une petite protéine G, Rac, au moment l’activation.L’objectif de ce projet est de caractériser les interactions spécifiques entre les sous-unités cytosoliques de NOX2 en cellules vivantes en utilisant le phénomène de transfert résonant d’énergie de type Förster (FRET) entre deux fluorophores, un donneur et un accepteur. Ici les fluorophores seront des protéines fluorescentes de la famille de la GFP. Elles sont fusionnées à deux sous-unités. L’efficacité du FRET dépend de la distance entre les fluorophores et permet ainsi de caractériser les interactions entre les protéines d’intérêt. Une méthode rapide d’identification des situations où le FRET est positif a été mise au point par cytométrie en flux. Des études détaillées et quantitatives ont ensuite été réalisées en utilisant l’imagerie de durée de fluorescence (FLIM) du donneur. Le FLIM, combiné à l’utilisation de donneurs présentant une durée de vie mono-exponentielle, permet de déterminer directement des efficacités de FRET apparentes et moléculaires, qui contiennent, toutes les deux, des informations qualitatives et quantitatives sur l’interaction et la structure des protéines impliquées. De ces données, il est possible d’extraire la fraction des donneurs interagissant avec un accepteur. Les informations obtenues à partir des données de FRET-FLIM permettent une meilleure compréhension de l’organisation et de la régulation de NOX2 tout en permettant une estimation des constantes de dissociation (Kd). Afin de confirmer ces résultats, des expériences de spectroscopie de corrélation de fluorescence à deux couleurs (FCCS) ont été réalisées. Cette méthode complétement indépendante n’est pas basée sur la distance entre fluorophores comme le FRET mais sur leur co-diffusion à travers un petit volume d’observation dans le cytoplasme cellulaire.L’approche FRET-FLIM nous a tout d’abord permis d’observer les interactions entre hétéro-dimères formés de deux sous-unites différentes en cellules vivantes et d’exclure la formation d’homo-dimères entre deux sous-unités identiques. Etant donné la bonne précision des mesures de FLIM, nous avons pu comparer les informations structurales obtenues en cellules avec les données structurales issues d’études sur les protéines purifiées in vitro et nous avons constaté qu’elles sont en bon accord. Nous avons ensuite aligné les structures disponibles pour proposer un premier modèle 3D du complexe cytosolique de la NADPH oxydase au repos dans le cytosol cellulaire.Les fractions de protéines en interaction sont pour tous les hétéro-dimères autour de 20% ce qui n’est pas en accord avec l’hypothèse courante qui propose que toutes les sous-unités cytosoliques soient sous forme de complexe. Toutefois nos premiers résultats de FCCS confirment ce résultat extrait des données de FRET-FLIM. Nous proposons donc que la complexation des sous-unités cytosolique pourrait être impliquée dans la régulation de la NADPH oxydase. Des études complémentaires seront nécessaires pour valider cette nouvelle hypothèse. Les constantes de dissociation Kd estimées à partir de nos résultats sont micromolaires et donc un ordre de grandeur plus élevé que les valeurs nanomolaires déterminées in vitro. Des mesures plus détaillées de FCCS pourront compléter et valider ces résultats. / The phagocyte NADPH oxidase (NOX2) is a key enzyme of the immune system generating superoxide anions, which are precursors for other reactive oxygen species. Any dysfunctions of NOX2 are associated with a plethora of diseases and thus detailed knowledge about its regulation is needed. This oxidase is composed of five subunits, the membrane-bound gp91phox and p22phox and the cytosolic p47phox, p67phox, and p40phox. The latter are assumed to be in a ternary complex that translocates together with the small GTPase Rac to the membranous subunits during activation.Our aim was to discover and to characterize specific interactions of the cytosolic subunits of NOX2 in live cells using a Förster Resonance Energy Transfer (FRET) based approach: Since FRET depends on the distance between two fluorophores, it can be used to reveal protein-protein interactions non-invasively by studying fluorescent protein tagged subunits. To have a rapid method on hand to reveal specific interactions, a flow cytometer based FRET approach was developed. For more detailed studies, FRET was measured by fluorescence lifetime imaging microscopy (FLIM), because it allows a direct determination of the apparent and molecular FRET efficiency, which contains both qualitative and quantitative information about the interaction and the structure of the interacting proteins. Furthermore, the FRET-FLIM approach enables an estimation of the fraction of bound donor. This information itself is important for a better understanding of the organisation and regulation of the NOX2, but it is also necessary for the calculation of the dissociation constant Kd from the FRET-FLIM data. To confirm the findings obtained by FRET-FLIM fluorescence cross correlation spectroscopy (FCCS) experiments were performed. This completely independent method is not based on distances like FRET but on the observation of the co diffusion of the fluorescently labelled samples when they move across a small observation volume inside the cells.The FRET-FLIM approach allowed us in a first step to discover heterodimeric interactions between all cytosolic subunits in live cells. Due to the good precision of the results, we were able to extract structural information about the interactions and to compare them with available structural data obtained from in vitro studies. The information from FRET-FLIM was coherent with in vitro data. We then aligned the available structures leading to the first 3D model of the cytosolic complex of the NADPH oxidase in the resting state in live cells.Additionally, the bound fraction for all heterodimeric interactions derived by FRET-FLIM is around 20 %, which is in contrast to the general belief that all cytosolic subunits are bound in complex. The first FCCS results support our findings. Therefore, we believe that the complexation of the cytosolic subunits could be involved in the regulation of the NADPH oxidase and should be investigated further. The estimated Kd derived from the FRET-FLIM approach is in the low micomolar range, which is an order of a magnitude higher than the nanomolar range of in vitro studies.In conclusion, we showed that our quantitative FRET-FLIM approach is not only able to distinguish between specific and unspecific protein-protein interactions, but gives also information about the structural organisation of the interacting proteins. The high precision of the FRET-FLIM data allow the determination of the bound fraction and an estimation of the Kd in live cells. FCCS is a complementary method, which can verify these quantitative findings. However, it cannot replace FRET-FLIM completely as it does not give any structural information.With respect to the biological outcome of this project, we can propose for the first time a 3D-model of the cytosolic complex of the NADPH oxidase covering the in vitro as well as the live cell situation. Additionally, the small bound fraction found here may raise new ideas on the regulation of this vital enzyme.