Background: Ageing-related failure of homeostasis mechanisms contributes to articular cartilage degeneration and osteoarthritis (OA), for which disease-modifying treatments are not available. Our objective was to identify molecules to prevent OA by regulating chondrocyte senescence and autophagy. Methods: Human chondrocytes with IL-6 induced senescence and autophagy suppression and SA-β-gal as a reporter of senescence and LC3 as reporter of autophagic flux were used to screen the Prestwick Chemical Library of approved drugs. Preclinical cellular, tissue and blood from OA and blood from OA and ageing models were used to test the efficacy and relevance of activating PPARα related to cartilage degeneration. Findings: Senotherapeutic molecules with pro-autophagic activity were identified. Fenofibrate (FN), a PPARα agonist used for dyslipidaemias in humans, reduced the number of senescent cells via apoptosis, increased autophagic flux, and protected against cartilage degradation. FN reduced both senescence and inflammation and increased autophagy in both ageing human and OA chondrocytes whereas PPARα knockdown conferred the opposite effect. Moreover, PPARα expression was reduced through both ageing and OA in mice and also in blood and cartilage from knees of OA patients. Remarkably, in a retrospective study, fibrate treatment improved OA clinical conditions in human patients from the Osteoarthritis Initiative (OAI) Cohort. Interpretation: These results demonstrate that FDA-approved fibrate drugs targeting lipid metabolism protect against cartilage degeneration seen with ageing and OA. Thus, these drugs could have immediate clinically utility for age-related cartilage degeneration and OA treatment.
Pellets were cultured for 14 days in serum-free chondrogenic medium (without dexamethasone). After 10 days of differentiation, MSCs were exposed to the OAs-cm from the same twelve individual donors. Cartilage formation was determined at day 14 by measuring sulfated glycosaminoglycan (sGAG) content. Results: Stimulation of the hIL6, hIL8 and hADAMTS5 promoter reporters with OAs-cm form twelve different patients resulted in an upregulation of promoter activity compared to control stimulation, whereas stimulation of the hWISP1 promoter reporter resulted in a down-regulation of the promoter activity compared to control. Interestingly, large heterogeneity was observed in the response of the promoter reporters between the OAs-cm from distinct patients with hIL6 (FC 33.9 ± 32.3), hIL8 (FC 32.2 ± 27.7), hADAMTS5 (FC 3.7 ± 2.4) and hWISP1 (FC 0.4 ± 0.24).Individual OAs-cm reduced sGAG content of differentiating MSCs by a range between 3% and 65%. Significant correlations were found between the fold change of the promoter activity and the percentage of inhibition on chondrogenesis. We observed a positive correlation with hIL6 (p¼0.01 and r¼0.67), hIL8 (p¼0.01 and r¼0.66) and with hADAMTS5 (p¼0.01 and r¼0.72), whereas a negative correlation was seen with hWISP1 (p¼0.003 and r¼-0.75). Conclusions: The current study demonstrates that we are able to measure profound heterogeneous patient-specific responses on different promoter reporters with OAs-cm from individual patients. Furthermore, significant correlations were found between the fold change of these promoter reporters and the inhibition of chondrogenic capacity of hMSCs, with positive correlations for hIL6, hIL8 and hADAMTS5 and a negative correlation with hWISP1. Our promoter constructs appear to be able to predict repair capacity of stem cells and could be of use for a personalized treatment strategy.
BackgroundIn osteoarthritis (OA), defects in cellular homeostasis, and in particular in autophagy, are evident and precede joint damage. In this sense, we have shown that there is a defect in autophagy in OA human chondrocytes and cartilage, and pharmacological activation of autophagy protects against joint damage. These data suggest that joint damage could be due, at least in part, to a failure of autophagy, by inducing an abnormal accumulation of cellular products related to disease.ObjectivesThese observations represent a unique opportunity to identify and validate potential biomarkers associated with autophagy defects that could facilitate the development of therapeutic strategies to prevent OA progression.MethodsA comparative analysis of 86 autophagy genes was performed in blood from non-OA and knee OA patients. Non-OA patients (Age: 61,17±1,370 years; BMI: 25,76±0,69; Sex: Females; n=12) and Knee OA patients (Age: 65,75±1,528 years; BMI: 30,25±0,88; Sex: Females; n=12, OA grade III-IV) were profiled using a human autophagy PCR array (PrimePCR autophagy human panel, BioRad) and analysed using the PrimePCR analysis software, Biorad. In addition, we performed a quantitative proteomic analysis of defective autophagy by genetic deletion of Atg5 in human OA chondrocytes by using iTRAQ (isobaric tags for relative and absolute quantitation) labelling coupled with on-line 2D LC/MS/MS. Protein identification and quantification were performed using Protein Pilot Software 4.0. Each MS/MS spectrum was searched in the Uniprot/Swissprot database for Homo sapiens.Results16 autophay-related genes were significantly down-regulated in blood from knee OA patients compared to non-OA patients. No significant up-regulation was observed in blood from Knee OA patients, however a trend-toward up-regulation was detected in several genes involved in the mTOR signalling pathway. Importanly, 5 key autophagy-related genes, such as, ATG16L2, ATG12, ATG7, ATG4B and MAP1LC3B involved in initiating autophagy, phagophore extension and autophagosome formation were significant downregulated in knee OA patients compared to non-OA patients (p<0.05). Interestingly, HSP90AA1 and HSPA8, a chaperone-mediated autophagy genes involved in stress response and protein folding, were significant downregulated (p<0.001) in blood from knee OA patients. In addition, several regulators of autophagy and apoptosis, such as BNIP3, BCL-2 and BCL2L1 were a significantly downregulated in OA patients (p<0.01). Total proteome screening in human OA chondrocytes with defective autophagy, showed a significant reduction of Heat shock protein HSP90-alpha (HSP90A1) (p<0.05), suggesting that reduced autophagy is associated to OA pathology and could be a potential biomarker for OA progression and development.ConclusionsThis approach represents an unique opportunity to identify and validate early-stage biomarkers associated with defective autophagy that could facilitate the development of therapeutic strategies to prevent joint damage.Disclosure of InterestNone declared
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