Background/Purpose: We previously found that intra-articular injections of liposomal preparations of adenosine completely prevent progression and reverse cartilage loss in post-traumatic OA. TGF-β signaling plays dual and opposing roles in cartilage health and chondrocyte life depending on the signals activated downstream. Activation of downstream signaling pathways for TGF-β leading to localization of phospho-SMAD2/3 associated with maintenance of cartilage. In contrast nuclear localization of phospho-SMAD1/5/8 results in chondrocyte hypertrophy. Here we report that intraarticular injections of liposomal adenosine and A2AR agonist reverse OA in a different OA model and explore the role of TGFb signaling in this phenomenon.

Methods: Obesity-induced OA model: C57Bl6 mice (5-6 for each group, 12 weeks old) were fed a 60% fat diet (HFF mice) for 3months, after which received intraarticular injections (10 µl) of empty liposomes (LIPO) or liposomes containing the A2AR agonist CGS21680 (LIPO-CGS) or LIPO-Adenosine into the knee every 10 days for 4 injections. PTOA was induced in Sprague Dawley rats following non-surgical rupture of anterior cruciate ligament (ACL). Four weeks later rats were injected in the knee with 100ul of saline, LIPO or LIPO-CGS every 10 days (6 injections). RNA was isolated from chondrocytes in knee cartilage of rats treated as described above (3 from each group X 3 replicates) and subjected to RNAseq analysis. TC28a2 human chondrocyte cell line was used for in vitro experiments.

Results:

LIPO-CGS and LIPO-Adenosine reversed OA in the obesity-induced OA model. Mouse knees had an OARSI score of 5.17±1.84 before treatment. Treatment with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33±0.81 and 1.83±0.98, respectively, p<0.001 vs pre-treatment). RNAseq revealed an increase in aggrecan, TGFb2 and 3 and TGFb receptor 2 expression in chondrocytes from knees treated with LIPO-CGS. TGF-β expression was increased in deep layers of cartilage in the Lipo-CGS-treated rats and there was notable nuclear localization of phospho-SMAD2/3 in these chondrocytes. In contrast, phospho-SMAD1/5/8 was expressed in the nuclei of chondrocytes in the saline and LIPO-treated rats but not in the LIPO-CGS treated rats. Identical changes were observed in the knees of obese mice. To determine whether the effect of A2AR stimulation on TGFb signaling was direct or indirect we studied the effect of CGS21680 on nuclear phospho-SMAD expression in TC28a2 cells and found that CGS21680 increased nuclear phospho-SMAD2/3 and reduced nuclear phospho-SMAD1/5/8, as detected by immunofluorescence.

Conclusion: Administration of an A2AR agonist to established OA knees reverses OA in rats and mice and shifts TGFb signaling from ALK1/SMAD1/5/8 to ALK5/SMAD2/3 in OA chondrocytes after activation of A2AR in 2 OA animal models. These findings suggest a novel approach to the treatment of OA with the potential to reverse the changes in cartilage that characterize OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a2a-adenosine-receptor-stimulation-switches-tgf-%ce%b2-signaling-to-promote-chondrocyte-proliferation-and-cartilage-regeneration/