Mechanisms of endothelial barrier phenotypes in sickle cell disease

描述（由申请人提供）：内皮在血管内形成薄层，作为控制血管对炎症激动剂反应的屏障。几种脉管系统疾病已被确定为镰状细胞病（SCD）发病率和死亡率的主要来源。我们假设血管内皮通透性增加是SCD的一种疾病，以前没有被认识到。因此，本项目的长期目标是明确SCD内皮屏障功能障碍的发病机制、急性并发症、预防和治疗。由于SCD在美国影响约10万人，在世界各地影响数百万人，我们的长期目标可能对全球健康负担产生重大影响。目前对SCD中涉及缺氧、游离血红素和缺血再灌注的血管病变过程尚不清楚。每一种触发因素（缺氧、缺血再灌注和血红素）都会产生氧化应激，众所周知，氧化应激会增加内皮通透性并导致组织水肿。在初步研究中，我们发现两种转基因SCD模型的内皮通透性和肺水肿增加。我们通过静脉注射游离血红素急性增加镰状小鼠的全身溶血和氧化应激。这导致SCD小鼠肺泡充血和猝死，而具有镰状性状的对照小鼠存活。为了理解为什么对过量血浆游离血红素的反应如此严重，我们检查了肺中急性期酶血红素加氧酶-1 （HO-1）的表达，这种酶被认为可以保护细胞免受血红素的侵害。我们发现HO-1表达在SCD小鼠和人的肺中没有升高，这表明肺使用另一种机制来中和SCD中游离血红素的毒性作用。我们鉴定了NAD(P)H醌氧化还原酶1 (NQO1)，它是一种清除超氧化物的多功能细胞保护酶。NQO1的表达受氧化还原敏感转录因子NF-E2相关因子2 （Nrf2）的调控。我们发现NQO1的表达在SCD小鼠和人的肺内皮中显著升高，这表明该酶在SCD中提供抗氧化剂的保护。基于这些初步研究，我们将验证“镰状细胞病中的氧化应激可能导致致命的内皮屏障功能障碍，这种功能可能通过血管保护治疗而减弱”的OVERALL假说。这一总体假设将在三个相互关联的特定目的中得到验证：[1]确定SCD的外周和细胞氧化负荷以及转基因小鼠的相关反应[2]确定Nrf2是否对镰状细胞病的内皮保护至关重要[3]确定镰状细胞病肺内皮屏障的完整性是否依赖于NQO1活性

DESCRIPTION (provided by applicant): Endothelium forms a thin lining inside blood vessels that acts a barrier to control the response of the vasculature to inflammatory agonists. Several disorders of the vasculature have been identified as major sources of morbidity and mortality in sickle cell disease (SCD). We hypothesize that increased permeability of the vascular endothelium is a disorder of SCD that has not previously been appreciated. Thus, the long- term goal of this project is to define the pathogenetic mechanisms, acute complications, prevention and treatment of endothelial barrier dysfunction in SCD. Since, SCD affects an estimated 100,000 people in the United States (US), and millions more around the world, our long-term goal may have a significant impact on the global health burden. Currently, the vascular disease process involving hypoxia, free heme and ischemia reperfusion in SCD are not well understood. Each of these triggers (hypoxia, ischemia reperfusion and heme) generates oxidative stress, which is well known to increase endothelial permeability and cause tissue edema. In preliminary studies we found increased endothelial permeability and pulmonary edema in two transgenic models of SCD. We acutely increased systemic hemolytic and oxidative stress by intravenously injecting sickle mice with free heme. This caused alveolar flooding and sudden death in SCD mice, while control mice with sickle trait survived. To understand why the response to excess plasma free hemin was so severe, we examined the lung for expression of the acute phase enzyme heme oxygenase-1 (HO-1), which is well known to protect cells from heme. We discovered that HO-1 expression is not elevated in the lungs of mice and humans with SCD, suggesting that the lung uses another mechanism to neutralize the toxic effects of free heme in SCD. We identified NAD(P)H quinone oxido-reductase 1 (NQO1), which is a multi-functional cytoprotective enzyme that scavenges superoxide. NQO1 expression is regulated by the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2). We found that expression of NQO1 is markedly elevated in the lung endothelium of both mice and humans with SCD, suggesting that this enzyme affords protection against oxidants in SCD. Based on these preliminary studies, we will test the OVERALL HYPOTHESIS that "Oxidative stress in sickle cell disease causes potentially fatal endothelial barrier dysfunction that may be attenuated with vasculoprotective therapy". This overall hypothesis will be tested in three inter-related Specific Aims: [1] Define the peripheral and cellular oxidative burden of SCD and the cognate response in transgenic mice [2] Determine whether Nrf2 is essential for protection of the endothelium in sickle cell disease [3] Determine whether lung endothelial barrier integrity in sickle cell disease is dependent on NQO1 activity