Identification of a complex between fibronectin and aggrecan G3 domain in synovial fluid of patients with painful meniscal pathology Gaetano J. Scuderi b , Naruewan Woolf a , Kaitlyn Dent a , S. Raymond Golish b , Jason M. Cuellar b,1 , Vanessa G. Cuellar b,1 , David C. Yeomans b , Eugene J. Carragee b , Martin S. Angst b , Robert Bowser a , Lewis S. Hanna a, ⁎ a Research and Development, Cytonics Corporation, 555 Heritage Drive, Suite 115, Jupiter, FL 33458, USA b Stanford University, Stanford, CA, USA abstract article info Article history: Received 10 February 2010 Received in revised form 6 April 2010 Accepted 22 April 2010 Available online 11 May 2010 Keywords: Fibronectin Aggrecan Biomarker Cartilage Synovial fluid Pain Objectives: We previously described a panel of four cytokines biomarkers in knee synovial fluid for acute knee pain associated with meniscal pathology. The cytokine biomarkers included interferon gamma (IFN-γ), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β). Validation studies using other immunologic techniques confirmed the presence of IL-6, MCP-1 and MIP-1β, but not IFN-γ. Therefore we sought the identity of the IFN-γ signal in synovial fluid. Methods: Knee synovial fluid was collected from patients with an acute, painful meniscal injury, as well as asymptomatic volunteers. A combination of high-pressure chromatography, mass spectrometry and immunological techniques were used to enrich and identify the protein components representing the IFN-γ signal. Results: A protein complex of fibronectin and the aggrecan G3 domain was identified in the synovial fluid of patients with a meniscal tear and pain that was absent in asymptomatic controls. This protein complex correlated to the IFN-γ signal. A novel enzyme-linked immunosorbent assay (ELISA) was developed to specifically identify the complex in synovial fluid. Conclusions: We have identified a protein complex of fibronectin and aggrecan G3 domain that is a candidate biomarker for pain associated with meniscal injury. © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Degenerative joint disease and joint injury are associated with increased turnover of articular cartilage proteins, inflammation and alterations to other joint tissue proteins [1,2]. Degenerative joint disease in the knee is often idiopathic, however it has also been strongly associated with prior injury such as meniscal damage. The inflammatory milieu induced by such injury may therefore lay the groundwork for future degeneration and osteoarthritis. The profile of inflammatory proteins within synovial fluid after acute knee injury may represent diagnostic or prognostic biomarkers for the degener- ative joint disease or osteoarthritis that may ensue. Expression and fragmentation of the extracellular matrix protein fibronectin has been shown to occur in the synovial fluid of arthritic patients and joint injury [3,4]. Fibronectin fragment induced knee injury in an animal model results in further cartilage damage and loss of proteoglycans [5]. Fibronectin also induces microglial activation and stimulation of cytokine production and activation of matrix metalloproteases [6]. It is well known that inflammatory cytokines are associated with fibronectin and its fragments in the pathophysiology of degenerative joint disease [7]. Aggrecan, a high molecular weight proteoglycan present in articular cartilage, undergoes extensive degradation and turnover during normal cartilage metabolism, aging and joint diseases [8]. Aggrecanases are activated during cartilage degradation and diseases [9]. Recently, it was demonstrated that patterns of aggrecan fragments differ between acute injury and chronic degeneration relative to healthy controls [10]. Therefore both fibronectin and aggrecan exhibit increased fragmentation in degenerative joint conditions and after articular cartilage damage. It is possible that fibronectin, aggrecan and their fragments interact in the synovial fluid to facilitate signaling cascades that augment joint and cartilage degeneration. Clinical Biochemistry 43 (2010) 808–814 Abbreviations: IFN-γ, interferon gamma; IL-6, interleukin 6; MCP-1, monocyte chemotactic protein 1; MIP-1β, macrophage inflammatory protein-1 beta; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; HPLC, high performance liquid chromatography; SEC, size exclusion chromatography; AEC, anion exchange chromatography; TMB, tetramethylbenzidine; LC-MS/MS, liquid chromatog- raphy based mass spectrometry; FN1, fibronectin; ACAN, aggrecan. ⁎ Corresponding author. E-mail address: Lewis.hanna@cytonics.com (L.S. Hanna). 1 Current address: New York University Hospital for Joint Diseases, New York City, NY, USA. 0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2010.04.069 Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem