References of "Mouithys-Mickalad, Ange"
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See detailEffect of different kinds of anoxia/reoxygenation on the mitochondrial function and the free radicals production of cultured primary equine skeletal myoblasts.
Ceusters, Justine ULg; Mouithys-Mickalad, Ange ULg; Franck, Thierry ULg et al

in Research in Veterinary Science (2013), 95

Horses are outstanding athletes, performing in many different disciplines involving different kinds of efforts and metabolic responses. Depending on exercise intensity, their skeletal muscle oxygenation ... [more ▼]

Horses are outstanding athletes, performing in many different disciplines involving different kinds of efforts and metabolic responses. Depending on exercise intensity, their skeletal muscle oxygenation decreases, and the reperfusion at cessation of the exercise can cause excessive production of free radicals. This study on cultured primary equine myoblasts investigated the effect of different kinds of anoxia/reoxygenation (A/R) on routine respiration, mitochondrial complex I specific activity and free radicals production. Our data revealed that short cycles of A/R caused a decrease of all the parameters, opposite to what a single long period of anoxia did. A preconditioning-like effect could explain our first pattern of results whereas mild uncoupling could be more appropriate for the second one. Anyway, it seems that mitochondrial complex I could play a major role in the regulation of the balance between metabolic and antioxidant protection of the muscular function of athletic horses. [less ▲]

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See detailComplexe I mitochondrial dysfunction in HDAC5 depleted cancer cells induces glucose-dependent metabolic reprogrammation.
Hendrick, Elodie ULg; Matheus, Nicolas ULg; Peixoto, Paul ULg et al

Poster (2013, September 13)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes, which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes, which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad-spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that specific depletion of HDAC5 using siRNA technology reduced cancer cells proliferation and survival1. Aims: The goal of this study is to further understand the metabolic response of cancer cells to HDAC5 depletion. Results: Screening transcriptomic study demonstrated that HDAC5 depletion induces a deregulation of genes encoding subunits of complex I of the mitochondrial respiratory chain leading to a significant increase of ROS production and inducing uncoupled mitochondrial respiration. In addition, this HDAC5 depletion-induced mitochondrial dysfunction provokes metabolic adaptation associated with increased importance of glucose. Indeed, interference with glucose supply in HDAC5-depleted cancer cells significantly increases apoptotic cell death suggesting that glucose deprivation might be combined to HDAC5 inhibition as a therapeutic strategy to kill cancer cells. Conclusion: Our study demonstrated for the first time that specific HDAC5 inhibition induces alteration of gene expression encoding mitochondrial proteins in cancer cells and provide insight into a valuable experimental strategy for manipulation of specific HDAC5 inhibition and glucose metabolism in therapy against cancer. 1.Peixoto, P. et al. HDAC5 is required for maintenance of pericentric heterochromatin, and controls cell-cycle progression and survival of human cancer cells. Cell death and differentiation, 2012; 1-14. elodie.hendrick@student.ulg.ac.be [less ▲]

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See detailAN IMMUNOLOGICAL METHOD TO COMBINE THE MEASUREMENT OF ACTIVE AND TOTAL HUMAN MYELOPEROXIDASE ON THE SAME SAMPLE FROM A COMPLEX MEDIUM
Franck, Thierry ULg; MINGUET, Grégory ULg; Mossay, Pierre et al

Conference (2013, September 12)

Active neutrophil myeloperoxidase (MPO) is a powerful producer of oxidant molecules in acute or chronic inflammation, and it is essential to measure its activity in biological samples. We combined two ... [more ▼]

