PDF(Pubmed)
Abstract：
Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1β, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.
摘要：
背景：慢性香烟烟雾暴露会降低WWOX的肺表达，已知WWOX在ARDS感染模型中保护内皮屏障。
方法：使用串联质谱（TMT-MS）对WWOX沉默的内皮细胞（ECs）进行蛋白质组学分析。WWOX沉默的EC以及从内皮Wwox基因敲除（ECWwoxKO）小鼠中分离的EC进行循环拉伸（18％伸长率，0.5Hz,4小时）。收获细胞裂解物和培养基上清液用于细胞信号的测定,蛋白质表达，和细胞因子释放。用WWOX和zyxin的双重沉默重复这些。对对照和ECWwoxKO小鼠进行高潮气量通气。收集支气管肺泡灌洗液和小鼠肺组织的细胞信号，细胞因子分泌，和组织学检测。
结果：TMT-MS揭示了在WWOX敲除过程中，zyxin表达上调，这预测了对机械牵张的炎症反应增强。WWOX沉默的ECs和从ECWwox小鼠分离的ECs显示出各种细胞因子（IL-6，KC/IL-8，IL-1β，和MCP-1）相对于对照。这与ERK和JNK磷酸化增加有关，但p38MAPK磷酸化减少。接受VILI的ECWwoxKO小鼠比相应的对照遭受更大程度的损伤。WWOX敲除过程中zyxin的沉默消除了拉伸诱导的IL-8分泌增加。
结论：ECs中WWOX功能的丧失与机械牵张过程中炎症反应的增强有关，这种炎症反应与MAPK磷酸化的增加有关，并且似乎依赖于酶素的上调。
方法：使用串联质谱（TMT-MS）对WWOX沉默的内皮细胞（ECs）进行蛋白质组学分析。WWOX沉默的EC以及从内皮Wwox基因敲除（ECWwoxKO）小鼠中分离的EC进行循环拉伸（18％伸长率，0.5Hz,4小时）。收获细胞裂解物和培养基上清液用于细胞信号的测定,蛋白质表达，和细胞因子释放。用WWOX和zyxin的双重沉默重复这些。对对照和ECWwoxKO小鼠进行高潮气量通气。收集支气管肺泡灌洗液和小鼠肺组织的细胞信号，细胞因子分泌，和组织学检测。
结果：TMT-MS揭示了在WWOX敲除过程中，zyxin表达上调，这预测了对机械牵张的炎症反应增强。WWOX沉默的ECs和从ECWwox小鼠分离的ECs显示出各种细胞因子（IL-6，KC/IL-8，IL-1β，和MCP-1）相对于对照。这与ERK和JNK磷酸化增加有关，但p38MAPK磷酸化减少。接受VILI的ECWwoxKO小鼠比相应的对照遭受更大程度的损伤。WWOX敲除过程中zyxin的沉默消除了拉伸诱导的IL-8分泌增加。
结论：ECs中WWOX功能的丧失与机械牵张过程中炎症反应的增强有关，这种炎症反应与MAPK磷酸化的增加有关，并且似乎依赖于酶素的上调。
