Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis.

E Skiöldebrand,S Ekman,P Önnerfjord,L Mattsson Hultén,U Rüetschi,A Lindahl,C Sihlbom,A Lundqvist,K Björkman,E Svala

doi:10.1111/evj.12666

Abstract

SummaryBackgroundClinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking.ObjectivesWe sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA.Study design In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well‐characterised horses.MethodsArticular cartilage explants were incubated with or without interleukin‐1β for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom‐developed inhibition enzyme‐linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes.ResultsSemitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N‐terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin‐1β‐stimulated explants.Main limitationsThe ELISA is based on polyclonal antisera rather than a monoclonal antibody.ConclusionsThe increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA.

Highlights

Many horses retire at a young age because of osteoarthritis (OA) [1,2], the most common reason for failure to train and race, and OA accounts for the greatest single economic loss in the horse racing industry and impacts on equine welfare [3]
The identification of cartilage oligomeric matrix protein (COMP) neoepitopes released from articular cartilage explants stimulated with IL-1b
The neopeptides were released at a higher abundance in the IL-1b-stimulated cartilage explants compared to nonstimulated (Table 1)

Summary

Introduction

Many horses retire at a young age because of osteoarthritis (OA) [1,2], the most common reason for failure to train and race, and OA accounts for the greatest single economic loss in the horse racing industry and impacts on equine welfare [3]. OA includes a low-grade inflammation with proinflammatory cytokines present during the early stages [4,8] that leads to the fragmentation of extracellular matrix (ECM) molecules and the formation and release of. Our hypothesis was that with the aid of a mass spectrometry (MS)-based approach using isobaric tandem mass tags (TMTs) in an in vitro cartilage inflammatory model to identify and quantify unique fragments of COMP, an increased concentration of identified fragments in synovial fluid would reflect early events in OA with no overlap to healthy joints. We investigated whether this neoepitope was uniquely found in the synovial fluid of horses with OA in vivo

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