Active neutrophil myeloperoxidase (MPO) is a powerful producer of oxidant molecules in acute or chronic inflammation, and it is essential to measure its activity in biological samples. We combined two immunological techniques, the SIEFED (specific immunologic extraction followed by enzymatic detection) and an ELISA, to measure the active and total contents of human MPO on the same sample and with the same calibration curve, to define an accurate ratio between the active and total enzyme. After the extraction of MPO from aqueous or biological samples by immobilized anti-MPO antibodies followed by a washing to eliminate unbound material, the active and the total contents of the enzyme were sequentially measured without interferences (patent: EP2017351-B1, 2010). Compared to a classical sandwich ELISA, there is one additional step corresponding to the in situ measurement of MPO activity, but this step does not affect the following measurement of the total MPO content. After validation, the combined technique was applied to a whole blood model of in vitro stimulation with phorbol-myristate-acetate (PMA), cytochalasin B/N-formyl-methionyl-leucyl-phenyl-alanine (CB/fMLP) or lipopolysaccharide/ tumor necrosis factor-alpha (LPS/TNF-α) (n=9). The active/total MPO ratio in whole blood reached 0.267±0.126 for non-stimulated condition and was significantly (p<0.05) higher for PMA (0.360±0.106), CB/fMLP (0.380±0.113) and LPS/TNF-α (0.432±0.124) stimulated conditions. These different ratios highlight the real oxidant potential of MPO, which depends on the stimulating conditions, witness of what could happen in pathological situations with diagnostic purpose. The combined SIEFED/ELISA method also appeared as a powerful tool to screen potential inhibitors that could interact directly with the enzyme, either on its active site or on another key position. [less ▲]

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See detailA water-soluble salt of curcumin (NDS27) inhibits myeloperoxidase and NADPH oxidase activities, two major enzymes of neutrophils.
Derochette, Sandrine ULg; Mouithys-Mickalad, Ange ULg; Deby-Dupont, Ginette et al

Poster (2013, September 11)

Neutrophils (PMNs) produce reactive oxygen species (ROS) to kill pathogenic agents. After appropriate stimulation, leading to the activation of protein kinase C (PKC), the cytosolic subunits of the NADPH ... [more ▼]

Neutrophils (PMNs) produce reactive oxygen species (ROS) to kill pathogenic agents. After appropriate stimulation, leading to the activation of protein kinase C (PKC), the cytosolic subunits of the NADPH oxidase (Nox2) are phosphorylated and translocated to the membrane flavocytochrome b558, forming the active enzyme which produces superoxide anion (O2●-). From O2●- derives H2O2 used by the PMNs myeloperoxidase (MPO) to form strong oxidant species. Many human and animal pathologies with fatal issue are associated with uncontrolled activation of PMNs. The modulation of enzymes implied in ROS production is thus a primary target to manage excessive inflammatory events. For this purpose, we evaluated the effects of NDS27, a water-soluble salt of curcumin combined with hydroxypropyl-β-cyclodextrin, on the activities of PKC, Nox2 and MPO. PKC activation was determined by western blotting with specific antibodies against phosphorylated PKC in extracts from PMNs after their incubation or not with NDS27. A cell-free assay was used to evaluate the effect of NDS27 before or after the assembly of Nox2 subunits. MPO activity was tested by the SIEFED technique in which NDS27 was pre-incubated with the enzyme and discarded before its activity measurement. An inhibition of PKC phosphorylation and Nox2 activity were observed at respectively 10-4 and 10-5 M of NDS27. The Nox2 inhibition was more pronounced when NDS27 was added before the assembly stimulation, suggesting a direct action of NDS27 on the subunits translocation. NDS27 also dose-dependently decreased the activity of MPO (21 % at 10-5 M), indicating an interaction with the enzyme structure. Our results demonstrated that NDS27 is a potent inhibitor of the two major enzymes responsible for ROS production in PMNs, and also acts on the activation cascade of Nox2. The modulatory effect of NDS27 towards the oxidant activity of PMNs opens therapeutic perspectives to control pathologies with excessive inflammatory reactions. [less ▲]

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See detailDisruption in energy metabolism and mitochondrial function in a cellular model of inflammation-induced acute kidney injury
Quoilin, Caroline ULg; Mouithys-Mickalad, Ange ULg; Lécart, Sandrine et al

Poster (2013, September)

