A PROPOSAL FOR CONTINUATION OF A RESEARCH PROGRAM TO INVESTIGATE THE ROLE OF CATHEPSIN K IN EQUINE OSTEOARTHRITIS (OA) AND CREATE NEW DIAGNOSTIC ASSAYS FOR THE EARLY DETECTION OF OA

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Funds are requested for support of an ongoing and highly productive research program in equine joint disease. Osteoarthritis (OA), the most common equine joint disease, is a major economic and welfare problem in athletic and aged Canadian horses. OA is characterized by destruction of the articular cartilage, bone remodeling and inflammation that causes pain and loss of joint function. The outcome is retirement from athletic activity and, if severe, euthanasia.*The extracellular matrix of articular cartilage is maintained by resident chondrocytes. The most abundant and mechanically essential structural cartilage matrix molecule is type II collagen. It forms an extensive network that holds cartilage together and gives it its tensile strength. Type II collagen molecules consist of three identical a chains that combine to form a triple helix. These molecules are cross-linked to form the microfibrils of the fibrillar collagen network. It has long been recognized that the destruction articular cartilage in OA is mediated by chondrocyte protease digestion of the matrix. Destruction of the type II collagen fibrillar network of cartilage is a key irreversible event in OA and clearly linked to cartilage loss. It was believed that the triple helical domain of intact type II collagen molecules is resistant to degradation by most proteases except collagenases such as MMP1, 8, 13 and 14. MMP 13 is considered of key importance in articular cartilage breakdown and a drug target for OA. Detection of specific molecular fragments, released following degradation of type II collagen by collagenases, has resulted in the development of biomarker assays to measure OA disease activity in vitro and in vivo. *We recently reported that cathepsin K (catK) is also capable of cleavage of the intact triple helix of type II collagen, and activity of this enzyme is highly upregulated in equine OA cartilage. Based on these observations, we believe that catK is as important, and potentially more important, than MMP 13 in the enzymatic degradation of articular cartilage. We identified unique equine catK type II collagen specific cleavage neoepitopes, including C2K77 (patent filed), and have raised polyclonal antibodies to C2K77 that can detect early equine OA in tissues.*In continuation of this research program, I propose to study the conditions that influence catK-mediated generation and release of these C2K77 from equine cartilage. The structures of these neoepitope-containing fragments released into body fluids will be elucidated to further understand the importance of catK in OA and to develop new immunoassays to detect early equine OA. *Specific objectives of the program are to: 1) Develop an ELISA immunoassay directed at a catK-generated type II collagen specific neoepitope C2K77 for quantitation of catK activity. 2) Identify chemical factors that upregulate catK activity in equine articular cartilage in vitro. 3) Establish the dominant fragment(s) generated in OA by characterization of sequence structures of all fragments containing the C2K77 produced in vivo by catK digestion of equine type II collagen and develop sandwich immunoassays. 4) Assess the new equine biomarker assays based on C2K77 for the detection of OA in body fluids. This program of investigation in a novel emerging field in equine OA research holds the promise to advance knowledge of catK involvement in type II collagen degradation and destruction of cartilage in equine OA. The development of a sensitive, specific diagnostic test(s) for early equine OA based on analysis of body fluids would be extremely important for the equine industry as OA is a major welfare and economic issue. The results of this research also have the potential to impact both human and canine research in OA.