Identification of fibronectin neoepitopes present in human osteoarthritic cartilage

Abstract

Fibronectin fragments are present at high concentrations in the cartilage of patients with rheumatoid arthritis and patients with osteoarthritis (OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in human OA cartilage and confirm their ability to elicit cartilage catabolism, 2) identify the cleavage sites in fibronectin and generate the corresponding neoepitope antibodies, and 3) explore the utility of fibronectin neoepitopes as biomarkers. Fibronectin fragments were purified from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing. Bovine nasal cartilage was treated with affinity-purified fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans and the aggrecan neoepitope 1771AGEG. Fibronectin neoepitopes were detected by Western blotting in cytokine-treated media of human cartilage explants, and by immunohistochemical analyses of human OA cartilage. Multiple fibronectin fragments were isolated from human OA cartilage, and all contained the N-terminus 272VYQP. These fragments induced aggrecanase-mediated cartilage catabolism in bovine cartilage explants. Fibronectin fragments with the N-terminus 272VYQP and fragments with the C-terminus VRAA271 were detected following cytokine treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA cartilage. Human OA cartilage contains fibronectin fragments with catabolic activity and a major cleavage site within fibronectin. This study is the first to characterize fibronectin neoepitopes in OA cartilage, suggesting that they may represent a novel biomarker of arthritis.