Background/Purpose: Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 10% of the US population. With age and injury, chondrocytes have diminished mitochondrial content and mitochondrial production of ATP contributing to OA pathogenesis. We have previously reported that chondrocytes release ATP, which is converted extracellularly to adenosine and maintains chondrocyte homeostasis via endogenous stimulation of the A2AR. Injured/inflamed chondrocytes have lower ATP levels and release less ATP resulting in diminished extracellular adenosine and A2AR stimulation. Mice and humans lacking the capacity to convert extracellular ATP to adenosine (ecto-5’nucleotidase deficient) develop spontaneous OA as do mice lacking A2AR (A2ARKO). We therefore studied the effect of A2AR stimulation on mitochondrial health and function in chondrocytes from WT and A2ARKO mice and in a human chondrocytic cell line.

Methods: A human chondrocyte cell line, T/C28-a2, or neonatal chondrocytes isolated from WT and A2ARKO mice (C57Bl6 background) were grown in culture, treated with IL-1β (5ng/ml) or medium (4h, 37^oC) and during the last hour of incubation, with either medium or the selective A2AR agonist (CGS21680, 1mM). Mitochondrial function was then analyzed by Seahorse Mito Stress Kits using the Seahorse apparatus. Mitochondrial health was assessed by analyzing mean pixel intensity (MPI) of tetramethylrhodamine (TMRM) live cell staining which correlates with reduced collapsibility of mitochondrial membrane potential. Mitochondrial content and ROS burden were assessed in live cell confocal imaging (MitoTracker; MitoSox) and by immunohistochemistry (anti-ATPase Ab; anti-8hydroxyguanosine Ab, 8OHg) in paraffin-embedded tissue from WT and A2ARKO mice.

Results:

The mitochondrial membrane potential and mitochondrial content were reduced in A2ARKO chondrocytes compared to WT. In WT chondrocytes mitochondrial content increased after IL-1β treatment and A2AR stimulation increased mitochondrial membrane potential as well. Histologic staining of knee cartilage for 8OHg, a marker of ROS-induced oxidation in mitochondria, of age-matched WT and A2ARKO mice revealed increased ROS burden in OA (A2AR KO) cartilage. In T/C28-a2 cells, neither IL-1β nor CGS21680 affected basal O2 consumption rates (OCR), maximal respiratory rate or ATP production but IL-1β-treated T/C28-a2 cells stimulated by CGS21680 increased all three measures of mitochondrial function (p<0.03, one-way ANOVA). Membrane potential (measured by TMRM MPI) decreased in T/C28-a2 cells after IL-1β or CGS treatment alone, but IL-1β + CGS21680 treatment significantly increased MPI, indicating enhanced mitochondrial health. Mitochondrial content is not significantly modulated by IL-1β, CGS or IL-1β+CGS in vitro, but IL-1β treatment significantly increased ROS burden (p<0.0001) and IL-1β+CGS did not affect ROS-burden in T/C28-a2 cells.

Conclusion: Mitochondrial function and biomass decline with age and after injury and diminished mitochondrial function contributes to the development of OA. A2AR stimulation enhances the function of mitochondria in inflamed chondrocytes and contributes to the maintenance of healthy chondrocytes and cartilage.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-a2a-receptor-a2ar-stimulation-enhances-mitochondrial-metabolism-and-mitigates-reactive-oxygen-species-mediated-mitochondrial-injury/