Abstract
IntroductionAggrecanase cleavage at the 392Glu-393Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal 393ARGS fragments, is an early key event in arthritis and joint injuries. Here, we use a quantitative immunoassay of aggrecan ARGS neoepitope fragments in human synovial fluid to determine if this cleavage-site specific method better identifies joint pathology than previously available less specific aggrecan assays.MethodsSynovial fluid (SF) from 26 people with healthy knees (reference) and 269 patients were analyzed in a cross-sectional study. Patient groups were acute inflammatory arthritis, acute knee injury, chronic knee injury and knee osteoarthritis (OA). Aggrecan ARGS fragments were assayed by ELISA using the monoclonal antibody OA-1. Total aggrecan content was analyzed by an ELISA using the monoclonal antibody 1-F21, and sulfated glycosaminoglycan by Alcian blue precipitation.ResultsAggrecan ARGS fragment concentrations in all groups differed from the reference group (P < 0.001). The acute inflammatory arthritis group had the highest median level, 177-fold greater than that of the reference group. Median levels (in pmol ARGS/ml SF) were: reference 0.5, acute inflammatory arthritis 88.5, acute knee injury 53.9, chronic knee injury 0.5 and OA 4.6. In contrast, aggrecan and sulfated glycosaminoglycan concentrations varied much less between groups, and only acute inflammatory arthritis and acute knee injury were found to have a two-fold increase in median levels compared to the reference.ConclusionsLevels of aggrecan ARGS fragments in human synovial fluid are increased in human arthritis, OA and after knee injury, likely reflecting an enhanced cleavage at the 392Glu-393Ala bond in the IGD by aggrecanase. An assay that specifically quantified these fragments better distinguished samples from joints with pathology than assays monitoring aggrecan or glycosaminoglycan concentrations. The newly developed ARGS fragment assay can be used to monitor aggrecanase activity in human joint disease and experimental models.
Highlights
Introduction Aggrecanase cleavage at the392Glu-393Ala bond in the interglobular domain (IGD) of aggrecan, releasing Nterminal 393ARGS fragments, is an early key event in arthritis and joint injuries
Aggrecan and sulfated glycosaminoglycan concentrations varied much less between groups, and only acute inflammatory arthritis and acute knee injury were found to have a two-fold increase in median levels compared to the reference
Levels of aggrecan ARGS fragments in human synovial fluid are increased in human arthritis, OA and after knee injury, likely reflecting an enhanced cleavage at the 392Glu393Ala bond in the IGD by aggrecanase
Summary
Proteolysis of aggrecan is an early and critical feature of cartilage degradation in arthritis and after knee injury, and is measurable as an elevation of aggrecan release from the cartilage into the synovial fluid (SF) [1,2,3,4] Proteases, such as matrix metalloproteases (MMPs), cathepsins and calpains, are involved [5], aggrecanase plays a major role in aggrecan degradation in murine [6,7] and human [4,8,9,10,11,12,13,14,15] joint disease. Fragments carrying the same neoepitope were found in serum from patients with rheumatoid arthritis, but not in healthy controls [15]
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