Background/Purpose: Disease-modifying treatments for Osteoarthritis (OA) are not available. Aging-related features such as failure of homeostasis mechanisms, including autophagy, cause extracellular matrix damage, chondrocyte senescence and death, which leads to articular cartilage degeneration and joint dysfunction. The objective of this study was to identify Senolytic and Pro-Autophagy molecules to prevent cartilage degeneration and OA.

Methods: Cellular senescence and defective autophagy was induced in immortalized human chondrocytes (T/C28-a2) with IL-6, a SASP factor, at 20ng/ml for 72 or 18 hours, respectively. Then, chondrocytes were incubated with Prestwick Chemical Library at 10μM for 72 hours. To identify senolytics, SA-β-gal activity was determined by Imagene Green C12FDG substrate reporter. To identify molecules activating autophagy flux, pBABE-mCherry-EGFP-LC3 reporter was used to generate stable expression in chondrocytes by retrovirus transfection. Imaging was done by using Operetta® High Content Screening system. Confirmatory assays with readouts for senescence, autophagy, inflammation and apoptosis were performed in primary human chondrocytes. The anabolic effect was evaluated by Safranin O staining and Nitric Oxide production in human cartilage. To establish the senomorphic or senolytic nature of the candidate, senescent cells and total cells were counted with Cell Analyzer 6000 Confocal Imaging System. Navitoclax 2,5μM and Rapamycin 10μM were used as controls for senolytic and senomorphic activity, respectively. The functional consequence of treatment with the candidate was evaluated in blood, chondrocytes and cartilage from non-OA and knee OA patients.

Results: Primary screen yielded 279 senolytic compounds. Secondary screen identified 37 compounds with both senolytic and pro-autophagy activity. Fenofibrate (FN), a PPARα agonistapproved for dyslipidemia was selected as candidate. FN reduced senescence (p<0.001) and increased autophagic flux (p<0.0001), protecting against defective autophagy and inflammation in response to IL-6 and IL-1b. This protection was confirmed in articular cartilage explants by a reduction of proteoglycans loss (p <0.05) and in primary human chondrocytes by a reduction of NO production and death by apoptosis (p<0.0001). Moreover, a senolytic effect was observed in human chondrocytes (p<0.05) by selective reduction the number of senescent cells by apoptosis. Furthermore, FN upregulated PPARα target genes and FoxO1 expression. These effects were also observed for structurally distinct PPARα agonists, suggesting that pharmacological modulation of PPARα may provide therapeutic benefits in OA. Remarkably, PPARα expression was reduced in blood and cartilage from knee OA patients.

Conclusion: Our cell-based imaging assay provides a unique opportunity to identify novel drugs and mechanisms to prevent cartilage pathology. Dual senolytic and pro-autophagy effects may provide benefits in cartilage degeneration associated to aging and inflammation. A positive outcome of ongoing efficacy studies focused on PPARα might provide the basis to propose proof-of-concept studies in patients with OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-novel-molecules-with-senolytic-and-autophagy-activity-as-osteoarthritis-therapeutics/