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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630101
• 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.

要求资金支持马匹联合疾病中正在进行的高产研究计划。骨关节炎（OA）是最常见的马关节疾病，是运动和老年马匹的主要经济和福利问题。 OA的特征是破坏关节软骨，骨骼重塑和炎症，导致疼痛和关节功能的丧失。结果是从运动活动中退休，如果严重的是安乐死。关节软骨的细胞外基质由居民软骨细胞维持。最丰富，最重要的结构软骨基质分子是II型胶原蛋白。它形成了一个广泛的网络，将软骨融合在一起并赋予其拉伸强度。 II型胶原蛋白分子由三个相同的A链组成，它们结合形成三重螺旋。这些分子交联以形成原纤维胶原网络的微纤维。长期以来，人们已经认识到，OA中的破坏关节软骨是由基质的软骨细胞蛋白酶消化介导的。在OA中破坏了软骨II型胶原蛋白原纤维网络是一个重要的不可逆事件，并且与软骨损失显然有关。人们认为，除了MMP1、8、8、13和14之外，大多数蛋白酶的完整II型胶原蛋白分子的三重螺旋结构域对大多数蛋白酶的降解具有抵抗力。MMP13被认为在关节软骨破坏和OA的OA中具有关键的重要性。在通过胶原酶降解II型胶原蛋白后释放的特定分子片段的检测导致生物标志物测定的发展，以在体外和体内测量OA疾病活动。我们最近报道说，组织蛋白酶K（CATK）也能够切割II型胶原蛋白的完整三螺旋，该酶的活性在马软骨中高度上调。基于这些观察结果，我们认为CATK在关节软骨的酶促降解中比MMP 13同样重要，并且可能更重要。我们确定了独特的马catk II胶原特异性裂解新发表型，包括C2K77（已提交的专利），并提高了对C2K77的多克隆抗体，该抗体可以检测到组织中的早期马OA。 CATK介导的这些C2K77从马软骨中产生和释放。这些释放到体液中的含新EPITOPE的片段的结构将被阐明，以进一步了解CATK在OA中的重要性，并开发新的免疫测定以检测早期的马OA。该程序的具体目标是：1）开发针对CATK生成的II型胶原蛋白特异性新皮质C2K77的ELISA免疫测定法，用于定量CATK活性。 2）确定在体外上调CATK活性的化学因子。 3）通过表征所有包含通过catk在体内产生的C2K77的序列结构来建立在OA中产生的主要片段，该片段是通过CATK消化在体内产生的，而Equine II型胶原蛋白胶原蛋白的消化并产生了三明治免疫测定。 4）根据C2K77评估新的马生物标志物测定法，以检测体液中的OA。在马OA研究中，这项新的新兴领域的调查计划有望促进对CATK参与的知识，以促进II型胶原蛋白降解和在马OA中软骨的破坏。基于对体液的分析，对早期马OA的敏感，特定的诊断测试的开发对于马行业来说非常重要，因为OA是主要的福利和经济问题。这项研究的结果还有可能影响OA中的人类和犬类研究。