FRI0540 Identification of biomarkers of oa associated to defective autophagy

Abstract

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