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Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism.

ABSTRACT: INTRODUCTION: The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is to compare the metabolic profile of traditional and new generation NO donors to see which one points out the osteoarthritic process in the best way. METHODS: Human healthy and OA chondrocytes were isolated from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with NO donors (NOC-12 or SNP). NO production was evaluated by the Griess method, and apoptosis was quantified by flow cytometry. Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities by enzymatic assay, mitochondrial membrane potential (??m) by JC-1 using flow cytometry, and ATP levels were measured by luminescence assays. Glucose transport was measured as the uptake of 2-deoxy-[(3)H]glucose (2-[(3)H]DG). Statistical analysis was performed using the Mann-Whitney U test. RESULTS: NOC-12 liberates approximately ten times more NO2- than SNP, but the level of cell death induced was not as profound as that produced by SNP. Normal articular chondrocytes stimulated with NOC-12 had reduced activity from complexes I, III y IV, and the mitochondrial mass was increased in these cells. Deleterious effects on ??m and ATP levels were more profound with SNP, and this NO donor was able to reduce 2-[(3)H]DG levels. Both NO donors had opposite effects on lactate release, SNP diminished the levels and NOC-12 lead to lactate accumulation. OA chondrocytes incorporate significantly more 2-[(3)H]DG than healthy cells. CONCLUSIONS: These findings suggest that the new generation donors, specifically NOC-12, mimic the OA metabolic process much better than SNP. Previous results using SNP have to be considered prudently since most of the effects observed can be induced by the interactions of secondary products of NO.

SUBMITTER: de Andres MC

PROVIDER: S-EPMC3978712 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism.

de Andrés María C MC Maneiro Emilia E Martín Miguel A MA Arenas Joaquín J Blanco Francisco J FJ

Arthritis research & therapy 20130101 5

<h4>Introduction</h4>The pathogenesis of osteoarthritis (OA) is characterized by the production of high amounts of nitric oxide (NO), as a consequence of up-regulation of chondrocyte-inducible nitric oxide synthase (iNOS) induced by inflammatory cytokines. NO donors represent a powerful tool for studying the role of NO in the cartilage in vitro. There is no consensus about NO effects on articular cartilage in part because the differences between the NO donors available. The aim of this work is t ...[more]

PMID: 24025112

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Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. C. jejuni NCTC11168 was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ÂºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 Î¼l sample onto both nutrient agar plates incubated at 37 Â°C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 Â°C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. 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Project description:Chondroitin sulfate (CS) is a symptomatic slow acting drug for osteoarthritis (OA) widely used for the treatment of this highly prevalent disease, characterized by articular cartilage degradation. However, little is known about its mechanism of action, and recent large scale clinical trials have reported variable results on OA symptoms. Herein, we aimed to study the modulations in the intracellular proteome and the secretome of human articular cartilage cells (chondrocytes) treated with three different CS compounds, with different origin or purity, by two complementary proteomic approaches. Osteoarthritic cells were treated with 200 ?g/ml of each brand of CS. Quantitative proteomics experiments were carried out by the DIGE and stable isotope labeling with amino acids in cell culture (SILAC) techniques, followed by LC-MALDI-MS/MS analysis. The DIGE study, carried out on chondrocyte whole cell extracts, led to the detection of 46 spots that were differential between conditions in our study: 27 were modulated by CS1, 4 were modulated by CS2, and 15 were modulated by CS3. The SILAC experiment, carried out on the subset of chondrocyte-secreted proteins, allowed us to identify 104 different proteins. Most of them were extracellular matrix components, and 21 were modulated by CS1, 13 were modulated by CS2, and 9 were modulated by CS3. Each of the studied compounds induces a characteristic protein profile in OA chondrocytes. CS1 displayed the widest effect but increased the mitochondrial superoxide dismutase, the cartilage oligomeric matrix protein, and some catabolic or inflammatory factors like interstitial collagenase, stromelysin-1, and pentraxin-related protein. CS2 and CS3, on the other hand, increased a number of structural proteins, growth factors, and extracellular matrix proteins. Our study shows how, from the three CS compounds tested, CS1 induces the activation of inflammatory and catabolic pathways, whereas CS2 and CS3 induce an anti-inflammatory and anabolic response. The data presented emphasize the importance of employing high quality CS compounds, supported by controlled clinical trials, in the therapy of OA. Finally, the present work exemplifies the usefulness of proteomic approaches in pharmacological studies.

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Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism.

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Nitric oxide compounds have different effects profiles on human articular chondrocyte metabolism.

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