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

项目摘要 血友病是一种X连锁遗传疾病，由于缺乏以下两种基因， 凝血因子VIII或因子IX。不受控制的内出血可能发生在手术过程中和钝力 创伤引起的滑膜血管破裂。关节积血电极导线复发 完全破坏关节软骨，称为血友病性关节病。虽然确切的机制 血液诱导的软骨损伤的机制尚不清楚，细胞凋亡被认为是受调节细胞的主要形式。 由于促炎性细胞因子浓度升高，软骨细胞中的死亡（RCD）。但其他 RCD的形式可能在诱导软骨以及周围滑膜损伤方面发挥作用。铁死亡 是一种最近发现的，铁依赖性，非凋亡形式的RCD与过度的细胞内 由自由羟基和多不饱和脂肪酸形成的脂质氢过氧化物的积累。这 一项提案探讨了铁下垂作为关节内过度运动引起关节组织损伤的潜在机制， 血液中释放的铁此外，铁凋亡抑制剂，如铁抑素-1，其防止铁凋亡的形成。 脂质氢过氧化物被探索作为对抗血液诱导的细胞死亡的潜在治疗剂。 这一新的F31提案将填补目前对血友病性关节病认识的巨大空白。更好 了解血液暴露的剂量反应和持续时间可能会产生潜在的治疗窗口， 消除关节出血后遗症干预。生理性关节负荷和滑膜的作用 血液诱导的软骨损伤也将使用滑膜关节模型系统， 现代生物工程和分子生物学技术（例如，代谢组学）。分离血液对 软骨、滑膜及其共培养物将为针对关节出血的软骨保护提供新的靶点。 假设1：血液诱导的软骨损伤部分是由于关节软骨细胞的铁凋亡。具体 目的1：A）对关节软骨的力学和生化特性进行血液剂量反应。 评估坏死、凋亡和铁凋亡在血液诱导的软骨损伤中的相对作用。B）研究 血液成分对关节组织损伤的各自贡献，并与铁凋亡诱导剂进行比较。C）、 使血液经受流体诱导的剪切，并监测血液中的红细胞活力和血细胞相关产物。 滑液D）评估铁抑素-1减轻血液诱导的软骨变化的能力。 假设2：血液相关的软骨损伤会因血液诱导的滑膜变化而加剧。具体 目的2：对滑膜和软骨-滑膜共培养物重复特定目的1。A）执行往复剪切， 滑膜对软骨和软骨对软骨。B）进行条件培养基实验， 从玻璃上软骨到滑膜培养或玻璃上滑膜到软骨培养的相互剪切载荷。 执行空载控制。

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.