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

摘要/摘要 镰状细胞病（SCD）是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病理学的核心，导致生命- 威胁的后果。一个主要的后果是闭塞的激活微血管与镰状红细胞 导致不可预测和频繁发作的急性疼痛，称为血管闭塞性危象（VOC）， 住院和生活质量差。许多患有SCD的个体遭受慢性疼痛，这些疼痛可能始于 婴儿期，并在整个生命中继续增加。阿片类药物是治疗的主要药物，但其副作用 以及对上瘾的恐惧仍然是一个主要问题。因此，一个主要的未满足的需求是预防和/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 游离血红素的施用在表达人镰状细胞的转基因镰状细胞小鼠中引起痛觉过敏（疼痛 血红蛋白（Hb）和表达正常人HbA的对照小鼠。我们的初步数据显示血红素 通过释放核DNA和瓜氨酸化组蛋白刺激肥大细胞胞外陷阱（cytotoxictraps，cytotoxictraps）。桅杆- 细胞活化促进镰状小鼠的痛觉过敏。我们假设血红素诱导的肥大细胞 激活导致瓜氨酸化组蛋白和有毒物质的释放， 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和慢性疼痛中发挥因果作用。靶向肥大细胞 将从源头上改善VOC和疼痛。我们将使用翻译方法测试我们的假设， 四个具体目标是确定血红素是否有助于慢性和/或急性痛觉过敏（Aim 1）， 血红素通过肥大细胞激活（Aim2）和血红素诱导的痛觉过敏促进慢性/急性疼痛 是由新的肥大细胞依赖性机制驱动的，导致轴突和血管损伤（Aim3）， 包括从肥大细胞释放炎性细胞因子、蛋白酶、DNA和瓜氨酸化组蛋白， 引起外周和DRG神经元轴突损伤的细胞，以及通过内质网激活内皮细胞 网状应激Aim4将需要确定是否靶向血红素诱导的肥大细胞的机制， 细胞活化减弱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]人源化转基因HbSS-BERK镰状小鼠，仅表达人镰状细胞 Hb，[ii]表达正常人HbA的HbAA-BERK对照小鼠，[iii]缺失的镰状小鼠，[a]肥大细胞或 [b]TLR4及其同类对照;以及预防血管紧张素Ⅱ的机制特异性药理学抑制剂。 闭塞和疼痛。我们的实验室提供小鼠模型和生物制剂。通过使用多个 在体内和体外的策略，涉及肥大细胞介导的痛觉过敏和他们的目标与新的 和/或FDA批准的药物，我们希望我们的观察将导致与临床相关的 功能结局-减少VOC和SCD中的急性及慢性疼痛。

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.