Age-Related Differences in Cartilage OP-1

关节软骨的退化是随着年龄的增长而发生的，在骨关节炎（OA）中会加速。然而，导致这些变化的机制目前尚不清楚。这个项目的重点是骨形态发生蛋白（BMP）家族的成员，BMP-7或成骨蛋白-1 (OP-1)，它具有诱导软骨合成代谢过程的能力。在此之前，我们首次发现OP-1信息和蛋白在成人关节软骨中表达，并且它们的水平随着年龄和软骨变性而下降。我们的假设是，随着年龄的增长，人类关节软骨表现出内源性OP-1含量、合成和代谢的减少。这可能导致软骨细胞对分解代谢过程的易感性升高，从而促进软骨变性。长期目标是了解内源性OP-1在衰老的人关节软骨中的生物学功能和调控，并与OA进行比较。这个项目的独特之处在于我们可以无限制地获取来自伊利诺伊州地区器官库的器官捐献者的人体软骨。我们可以通过记录很少发生OA的关节（踝关节）的变化与OA发病率较高的关节（膝关节）的变化来区分正常（无病理）和病理进行性（退行性）衰老。本研究的目标将通过三个具体目标来检验：1)通过研究软骨内源性OP-1来评估关节软骨对分解代谢刺激（IL-1和机械损伤）的反应，并研究这种反应在正常和病理性进行性衰老之间是否存在差异；2)研究不同年龄成人关节软骨内源性OP-1在接近稳定状态和暴露于分解代谢刺激（IL-1和机械损伤）下的代谢情况；3)研究内源性OP-1的改变是否在正常和病理性进行性衰老过程中对软骨稳态的损害有致病作用。我们打算使用特异性中和抗体、结合蛋白和/或反义寡核苷酸来干扰OP-1的功能或表达。为了达到这些特定的目的，我们开发了一种新的灵敏的ELISA方法。这种方法使我们能够分析软骨提取物和培养基中OP-1蛋白的浓度。此外，一种新的新表位抗体已被开发并鉴定为OP-1的成熟和成熟结构域之间的特定切割位点。这种抗体是至关重要的，因为两种形式的OP-1（前和成熟）已经在人关节软骨中被鉴定出来，新表位抗体将提供一种工具来评估内源性OP-1在不同实验条件下的加工和激活变化。这项研究将有助于理解软骨修复/合成代谢的机制。

Degeneration of articular cartilage occurs with aging and accelerated in osteoarthritis (OA). However, the mechanisms responsible for these changes are currently unknown. The focus of this project is the member of the bone morphogenetic protein (BMP) family, BMP-7 or osteogenic protein-1 (OP-1), that has an ability to induce anabolic processes in cartilage. Previously, we showed for the first time that OP-1 message and protein are expressed in adult human articular cartilage and that their levels declined with aging and cartilage degeneration. Our hypothesis is that, with aging, human articular cartilage exhibits a decrease in endogenous OP-1 content, synthesis and metabolism. This may lead to an elevated susceptibility of the chondrocytes to catabolic processes thus contributing/promoting cartilage degeneration. The long-term objective is to understand the biological function and regulation of endogenous OP-1 in aging human articular cartilage in comparison to OA. The uniqueness of this project is that we have unlimited access to human cartilages from organ donors collected through the Regional Organ Bank of Illinois. We can distinguish between normal (no-pathologically) and pathologically progressive (degenerative) aging by documenting changes in a joint that rarely develops OA (ankle joint) compared to changes in a joint with the higher prevalence of OA (knee joint). The objectives of the present study will be tested by three specific aims: 1) Assess the response of articular cartilage to catabolic stimuli (IL-1 and mechanical injury) by studying cartilage endogenous OP-1 and investigate whether this response is different between normal and pathologically progressive aging; 2) Investigate the metabolism of endogenous OP-1 at its near steady state and under exposure to catabolic stimuli (IL-1 and mechanical injury) in adult articular cartilage of different ages; 3) Study whether changes in endogenous OP-1 have a causative role in impairing cartilage homeostasis in normal and pathologically progressive aging. We intend to perturb OP-1 function or expressing using specific neutralizing antibody, binding proteins, and/or antisense oligonucleotides. In order to accomplish these specific aims, a novel sensitive ELISA method has been developed by us. This method allows us to analyze the concentrations of OP-1 protein in cartilage extracts and culture medium. Also a new neoepitope antibody to the specific cleavage site between pro- and mature domains of OP-1 has been developed and characterized. This antibody is critical, since both forms of OP-1 (pro- and mature) have been identified in human articular cartilage and the neoepitope antibody will provide a tool to assess changes in processing and activation of endogenous OP-1 under different experimental conditions. This study will contribute to the understanding of the mechanisms of cartilage repair/anabolism.