In OA chondrocytes, there is diminished mitochondrial production of ATP and diminished extracellular adenosine resulting in diminished adenosine A2A receptor (A2AR) stimulation and altered chondrocyte homeostasis which contributes to the pathogenesis of OA. We tested the hypothesis that A2AR stimulation maintains or enhances mitochondrial function in chondrocytes. The effect of A2AR signaling on mitochondrial health and function was determined in primary murine chondrocytes, a human chondrocytic cell line (T/C‐28a2), primary human chondrocytes, and a murine model of OA by transmission electron microscopy analysis, mitochondrial stress testing, confocal live imaging for mitochondrial inner membrane polarity, and immunohistochemistry. In primary murine chondrocytes from A2AR−/− null mice, which develop spontaneous OA by 16 weeks, there is mitochondrial swelling, dysfunction, and reduced mitochondrial content with increased reactive oxygen species (ROS) burden and diminished mitophagy, as compared to chondrocytes from WT animals. IL‐1‐stimulated T/C‐28a2 cells treated with an A2AR agonist had reduced ROS burden with increased mitochondrial dynamic stability and function, findings which were recapitulated in primary human chondrocytes. In an obesity‐induced OA mouse model, there was a marked increase in mitochondrial oxidized material which was markedly improved after intraarticular injections of liposomal A2AR agonist. These results are consistent with the hypothesis that A2AR ligation is mitoprotective in OA.

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced oA cartilage damage in a murine model of obesity-induced oA. the same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established posttraumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat oA. Osteoarthritis (OA) is a common disease affecting 151 million people worldwide and its incidence is expected to increase in industrialized countries due to aging and increased obesity of the population, a condition that together with previous joint injury represent the most common risk factors 1. OA can affect any joint, but most commonly affects the knee, hip and hand. The prevalence of OA is greatest in the knee joint, in both women (47%) and men (40%), and there is no therapy currently available that can reverse or halt the progression of OA 2,3 short of total joint replacement. Total knee replacements are the most common joint surgeries and it has been estimated that there will be a fivefold increase in the number of patients undergoing this surgical procedure up to 3.5 million by 2030 4,5. In search of effective therapies, a number of different approaches have been taken including a focus on both growth factors, such as transforming growth factor-beta (TGFβ), and other molecular pathways involved in regulating cartilage development and homeostasis. TGFβ molecular signaling exerts dual and opposing roles in cartilage and chondrocyte health depending on the receptor and signal activated downstream. It has been shown that inhibition of high levels of systemic TGFβ attenuates anterior cruciate ligament rupture-induced OA in mice by preventing loss of proteoglycan from the cartilage and protecting the subchondral bone from structural alteration 6. Moreover, the effect of TGFβ on joint health depends on which receptor it binds. Activation of

Autophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

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Methods: Ext1 was stably knocked down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Gene expression of chondrogenic markers (Col2a1, Acan andSox9) and direct Wnt target genes (Axin2 andLef1) was measured by RT q-PCR, at day 1, 7 and 14 of the chondrogenic differentiation process. Total proteoglycan content was evaluated by Alcian blue staining and DMMB assay. Activation of canonical Wnt signaling was