Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

peer reviewedBy oxymetry and electron paramagnetic resonance (EPR), we investigated the effects of repeated anoxia/re-oxygenation (A/R) periods on the respiration and production of free radicals by synoviocytes (rabbit HIG-82 cell line and primary equine synoviocytes) and equine articular chondrocytes. Three periods of 20 min anoxia followed by re-oxygenation were applied to 10(7)cells; O(2) consumption was measured before anoxia and after each re-oxygenation. After the last A/R, cellular free radical formation was investigated by EPR spectroscopy with spin trapping technique (n=3 for each cell line). Both types of synoviocytes showed a high O(2) consumption, which was slowered after anoxia. By EPR with the spin trap POBN, we proved a free radical formation. Results were similar for equine and rabbit synoviocytes. For chondrocytes, we observed a low O(2) consumption, unchanged by anoxia, and no free radical production. These observations suggest an oxidant activity of synoviocytes, potentially important for the onset of osteoarthritis

C-60-containing nanostructured polymeric materials with potential biomedical applications

Star-shaped polymers with a fullerene (C-60) Core and an unexpanded structure were successfully prepared by reaction Of C60 with amino end-capped polyesters HxC60(NHPCLn)(x) and polyethers HxC60(NHPEG(n))(x), respectively. Upon irradiation of these C-60-derivatives, a large amount of singlet oxygen was released. Compared to previously synthesized star-shaped azafulleroids with an expanded structure, the photosensitivity of HxC60(NHPCLn)(x) is higher as assessed by a higher production of singlet oxygen. The cytotoxicity of the photoactive water-soluble HxC60(NHPEG(n))(x) derivatives was tested against THP-1 cells and expressed in terms of cell viability. Moreover, they were processed as micro-/nanosized fibers by electrospinning, which however required the addition of poly(F-caprolactone) (PCL). The diameter distribution of the fibers was trimodal, where the fraction with the 270 nm average diameter was the major population. Because of their photoactivity, the herein reported star-shaped C-60-derivatives are promising candidates for photodynamic cancer therapy and treatment of multidrug resistant pathogens. (c) 2007 Elsevier Ltd. All rights reserved

Production of Free Radicals and Oxygen Consumption by Primary Equine Endothelial Cells During Anoxia-Reoxygenation

The endothelium plays an active role in ischemia/reperfusion injuries. Herein, we report the effect of a single or successive cycles of anoxia/reoxygenation (A/R) on the mitochondrial respiratory function of equine endothelial cells (cultured from carotids) monitored by high resolution oxymetry, and on their production of reactive oxygen species (ROS). ROS were measured by electron paramagnetic resonance (ESR) using POBN and DMPO spin traps, and by gas chromatography (GC) of ethylene released by ROS-induced α-keto-γ-(methylthio)butyric acid (KMB) oxidation. The oxygen consumption significantly decreased with the number of A/R cycles, and POBN-ESR spectra were specific of adducts formed in the cells from superoxide anion. After a one-hour A/R cycle, high intensity DMPO-ESR spectra were observed and assigned to superoxide anion trapping; the GC results confirmed an important production of ROS compared to normoxic cells. These results show that A/R induces mitochondrial alterations in endothelial cells, and strongly stimulates their oxidative activity as demonstrated by ESR and GC methods

Targeting Myeloperoxidase Activity and Neutrophil ROS Production to Modulate Redox Process: Effect of Ellagic Acid and Analogues.

peer reviewedMalaria is an infectious disease caused by a Plasmodium genus parasite that remains the most widespread parasitosis. The spread of Plasmodium clones that are increasingly resistant to antimalarial molecules is a serious public health problem for underdeveloped countries. Therefore, the search for new therapeutic approaches is necessary. For example, one strategy could consist of studying the redox process involved in the development of the parasite. Regarding potential drug candidates, ellagic acid is widely studied due to its antioxidant and parasite-inhibiting properties. However, its low oral bioavailability remains a concern and has led to pharmacomodulation and the synthesis of new polyphenolic compounds to improve antimalarial activity. This work aimed at investigating the modulatory effect of ellagic acid and its analogues on the redox activity of neutrophils and myeloperoxidase involved in malaria. Overall, the compounds show an inhibitory effect on free radicals as well as on the enzyme horseradish peroxidase- and myeloperoxidase (HRP/MPO)-catalyzed oxidation of substrates (L-012 and Amplex Red). Similar results are obtained with reactive oxygen species (ROS) produced by phorbol 12-mystate acetate (PMA)-activated neutrophils. The efficiency of ellagic acid analogues will be discussed in terms of structure-activity relationships

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture.

We investigated the oxygen (O2) uptake of equine articular chondrocytes to assess their reactions to anoxia/re-oxygenation. They were cultured under 5% or 21% gas phase O2 and at glucose concentrations of 0, 1.0 or 4.5 g/L in the culture medium (n = 3). Afterwards, the O2 consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25 min. The glucose consumption and lactate release were measured at the end of the re-oxygenation period. The chondrocytes showed a minimal O2 consumption rate, which was hardly changed by anoxia. Independently from the O2 tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5 g/L glucose (n = 3). Lactate release was also independent from O2 tension, but lower for cells at 4.5 g/L glucose (n = 3). Our observations indicated that O2 consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re-oxygenation. But the chondrocytes metabolism was modified by an excess of O2 and glucose

