Introduction: Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints. Methods: Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity. Results: According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17. Conclusions: According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.

Extracellular vesicles (EVs) are emerging as key factors in maintaining cellular homeostasis, critical mediators of intercellular communication, potential biomarkers, and therapeutic tools. While small EVs have been extensively characterized, the molecular signatures of large EVs (including those generated during regulated cell death pathways) remain poorly defined. Here, we investigated the characteristics of large EVs released during apoptosis and pyroptosis by human monocytic cell lines (THP-1 and U937). Apoptosis was induced by staurosporine and blocked using the pan-caspase inhibitor Q-VD-OPh, whereas pyroptosis was triggered by LPS/nigericin and inhibited with a selective NLRP3 inhibitor. We found that both forms of regulated cell death markedly enhanced the release of large EVs. Both apoptotic and pyroptotic large EVs showed increased Annexin V binding and decreased CD9 expression compared with those released by healthy cells. Large EVs derived from apoptotic and pyroptotic cells exhibited distinct proteomic profiles. Pyroptotic large EVs carried interacting protein networks of RNA-binding proteins and chromatin-associated proteins many of which are known damage-associated molecular patterns or alarmins. In contrast, we found that a subpopulation of apoptotic large EVs was characterized by the presence of dsDNA, and active caspase-3/7. Together, our data shed light on the specific protein cargo of large EVs released by cells during apoptosis and pyroptosis. This study identifies candidate markers of large EVs released by dying cells and may enhance our understanding of the role of EVs in regulated cell death.