Sepsis is a very complex clinical condition characterized by stimulation of a systemic inflammatory response due to an infection. It has a profound deleterious effect on kidney functions leading to sepsis ... [more ▼]

Sepsis is a very complex clinical condition characterized by stimulation of a systemic inflammatory response due to an infection. It has a profound deleterious effect on kidney functions leading to sepsis-induced acute kidney injury (AKI). This failure seems to occur through complex mechanisms involving the immune system response, inflammatory pathways, cellular dysfunction and hemodynamic instability. To study the role of cellular energetic metabolism dysfunction and mitochondrial impairment in the occurrence of AKI during sepsis, we developed an inflammation-induced in vitro model using proximal tubular epithelial cells (HK-2) exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between endotoxic stress and mitochondrial respiratory chain assembly defects. Firstly, we have shown that renal cells subjected to LPS are no longer capable to use adequately the available oxygen to maintain their metabolic functions. One hypothesis of this down-regulation suggests that impairment in mitochondria oxidative phosphorylation could prevent cells from using oxygen for adenosine triphosphate (ATP) production and potentially could cause sepsis-induced organ failure. Our study has then investigated this possible mitochondrial impairment to explain the decreased O2 consumption rate observed in LPS-treated HK-2 cells. After exposure to LPS, functionality of mitochondria was affected without any disturbance in their spatial organization. LPS seemed rather to interrupt mitochondrial oxidative phosphorylation by blocking cytochrome c oxidase activity. As a consequence, disruptions in the electron transport and the proton pumping across the system occurred, leading to a decrease of the mitochondrial membrane potential, an electron leakage as the form of superoxide anion, a release of cytochrome c in the cytosol and a decrease in ATP production. This irreversible defect in the production of cellular energy would support the concept that kidney failure in sepsis may occur on the basis of cytopathic hypoxia. [less ▲]

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See detailThe challenge of understanding myopathies in horses using permeabilized muscle cells
Votion, Dominique ULg; Mouithys-Mickalad, Ange ULg; Ceusters, Justine ULg et al

in In proceedings 9th Conference on Mitochondrial Physiology (2013, September)

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See detailIdentification of methylenecyclopropyl acetic acid in serum of European horses with atypical myopathy
Votion, Dominique ULg; van Galen, G; Sweetan et al

in Equine Veterinary Journal (2013)

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See detailMitochondrial dysfunction in HDAC5-depleted cancer cells induces glucose-dependent metabolic adaptation
Hendrick, Elodie ULg; Matheus, Nicolas ULg; Peixoto, Paul ULg et al

Poster (2013, May 17)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes, which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes, which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad-spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis1. Aims: The goal of this study is to further investigate the molecular mechanisms by which HDAC5 depletion induces both autophagy and apoptosis in cancer cells. Results: Screening transcriptomic study demonstrated that HDAC5 depletion induces a deregulation of genes encoding subunits of complex I of the mitochondrial respiratory chain leading to a significant increase of ROS production. This ROS accumulation promotes autophagy including mitophagy. Indeed, pretreatment with NAC, a ROS scavenger, blocked autophagy triggered by HDAC5 silencing. This autophagy seems to be protective as its blocking with NAC, chloroquine or bafilomycin A1 enhances pro-apoptotic effect of HDAC5 depletion. In addition, mitochondrial dysfunction provokes metabolism adaptation associated with increase of the importance of glucose metabolism in HDAC5 depleted cancer cells. Indeed, low-glucose culture of HDAC5-depleted cells significantly increases apoptotic cell death suggesting that glucose deprivation might be combined to HDAC5 inhibition as a therapeutic strategy to kill cancer cells. Conclusion: Our study demonstrated for the first time that specific HDAC5 inhibition induces alteration of gene expression encoding mitochondrial proteins in cancer cells and provide insight into a valuable experimental strategy for manipulation of specific HDAC5 inhibition and glucose metabolism in therapy against cancer. 1.Peixoto, P. et al. HDAC5 is required for maintenance of pericentric heterochromatin, and controls cell-cycle progression and survival of human cancer cells. Cell death and differentiation, 2012; 1-14. [less ▲]

