Abstract
ObjectiveMitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.MethodsChondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.ResultsCultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm2*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.ConclusionsThe superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.
Highlights
Articular cartilage is an avascular connective tissue that contains only one specialized cell type, the chondrocyte, which is anchored in the dense extracellular matrix and is supplied with oxygen and nutrients by diffusion from surrounding tissues, principally, from the synovial fluid [1]
In order to investigate mitochondrial reactive oxygen species (ROS) signaling in articular chondrocytes, we generated a pan-tissue mt-circularly permuted yellow fluorescent protein (cpYFP) transgenic mouse model
In cultured chondrocytes isolated from the transgenic mice, we stained the mitochondria with TMRM, whose fluorescence signal is spectrally separable from that of mt-cpYFP
Summary
Articular cartilage is an avascular connective tissue that contains only one specialized cell type, the chondrocyte, which is anchored in the dense extracellular matrix and is supplied with oxygen and nutrients by diffusion from surrounding tissues, principally, from the synovial fluid [1]. The median oxygen tension in systemic arterioles is ,7 kPa and falls to ,3– 4 kPa in precapillary arterioles and capillaries [9]. Chondrocytes, especially those in the superficial zone, are subjected to similar oxygen tension as that for the cells in blood-supplied tissues. Mitochondria, the major cellular consumer of oxygen, are very likely to play important physiological roles in articular chondrocytes, and more importantly, mitochondrial dysfunction has been reported to be involved in the process of cartilage degeneration [12,13,14]
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