Age-Related Differences in Cartilage OP-1

关节软骨的退变随着年龄的增长而发生，在骨关节炎(OA)中加速。然而，导致这些变化的机制目前尚不清楚。该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。此前，我们首次证明OP-1信息和蛋白在成人关节软骨中表达，并且其水平随着年龄的增长和软骨的退变而下降。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敏感性增加，从而促进/促进软骨退化。长期目标是了解内源性OP-1在人关节软骨老化过程中的生物学功能和调控，并与骨性关节炎进行比较。这个项目的独特之处在于，我们可以无限制地从伊利诺伊州地区器官银行收集器官捐赠者的人体软骨。我们可以通过记录很少发生关节(踝关节)的变化与患病率较高的关节(膝关节)的变化来区分正常(非病理)和病理性渐进性(退行性)衰老。本研究的目的有三个方面：1)通过研究软骨内源性OP-1对分解代谢刺激(IL-1和机械损伤)的反应，并探讨这种反应在正常和病理渐进性老化中的差异；2)研究不同年龄的成人关节软骨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的变化是否在正常和病理渐进性衰老中软骨内稳态的破坏中起作用。我们打算使用特定的中和抗体、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法。这种方法使我们能够分析软骨提取液和培养液中OP-1蛋白的浓度。此外，还开发了一种新的针对OP-1前结构域和成熟结构域之间的特定切割位点的新表位抗体并进行了鉴定。这种抗体是至关重要的，因为在人类关节软骨中已经发现了两种形式的OP-1(前和成熟)，并且新的表位抗体将提供一个工具来评估不同实验条件下内源性OP-1的加工和激活的变化。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.