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See detailJNK/ROS signaling pathway is responsible for induction of autophagy in HDAC5 depleted cancer cells
Hendrick, Elodie ULg; Mathéus, Nicolas; Peixoto, Paul ULg et al

Poster (2013, February 02)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis (ref papier). The study of autophagy in cancer is a new research field that has recently generated tremendous attention due to the recognition that autophagy can have either pro-survival or pro-death functions depending on its level of activation. In addition, more and more studies indicate that a complex relationship exists between autophagy and apoptosis, and that the interplay between these two processes determines whether a cell will live or die. Aims: The goal of this study is to further understand the role of autophagy induced by HDAC5 depletion. Current investigations include determining the molecular mechanisms by which HDAC5 depletion induces autophagy and exploring regulatory relationship between autophagy and apoptosis on cancer cell death in absence of HDAC5. Results: The set up of the autophagy in absence of HDAC5 was demonstrated by the conversion of LC3 and development of autophagosomes by electronic microscopy. Transcriptomic study demonstrated a deregulation of a set of genes involved in ROS detoxification in HDAC5 depleted cancer cells leading to significant increase of ROS levels. Further investigations showed that pretreatment with NAC, a ROS scavenger, effectively blocked the accumulation of ROS and autopahgy triggered by HDAC5 silencing. Moreover, HDAC5 depletion induces activation of JNK, and knockdown of JNK by siRNA inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by HDAC5 depletion indicating that JNK activation may be a upstream signaling of ROS and should be a core component in HDAC5 silencing-induced autophagic signaling pathway. Finally, blocking of autophagy induced by HDAC5 silencing with NAC or chloroquine and bafilomycin enhanced pro-apoptotic effect. Conclusion: Autophagy functions as a prosurvival mechanism to mitigate HDAC5 depletion-induced apoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of specific HDAC5 inhibition. [less ▲]

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See detailJNK/ROS Signaling Pathway Is Responsible for Induction of Autophagy in HDAC5 depleted Cancer Cells
Hendrick, Elodie ULg; Mathéus, Nicolas; Peixoto, Paul ULg et al

Conference (2013, January 29)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis (ref papier). The study of autophagy in cancer is a new research field that has recently generated tremendous attention due to the recognition that autophagy can have either pro-survival or pro-death functions depending on its level of activation. In addition, more and more studies indicate that a complex relationship exists between autophagy and apoptosis, and that the interplay between these two processes determines whether a cell will live or die. Aims: The goal of this study is to further understand the role of autophagy induced by HDAC5 depletion. Current investigations include determining the molecular mechanisms by which HDAC5 depletion induces autophagy and exploring regulatory relationship between autophagy and apoptosis on cancer cell death in absence of HDAC5. Results: The set up of the autophagy in absence of HDAC5 was demonstrated by the conversion of LC3 and development of autophagosomes by electronic microscopy. Transcriptomic study demonstrated a deregulation of a set of genes involved in ROS detoxification in HDAC5 depleted cancer cells leading to significant increase of ROS levels. Further investigations showed that pretreatment with NAC, a ROS scavenger, effectively blocked the accumulation of ROS and autopahgy triggered by HDAC5 silencing. Moreover, HDAC5 depletion induces activation of JNK, and knockdown of JNK by siRNA inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by HDAC5 depletion indicating that JNK activation may be a upstream signaling of ROS and should be a core component in HDAC5 silencing-induced autophagic signaling pathway. Finally, blocking of autophagy induced by HDAC5 silencing with NAC or chloroquine and bafilomycin enhanced pro-apoptotic effect. Conclusion: Autophagy functions as a prosurvival mechanism to mitigate HDAC5 depletion-induced apoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of specific HDAC5 inhibition. [less ▲]