Cardiomiopatia hipertrófica felina

A Cardiomiopatia Hipertrófica (CMH) é uma doença é a doença cardíaca primária mais comum em gatos causada por uma mutação genética autossómica de dominância incompleta com apresentações clínicas variadas que apresenta uma grande dispersão mundial e racial. O carácter hereditário desta doença cardíaca resulta de uma mutação genética autossómica dominante com penetração incompleta, sendo mais exuberante em situações de homozigotia. A mutação mais comum ocorre em genes que codificam para a Proteína C3 de Ligação à Miosina, com alterações estruturais do mesmo e consequente hipertrofia concêntrica parcial ou total do ventrículo esquerdo. Com o aumento do septo interventricular, dos músculos papilares e/ou da parede do ventrículo esquerdo, ocorre comprometimento tanto do enchimento ventricular como do seu relaxamento, o que por sua vez contribui para a progressão da hipertrofia. A diminuição da câmara cardíaca, a diminuição do débito cardíaco, o aumento compensatório do ritmo, o aumento da pressão de enchimento diastólico e o aumento do átrio esquerdo predispõem para uma insuficiência cardíaca congestiva, formação trombos e até morte súbita. A avaliação clínica de um gato com suspeita de Cardiomiopatia Hipertrófica deve ser exaustiva, mesmo que o diagnóstico definitivo apenas possa ser confirmado através de ecocardiografia e de métodos de biologia molecular no sentido de identificar a mutação genética. A ecocardiografia pode ainda ser útil para direcionar a terapêutica em função do grau de hipertrofia estabelecer prognóstico. Atualmente, em medicina humana, existem outras formas de terapêutica, como o novo MYK 461, que se revelam bastante promissores.The Hypertrophic Cardiomyopathy is the heart disease more common in cats caused by a genetic autossomic mutation with imcomplete penetrance, having several clinic findings worldwide and inbreed. This disease assumes an hereditary character associated with a genetic mutation autossomic dominant with incomplete penetrance, being more exuberant in homozigotic. It’s a common mutation in genes that codify for the Myosin Binding Protein C (MYBPC3), with structural alteration of itself and leading to the partial or total concentric hypertrophy os left ventricule. With the increase of the interventricular septum and/or papillary muscles and/or left ventricle hall there is a compromise of the ventricular filling and it’s relaxation, which in other hand contributes to the progression of the hypertrophy. The decrease of the cardiac chamber, the decrease of cardiac output, the compensatory increase of rhythm, the increase of pressure in diastolic filling and the increase of the left atrium predispose to congestive heart failure and even sudden death. Cats clinical evaluation under the suspect of Hypertrophic Cardiomyopathy should be exhaustive, even if the definite diagnostic can only be confirmed throght Ultrasound and bio molecular methods looking for identification of genetic mutations. Ultrasound can also be useful to direct the therapeutics accordingly to the level of Hypertrophy, and better prognostics. Nowadays, there are other new therapeutic drugs, like MYK 461, that are showing promissing results

Effects of Juglone on Neutrophil Degranulation and Myeloperoxidase Activity Related to Equine Laminitis.

Experimental laminitis, characterized by a failure of the dermal-epidermal interface of the foot, can be induced in horses by the oral administration of a black walnut extract (BWE). In the early phase of this severe and painful disease, an activation of neutrophil occurs, with the release of myeloperoxidase (MPO), a pro-oxidant enzyme of neutrophils, in plasma, skin, and laminar tissue. Juglone, a naphthoquinone derivative endowed with redox properties, is found in walnuts and has been incriminated in this neutrophil activation. We report for the first time the inhibitory activity of juglone on the degranulation of neutrophils induced by cytochalasin B and formyl-methionyl-leucyl-phenylalanine as monitored by the MPO release (>90% inhibition for 25 and 50 μM). Moreover, it also acts on the peroxidase activity of MPO by interacting with the intermediate "π cation radical," as evidenced by the classical and specific immunological extraction followed by enzymatic detection (SIEFED) assays. These results are confirmed by a docking study showing the perfect positioning of juglone in the MPO enzyme active site and its interaction with one of the amino acids (Arg-239) of MPO apoprotein. By chemiluminescence and electron paramagnetic resonance techniques, we demonstrated that juglone inhibited reactive oxygen species (ROS) and superoxide anion free radical produced from phorbol myristate acetate (PMA)-activated polymorphonuclear neutrophils (PMNs). These results indicate that juglone is not the trigger for equine laminitis, at least if we focus on the modulation of neutrophil activation

Free Radical Inhibition Using a Water-Soluble Curcumin Complex, NDS27: Mechanism Study Using EPR, Chemiluminescence, and Docking.

peer reviewedThere is a growing interest in the use of natural compounds to tackle inflammatory diseases and cancers. However, most of them face the bioavailability and solubility challenges to reaching cellular compartments and exert their potential biological effects. Polyphenols belong to that class of molecules, and numerous efforts have been made to improve and overcome these problems. Curcumin is widely studied for its antioxidant and anti-inflammatory properties as well as its use as an anticancer agent. However, its poor solubility and bioavailability are often a source of concern with disappointing or unexpected results in cellular models or in vivo, which limits the clinical use of curcumin as such. Beside nanoparticles and liposomes, cyclodextrins are one of the best candidates to improve the solubility of these molecules. We have used lysine and cyclodextrin to form a water-soluble curcumin complex, named NDS27, in which potential anti-inflammatory effects were demonstrated in cellular and in vivo models. Herein, we investigated for the first time its direct free radicals scavenging activity on DPPH/ABTS assays as well as on hydroxyl, superoxide anion, and peroxyl radical species. The ability of NDS27 to quench singlet oxygen, produced by rose bengal photosensitization, was studied, as was the inhibiting effect on the enzyme-catalyzed oxidation of the co-substrate, luminol analog (L012), using horseradish peroxidase (HRP)/hydrogen peroxide (H(2)O(2)) system. Finally, docking was performed to study the behavior of NDS27 in the active site of the peroxidase enzyme