Mitochondrial DNA Integrity and Endothelial Free Radical Stress

在几种重要的肺部疾病中，包括肺气肿、ARDS后的血管重塑和 可能是原发性肺动脉高压，肺动脉高压有显著的细胞毒性反应。 微血管内皮细胞（MV EC）导致毛细血管密度减少。虽然毫无疑问 活性氧在这种反应中起着重要作用，ROS的特定靶点， 作为一个哨兵分子-触发细胞死亡时，氧化应激是如此严重，以排除有效的 恢复或以突变威胁生物体-尚不清楚。在这方面，ROS的一个有趣的目标是 线粒体DNA。线粒体基因组对氧化损伤的敏感性至少高出30倍 比核DNA，我们在最初的资助期间的工作支持的假设，氧化 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 途径可能成为干预氧化剂诱导的MV EC死亡和毛细血管 稀疏化然而，关于线粒体DNA修复的细节在MV EC中的信息非常缺乏 和其他细胞。例如，虽然怀疑碱基切除修复机制是主导的， 保护线粒体基因组免受氧化损伤的途径，其他DNA修复的存在 通路成分表明，一个更复杂的修复范式可能是有效的。此外，本发明还提供了一种方法， 无论是参与线粒体碱基切除修复的酶的身份， 决定因素是已知的。在此背景下，本建议的目的是：（1）确定占主导地位的 途径修复MV EC中线粒体基因组的氧化损伤;（2）确定MV EC中线粒体基因组氧化损伤的限速途径。 线粒体DNA修复的功能步骤;（3）建立了MV线粒体DNA修复的关键酶 EC。总的来说，这些研究将提供第一个详细了解的途径，捍卫 线粒体基因组在这一重要的肺细胞群体，并确定线粒体DNA修复的适用性 酶作为干预的孤立目标。重要的是，这些研究的结果也将确定 临床前阶段，翻译实验的能力增强mtDNA修复抑制 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.