Targeting mechanisms contributing to vascular dysfunction and pain in sickle cell disease

摘要/摘要 镰状细胞病(SCD)，一种隐性遗传性疾病，由血红蛋白链的点突变引起 红细胞(RBC)。微血管功能障碍是SCD病理生物学的核心，导致生命- 危险的后果。一个主要后果是镰刀状红细胞阻塞激活的微血管。 导致不可预测和频繁的急性疼痛发作，称为血管闭塞危象(VOC)，频繁 住院和生活质量差。许多SCD患者患有慢性疼痛，可能始于 婴儿期，并在一生中不断增加。阿片类药物是治疗的主要药物，但其副作用 对上瘾的恐惧仍然是一个主要的担忧。因此，一个尚未得到满足的主要需求是更多地预防和/或治疗疼痛。 有效地。VOC与释放游离血红素的溶血增加有关。我们的初步数据显示 在表达人镰刀的转基因镰刀小鼠中，注射游离血红素会引起痛觉过敏(疼痛) 血红蛋白(Hb)和对照组小鼠表达正常的人HBA。我们的初步数据显示血红素 通过释放核DNA和瓜氨酸组蛋白来刺激肥大细胞外陷阱(ETS)。桅杆- 细胞激活促进镰刀鼠的痛觉过敏。我们假设血红素诱导的肥大细胞 激活导致瓜氨酸组蛋白和有毒物质的释放，并有助于 炎症、血管功能障碍和轴突损伤导致血管闭塞和痛觉过敏 SCD(模式I)。肥大细胞可能在SCD的VOC和慢性疼痛中起作用。靶向肥大细胞 将从源头上缓解挥发性碳水化合物和疼痛。我们将使用翻译方法来验证我们的假设 四个具体目标旨在确定，血红素是否导致慢性和/或急性痛觉过敏(Aim1)， 亚铁血红素通过肥大细胞激活(AIM2)和亚铁血红素诱导的痛觉过敏导致慢性/急性疼痛 是由导致轴突和血管损伤的新的肥大细胞依赖机制(Aim3)驱动的， 包括从肥大释放炎性细胞因子、蛋白酶、DNA和瓜氨酸组蛋白等 导致外周和背根节神经元轴突损伤的细胞，以及通过内质网激活内皮细胞 网织应激。AIM4将需要确定是否针对血红素诱导的肥大机制- 细胞激活可减轻痛觉过敏和血管闭塞。我们将使用遗传学和药理学 方法，即[I]人源化的专门表达人镰刀的HbSS-Berk镰刀小鼠 Hb，[II]HbAA-Berk对照组小鼠表达正常的人HBA，[III]缺失的镰刀状小鼠，[a]肥大细胞或 [B]TLR4及其同源对照；以及机制特异的药物抑制物，以防止血管紧张素转换酶(VAS)。 闭塞和疼痛。我们的实验室里有老鼠模型和生物制品。通过使用多个 肥大细胞介导的痛觉过敏的体内外策略及其与新的靶向 和/或FDA批准的药物，我们预计我们的观察将导致翻译相关 功能结果-SCD患者的VOC和急慢性疼痛的减少。

Summary/Abstract Sickle cell disease (SCD), a recessive inherited disorder caused by a point mutation in the hemoglobin chain of red blood cells (RBCs). Microvascular dysfunction is central to the pathobiology of SCD, leading to life- threatening consequences. A major consequence is occlusion of activated microvasculature with sickle RBCs leading to unpredictable and frequent episodes of acute pain called vaso-occlusive crises (VOC), frequent hospitalization and poor quality of life. Many individuals with SCD suffer chronic pain that may start during infancy and continue to increase throughout life. Opioids are the mainstay for treatment but their side-effects and fear of addiction remain a major concern. Hence, a major unmet need is to prevent and/or treat pain more effectively. VOC is associated with increased hemolysis that releases free heme. Our preliminary data reveal that administration of free heme causes hyperalgesia (pain) in transgenic sickle mice expressing human sickle hemoglobin (Hb) and in control mice expressing normal human HbA. Our preliminary data shows that heme stimulates mast-cell extracellular traps (ETs) by releasing nuclear DNA and citrullinated histones. Mast- cell activation promotes hyperalgesia in sickle mice. We hypothesize that heme-induced mast-cell activation leads to release of citrullinated histones and noxious substances and contributes to inflammation, vascular dysfunction and axonal injury leading to vasoocclusion and hyperalgesia in SCD (Schema I). Mast cells may play a causal role in VOC and chronic pain in SCD. Targeting mast cells will ameliorate VOC and pain at its source. We will test our hypothesis using a translational approach with four specific aims to establish whether, heme contributes to chronic and/or acute hyperalgesia (Aim1), heme contributes to chronic/acute pain via mast-cell activation (Aim2), and heme-induced hyperalgesia is driven by novel mast cell–dependent mechanisms leading to axonal and vascular injury (Aim3), including, release of inflammatory cytokines, proteases, ETs with DNA and citrullinated histones from mast cells that cause axonal injury in the periphery and DRG neurons, and endothelial activation via endoplasmic reticulum stress. Aim4 will entail determining whether targeting the mechanisms of heme-induced mast- cell activation attenuates hyperalgesia and vaso-occlusion. We will use genetic and pharmacological approaches, namely [i] humanized transgenic HbSS-BERK sickle mice exclusively expressing human sickle Hb, [ii] HbAA-BERK control mice expressing normal human HbA, [iii] sickle mice deleted for, [a] mast cells or [b] TLR4 and their congenic controls; and mechanism-specific pharmacological inhibitors to prevent vaso- occlusion and pain. Mouse models and biologicals are available in our laboratories. By using multiple strategies in vivo and in vitro, involving mast cell–mediated hyperalgesia and their targeting with novel and/or FDA-approved drugs, we expect that our observations will lead to translationally relevant functional outcomes—reduction of VOC and acute as well as chronic pain in SCD.