Mitochondrial DNA Integrity and Endothelial Cell Free Radical Stress

线粒体基因组中的突变和缺失与一系列疾病有关，但线粒体（mt）DNA是急性自由基介导的肺部疾病中反应性物质的靶点这一概念尚未得到考虑。由于它们的病理相关性，我们在实验中使用大鼠肺动脉（PA），肺微血管（MV）和肺静脉（PV）EC来探讨黄嘌呤氧化酶（XO）诱导的mtDNA损伤与细胞活力丧失之间的关系。XO诱导的线粒体DNA损伤的等级顺序与XO介导的细胞毒性的等级顺序相同。有趣的是，XO治疗后的生存率与mtDNA修复能力呈负相关;最具抗性的表型MVEC迅速修复XO诱导的mtDNA损伤，其次是PA和PVEC。线粒体DNA的重要性进一步表明，发现转染PAEC与线粒体DNA修复酶，ogg-1，减少XO诱导的线粒体DNA损伤和伴随的细胞毒性。最后，我们确定了与人类疾病相关的脆弱mtDNA序列易于在PAEC中发生XO介导的损伤。基于这些考虑，我们提出了测试的工作假设，线粒体DNA作为一个哨兵分子，其中过度或持续的特定序列的损伤导致肺血管EC死亡，由于激活线粒体途径的凋亡。我们将以大鼠肺血管内皮细胞为模型系统，检验以下假设：（1）线粒体DNA损伤的修复能力是内皮细胞对反应性物种介导的线粒体基因组损伤和凋亡细胞死亡的表型敏感性的预测;（2）线粒体DNA修复能力的增加可以保护细胞免受反应性物种诱导的线粒体DNA损伤;线粒体功能障碍和细胞凋亡的线粒体途径的激活;和（3）mtDNA“重链”中功能重要的序列倾向于反应性物质-诱导的损伤和对这些序列的损伤与抑制的线粒体基因表达和ATP含量有关。拟议的研究将提供新的洞察因素EC响应自由基。如果肺EC表型对自由基应激表现出不同的敏感性，这将对理解急性肺损伤的细胞基础具有重要意义。

Mutations and deletions in the mitochondrial genome are linked to a spectrum of diseases, but the concept that mitochondrial (mt) DNA is a target of reactive species in acute, free radical-mediated pulmonary disorders has not been considered. Because of their pathological relevance, we used rat pulmonary artery (PA), pulmonary microvascular (MV), and pulmonary vein (PV) ECs in experiments to explore the relation between xanthine oxidase (XO)-induced mtDNA damage and loss of cell viability. The rank order of XO-induced mtDNA damage was the same as the rank order of XO-mediated cytotoxicity. Interestingly, survival after XO treatment was inversely related to mtDNA repair capacity; the most resistant phenotype, MVECs, repaired XO-induced mtDNA damage rapidly, followed by PA and PVECs. The importance of mtDNA was further suggested by the finding that transfection of PAECs with the mtDNA repair enzyme, ogg-1, reduced XO-induced mtDNA damage and attendant cytotoxicity. Finally, we determined that the fragile mtDNA sequence incriminated in human diseases was prone to XO- mediated damage in PAECs. Based on these considerations, we proposed to test the working hypothesis that mtDNA serves as a sentinel molecule in which excessive or persistent damage to specific sequences causes pulmonary vascular EC death due to activation of mitochondrial pathway of apoptosis. Using rat pulmonary vascular ECs as model systems, we will test the hypothesis that: (1) The capacity to repair different patterns of mtDNA lesions is predictive of EC phenotype sensitivity to reactive species-mediated damage to the mitochondrial genome and to apoptotic cell death; (2) Increased mtDNA repair capacity protects against reactive species-induced mtDNA damage; mitochondrial dysfunction, and activation of the mitochondrial pathway of apoptosis; and (3) Functionally-significant sequences within the mtDNA "heavy strand" are prone to reactive species-induced damage and damage to these sequences is linked to depressed mitochondrial gene expression and ATP content. The proposed research will provide new insight into factors governing EC responses to free radicals. If lung EC phenotypes exhibit divergent sensitivities to free radical stress, this will have critical implications for understanding the cellular basis of acute lung injury.