Nitric oxide regulates mitochondrial respiration and functions of articular chondrocytes

227

178

Objective. Chondrocyte cell death, possibly related to increased production of endogenous nitric oxide (NO), has been observed during the pathogenesis of osteoarthritis and rheumatoid arthritis. The purpose of this study was to investigate the potential role of NO in causing chondrocyte cell death and to determine the contribution of other reactive oxygen species (ROS).Methods. Cell death and cytotoxicity were evaluated in human articular chondrocytes in response to various NO donor compounds with and without agents that stimulate or inhibit the production of additional ROS using both the alginate bead and the monolayer culture systems. Cell death was quantified by a total cell count with fluorescent labels, and cytotoxicity was measured as a function of cellular NADH-and NADPHdependent dehydrogenase activity. To determine if the redox status of the chondrocyte could influence the observed effect of NO, cells were preincubated for 24 hours in L-cystine-and glutathione (GSH)-depleted media to reduce intracellular GSH levels, a major defense mechanism against oxidative stress. Apoptosis was analyzed with the quantification of histoneassociated DNA fragments. Conclusion. These results show that NO by itself is not cytotoxic to cultured chondrocytes and can even be protective under certain conditions of oxidative stress. Chondrocyte cell death from NO occurs under conditions where other ROS are also generated. Results. Treatment of chondrocytes with peroxynitrite (ONOONitric oxide (NO) participates in the normal functioning of a wide array of mammalian processes, including vasodilation, inhibition of platelet aggregation, neurotransmission, and macrophage-mediated immunity (1-4). Paradoxically, however, NO has also been proposed to be the cytotoxic species responsible for an increasing number of pathologic disorders, including rheumatic (5) and neurodegenerative (6) diseases. Expression of inducible nitric oxide synthase (iNOS) has been associated with chondrocytes during the pathogenesis of osteoarthritis (OA) (7) and rheumatoid arthritis (RA) (8); however, the exact role of NO in this regard is not fully understood. While it has been reported that NO causes chondrocyte cell death (9,10), other more recent reports have proposed NO to be a physiologic regulator of mitochondrial respiration in chondrocytes (11,12). Moreover, production of high levels of endogenous NO by overexpression of the iNOS gene in transfected chondrocytes did not cause cell death (13).The direct investigation of the function of exogenous NO production on chondrocytes has been hampered by the lack of uniformity that exists between the different types of NO donor compounds. Knowledge of the precise reactive nitrogen species (RNS) that is

supporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Del Carlo and Loesermentioning

confidence: 99%

mentioning

confidence: 95%

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Nitric oxide–mediated chondrocyte cell death requires the generation of additional reactive oxygen species

Carlo

Loeser

2002

227

178

Mitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes

Maneiro¹,

Martín²,

Andrés³

et al. 2003

220

189

Objective. Osteoarthritis (OA) is a degenerative rheumatic disease that is associated with extracellular matrix degradation and chondrocyte apoptosis in the articular cartilage. The role of mitochondria in degenerative diseases is widely recognized. We undertook this study to evaluate mitochondrial function in normal and OA chondrocytes and to examine age-related changes in mitochondria.Methods. Mitochondrial function was evaluated by analyzing respiratory chain enzyme complexes and citrate synthase (CS) activities as well as changes in mitochondrial membrane potential (⌬⌿m). The activities of mitochondrial respiratory chain complexes (complex I: rotenone-sensitive NADH-coenzyme Q 1 reductase; complex II: succinate dehydrogenase; complex III: antimycin-sensitive ubiquinol cytochrome c reductase; and complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from OA and normal cartilage. ⌬⌿m was measured by JC-1 using flow cytometry. Statistical analysis was performed using the Mann-Whitney U test and Student's t-test as well as several models of multiple linear regression.Results. OA articular chondrocytes had reduced activities of complexes II and III compared with cells from normal cartilage. However, the mitochondrial mass was increased in OA. Cultures of OA chondrocytes contained a higher proportion of cells with de-energized mitochondria. We found no relationship between mitochondrial function and donor age either in normal or in OA chondrocytes. Conclusion. These findings suggest the involvement of mitochondrial function in the pathophysiology of OA. Cartilage degradation by OA and cartilage aging may be two different processes.Osteoarthritis (OA) is a degenerative joint disease that is characterized by articular cartilage degradation. The final phase of OA seems to reflect a failure of the reparative process, resulting in degradation of the matrix, cell death, and total loss of cartilage integrity. The chondrocyte is the only cell type that is present in mature cartilage, and it is responsible for repairing the damaged tissue (1,2).Mitochondria are complex organelles that oxidize a wide range of metabolic intermediates. It has been reported that in living cells, these organelles have a characteristic appearance that depends on the tissue type and the oxidative state of the cell. Multienzyme complexes located both in the inner mitochondrial membrane and in the mitochondrial matrix oxidize tricarboxylic acid-cycle intermediates derived from primary nutrients. ATP is generated by the activity of an electrogenic proton pump that spans the inner mitochondrial membrane. The energy for synthesis of ATP is derived from two components: a pH gradient and the electric potential across the mitochondrial membrane.Mitochondrial impairment and defective oxidative phosphorylation have been linked to some human disorders (3,4). Mitochondria are important in regulating cell survival (5-9), and the classic signs of cell death

Mediation of spontaneous knee osteoarthritis by progressive chondrocyte ATP depletion in Hartley guinea pigs

Johnson

Svensson

Etten

et al. 2004

Objective. Because articular chondrocytes reside in a hypoxic milieu, anaerobic glycolysis is central in generating ATP to support chondrocyte matrix synthesis and viability, with mitochondrial oxidative phosphorylation possibly providing physiologic reserve ATP generation. Nitric oxide (NO) potently suppresses mitochondrial oxidative phosphorylation. Because enhanced cartilage NO generation occurs in osteoarthritis (OA), we systematically tested for mitochondrial dysfunction in the pathogenesis of OA.Methods. We assessed chondrocytes for ATP depletion and for in situ changes in mitochondrial ultrastructure prior to and during the evolution of spontaneous knee OA in male Hartley guinea pigs, a model in which chondrocalcinosis also supervenes.Results. Spontaneous NO release from knee cartilage samples in organ culture doubled between ages 2 months and 8 months as knee OA developed. Concomitantly, chondrocyte intracellular ATP levels declined by ϳ50%, despite a lack of mitochondrial ultrastructure abnormalities in knee chondrocytes. As ATP depletion progressed with aging in knee chondrocytes, an increased ratio of lactate to pyruvate was observed, consistent with an adaptive augmentation of glycolysis to mitochondrial dysfunction. Furthermore, we observed progressive elevation of chondrocyte ATP-scavenging nucleotide pyrophosphatase/phosphodiesterase (NPP) activity and extracellular levels of the NPP enzymatic end product inorganic pyrophosphate (PPi), which stimulate chondrocalcinosis.Conclusion. Profound chondrocyte ATP depletion develops in association with heightened NO generation in guinea pig knee OA. Increased NPP activity and concordant increases in extracellular PPi, which are strongly associated with human aging-associated degenerative arthropathy and directly stimulate chondrocalcinosis, may be primarily driven by chondrocyte ATP depletion. Our findings implicate a decreased mitochondrial bioenergetic reserve as a pathogenic factor in both degenerative arthropathy and chondrocalcinosis in aging.