Mechanisms of endothelial barrier phenotypes in sickle cell disease

描述(申请人提供)：内皮细胞在血管内形成一层薄的衬里，作为屏障来控制血管系统对炎性激动剂的反应。几种血管系统疾病已被认为是镰状细胞病(SCD)发病率和死亡率的主要来源。我们假设血管内皮细胞通透性增加是SCD的一种疾病，以前还没有认识到这种疾病。因此，本项目的长期目标是明确SCD的发病机制、急性并发症、预防和治疗内皮屏障功能障碍。据估计，美国有100,000人受到SCD的影响，全球还有数百万人受到SCD的影响，因此我们的长期目标可能会对全球健康负担产生重大影响。目前，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。这些触发因素中的每一个(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细胞的通透性，并导致组织水肿。在初步研究中，我们发现在两个SCD转基因模型中，内皮通透性增加和肺水肿。我们通过静脉注射游离血红素使镰刀鼠的全身溶血和氧化应激显著增加。这导致了SCD小鼠的肺泡泛滥和猝死，而具有镰刀特征的对照组小鼠存活了下来。为了了解为什么对过量血浆游离氯化血红素的反应如此严重，我们检查了肺组织中急性时相酶血红素加氧酶-1(HO-1)的表达，这种酶众所周知可以保护细胞免受血红素的影响。我们发现，HO-1在SCD小鼠和人类的肺中的表达并不升高，这表明肺使用另一种机制来中和SCD中游离血红素的毒性效应。我们鉴定了NAD(P)H-苯醌氧化还原酶1(NQO1)，它是一种清除超氧化物歧化的多功能细胞保护酶。NQO1的表达受氧化还原敏感的转录因子NF-E2相关因子2(Nrf2)的调控。我们发现NQO1在SCD小鼠和人类的肺内皮细胞中的表达都显著增加，这表明该酶对SCD中的氧化剂具有保护作用。在这些初步研究的基础上，我们将检验“镰状细胞疾病中的氧化应激导致可能致命的内皮屏障功能障碍，而血管保护疗法可能会减轻这种功能障碍”这一总体假设。这一总体假设将在三个相互关联的特定目标中进行检验：[1]确定SCD的外周和细胞氧化负担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]确定镰状细胞病的肺内皮屏障完整性是否依赖于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