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，由MV引起的机械拉伸可能加剧肺损伤并导致超额死亡率，这一过程称为呼吸机相关性肺损伤（VALI）。先前的动物模型研究表明，高潮气量（HVT 20 ml/kg）通气可同时激活黄嘌呤氧化还原酶（XOR），促进肺泡内皮细胞和上皮细胞凋亡，导致肺泡毛细血管通透性增加。我们已经证明，别嘌呤醇对XOR的药理学抑制可以消除HVT诱导的血管渗漏增加，这表明通过该途径的信号传导是体内通透性变化所必需的。此外，我们还通过体外VALI模型证明，内皮细胞（EC）的循环拉伸（CS）在18%，20cycles/min，而不是5%或静态时，足以促进XOR活性和细胞凋亡。目的：别嘌呤醇预处理EC对体外抑制CS诱导的细胞凋亡的作用；然而，异或增强机械应力诱导的细胞凋亡的机制尚未得到很好的描述，这也是本研究的重点。我们将验证XOR在机械应力环境下内皮细胞凋亡的上调是细胞凋亡分子抑制剂cIAP2下调的结果。方法：将肺微血管内皮细胞（EC）暴露于病理性循环拉伸（18%）与生理性拉伸（5%）或静态条件下，增加时间间隔（2,4和6小时），不存在或存在XOR抑制剂（别嘌呤醇、非布司他或RNAi），然后收获细胞。终点包括mRNA分析、Western blotting、caspase活性和核形态学特征凋亡变化分析。

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.