Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes

SummaryObjectiveHypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions.MethodsHuman cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-β (IL-1β), tumour necrosis factor α (TNF-α) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2− production, NOX subunit expression and nitrosylation were also assessed.ResultsiNOS expression and nitrite (but not peroxynitrite) production were significantly increased under H/R conditions when compared with to normoxia (P < 0.05). H/R conditions decreased O2− production from ∼0.20 to ∼0.12 nmol min−1 mg proteins−1 (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2− production was restored at normoxic levels (0.21 nmol min−1 mg of proteins−1).ConclusionsOur results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia

Lipocalin 2 as a potential systemic biomarker for central serous chorioretinopathy

No systemic biomarker of Central Serous Chorioretinopathy (CSCR) has been identified. Lipocalin 2 (LCN2 or NGAL), alone or complexed with MMP-9 (NGAL/MMP-9), is increased in several retinal disorders. Serum levels of LCN2 and NGAL/MMP-9 were measured in CSCR patients (n = 147) with chronic (n = 76) or acute/recurrent disease (n = 71) and in age- and sex-matched

Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth

Designing synthetic microenvironments for cellular investigations is a very active area of research at the crossroads of cell biology and materials science. The present work describes the design and functionalization of a three-dimensional (3D) culture support dedicated to the study of neurite outgrowth from neural cells. It is based on a dense self-assembled collagen matrix stabilized by 100-nm wide interconnected native fibrils without chemical crosslinking. The matrices were made suitable for cell manipulation and direct observation in confocal microscopy by anchoring them to traditional glass supports with a calibrated thickness of ∼50 μm. The matrix composition can be readily adapted to specific neural cell types, notably by incorporating appropriate neurotrophic growth factors. Both PC-12 and SH-SY5Y lines respond to growth factors (nerve growth factor and brain-derived neurotrophic factor, respectively) impregnated and slowly released from the support. Significant neurite outgrowth is reported for a large proportion of cells, up to 66% for PC12 and 49% for SH-SY5Y. It is also shown that both growth factors can be chemically conjugated (EDC/NHS) throughout the matrix and yield similar proportions of cells with longer neurites (61% and 52%, respectively). Finally, neurite outgrowth was observed over several tens of microns within the 3D matrix, with both diffusing and immobilized growth factors

Transformation of Aluminosilicate Nanotubes into a Surface Active Hybrid Phase by Decylphosphonic Acid

International audienceThe surface functionalization of imogolite nanotubes enables to tune their properties and opens many possibilities, e.g. for the design and tailoring of polymer matrices and membranes. It has been unambiguously shown that imogolite reacts with the phosphonic acid moiety and that the properties of the obtained materials are modified. However, the impact of the grafting reaction on the structure of imogolite has never been clearly established. In this article, we take one of the most spread imogolite grafting protocols and study the impact of the reaction on the structure of the nanotube. By combining X-ray scattering, FT-IR, and magic-angle spinning nuclear magnetic resonance spectroscopy, we observed the formation of a lamellar phase but no evidence for grafted tubes. The obtained composite material exhibits unexpected properties at the oilwater interface

Contribution to accurate Spherical Gold Nanoparticles analysis (size, size distribution) by SpICPMS and SAXS

International audienceSmall-Angles X-Ray Scattering (SAXS) has been established as a metrological method for the determination of nanoparticles size and size distribution. Modern SAXS Laboratory experiments, by involving synchrotron-based instrumentation at lower price and very stable X-ray source, are more and more used in nanomaterials domain. In the frame of the EMPIR Innanopart project, we have developed a methodology for the size, size distribution and concentration determination of spherical nanoparticles. This protocol involves a precise sample preparation, and a set of homemade software tools for the data processing-from the acquisition, the absolute scaling, to the analysis. spICPMs is not a metrological traceable technique but has many strengths to become a useful complement of nanoparticle characterization methods such as SAXS and microscopy. It can also measure highly diluted nanoparticles suspensions which is not the case of Dynamic Light scattering (DLS) or SAXS. Finally, ICPMS analyzes inorganic ions in liquid solution in a very large range of concentration, which should allow linear diameter measurement range over at least 3 orders of magnitude. In this work, we confront spICPMS with SAXS in order to investigate the method and the developed protocols on a set of commercial spherical Gold Nanoparticles. Comparison between SAXS and spIPCMS method for the determination of size of spherical Gold Nanoparticle