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See detailJNK/ROS signaling pathway is responsible for induction of autophagy in HDAC5 depleted cancer cells
Hendrick, Elodie ULg; Mathéus, Nicolas; Peixoto, Paul ULg et al

Poster (2013, January 28)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis (ref papier). The study of autophagy in cancer is a new research field that has recently generated tremendous attention due to the recognition that autophagy can have either pro-survival or pro-death functions depending on its level of activation. In addition, more and more studies indicate that a complex relationship exists between autophagy and apoptosis, and that the interplay between these two processes determines whether a cell will live or die. Aims: The goal of this study is to further understand the role of autophagy induced by HDAC5 depletion. Current investigations include determining the molecular mechanisms by which HDAC5 depletion induces autophagy and exploring regulatory relationship between autophagy and apoptosis on cancer cell death in absence of HDAC5. Results: The set up of the autophagy in absence of HDAC5 was demonstrated by the conversion of LC3 and development of autophagosomes by electronic microscopy. Transcriptomic study demonstrated a deregulation of a set of genes involved in ROS detoxification in HDAC5 depleted cancer cells leading to significant increase of ROS levels. Further investigations showed that pretreatment with NAC, a ROS scavenger, effectively blocked the accumulation of ROS and autopahgy triggered by HDAC5 silencing. Moreover, HDAC5 depletion induces activation of JNK, and knockdown of JNK by siRNA inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by HDAC5 depletion indicating that JNK activation may be a upstream signaling of ROS and should be a core component in HDAC5 silencing-induced autophagic signaling pathway. Finally, blocking of autophagy induced by HDAC5 silencing with NAC or chloroquine and bafilomycin enhanced pro-apoptotic effect. Conclusion: Autophagy functions as a prosurvival mechanism to mitigate HDAC5 depletion-induced apoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of specific HDAC5 inhibition. [less ▲]

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See detailPhotochemical properties and activity of water-soluble polymer/C60 nanohybrids for photodynamic therapy
Hurtgen, Marie ULg; Debuigne, Antoine ULg; Hoebeke, Maryse ULg et al

in Macromolecular Bioscience (2013), 13(1), 106-115

Water-soluble star-like poly(vinyl alcohol)/C60 and poly{[poly(ethylene glycol) acrylate]-co- (vinyl acetate)}/C60 nanohybrids are prepared by grafting macroradicals onto C60 and are assessed as ... [more ▼]

Water-soluble star-like poly(vinyl alcohol)/C60 and poly{[poly(ethylene glycol) acrylate]-co- (vinyl acetate)}/C60 nanohybrids are prepared by grafting macroradicals onto C60 and are assessed as photosensitizers for photodynamic therapy. The photophysical and biological properties of both nanohybrids highlight key characteristics influencing their overall efficiency. The macromolecular structure (linear/graft) and nature (presence/absence of hydroxyl groups) of the polymeric arms respectively impact the photodynamic activity and the stealthiness of the nanohybrids. The advantages of both nanohybrids are encountered in a third one, poly[(Nvinylpyrrolidone)- co-(vinyl acetate)]/C60, which has linear grafts without hydroxyl groups, and shows a better photodynamic activity. [less ▲]

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See detailIntra- and extracellular antioxidant capacities of the new water-soluble form of curcumin (NDS27) on stimulated neutrophils and HL-60 cells
Derochette, Sandrine ULg; Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg et al

in Chemico-Biological Interactions (2013), 201(1-3), 49-57

Phagocytic cells, especially neutrophils (PMNs) are specialized in the production of reactive oxygen species (ROS) to kill pathogenic agents, but an excessive ROS production is associated with tissue ... [more ▼]

