Ferroptosis as a Potential Mechanism of Blood-Induced Chondrocyte Cell Death

项目摘要 血友病是一种X连锁的遗传疾病，由于两者中的任何一种都缺乏，导致血液无法正常凝结 凝血因子VIII或因子IX。在手术中和钝力作用下可能会发生失控的内出血。 外伤性滑膜血管断裂。反复发作的关节出血导致 完全破坏关节软骨，称为血友病关节病。而确切的机制 血液诱导软骨损伤的机制尚不清楚，细胞凋亡被认为是调节细胞的主要形式 软骨细胞死亡(RCD)是由于促炎细胞因子浓度升高所致。然而，其他 各种类型的RCD可能在导致软骨和周围滑膜损伤方面发挥作用。铁性下垂 是一种新近发现的铁依赖的非凋亡形式的RCD，与细胞内过量相关 由自由基羟基和多不饱和脂肪酸形成的过氧化脂质的积累。这 Proposal探讨了铁性下垂作为关节内过度损伤所致关节组织损伤的潜在机制 铁从血液中释放出来。此外，铁性下垂抑制剂，如铁抑素-1，可防止形成 脂质过氧化氢，被认为是对抗血液诱导的细胞死亡的潜在疗法。 这项新的F31建议将填补目前对血友病关节病认识的一大空白。更好 了解血液暴露和持续时间的剂量反应可能会产生潜在的治疗窗口 消除关节出血后遗症的干预措施。生理关节负荷与滑膜的作用 关于血液诱导的软骨损伤也将使用滑膜关节模型系统来解决，该系统与 现代生物工程和分子生物学技术(例如代谢组学)。隔离血液对人体健康的影响 软骨、滑膜和它们的混合培养将提供新的靶点，旨在保护软骨免受关节出血的伤害。 假设1：血液引起的软骨损伤部分是由于关节软骨细胞的铁性下垂。特定的 目的1)对关节软骨的力学和生化特性进行血液剂量效应研究。 评估坏死、细胞凋亡和铁性下垂在血液诱导的软骨损伤中的相对贡献。B)研究 血液成分对关节组织损伤的各自贡献及与铁性下垂诱导剂的比较。c) 对血液进行液体诱导切变，并监测红细胞活性和血细胞相关产物 滑液。D)评估铁抑素-1减轻血液引起的软骨改变的能力。 假设2：血液引起的滑膜改变会加重与血液有关的软骨损伤。特定的 目的2：重复滑膜和软骨-滑膜共培养的特异性目标1。A)执行以下各项的往复剪切 软骨上的滑膜和软骨上的软骨。B)执行传输介质的条件介质实验 从玻璃上的软骨到滑膜培养或从玻璃上的滑膜到软骨培养的相互剪切载荷。 执行不加载控制。

Project Summary Hemophilia is an X-linked genetic disorder that prevents blood from clotting normally due to a deficiency in either coagulation factor VIII or factor IX. Uncontrolled internal bleeding can occur during surgery and from blunt force trauma-induced breakage of blood vessels in the synovial membrane. Recurrent episodes of hemarthrosis lead to complete destruction of the articular cartilage, known as hemophilic arthropathy. While the exact mechanisms of blood-induced cartilage damage remain unclear, apoptosis is implicated as the primary form of regulated cell death (RCD) in chondrocytes due to elevated concentrations of pro-inflammatory cytokines. However, other forms of RCD may play roles in inducing damage to cartilage as well as the surrounding synovium. Ferroptosis is a recently discovered, iron-dependent, nonapoptotic form of RCD associated with excessive intracellular accumulation of lipid hydroperoxides formed from free hydroxyl radicals and polyunsaturated fatty acids. This proposal explores ferroptosis as a potential mechanism of joint tissue damage caused by excess intra-articular iron released from blood. In addition, ferroptosis inhibitors such as Ferrostatin-1, which prevent the formation of lipid hydroperoxides, are explored as potential therapeutics against blood-induced cell death. This new F31 proposal will fill a large gap in the current understanding of hemophilic arthropathy. Better understanding of the dose-response of blood exposure and duration may yield potential therapeutic windows of intervention that abrogate the sequela of joint bleeding. The role of physiologic joint loading and the synovium on blood-induced cartilage damage will also be addressed using a synovial joint model system coupled with modern bioengineering and molecular biology techniques (e.g., metabolomics). Isolating the effects of blood on cartilage, synovium, and their co-culture will inform new targets aimed at chondroprotection from joint bleeding. Hypothesis 1: Blood-induced cartilage damage is due in part to ferroptosis of articular chondrocytes. Specific Aim 1: A) Perform dose-response to blood on mechanical and biochemical properties of articular cartilage. Assess relative contribution of necrosis, apoptosis and ferroptosis in blood-induced cartilage damage. B) Study respective contribution of blood constituents to joint tissue damage and compare with ferroptosis inducers. C) Subject blood to fluid-induced shear and monitor erythrocyte viability and blood-cell related products in the synovial fluid. D) Assess ability of Ferrostatin-1 to mitigate blood-induced changes to cartilage. Hypothesis 2: Blood related cartilage damage is exacerbated by blood-induced changes to synovium. Specific Aim 2: Repeat Specific Aim 1 on synovium and cartilage-synovium co-culture. A) Perform reciprocating shear of synovium-on-cartilage and cartilage-on-cartilage. B) Perform conditioned media experiments that transfer media from reciprocal shear loading of cartilage-on-glass to synovium culture or synovium-on-glass to cartilage culture. Perform no-loading controls.