Mitochondrial DNA Integrity and Endothelial Cell Free Radical Stress

线粒体基因组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的活性物种的概念尚未被考虑。本实验以大鼠肺动脉(PA)、肺微血管(MV)和肺静脉(PV)内皮细胞为实验材料，探讨黄嘌呤氧化酶(XO)诱导的线粒体DNA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介导的细胞毒作用等级顺序相同。有趣的是，XO处理后的存活率与mtDNA修复能力呈负相关，抗性最强的表型MVECs能迅速修复XO引起的mtDNA损伤，其次是PA和PVECs。MtDNA修复酶Ogg-1转染PAECs可降低XO诱导的mtDNA损伤及相应的细胞毒作用，进一步提示了mtDNA的重要性。最后，我们确定与人类疾病有关的脆性mtDNA序列容易受到XO介导的PAECs的损伤。基于这些考虑，我们建议检验mtDNA作为一种哨兵分子的工作假设，在该分子中，特定序列的过度或持续损伤通过激活线粒体凋亡途径而导致肺血管内皮细胞死亡。以大鼠肺血管内皮细胞为模型系统，我们将检验以下假设：(1)修复不同模式的mtDNA损伤的能力可以预测EC对活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；(2)增加的mtDNA修复能力可以保护细胞免受活性物种诱导的mtDNA损伤；线粒体功能障碍和线粒体凋亡途径的激活；以及(3)mtDNA“重链”内的功能显著序列容易受到活性物种诱导的损伤，这些序列的损伤与线粒体基因表达和ATP含量下降有关。这项拟议的研究将为控制EC对自由基反应的因素提供新的见解。如果肺内皮细胞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学基础具有重要意义。

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.