Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.
Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins.
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DOI：
10.1161/atvbaha.117.310605
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发表时间：
2018-03
期刊：
影响因子：
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通讯作者：
Jacobs ER
中科院分区：
文献类型：
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作者：
Ma C;Beyer AM;Durand M;Clough AV;Zhu D;Norwood Toro L;Terashvili M;Ebben JD;Hill RB;Audi SH;Medhora M;Jacobs ER
我们探讨了高氧后肺内皮细胞（PEC）功能中线粒体结构和功能的改变机制。观察暴露于高氧或常氧环境下的PECs的线粒体结构，并量化线粒体断裂。western blot检测前裂变、融合蛋白及自噬相关蛋白的表达。采用mito-roGFP法测定线粒体氧化状态。TMRM估计治疗组的线粒体极化。使用mito-TEMPOL研究线粒体来源的ROS在线粒体断裂中的作用，使用ENDO III（一种修复线粒体DNA损伤的蛋白质）研究线粒体DNA （mtDNA）损伤。通过过表达或沉默Drp-1来检测该蛋白在细胞存活或transwell抗性中的作用。暴露48小时后，高氧以时间依赖性的方式增加了PEC线粒体的碎片化。高氧PECs表现出Drp-1（丝氨酸616）磷酸化增加，Mfn1减少，但OPA-1增加。促自噬蛋白p62、PINK-1、LC3B升高。将细胞恢复到常氧状态24小时，可以逆转增加的mt碎片和促裂变蛋白表达的变化。抗氧化剂mito-TEMPOL、Drp-1沉默或线粒体内切酶ENDO III抑制或保护可减轻高氧诱导的线粒体结构和/或细胞存活的变化。高氧诱导氧化和线粒体去极化以及transwell抗性受损。mito-TEMPOL或ENDO-III减轻了抗性的降低，并通过过表达Drp-1来复制。由于高氧诱发mt-DNA断裂，细胞存活和/或transwell耐药被ENDO III和mito-TEMPOL以及Drp-1沉默所阻止，这些数据链接高氧诱导的mt-DNA损伤、Drp-1表达、mt-碎片化和PEC功能障碍。
We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by western blot. Mitochondrial oxidative state was determined using mito-roGFP. TMRM estimated mitochondrial polarization in treatment groups. The role of mitochondrially-derived ROS in mt-fragmentation was investigated with mito-TEMPOL, and mitochondrial DNA (mtDNA) damage studied by using ENDO III, a protein that repairs mDNA damage. Drp-1 was over-expressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1, but increases in OPA-1. Pro-autophagy proteins p62, PINK-1 and LC3B were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure and/or cell survival were mitigated by anti-oxidants mito-TEMPOL, Drp-1 silencing or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO-III, and reproduced by over-expression of Drp-1. Because hyperoxia evoked mt-fragmentation, cell survival and/or transwell resistance are prevented by ENDO III and mito-TEMPOL, and Drp-1 silencing, these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation and PEC dysfunction.