Mitochondrial DNA Integrity and Endothelial Free Radical Stress

几种重要的肺部疾病，包括肺气肿，ARDS后的血管重塑，以及 可能是原发性肺动脉高压，有明显的细胞毒性反应。 微血管内皮细胞(MVECs)导致毛细血管密度降低。虽然毫无疑问 活性氧在这一反应中发挥着重要作用，这是ROS的特定靶标(S) 作为一种前哨分子，当氧化应激严重到排除有效的氧化应激时，触发细胞死亡 恢复或威胁到有机体的突变--尚不清楚。在这方面，ROS的一个有趣的目标是 线粒体(MT)DNA。线粒体基因组对氧化损伤的敏感性至少要高出30倍。 而我们在初始资助期的工作支持氧化作用的假说 线粒体DNA损伤是肺EC死亡的直接触发因素。如果这一假设成立，那么线粒体DNA修复 通路可能成为干预氧化剂诱导的血管内皮细胞死亡和毛细血管的新靶点 稀疏性。然而，对于mtdna在MV ECs中的修复细节，目前还缺乏相关信息。 和其他细胞。例如，尽管人们怀疑碱基切除修复机制是主要的 保护线粒体基因组免受氧化损伤的途径，以及其他DNA修复的存在 通路成分表明，一种更复杂的修复模式可能是可行的。此外, 参与线粒体碱基切除修复的酶的身份和速率限制 决定因素是已知的。在这种背景下，这项提议的目的是：(1)确定主导 微血管内皮细胞线粒体基因组氧化损伤修复途径；(2)确定限速 MtDNA修复的功能步骤；以及，(3)建立MV中修复mtDNA的关键操作酶 ECS。总而言之，这些研究将首次详细了解保护人类健康的途径 线粒体基因组在这一重要的肺细胞群中的表达并确定mtDNA修复的适宜性 酶作为单独的干预目标。重要的是，这些研究的结果也将确定 关于线粒体DNA修复增强抑制能力的临床前转译实验阶段 相关动物模型毛细血管稀疏。

In several important lung diseases, including emphysema, vascular remodeling after ARDS, and possibly primary pulmonary hypertension, there is a prominent cytotoxic response of pulmonary microvascular endothelial cells (MV ECs) leading to a diminution in capillary density. While there is no doubt that reactive oxygen species play an important role in this response, the specific target(s) of ROS that serve as a sentinel molecule - triggering cell death when the oxidant stress is so severe as to preclude effective recovery or threaten the organism with mutation - is not known. In this regard, an intriguing target of ROS is mitochondrial (mt) DNA. The mitochondrial genome is at least 30-fold more sensitive to oxidative damage than nuclear DNA, and our work during the initial funding period supports the hypothesis that oxidative mtDNA damage is a proximate trigger for lung EC death. If this hypothesis is valid, then mtDNA repair pathways could emerge as a new target for intervention in oxidant-induced MV EC death and capillary rarefaction. However, there is a stark lack of the information about the details of mtDNA repair in MV ECs and other cells. For example, while it is suspected that the base excision repair mechanism is the dominant pathway defending the mitochondrial genome from oxidative damage, the presence of other DNA repair pathway components suggests that a more complicated repair paradigm could be operative. In addition, neither the identities of the enzymes participating in mitochondrial base excision repair nor the rate limiting determinants are known. Against this background, the Aims of this proposal are to: (1) Identify the dominant pathway repairing oxidative damage to the mitochondrial genome in MV ECs; (2) Determine the rate-limiting functional steps in mtDNA repair; and, (3) Establish the critical operational enzymes repairing mtDNA in MV ECs. Collectively, these studies will provide the first detailed understanding of pathways defending the mitochondrial genome in this important lung cell population and determine the suitability of mtDNA repair enzymes to serve as isolated targets for intervention. Importantly, the outcome of these studies also will set the stage for pre-clinical, translational experiments on the ability of augmented mtDNA repair to suppress capillary rarefaction in relevant animal models.