Oxidant State and Nitric Oxide Metabolism in the Acute Chest Syndrome

镰状细胞病（SCD）肺血管局部停滞和血栓形成的发病机制的核心过程是血管内皮及其代谢功能的改变。通过体外SCD模型，我们观察到暴露于急性胸综合征（ACS）患者的血浆会导致培养的肺动脉内皮细胞（PAEC）中的氮氧化物（NO2）增加10倍。在随后的研究中，我们发现：1)NO的增加与eNOS蛋白表达和酶活性的增加有关，但与组成型一氧化氮合酶（NOSIII）转录本的变化无关，也与iNOS成分的变化无关；2) PAEC抗氧化分子明显减少；3) PAEC超氧化物产量增加；4)转基因镰状细胞小鼠在体外和肺中诱导ACS时ONOO的大量形成；5)急性冠脉综合征患者血浆氧化应激标志物（F2:-异前列腺素）升高。这些发现表明，一氧化氮（包括ONOO-）的产生及其分泌的氧化状态有助于在与SCD相关的急性和慢性肺部疾病中观察到的细胞和组织损伤。为了在体内研究这一假设，我们将重点关注：1)通过将功能测定、肺组织学和动物死亡率与基线和缺氧诱导的危象期间NO代谢的变化相关联，确定NO在镰状细胞转基因小鼠血管反应和血管封闭事件中的作用；2)使用以下方法确定药物操作对镰状细胞转基因小鼠acs样危象的影响：a) NOS底物、NOS辅助因子、NOS抑制剂和吸入NO或抗氧化剂，并在基线和缺氧引起的危机期间检查功能分析、肺组织学和动物死亡率；3)通过将镰状细胞转基因小鼠与：a) NOSIII（或NOll）缺陷小鼠杂交，确定NO代谢和氧化应激的重要性；b)谷胱甘肽过氧化物酶（GPI）缺陷小鼠；或c)葡萄糖-6-磷酸脱氢酶（G6PD）突变小鼠，在基线和诱导危机期间检查功能测定、肺组织学和动物死亡率。这些研究将探讨一氧化氮代谢和氧化状态的改变对急性和慢性肺部疾病（SCD）发病机制的影响。这些研究也可能证明与肺外VOC相关，并可能导致预防或逆转EC激活/功能障碍的治疗，这似乎是该疾病过程的核心。

Central to processes important in the pathogenesis of local stasis and thrombosis in the pulmonary vasculature in sickle cell disease (SCD) is the vascular endothelium and alterations in its metabolic functions. Using ex vivo model of SCD, we have observed that exposure to plasma from patients with acute chest syndrome (ACS) causes a ten-fold increase in nitrogen oxides (NO2) from cultured pulmonary artery endothelial cell (PAEC). In subsequent studies, we found: 1) this increase in NO, is associated with increased eNOS protein expression and enzymatic activity but not with a change in transcripts of constitutive nitric oxide synthase (NOSIII) nor any change in iNOS components; 2) a marked reduction in PAEC antioxidant molecules; 3) increased production of PAEC superoxide; 4) substantial formation of ONOO- both ex vivo and in lungs of a transgenic sickle cell mouse during induced ACS; and 5) an increase in markers of oxidative stress (F2:-isoprostanes) in plasma of patients during ACS. These findings suggest that NO, (including ONOO-) production and the oxidative state into which it is secreted contribute to the cellular and tissue injury observed in acute, as well as chronic, pulmonary disease associated with SCD. To investigate this hypothesis in vivo, we will focus on: 1) defining the role of NO in vascular responses and vasoocclusive events in a sickle cell transgenic mouse by correlating functional assays, lung histology and animal mortality with changes in NO metabolism at baseline and during crisis induced by hypoxia: 2) defining the effect of pharmacological manipulation of ACS-like crisis in a sickle cell transgenic mouse using: a) NOS substrate, NOS cofactors, NOS inhibitors and inhaled NO or antioxidants and examining functional assays, lung histology and animal mortality at baseline and during crisis induced by hypoxia: and 3) defining the importance of NO metabolism and oxidative stress in a sickle cell transgenic mouse by interbreeding it with: a) a NOSIII (or NOll) deficient mouse; b) a glutathione peroxidase (GPI) deficient mouse: or c) a glucose-6-phosphate dehydrogenase (G6PD) mutant mouse and examining functional assays, lung histology and animal mortality at baseline and during induced crisis. These studies will investigate the contribution of altered NO metabolism and the oxidative state to the pathogenesis of the acute and chronic pulmonars." disease associated with SCD. These studies may also prove germane to extra-pulmonary VOC and may lead to therapies to prevent or reverse EC activation/dysfunction that appears central to this disease process.