XOR Regulation of Endothelial Cell Apoptosis in the Model of VALI

描述（由申请人提供）：机械通气（MV）是支持急性肺损伤（ALI）患者所必需的;然而，众所周知，MV诱导的机械牵拉可能会加重肺损伤并导致死亡率过高，这一过程称为呼吸机相关肺损伤（VALI）。先前的动物模型研究已经证明，高潮气量（HVT 20 ml/kg）通气伴随着激活酶黄嘌呤氧化还原酶（XOR）并促进肺泡内皮和上皮细胞的凋亡，导致肺泡毛细血管通透性增加。我们已经表明，用别嘌呤醇对XOR的药理学抑制消除了HVT诱导的血管渗漏增加，表明通过该途径的信号传导对于体内渗透性变化是必要的。此外，我们还证明了使用我们的VALI体外模型，以18%的循环拉伸（CS）内皮细胞（EC）20个循环/分钟，而不是5%或静态，足以促进XOR活性和凋亡。目的：别嘌呤醇预处理EC在体外防止CS诱导的细胞凋亡，然而，XOR增强机械应力诱导的细胞凋亡的机制没有得到很好的描述，是本研究的重点。我们将测试的假设，XOR的上调内皮细胞凋亡的机械应力的设置是一个结果的下调的分子抑制剂的凋亡，即cIAP 2。方法：将肺微血管内皮细胞（EC）暴露于病理性周期性拉伸（18%）与生理性拉伸（5%）或静态条件，持续增加的时间间隔（2、4和6小时），其中不存在或存在XOR抑制剂（别嘌呤醇、非布司他或RNAi），之后收获细胞。终点包括mRNA分析、蛋白质印迹法、半胱天冬酶活性和特征性凋亡变化的核形态学分析。 公共卫生相关性：在美国，ALI和ARDS的发病率很高，每年有20万例，总死亡率为35%至60%。与肺泡过度扩张和机械通气的周期性打开和关闭相关的机械应力会导致致病事件，但对这些事件的了解甚少。对VALI潜在的病理生理机制的剖析将为旨在降低死亡率的新治疗方法的开发提供关键动力。

DESCRIPTION (provided by applicant): Mechanical ventilation (MV) is necessary to support patients with acute lung injury (ALI); however, it is well recognized that mechanical stretch induced by MV may exacerbate lung injury and contribute to excess mortality, a process called ventilator-associated lung injury (VALI). Previous animal model studies have demonstrated that high tidal volume (HVT 20 ml/kg) ventilation concomitantly activates the enzyme xanthine oxidoreductase (XOR) and promotes apoptosis of alveolar endothelial and epithelial cells resulting in increases in alveolar capillary permeability. We have shown that pharmacologic inhibition of XOR with allopurinol abrogates HVT- induced increase in vascular leakage indicating that signaling through this pathway is necessary for permeability changes in vivo. In addition, we have also demonstrated using our in vitro model of VALI that cyclic stretch (CS) of endothelial cells (EC) at 18% for 20cycles/min but not 5% or static, is sufficient to promote XOR activity and apoptosis. Objective: Allopurinol pre-treatment of EC in vitro prevents CS induced apoptosis; however, the mechanisms by which XOR potentiates mechanical stress induced apoptosis are not well described and are the focus of this study. We will test the hypothesis that XOR's upregulation of endothelial cell apoptosis in the setting of mechanical stress is a result of the downregulation of molecular inhibitors of apoptosis, namely cIAP2. METHODS: Pulmonary microvascular endothelial cells (EC) will be exposed to pathologic cyclic stretch (18%) versus physiologic stretch (5%) or static conditions for increasing time intervals (2, 4, and 6 hours) with the absence or presence of XOR inhibitor (allopurinol, febuxostat, or RNAi) after which the cells were harvested. Endpoints included mRNA analysis, Western blotting, caspase activity, and analysis of nuclear morphology for characteristic apoptotic changes. PUBLIC HEALTH RELEVANCE: ALI and ARDS account for significant morbidity with 200,000 cases a year in the US and an overall mortality ranging from 35 to 60%. Mechanical stress related to alveolar over-distension and cyclical opening and closing from mechanical ventilation leads to pathogenic events that are poorly understood. Dissection of the pathophysiologic mechanisms underlying VALI will provide a critical impetus for the development of new treatments aimed at decreasing mortality.