Investigating the Role of Xanthine oxidase in hemolytic disease

项目摘要/摘要 溶血性疾病，包括镰状细胞病(SCD)、疟疾和败血症，影响着全世界数百万人 每年。每年大约每360名非裔美国人中就有一人被诊断出患有SCD，估计有214人 每年报告的疟疾病例有100万例，而败血症是重症监护病房的主要死亡原因。 循环中游离血红素水平升高(血红素危象)是溶血性疾病的一个关键特征，导致 直接和间接产生活性氧物种(ROS)。ROS的过度生产可能会导致公开的 血管内皮损伤和器官损伤。黄嘌呤氧化酶(XO)是一种这样的ROS的酶来源，它已经被 在一些溶血性疾病中显示升高，包括SCD(4倍)、疟疾(5倍)和败血症(9- 折叠)。XO主要在肝脏中产生，但在应激条件下，如缺氧、缺血和 炎症XO可以释放到循环中。在这里，XO可以将远端的糖胺聚糖结合在根尖上 它位于血管内皮细胞表面，有可能直接在血管内皮细胞表面产生ROS。而当 已有研究表明，XO活性在溶血性疾病中升高，XO在血红素危象中所起的作用 没有很好地定义。文献表明，XO活性增加通过产生氢是有害的 氧化过程中的过氧化氢(H_2O_2)和超氧自由基(O_2·-)。 嘌呤降解途径。然而，我们的初步结果表明，XO可能反而具有保护作用 在严重的血红素危机期间。我们开发并验证了一种新的血红素危机的“两次击中”模型，以便研究 XO在溶血性疾病中的作用机制。我们通过结合使用该模型来检验XO的角色 FDA批准的特定抑制剂非布索坦对XO有药理抑制作用。非布索他汀预处理 与未治疗的小鼠相比，小鼠的存活率更差，并表现出器官损伤加速和 炎症反应。我们的初步数据导致了以下目标的形成：1)确立 肝脏XO因血管内血红素危象而释放到循环中，以及2)确定XO是否起作用 降解血红素，保护血管内皮细胞免受损伤。对肝脏特异性XO基因敲除小鼠Aim 1进行评估 一种带有白蛋白启动子的腺相关病毒可以过表达肝脏特异性XO 已生成。XO肝脏特异性基因敲除和过度表达的小鼠模型将受到血红素危机的挑战 他们的反应将以24小时存活率、器官损伤和内皮损伤为特征。至 评估目标2一系列生化分析以测量氯化血红素吸光度、反应动力学和 游离铁的释放将完成。此外，通过XO潜在的血红素降解对初级 人肺血管内皮细胞的通透性和活性也将完成。加在一起，这些 AIMS将表明XO是否通过作为血红素的额外来源在血红素危机期间起到保护作用 退化。这些目标的完成对一些溶血性疾病有广泛的影响，并具有 有可能确定一种新的目标，用于治疗SCD、疟疾和败血症等疾病中的血红素危机。

PROJECT SUMMARY/ABSTRACT Hemolytic diseases, including sickle cell disease (SCD), malaria, and sepsis, affect millions of people worldwide each year. Approximately one in 360 African Americans is diagnosed with SCD each year, an estimated 214 million cases of malaria are reported each year, and sepsis is the leading cause of death in intensive care units. Increased levels of circulating free heme (heme crisis) is a key characteristic of hemolytic disease that results in direct and indirect production of reactive oxygen species (ROS). The overproduction of ROS can cause overt endothelial and organ damage. Xanthine oxidase (XO) is one such enzymatic source of ROS that has been shown to be elevated in a number of hemolytic diseases including SCD (4-fold), malaria (5-fold), and sepsis (9- fold). XO is produced primarily in the liver, but under stress conditions such as hypoxia, ischemia, and inflammation XO can be released into circulation. Here XO can bind distal glycosaminoglycans on the apical surface of vascular endothelium and has the potential to generate ROS directly at the endothelial surface. While it has been shown that XO activity is increased in hemolytic disease, the role XO plays during heme crisis has not been well defined. The literature suggests increased XO activity is harmful via the generation of hydrogen peroxide (H2O2) and superoxide (O2•-) during the oxidation of hypoxanthine and xanthine in the final steps of the purine degradation pathway. However, our preliminary results suggest XO may instead have a protective role during severe heme crisis. We developed and validated a novel “two hit” model of heme crisis in order to study XO’s mechanism of action in hemolytic disease. We examined the role of XO by using this model in combination with pharmacological inhibition of XO by the specific, FDA approved inhibitor febuxostat. Febuxostat pre-treated mice had a worsened survival rate compared to non-treated mice and showed accelerated organ damage and inflammatory response. Our preliminary data led to the formation of the following aims: 1) Establish the role of hepatic XO released into circulation in response to intravascular heme crisis, and 2) Determine if XO serves to degrade heme and protect against endothelial damage. To assess Aim 1 a liver-specific XO knockout mouse and an adeno-associated virus with an albumin promotor for liver-specific XO overexpression have been generated. The XO liver-specific knockout and overexpression mouse models will be challenged by heme crisis and their response will be characterized by 24-hour survival rate, organ damage, and endothelial damage. To assess Aim 2 a series of biochemical assays to measure changes in hemin absorbance, reaction kinetics, and release of free iron will be completed. In addition, the effects of potential heme degradation via XO on primary human pulmonary vascular endothelial cell permeability and viability will also be completed. Together, these aims will indicate whether XO has a protective role during heme crisis by serving as an additional source of heme degradation. Completion of these aims has a broad impact on a number of hemolytic diseases and has the potential to identify a novel target for treatment of heme crisis in diseases such as SCD, malaria, and sepsis.