N-Ethylmaleimide Sensitive Factor (NSF) Inhibition Prevents Vascular Instability following Gram-Positive Pulmonary Challenge.
N-Ethylmaleimide Sensitive Factor (NSF) Inhibition Prevents Vascular Instability following Gram-Positive Pulmonary Challenge.
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DOI：
10.1371/journal.pone.0157837
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Miller EJ
中科院分区：
文献类型：
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作者：
Lee JY;Linge HM;Ochani K;Lin K;Miller EJ
急性呼吸窘迫综合征（ARDS）是危重患者发病率和死亡率的重要来源。肺炎和败血症是ARDS的主要原因，其病理生理学包括肺微血管通透性增加和血流动力学不稳定，导致器官功能障碍。我们假设N-乙基马来酰亚胺敏感因子（NSF）通过调节肌球蛋白轻链（MLC）磷酸化来调节炎症介质（如血管生成素-2（Ang-2））的胞吐作用和细胞骨架稳定性。因此，我们在体内和体外用革兰氏阳性细菌细胞壁组分、脂磷壁酸（LTA）和肽聚糖（PGN）挑战肺细胞，并检查NSF抑制的作用。用NSF抑制剂TAT-NSF 700预处理小鼠（以防止Ang-2释放）。30 min后，将LTA和PGN（或单独的生理盐水）注入气管内。在滴注前和滴注后6小时在清醒小鼠中评估脉搏血氧测定法。死后，收集组织用于炎症和Ang-2的研究。在体外，评估肺内皮细胞对LTA和PGN的反应。肺挑战诱导的迹象，空气和全身炎症，如中性粒细胞计数和蛋白质浓度的变化，在支气管肺泡灌洗液和组织Ang-2浓度，并降低生理参数，包括氧饱和度和脉搏扩张。TAT-NSF 700预处理可减轻LTA-PGN引起的肺组织Ang-2、氧饱和度和脉搏扩张的变化。在体外，LTA-PGN诱导Ang-2的快速（<2 min）释放，其被TAT-NSF 700或抗TLR 2抗体显著减弱。此外，TAT-NSF 700在1-10 nM的低浓度下减少LTA-PGN诱导的MLC磷酸化。TAT-NSF 700通过抑制MLC磷酸化（内皮细胞收缩的重要组成部分）减少Ang-2的释放，改善肺激发后的氧饱和度和脉搏扩张。提示在肺炎和脓毒症中抑制NSF可能有利于预防与ARDS相关的肺微血管和血流动力学不稳定。
The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the effects of NSF inhibition. Mice were pre-treated with an inhibitor of NSF, TAT-NSF700 (to prevent Ang-2 release). After 30min, LTA and PGN (or saline alone) were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Post mortem, tissues were collected for studies of inflammation and Ang-2. In vitro, pulmonary endothelial cells were assessed for their responses to LTA and PGN. Pulmonary challenge induced signs of airspace and systemic inflammation such as changes in neutrophil counts and protein concentration in bronchoalveolar lavage fluid and tissue Ang-2 concentration, and decreased physiological parameters including oxygen saturation and pulse distention. TAT-NSF700 pre-treatment reduced LTA-PGN induced changes in lung tissue Ang-2, oxygen saturation and pulse distention. In vitro, LTA-PGN induced a rapid (<2 min) release of Ang-2, which was significantly attenuated by TAT-NSF700 or anti TLR2 antibody. Furthermore, TAT-NSF700 reduced LTA-PGN-induced MLC phosphorylation at low concentrations of 1–10 nM. TAT-NSF700 decreased Ang-2 release, improved oxygen saturation and pulse distention following pulmonary challenge by inhibiting MLC phosphorylation, an important component of endothelial cell retraction. The data suggest that inhibition of NSF in pneumonia and sepsis may be beneficial to prevent the pulmonary microvascular and hemodynamic instability associated with ARDS.