Deconstructing Cartilage Mechanotransduction by Piezo Channels

骨性关节炎(OA)通过影响滑膜关节而疼痛和虚弱，在超过12%的 美国总人口年龄在25-74岁之间。OA的患病率随着年龄的增长而显著增加， 在超过70%的65岁以上人口中有放射证据。在我们这个日益增长的社会阶层中，办公自动化是 是导致残疾、虚弱和社会孤立的重要因素。尽管存在巨大的社会经济影响 对于骨性关节炎，目前还没有可以改变疾病的治疗方法。办公自动化的显著特点是进步性、 关节软骨的形态、成分和力学性能的退行性改变。 关节软骨细胞中的机械转导是疾病发病机制的关键组成部分，考虑到这一联系 直接感觉细胞的机械环境和由此导致的软骨代谢失衡之间 在办公自动化中。我们最近发现了机械敏感的压电离子通道--实际上是 PIEZO1和Piezo2均在关节软骨中表达-为软骨细胞机械转导奠定了基础 对有害的机械应力的反应。 本研究的总体目标是明确压电体介导的机械转导的机制。 对软骨细胞进行更深入的研究，以便将这些见解用于疾病的发展- 关节负荷性损伤的改进方法，包括骨关节炎。除了我们最近发现的 软骨细胞压电体介导的机械转导，我们发现 致炎细胞因子IL-1α的病理生理浓度增加Piezo1基因 Piezo1在增龄性骨关节炎软骨中也有表达增加 猪和人。因此，这笔赠款的具体目的是：(1)确定协同作用的机制 Piezo1/2在软骨细胞机械转导中的作用；(2)解构Piezo介导的 炎症条件下软骨细胞的机械转导；(3)阐明Piezo-2在软骨细胞中的作用。 器官型软骨移植和体内的机械转导。目标1将依赖于细胞研究。 我们将在电生理、通道传输、有限元等水平上探索Piezo1/2的协同作用 模型和超微结构，后者也检查了骨关节炎和对照组的人类软骨。在Aim 2小学 猪软骨细胞将被IL-1α刺激，以解构压电介导的机械转导。 目标3将依赖于猪的骨软骨外植体和软骨细胞特异性和可诱导的Piezo1/2-/-小鼠 我们已经产生了。不同的机械应力模式将应用于细胞、外植体和动物，以及 压电体介导的机械转导的功能丧失研究将在两种机械意图下进行 和翻译/治疗方向。拟议的目标将扩展我们的初步发现，使其具有机械性- 深入的研究将以非增量的方式增加我们对办公自动化的理解，这将启发 新的治疗骨性关节炎的药物(DMOADs)的开发。

Osteoarthritis (OA) is painful and debilitating by affecting the synovial joints, and is found in over 12% of the total United States population 25-74 years of age. The prevalence of OA increases significantly with age, with radiographic evidence in over 70% of the population over age 65. In this growing segment of our society, OA is a significant contributor to disability, frailty and social isolation. Despite the tremendous socioeconomic impact of OA, there are no disease-modifying therapies available. OA is distinctively characterized by the progressive, degenerative changes in the morphology, composition, and mechanical properties of articular cartilage. Mechanotransduction in articular chondrocytes is a key component of disease pathogenesis, given the link between direct sensing of the cells’ mechanical environment and the resulting metabolic imbalance of cartilage in OA. We have recently identified the mechanosensitive PIEZO ion channels - in fact a synergy between PIEZO1 and PIEZO2, both expressed in articular cartilage - to underlie chondrocyte mechanotransduction in response to injurious mechanical stress. The overall objective of this study is to define the mechanisms of Piezo-mediated mechanotransduction in chondrocytes more in-depth so that these insights can be leveraged toward the development of disease- modifying approaches in joint-loading-induced injuries, including OA. In addition to our recent discovery of chondrocytic Piezo-mediated mechanotransduction, we found that treatment of chondrocytes with pathophysiologically-relevant concentrations of IL-1α, a pro-inflammatory cytokine, increased Piezo1 gene expression, and that increased expression of Piezo1 was also present in osteoarthritic cartilage from aging pigs and humans. Thus, the Specific Aims of this grant are: (1) to determine the mechanisms of synergistic functioning of Piezo1/2 in chondrocyte mechanotransduction; (2) to deconstruct Piezo-mediated mechanotransduction in chondrocytes under inflammatory conditions; (3) to elucidate the role of Piezo- mediated mechanotransduction in organotypic cartilage explants and in-vivo. Aim 1 will rely on cellular studies. We will explore synergisms of Piezo1/2 at the levels of electrophysiology, channel trafficking, finite element modeling, and ultra-structure, the latter also examining human cartilage from OA vs controls. In Aim 2 primary porcine chondrocytes will be stimulated with IL-1α for deconstruction of Piezo-mediated mechanotransduction. Aim 3 will rely on porcine osteochondral explants and chondrocyte-specific and inducible Piezo1/2-/- mice which we have generated. Various modes of mechanical stress will be applied to cells, explants, and animals, and loss-of-function studies of Piezo-mediated mechanotransduction will be conducted with both mechanistic intent and translational/therapeutic direction. The proposed Aims will extend our initial discovery with mechanistic in- depth studies that will increase our understanding of OA in a non-incremental manner, and this will inspire the development of new Disease-Modifying OA Drugs (DMOADs).