Background/Purpose: Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 10% of the US population and one of the contributing factors in OA pathogenesis is inflammaging, chronic low grade inflammation. With injury and aging there is dysfunction of chondrocyte mitochondria, thought to be a central event in inflammaging, resulting in reduction of intracellular and extracellular ATP and extracellular adenosine.  Reductions in extracellular adenosine or loss of A2AR leads to the development of OA. We therefore explored determined whether A2AR ligation regulates mitochondrial health in vivo and in vitro.

Methods: Primary murine WT and A2ARKO as well as primary human chondrocytes were isolated from neonatal mice or differentiated from surgical discard, treated with medium (CTRL) or IL-1β (5ng/mL for 4h) with and without the A2AR agonist, CGS21680 (1uM for 1h, CGS) or CGS alone and then stained with TMRM to evaluate mitochondrial length, network volumes and membrane polarity. RNAseq, RT-PCR and transmission electron microscopy (TEM) were performed in untreated murine chondrocytes and proteins of interest were evaluated by WB. Mitochondrial markers were also evaluated in rat post-traumatic OA (PTOA) and mouse obesity-induced OA (OB-OA) modelsin vivo. Human chondrocytic cells (T/C28-a2) were grown in culture, treated as stated above and submitted for TEM to study mitochondrial ultrastructure. Primary human chondrocytes were cultured from discarded surgical specimens.

Results: WT chondrocytes treated with CGS +/- IL-1β had increased mitochondrial lengths than cells that received medium or IL-1β alone (p< 0.0001, ONE-Way ANOVA). There were no significant differences in mitochondrial lengths of untreated WT or null chondrocytes but loss of A2AR signaling prevented mitochondrial length expansion after stimuli (p< 0.04, ONE-Way ANOVA). Depolarization upregulates total PINK1 expression in A2AR-/-  (p< 0.005, unpaired, two-tailed t-test) but not Parkin levels. ROS burden was reduced and mitochondrial markers were improved in PTOA and OB-OA models in vivo. In fully differentiated human chondrocytes, A2AR ligation after stimulation with IL-1β increased TMRM and mitochondrial volumes (p< 0.0001, ONE-Way ANOVA). T/C28-a2 cells treated with CGS after IL-1β exposure had significantly reduced cristae widths (p< 0.0001) and an increased number of cristae junctions per mitochondria (p=0.02, ONE-Way ANOVA).

Conclusion: A2AR ligation enhances mitochondrial function and biomass in murine chondrocytes in vitroand in vivo in models of OA and, thereby diminishes the signs of inflammaging, maintains chondrocyte homeostasis and reverses OA in vivo.

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