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Impact of Mechanical Load on the Expression Profile of Synovial Fibroblasts from Patients with and without Osteoarthritis.

ABSTRACT: Osteoarthritis (OA) affects the integrity of the entire joint including the synovium. The most abundant cells in the synovium are fibroblasts (SF). Excessive mechanical loading might contribute to OA pathogenesis. Here, we investigate the effects of mechanical loading on SF derived from non-OA (N-SF) and OA patients (OA-SF). We treated N-SF and OA-SF with or without mechanical loading for 48h after 24h of preincubation. Then we assessed gene and protein expression of proinflammatory factors (TNF?, COX-2, PG-E2, IL-6), extracellular matrix (ECM) components (COL1, FN1) and glycosaminoglycans (GAGs) via RT-qPCR, ELISA, DMMB assay and HPLC. Mechanical loading significantly increased TNF? and PG-E2 secretion by N-SF and OA-SF, whereas in OA-SF IL-6 secretion was reduced. COL1 and FN1 secretion were downregulated in N-SF during loading. OA-SF secreted less COL1 compared to N-SF under control conditions. In contrast, OA-SF in general expressed more FN1. GAG synthesis was upregulated in N-SF, but not in OA-SF during loading with OA-SF displaying a higher charge density than N-SF. Mechanical loading enhanced proinflammatory factor expression and GAG synthesis and decreased secretion of ECM components in N-SFs, indicating a contributing role of SF to OA development.

SUBMITTER: Schroder A

PROVIDER: S-EPMC6387339 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.

Project description: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 beta-D-hexosaminidase, beta-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. beta-D-Glucuronidase, beta-D-glucosaminidase and beta-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, beta-D-hexosaminidase, beta-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 beta-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-beta1 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 TNFalpha, IL-1beta 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.

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Impact of Mechanical Load on the Expression Profile of Synovial Fibroblasts from Patients with and without Osteoarthritis.

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