Session Title: Biology and Pathology of Bone and Joint - Poster I
Session Type: ACR Poster Session C
Session Time: 9:00AM-11:00AM
Background/Purpose: Effective treatments for Osteoarthritis (OA) are not available. In aging-related diseases, including OA, failure of cellular homeostasis mechanisms, such as autophagy can cause extracellular matrix destruction and cell death. Chondrocytes are essential for the maintenance of cartilage integrity. With aging, chondrocyte function is diminished, contributing to a cellular senescence phenotype often observed in OA chondrocytes. In addition, a defect in autophagy is observed in both aging and cartilage degeneration. The objective of this study was to identify anti-senescence and pro-autophagy molecules by a cell-based high-throughput screening (HTS) in human chondrocytes.
Methods: To induce cellular senescence or reduced autophagy, immortalized human chondrocytes (TC28a2) were seeded (3000 cells/well) in 384 well plates, and treated with IL-6 (10 ng/ml) for 72 or 18 hours, respectively. Then, chondrocytes were incubated with Prestwick Chemical Library (1120 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 μM for 48 hours. To identify anti-senescence hits, nuclei was stained with Hoechst 33342 (10 μM), while β–galactosidase subcellular structures was stained by using Imagene Green C12FDG substrate (10 μM). To evaluate autophagic flux, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Relative intensity of C12FDG in cytoplasm and number of autophagosomes/autolysosomes per area of cytoplasm were determined to quantitate β–galactosidase activity and autophagy flux respectively. For compound validation, senescence markers, autophagic flux and apoptosis were evaluated.
Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated β-galactosidase activity. 308 compounds with anti-senescence effects were identified. The anti-senescence hits were analyzed by monitorizing autophagic flux. 42 compounds with both anti-senescence and pro-autophagy effects were selected. Then, 2 compounds were selected for further validation. These compounds significantly reduced senescence (p <0.01) and increased autophagic flux (p <0.01) in response to IL-6. Furthermore, they conferred protection against cell death by apoptosis in human chondrocytes.
Conclusion: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.
To cite this abstract in AMA style:Carames B, Nogueira-Recalde U, Dominguez E, Loza MI, Blanco FJ. Identification of Novel Molecules Targeting Cartilage Aging As Osteoarthritis Therapeutics [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/identification-of-novel-molecules-targeting-cartilage-aging-as-osteoarthritis-therapeutics/. Accessed March 25, 2019.
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