Investigating the Role of Xanthine oxidase in hemolytic disease

研究黄嘌呤氧化酶在溶血性疾病中的作用

• 批准号: 10090460
• 负责人: Heidi Marie Schmidt
• 金额: $ 4.55万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10090460
• 关键词: Address; Affect; African American; Albumins; Animal Model; Apical; Attenuated; Binding; Biochemical; Biological Assay; Blood Circulation; Blood Vessels; Brain Hypoxia-Ischemia; Cause of Death; Cell Count; Characteristics; Clinical; Data; Degradation Pathway; Dependovirus; Development; Diagnosis; Disease; Distal; Doctor of Philosophy; Effectiveness; Endothelium; Equilibrium; Erythrocytes; FDA approved; Functional disorder; Generations; Glycosaminoglycans; Heme; Hemin; Hemolysis; Hemopexin; Hepatic; Homeostasis; Hour; Human; Hydrogen Peroxide; Hypoxanthines; Hypoxia; In Vitro; Incubated; Individual; Inflammation; Inflammatory; Inflammatory Response; Injury; Intensive Care Units; Iron; Kidney; Kinetics; Knock-out; Knockout Mice; Knowledge; Lead; Literature; Liver; Lung; Malaria; Measures; Mediating; Mentors; Modeling; Mus; Organ; Oxidants; Pathway interactions; Patients; Permeability; Pharmacology; Physiological; Plasma; Play; Positioning Attribute; Production; Purines; Reaction; Reactive Oxygen Species; Reporting; Resources; Role; Sepsis; Series; Sickle Cell Anemia; Source; Stress; Suggestion; Superoxides; Surface; Survival Rate; Tail; Testing; Tissues; Translating; United States; Universities; Up-Regulation; Vascular Endothelial Cell; Vascular Endothelium; Veins; Xanthine Oxidase; Xanthines; design; febuxostat; improved; in vivo; inhibitor/antagonist; mouse model; new therapeutic target; novel; novel therapeutics; overexpression; oxidation; preservation; programs; promoter; protective effect; response; stressor; supportive environment; xanthine oxidase inhibitor

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.

项目总结/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.每年约有360名非洲裔美国人中有1人被诊断为SCD，估计有214人 每年报告的疟疾病例达100万例，败血症是重症监护病房的主要死亡原因。 循环游离血红素水平升高（血红素危象）是溶血性疾病的关键特征， 直接和间接产生活性氧（ROS）。ROS的过度产生会导致明显的 内皮和器官损伤。黄嘌呤氧化酶（XO）是ROS的一种这样的酶源，其已经被广泛应用。 显示在许多溶血性疾病中升高，包括SCD（4倍）、疟疾（5倍）和败血症（9- 10倍）。 折叠）。XO主要在肝脏中产生，但在应激条件下，如缺氧、缺血和缺氧。 炎症XO可以被释放到循环中。XO可以结合远端的糖胺聚糖， 在血管内皮细胞的表面，具有直接在内皮细胞表面产生ROS的潜力。而 已经表明，XO活性在溶血性疾病中增加，XO在血红素危象中所起的作用， 没有被很好地定义。文献表明，XO活性的增加是有害的，通过产生氢 过氧化物（H2 O2）和超氧化物（O2·-）在次黄嘌呤和黄嘌呤的氧化过程中的最后步骤， 嘌呤降解途径然而，我们的初步结果表明，XO可能具有保护作用， 在严重的血红素危机中。我们开发并验证了一种新的血红素危机的“两次打击”模型，以研究 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查XO的作用 通过FDA批准的特异性抑制剂非布司他药理学抑制XO。非布司他预处理 与未治疗的小鼠相比，小鼠的存活率更差，并显示出加速的器官损伤， 炎症反应。我们的初步数据导致了以下目标的形成：1）建立 响应血管内血红素危象，肝XO释放到循环中，和2）确定XO是否用于 降解血红素并保护内皮免受损伤。为了评估Aim 1，肝脏特异性XO敲除小鼠 和一种带有白蛋白启动子的腺相关病毒，用于肝脏特异性XO过表达， 生成的. XO肝脏特异性基因敲除和过表达小鼠模型将受到血红素危机的挑战 并且它们的反应将以24小时存活率、器官损伤和内皮损伤为特征。到 评估目标2一系列生化测定，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渗透性和活力也将完成。所有这些 目的将表明XO是否通过作为血红素的额外来源在血红素危机期间具有保护作用 降解这些目标的实现对许多溶血性疾病具有广泛的影响， 有可能确定一个新的目标，治疗血红素危机的疾病，如SCD，疟疾和败血症。