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

关于继续开展一项研究计划的提案，以调查组织蛋白酶 K 在马骨关节炎 (OA) 中的作用并为 OA 的早期检测创建新的诊断方法

• 批准号: RGPIN-2014-03836
• 负责人: Laverty, Sheila
• 金额: $ 3.86万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=656790
• 关键词: PROPOSAL; CONTINUATION; RESEARCH; PROGRAM; INVESTIGATE

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.

请求资金支持马关节疾病的一项正在进行的高产研究计划。骨关节炎（OA），最常见的马关节疾病，是一个主要的经济和福利问题，在运动和老年加拿大马。骨性关节炎的特点是关节软骨破坏、骨重塑和炎症，导致疼痛和关节功能丧失。其结果是退出体育活动，如果严重的话，甚至是安乐死。*关节软骨的细胞外基质由常驻软骨细胞维持。最丰富和机械必需的结构软骨基质分子是II型胶原。它形成了一个广泛的网络，将软骨连接在一起，并赋予其抗拉强度。II型胶原蛋白分子由三条相同的a链组成，它们结合形成三螺旋结构。这些分子交联形成纤维胶原网络的微原纤维。人们早就认识到骨性关节炎中关节软骨的破坏是由软骨细胞蛋白酶消化基质介导的。软骨II型胶原纤维网络的破坏是OA中一个关键的不可逆事件，显然与软骨损失有关。完整II型胶原分子的三螺旋结构域可以抵抗除MMP1、8、13和14等胶原酶外的大多数蛋白酶的降解。mmp13被认为在关节软骨破裂中起关键作用，也是OA的药物靶点。检测特定的分子片段，在II型胶原蛋白被胶原酶降解后释放，导致生物标志物检测的发展，以测量OA疾病在体外和体内的活动性。*我们最近报道了组织蛋白酶K （catK）也能够切割II型胶原的完整三螺旋结构，并且该酶的活性在马OA软骨中高度上调。基于这些观察，我们认为catK在关节软骨酶降解中与mmp13一样重要，甚至可能更重要。我们发现了独特的马catK II型胶原特异性切割新表位，包括C2K77（专利申请），并提出了针对C2K77的多克隆抗体，可以检测组织中的早期马OA。*在本研究计划的继续，我建议研究影响catk介导的马软骨中这些C2K77的产生和释放的条件。这些释放到体液中的新表位片段的结构将被阐明，以进一步了解catK在OA中的重要性，并开发新的免疫检测方法来检测早期马OA。*该计划的具体目标是：1)开发针对catK生成的II型胶原特异性新表位C2K77的ELISA免疫测定，用于定量catK活性。2)体外鉴定马关节软骨catK活性上调的化学因子。3)通过对马ⅱ型胶原catK消化在体内产生的含C2K77的所有片段序列结构的表征，建立OA中产生的优势片段，并建立夹心免疫分析法。4)评估基于C2K77的新型马生物标志物检测体液中OA的效果。马OA研究中一个新兴领域的研究项目有望推进catK参与马OA中II型胶原蛋白降解和软骨破坏的知识。由于OA是一个重大的福利和经济问题，开发一种基于体液分析的、敏感的、特异性的早期马OA诊断测试对马行业来说非常重要。这项研究的结果也有可能影响人类和犬类OA的研究。