Phagocytic cells, especially neutrophils (PMNs) are specialized in the production of reactive oxygen species (ROS) to kill pathogenic agents, but an excessive ROS production is associated with tissue damages and inflammatory diseases. Phagocytes are thus prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenols to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27, a highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs and human promyelocytic leukemia cells (HL-60). NDS27 was either pre-incubated with cells and eliminated before their activation (intracellular effect) or let in the medium (extracellular effect). Our results indicate that NDS27 significantly and dose-dependently (10 6 M–10 4 M) inhibited the ROS production in both cell types without affecting their viability. NDS27 was able to cross and interact with cell membrane, especially for HL-60 cells, while we observed a better intracellular antioxidant effect with PMNs. The activity of myeloperoxidase (MPO) released by PMNs and HL-60 cells, was decreased by NDS27, but more efficiently for PMNs. These results suggested that the greater efficiency of NDS27 in PMNs is due to an inhibitory effect on cells which are more mature for ROS production, probably by targeting the enzymes implied in respiratory burst like MPO. The modulatory effect of NDS27 on the oxidant activity of cells involved in immune and inflammatory responses opens perspectives for a therapeutic control of pathologies with excessive inflammatory reactions. [less ▲]

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See detailCurcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils
Derochette, Sandrine ULg; Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg et al

in Chemico-Biological Interactions (2013), 206

In neutrophils (PMNs), superoxide anion (O2●-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation ... [more ▼]

In neutrophils (PMNs), superoxide anion (O2●-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10-4 to 10-6 M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2●- production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10-5 M, while resveratrol appeared to be an O2●- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production. [less ▲]

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See detailEffect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts.
Ceusters, Justine ULg; Mouithys-Mickalad, Ange ULg; Franck, Thierry ULg et al

in Mitochondrion (2013), 13(5),

Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction ... [more ▼]

Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function. [less ▲]

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See detailTriphenylphosphonium salts of 1,2,4-benzothiadiazine 1,1-dioxides related to diazoxide targeting mitochondrial ATP-sensitive potassium channels
Constant-Urban, C.; Charif, M.; Goffin, Eric ULg et al

in Bioorganic & Medicinal Chemistry Letters (2013), 23

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See detailDifferentiation between stoichiometric and anticatalytic antioxidant properties of benzoic acid analogues: A structure/redox potential relationship study.
Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg; Robert, Thierry ULg et al

in Chemico-biological interactions (2013), 206(2), 194-203

We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities ... [more ▼]

We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities of phenolic acid derivatives were studied by measuring their capacity to scavenge the radical cation 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+) and reactive oxygen species (ROS) produced by stimulated neutrophils. The anticatalytic antioxidant capacity of the molecules was evaluated on the activity of myeloperoxidase (MPO), an oxidant enzyme present in and released by the primary granules of neutrophils. The ROS produced by PMA-stimulated neutrophils were measured by lucigenin-enhanced chemiluminescence (CL) and the potential interaction of the molecules with MPO was investigated without interferences due to medium by Specific Immuno-Extraction Followed by Enzyme Detection (SIEFED). The antioxidant activities of the phenolic compounds were correlated to their redox potentials measured by differential pulse voltammetry (DPV), and discussed in relation to their molecular structure. The ability of the phenolic molecules to scavenge ABTS radicals and ROS derived from neutrophils was inversely correlated to their increased redox potential. The number of hydroxyl groups (three) and their position (catechol) were essential for their efficacy as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential. Likewise, for the inhibition of MPO activity the number of OH groups and mainly the elongation of the carboxylic group were essential, probably by facilitating the interaction with the active site or the structure of the enzyme. The redox potential measurement, combined with ABTS and CL techniques, seems to be a good technique to select stoichiometric antioxidants but not anticatalytic ones, as seen for MPO, what rather involves a direct interaction with the enzyme. [less ▲]

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See detailEffects of diazoxide, benzothiadiazine and benzopyrane derivatives on mitochondrial proton and electron leaks of cardiomyocytes (H9C2 cell line).
Mouithys-Mickalad, Ange ULg; Ceusters, Justine ULg; Charef, M et al

Poster (2013)

Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of ... [more ▼]

Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of adenosine triphosphate (ATP) and reactive oxygen species (ROS). In many cancer cells those organelles become dysfunctional leading to a shift of energy metabolism from oxidative phosphorylation to active glycolysis and an increase of ROS generation. According to Warberg’ theory, cancer damage might occur at the mitochondrial level, affecting tiny structures within each cell implicated in the energy production through ATP. New insight is that mitochondria might be a good therapeutic target for metabolic syndromes, ischemia/reperfusion injury and organs transplantation. Therefore, search for novel molecules able to keep mitochondria functional are of relevant interest. Methodology: Cardiomyocytes (H9C2 cells) were from ATCC (USA) and grown till confluence. The basal cellular respiratory rate, proton and electron leaks as well as ATP production were measured with the High Resolution Oxygraphy (Oroboros, Austria). All compounds: diazoxide (DIAZ), diazoxide –related analogs (1: BPDZ-259, 2: BPDZ-444), and benzopyran derivatives (3: BPDZ-490, 4: BPDZ-711) were tested at final concentration of 10-5 M, except when specified and compared to control samples (cells with or without DMSO). Results and conclusion: The basal respiratory rate of H9C2 cells (5x106/mL) was changed depending on the chemical structure of the tested compounds: e.g. compound 3 strongly enhanced the routine respiration, while 4 displayed a marked lowering effect. In contrast, the addition of similar concentration of benzothiadiazin derivatives (1, 2) had no effect on routine respiration but also on the other respiratory parameters such as oligomycin-induced leak and ATP production. Similar profile was obtained with the reference molecule: diazoxide. Overall, our findings indicate that both diazoxide-like analogues (1 and 2) and diazoxide were without significant effect on basal respiration, ATP production, even on maximal respiration. Interestingly, two derivatives show opposite effects: compound 3 behaves as a uncoupling agent and the other one (4) exhibits a real lowering effect on respiration but that was reversible. The latter effect might be of interest if this kind of molecules could be used for further use as an agent for organ conservation during transplantation. Our results also demonstrate that diazoxide, a well-known Mito-KATP opener, did not exert its effect beside of clinical situation like ischemia/reperfusion injury. [less ▲]

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See detailAn in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.
Ceusters, Justine ULg; Serteyn, Didier ULg; MINGUET, Grégory ULg et al

in Veterinary Immunology and Immunopathology (2012), 150(3-4), 221-7

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate ... [more ▼]

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-alpha in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-alpha, LPS and LPS/TNF-alpha, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-alpha significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity. [less ▲]

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See detailESR spectroscopy for the study of an inflammation-induced AKI cellular model
Quoilin, Caroline ULg; Mouithys-Mickalad, Ange ULg; Gallez, Bernard et al

Poster (2012, November)

The kidney is faced to an impairment of oxygen extraction during sepsis which is well-known to be a risk factor for the development of acute kidney injury (AKI). Recent research activities in the ... [more ▼]

The kidney is faced to an impairment of oxygen extraction during sepsis which is well-known to be a risk factor for the development of acute kidney injury (AKI). Recent research activities in the mechanisms involved in the development of AKI in sepsis emphasize the central role of hemodynamic and inflammatory events. More particularly, two mechanisms are suggested to explain the inability of the injured kidney to extract oxygen: tissue hypoxia and cellular energetic metabolism dysfunction. Our working hypothesis of the pathophysiology of AKI is based on cellular respiratory dysfunction due to the inflammatory response inherent to sepsis. To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) by ESR oximetry. This method has shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells. This decrease was accompanied by increased nitric oxide (NO) production as measured by a spin trapping technique using ESR spectroscopy. This method is based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+) forming a water-soluble NO-FeMGD complex detected by ESR. Since inducible NO synthase (iNOS) has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-NMMA (L-NG-monomethyl Arginine citrate) was tested in this in vitro model. L-NMMA blocked NO generation and permitted the HK-2 cells to recover a normal cellular respiration. Overall, ESR spectroscopy and the model of HK-2 cells exposed to LPS displays some key features of inflammation-induced acute kidney injury. [less ▲]

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