POS0402 LYSYL OXIDASE (LOX) AND LYSYL OXIDASE-LIKE 2 (LOXL2) CONTRIBUTE TO CARTILAGE CALCIFICATION DURING OSTEOARTHRITIS

Background Cartilage pathological calcification is a hallmark of osteoarthritis (OA), a degenerative disease characterized by articular cartilage degradation that leads to joint pain and impaired movement. We previously demonstrated that lysyl oxidase-like enzymes (LOX(L), which include five enzymes, LOX and LOXL1-4), that catalyze the crosslinking of collagen and elastin fibers of the extracellular matrix (ECM), play a crucial role in cartilage calcification (Bernabei et al, in preparation). Indeed, the pan-LOX(L) inhibitor BAPN decreased crystal production in chondrocytes, but the identification of the crucial LOX(L) involved in this process remained to be established. Objectives To identify the specific LOX(L) involved in cartilage calcification during OA. Methods In vitro, murine chondrocytes (primary cells or ATDC5 cells) were cultured in normal medium ((Nt), DMEM high glucose + 10% FBS) or calcifying medium ((CM), BGJb +10% FBS+ 20mM β-glycerophosphate + 50μg/ml ascorbic acid). The expression of Lox was silenced using siRNA at 100nM ((siLox), OriGene). Gene expression was measured by qPCR. Calcium-containing crystals were evidenced by alizarin red staining and quantified by cetylpiridinium dissolution of crystals. In vivo, mice were subjected to meniscectomy (MNX) in the right knee or sham operated in the left. Ex vivo, cartilage explants from undamaged or damaged cartilage regions were obtained from 7 OA patients undergoing knee replacement surgery. Immunohistochemistry (IHC) was performed on murine knees and human cartilage sections ((anti-LOX or anti-LOXL2 rabbit polyclonal antibodies (Proteintech or Genetex respectively). Calcification was detected by Alizarin red staining on histological sections, and proteoglycan loss by safranin-O staining. Mouse gene expression data from primary articular chondrocytes treated with interleukin-1β (IL-1β) for 24h were obtained from GEO (identifier: GSE104793). Results Lox expression was increased in murine chondrocytes stimulated with calcification medium (CM), but not Loxl2. Similarly, transcriptome data from primary mouse articular chondrocyte treated with IL-1β, revealed that both Lox and Loxl2 were markedly upregulated in treated cells. We then evaluated Lox and Loxl2 modulation by IHC in calcified cartilage in the MNX OA model. Both Lox and Loxl2 were increased in MNX cartilage compared to sham cartilage and at an even higher extent in osteophytes and in newly formed calcified deposits appearing in MNX knees. In human OA cartilage we found massive calcification and proteoglycan loss in damaged areas compared to undamaged. In line with murine data, we revealed increased LOX and LOXL2 expression in damaged human cartilage, both in chondrocytes and in the extracellular matrix in correspondence to calcified regions. Next, we performed in vitro Lox RNA silencing using siLox or siControl RNA transfected ATDC5 cells (siLox cells or siCtrl cells). Lox expression was decreased by more than 80% in siLox cells compared to siCtrl cells. Most importantly, in CM, siLox cells calcified less as demonstrated by alizarin red quantification. Furthermore, gene expression data showed inhibition of calcification gene Annexin 5 by Lox silencing. In agreement, we found an increase of early differentiation genes Sox9 and Col2, along with significant downregulation of hypertrophic marker Col10. However, no effect by Lox silencing was found on fibrotic genes (Col1, Col3) and on pro-calcifying cytokine interleukin-6 (Il-6). Additionally, we revealed a trend towards decreased mitochondrial reactive oxygen species in siLox cells. Finally, Lox silencing decreased gene expression of the ECM catabolic enzyme Mmp13. Conclusion Our data revealed that LOX and LOXL2 are increased in calcified areas of murine and human cartilage. Additionally, in vitro inhibition of Lox prevents at least in part, calcium-crystal deposition, chondrocyte hypertrophy, and Mmp13 catabolic enzyme expression. Altogether, our results suggest that both LOX and LOXL2 might play a role in cartilage calcification and OA. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests None Declared.