Oxidative stress in vaso-occlusion pathophysiology of sickle cell disease

项目摘要 镰状细胞病（SCD）是美国最常见的遗传性血液病。的 SCD中器官功能障碍的病理生理学超出了异常流变学和溶血，包括 血管功能异常、血栓形成和炎症。越来越多的证据表明氧化应激 是SCD中血管闭塞（VO）的重要生化触发因素。活性氧（ROS）增加 可通过血小板和白细胞活化及微血管功能障碍导致VO。是否 氧化应激的药理学减少防止VO和改善微血管灌注是未知的。 这项建议的目标是使用最先进的微血管灌注和分子成像技术 与先进的血浆生化分析一起测试两种药物的机制和治疗效果， 涉及减少SCD中ROS的有希望的抗氧化剂：（a）N-乙酰胱氨酸（NAC），和（B）夹竹桃苷。在 目的1.建立Townes小鼠SCD模型及对照小鼠。无创微血管 常氧条件下肾脏和骨骼肌的超声造影灌注成像（CEU） 在用NAC、夹竹桃苷或赋形剂预处理6周的小鼠中进行缺氧后条件。 将使用参数灌注分析（微血管血容量和流量）和弥散建模 以更好地表征导致SCD模型中异常血流或治疗效果的血管事件。 在目标2中，激活的血管性血友病因子（vWF）和血小板的CEU分子成像，以及PET分子成像。 氧化应激成像将用于进一步评估微血管分子事件， 异常灌注或NAC或夹竹桃苷的治疗效果。在这两个目标中，先进的LC-MS/MS 用于无偏代谢组学和VWF氧化修饰及其主要调控因子的靶向鉴定 蛋白酶（ADAMTS-13）以及其他关键ROS的测定，以评估药物作用。在目标3中， 将在初步临床试验中进行肢体骨骼肌、肾脏和心肌的灌注成像 检验用NAC减少氧化应激改善患者微血管灌注的假设 关于SCD本试验设计为NAC或安慰剂的双交叉，各6周。的结果 这一建议将填补我们对SCD病理生物学知识的一个关键空白， 新的治疗干预措施。它还将为PI提供丰富的研究培训计划（包括 多学科指导和咨询团队，全面的课程和职业发展 该机构的可用资源），这将为成为一名独立的调查员奠定基础， 能够在临床前和临床研究中使用先进的成像和其他分析技术， 血液病微血管病理生理学

PROJECT SUMMARY Sickle cell disease (SCD) is the most common genetic hematologic disorder in the United States. The pathophysiology of organ dysfunction in SCD extends beyond abnormal rheology and hemolysis, and includes abnormal vascular function, thrombosis, and inflammation. There is increasing evidence that oxidative stress is an important biochemical trigger for vaso-occlusion (VO) in SCD. Increased reactive oxygen species (ROS) can lead to VO through platelet and leukocyte activation, and microvascular dysfunction. Whether pharmacologic reduction in oxidative stress prevents VO and improves microvascular perfusion is not known. The goal of this proposal is to use state-of-the-art microvascular perfusion and molecular imaging techniques together with advanced plasma biochemical analysis to test mechanism and therapeutic impact of two promising anti-oxidants implicated in reducing ROS in SCD: (a) N-acetylcystine (NAC), and (b) apocynin. In Aim 1, the Townes murine model of SCD and control mice will be studied. Non-invasive microvascular perfusion imaging with contrast-enhanced ultrasound (CEU) of the kidney and skeletal muscle under normoxic and post-hypoxic conditions will be performed in mice pre-treated for 6 weeks with NAC, apocynin, or vehicle. Parametric perfusion analysis (microvascular blood volume and flux rate) and dispersion modeling will be used to better characterize the vascular events responsible for abnormal flow in the SCD model or treatment effect. In Aim 2, CEU molecular imaging of activated von Willebrand Factor (vWF) and platelets, and PET molecular imaging of oxidative stress will be used to further assess the microvascular molecular events responsible for abnormal perfusion or treatment effect from the NAC or apocynin. In both of these Aims, advanced LC-MS/MS for unbiased metabolomics and targeted identification of oxidative modification of VWF and its main regulatory protease (ADAMTS-13), as well as other key ROS, will be performed to evaluate drug effect. In Aim 3, CEU perfusion imaging of limb skeletal muscle, kidney, and myocardium will be performed in a pilot clinical trial testing the hypothesis that reducing oxidative stress with NAC improves microvascular perfusion in patients with SCD. This trial is designed as a double cross-over with NAC or placebo for 6 weeks each. The results of this proposal will address a critical gap in our knowledge of the pathobiology of SCD and provide insight into new therapeutic interventions. It will also provide the PI with a rich research training program (including multidisciplinary mentorship and advisory teams, comprehensive coursework, and career advancement resources available at the institution) that will lay the foundation to become an independent investigator who is able to use advanced imaging and other analytic techniques in pre-clinical and clinical studies to examine microvascular pathophysiology in hematologic disease.