Redox Biology in COPD

描述（由申请人提供）： 项目概述/摘要众所周知，氧化应激有助于慢性阻塞性肺疾病（COPD）的发病机制。然而，氧化环境的变化导致疾病的确切机制仍然是一个“黑匣子”，检测靶蛋白中特定氧化修饰的方法受到限制。氧化剂的不利分子作用主要归因于它们在造成损害中的作用。然而，氧化剂是在正常细胞和组织稳态中具有重要调节功能的分子的概念也已经出现。氧化剂的后一种信号传导功能归因于半胱氨酸氧化，例如S-亚硝基化（PSNO）和S-谷胱甘肽化（PSSG）。我们实验室最近的工作已经确定，PSNO和PSSG是重要的生物化学事件，其限制了肺上皮细胞中促炎和促存活转录因子核因子κ B（NF-κ B）的活化。重要的是，在COPD患者中观察到NF-kB的活化，并认为其有助于在这些患者中观察到的炎症过程。迄今为止，关于COPD患者肺中PSNO和PSSG的稳态变化以及这些事件之间的相互作用的信息仍然很大程度上缺乏，这是由于难以在组织中原位检测这些氧化修饰。我们的实验室最近开发了两种新的检测方法，首次能够原位检测石蜡包埋切片中的PSNO和PSSG。这些最近的观察和新的方法使我们假设，PSNO和PSSG的稳态在COPD患者的肺组织中受到影响，并且这些变化与NF-κ B激活的区域模式相关。在具体目标#1中，我们将确定COPD患者肺活检中PSNO和PSSG的程度。我们将利用最近开发的化学和催化半胱氨酸衍生化策略，使用共聚焦激光扫描显微镜监测不同严重程度COPD患者石蜡包埋活检标本中PSNO和PSSG的模式。我们还将评估来自接受肺癌切除手术的患者的正常组织。我们还将进一步验证我们的检测方法并实施内部标准，以提高其诊断效用。我们将测量谷氧还蛋白-1（Grx 1）和S-亚硝基谷胱甘肽（GSNO）还原酶的表达，因为这些酶分别代表PSSG和PSNO含量的主要调节剂。在具体目标#2中，我们将确定是否可以在COPD患者中检测到NF-kB通路成员的PSNO和PSSG的变化。这些特定目的的完成代表了对新检测方法用于原位检测组织中PSNO和PSSG的实用性的重要验证。该项目的完成将对COPD患者中各种半胱氨酸氧化的程度产生新的见解，并提供其与NF-κ B激活状态之间关系的见解。(End摘要） 公共卫生相关性： 众所周知，氧化剂会导致慢性阻塞性肺疾病（COPD）患者的肺损伤。然而，氧化剂的确切细胞效应仍然不清楚，这是有问题的，因为它不能使研究人员确定抗氧化剂治疗是否足够有效。该实验室开发了新的检测方法，使研究人员首次能够研究肺组织切片中氧化剂的微妙作用。因此，该资助提案的主要目标是测试这两种新测定法在检测正常肺以及从COPD严重程度增加的患者制备的肺组织中的氧化变化方面的实用性。如果成功的话，这些新的检测方法可能在检测肺部疾病患者的氧化应激程度以及确定抗氧化疗法的有效性方面具有诊断用途。完成这项拨款申请也可能为氧化剂调节炎症严重程度的机制提供新的见解。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT It is well known that oxidative stress contributes to the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). However, the exact mechanism whereby changes in the oxidative environment contribute to disease has remained a "black box", and methods to detect specific oxidative modifications in target proteins have been limited. The adverse molecular actions of oxidants have largely been attributed to their role in causing damage. Yet, the concept that oxidants are molecules with regulatory functions important in normal cell and tissue homeostasis has also emerged. The latter signaling function of oxidants has been attributed to cysteine oxidations, such as S-nitrosylation (PSNO), and S-glutathionylation (PSSG). Recent work from our laboratory has determined that PSNO and PSSG are important biochemical events that limit the activation of the pro-inflammatory and pro-survival transcription factor, nuclear factor kappa B (NF-kB) in lung epithelial cells. Importantly, activation of NF-kB has been observed in patients with COPD and is believed to contribute the inflammatory process observed in these patients. To this date, information about changes in the homeostasis of PSNO and PSSG in lungs of patients with COPD, and the interplay between these events, remains largely absent, due to the difficulties to detect these oxidative modifications in situ in tissues. Our laboratory has recently developed two new assays that, for the first time, enable the detection of PSNO and PSSG in paraffin-embedded sections in situ. These recent observations and novel approaches lead us to hypothesize that the homeostasis of PSNO and PSSG is affected in lung tissues from patients with COPD, and that these changes correlate with regional patterns of NF-kB activation. In Specific Aim #1, we will determine the extent of PSNO and PSSG in lung biopsies of patients with COPD. We will utilize the recently developed chemical and catalytic cysteine derivatization strategies to monitor patterns in PSNO and PSSG in paraffin-embedded biopsy specimens from patients with varying severities of COPD using confocal laser scanning microscopy. We will also evaluate normal tissue derived from patients undergoing surgery for lung cancer resection. We will also further validate our assays and implement internal standards in order to improve their diagnostic utility. We will measure expression of glutaredoxin-1 (Grx1) and S-nitrosoglutathione (GSNO) reductase, as these enzymes represent the major regulators of PSSG and PSNO content, respectively. In Specific Aim #2 we will determine whether changes in PSNO and PSSG of NF-kB pathway members can be detected in patients with COPD. Completion of these Specific Aims represents an important validation of the utility of the new assays to detect PSNO and PSSG in tissues in situ. Finalization of this project will yield new insights into the extent to which various cysteine oxidations occur in patients with COPD, and provide insights into their relationship with the status of activation of NF-kB. (End of Abstract) PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE it is well known that oxidants contribute to damage of lungs of patients with Chronic Obstructive Pulmonary Disease (COPD). However, the exact cellular effects of oxidants have remained unclear which is problematic, because is does not enable investigators to determine whether antioxidant therapies were adequately effective. This laboratory has developed new assays which allow investigators for the first time to study the subtle action of oxidants in tissue slides prepared from lungs. This grant proposal therefore has as its major goal to test the utility of these two new assays to detect oxidative changes in normal lung as well as in lung tissue prepared from patients with increasing severities of COPD. If successful, these new assays may have diagnostic use in testing the extent of oxidative stress in patients with lung disease, and also in determining the effectiveness of antioxidant therapies. Completion of this grant proposal may also offer new insights into the mechanisms whereby oxidants regulate the severity of inflammation.