MtDNA repair: An isolated pharmacologic target in acute lung injury

描述（由申请人提供）：许多研究探索抗氧化剂作为急性肺损伤（ALI）的治疗药物，但其临床试验结果令人失望。对这种失败的一种解释是，ALI中产生的氧化剂不仅具有细胞毒性，而且它们还在肺细胞增殖和分化所需的途径中起第二信使的作用。因此，非选择性抗氧化剂可能会破坏细胞存活和恢复所需的信号。一个相关的可能性是整合ROS作用于肺血管的关键分子尚未被确定；抗氧化剂可能无法保护这些控制细胞死亡程序激活的假定哨兵分子免受损害。基于从细胞培养研究中获得的多行令人信服的证据，在此，我们提出验证线粒体（mt） DNA是一个哨兵分子，决定氧化应激反应中肺细胞的命运，mtDNA修复途径是ALI干预的目标。Specific Aim 1将开始将细胞培养的发现转化为实际应用，通过测试使用新型融合蛋白构建体增加mtDNA糖基酶活性的假设，保护和逆转患有铜绿假单胞菌或呼吸机诱导的ALI的啮齿动物的ALI。这些实验是我们开发mtDNA修复融合蛋白作为治疗ALI的平台药物分子的长期目标的重要一步。尽管mtDNA修复的增强抑制ros介导的细胞死亡，但这种有益作用的机制仍然难以捉摸。传统观念认为，加速mtDNA修复可以防止氧化mtDNA损伤的积累，从而减弱线粒体产生促凋亡ROS和线粒体驱动细胞死亡的激活，但目前并不是所有的数据都可用

DESCRIPTION (provided by applicant): Numerous studies have explored anti-oxidants as therapeutic agents in acute lung injury (ALI), but their outcome in clinical trials has been disappointing. One explanation for this failure is that oxidants generated in ALI are not only cytotoxic, but they also function as second messengers in pathways required for lung cell proliferation and differentiation. Thus, non-selective antioxidants may disrupt signaling required for cell survival and recovery. A related possibility is that key molecule(s) integrating ROS actions on the pulmonary vasculature have yet to be identified; antioxidants may fail to protect against damage to these putative sentinel molecule(s) governing activation of cell death programs. Based on multiple lines of compelling evidence obtained from cell culture studies, herein we propose to test the concept that mitochondrial (mt) DNA is a sentinel molecule dictating lung cell fate in response to oxidant stress and that the mtDNA repair pathway is a target for intervention in ALI. Specific Aim 1 will begin translating findings from cell culture ino practical application by testing the hypothesis that increasing mtDNA glycosylase activity using novel fusion protein constructs protects against, and reverses, ALI in rodents with either Pseudomonas aeruginosa- or ventilator- induced ALI. These experiments comprise an important step towards our longer term goal of developing mtDNA repair fusion proteins as platform drug molecules for treating ALI. Even though enhancement of mtDNA repair suppresses ROS-mediated cell death, the mechanism of this salutary effect has remained elusive. Traditional concepts hold that accelerated mtDNA repair prevents accumulation of oxidative mtDNA damage, thereby attenuating mitochondrial generation of pro-apoptotic ROS and attendant activation of mitochondrially-driven cell death, but not all currently available data support this model and some studies suggest that mtDNA repair, per se, is unimportant. Accordingly, Specific Aim 2 will use wild type and mutant Ogg1 proteins differing in their capacities to repair - but not to bind - mtDNA to determine whether repair of RS-induced mtDNA damage is required for prevention of RS-induced EC cytotoxicity, barrier dysfunction, and formation an extracellular release of injury-propagating mtDNA fragments.