Experimental and Computational Assessment of the Role of NOX4 in Mitochondrial Dysfunction Associated with ARDS

项目总结 急性呼吸窘迫综合征(ARDS)是一种以顽固性低氧为特征的危及生命的疾病。 失血症。煽动肺损伤导致肺内皮细胞和上皮细胞损伤，导致血管功能亢进 渗透性和水肿性。关于ARDS对特定细胞前体的影响，存在着丰富的信息。 CESS，包括充分的证据表明氧化应激在其启动中起关键作用，线粒体是主要的 目标。然而，活性氧物种(ROS)的来源和氧化应激的机制 在ARDS中诱导线粒体功能障碍的机制还知之甚少。此外，一种机械性的和定量的 整合在不同生物尺度上获得的新的和现有的生物能量数据以确定 它们的功能暗示是缺乏的。我们提出的工作解决了这些重要的知识差距。 NOX4是一种NADPH氧化酶亚型，其独特之处在于其ROS的产生速度依赖于细胞内的氧 (O2)浓度。这一点很重要，因为ARDS患者的主要治疗方法是提供高比例的 而大鼠暴露在高浓度氧气(高氧)中是人类ARDS的一个模型。对氧气的依赖- 在ROS动力学研究中，NOX4是启动或放大肺损伤的有力候选者。NOX4的激活还可以 增加线粒体ROS的产生，反之亦然，导致两者之间明显的串扰 重要的ROS来源和积极的反馈循环。这可能会导致线粒体损伤和释放 促炎症线粒体损伤相关的分子模式，这反过来可以刺激炎症 细胞死亡，增加血管通透性，最终会导致ARDS的一个主要特征--浮肿。 在我们强大的初步和已发表的数据的推动下，我们的总体假设是，NOX4是一个必不可少的 氧化应激导致线粒体功能障碍，并改变线粒体生物能量学 是ARDS发病机制中的关键途径。使用强大的垂直集成实验组合 方法，一种独特的NOX4基因敲除(KO)大鼠模型和两种人类ARDS模型(高氧血症和内源性ARDS)。 ，我们的数据将说明NOX4‘S在氧化应激和线粒体功能障碍中的作用 这是急性呼吸窘迫综合征的特征。一个大规模的计算模型将为整合提供一个量化的框架 在不同的生物尺度上获得的数据，以确定特定有丝分裂的变化的功能影响- ARDS患者的线粒体和胞浆突起改变，包括NOX4 KO和NOX4 KO之间的差异改变 WT大鼠。因此，具体目的是：1)证明NOX4产生的ROS在有丝分裂中的重要作用。 干性功能障碍和微血管高通透性是ARDS的特征，以及2)形成一种机制 肺组织生物能量学计算模型预测线粒体改变的功能意义-- 枯燥的过程。主要成果是：1)从机制和数量上理解NOX4在 修改ARDS中的线粒体功能，以及ii)整合生物素-蛋白质的综合计算模型。 ERGERTICS数据，并确定潜在的治疗靶点，以预防ARDS或减缓其进展。

PROJECT SUMMARY Acute Respiratory Distress Syndrome (ARDS) is a life-threatening disorder characterized by refractory hypox- emia. Inciting lung injury results in damage to pulmonary endothelial and epithelial cells causing vascular hyper- permeability and edema. A wealth of information exists regarding the effect of ARDS on specific cellular pro- cesses, including ample evidence of a key role for oxidative stress in its initiation, with mitochondria as a primary target. However, the sources of reactive oxygen species (ROS) and the mechanisms by which oxidative stress induces mitochondrial dysfunction in ARDS are poorly understood. Furthermore, a mechanistic and quantitative framework for integrating new and existing bioenergetic data acquired at different biological scales to ascertain their functional implications is lacking. Our proposed work addresses these important knowledge gaps. NOX4, an NADPH oxidase isoform, is unique in that its rate of ROS production is dependent on cellular oxygen (O2) concentration. This is important since a primary therapy for patients with ARDS is provision of high fractions of O2, and rat exposure to high fractions of inspired O2 (hyperoxia) is a model of human ARDS. With O2-depend- ent ROS kinetics, NOX4 is a strong candidate to initiate or amplify lung injury. Activation of NOX4 can also increase mitochondrial ROS production and vice-versa, resulting in an apparent crosstalk between these two important ROS sources and a positive feedback cycle. This can lead to mitochondrial damage and release of pro-inflammatory mitochondrial damage-associated molecular patterns, which in turn can stimulate inflammation and cell death, increase vascular permeability, and can ultimately result in edema, a cardinal feature of ARDS. Motivated by our strong preliminary and published data, our overall hypothesis is that NOX4 is an essential driver of oxidative stress that induces mitochondrial dysfunction, and that altered mitochondrial bioenergetics is a key pathway in the pathogenesis of ARDS. Using a powerful combination of vertically integrated experimental approaches, a unique NOX4 knockout (KO) rat model, and two models of human ARDS (hyperoxia and intratra- cheal lipopolysaccharide), our data will address NOX4’s role in oxidative stress and mitochondrial dysfunction that characterize ARDS. A large-scale computational model will provide a quantitative framework for integrating data acquired at different biological scales to ascertain the functional implications of a change in specific mito- chondrial and cytosolic processes altered in ARDS, including those differentially altered between NOX4 KO and WT rats. Thus, the specific aims are to 1) demonstrate the essential role of NOX4-generated ROS in mitochon- drial dysfunction and microvascular hyperpermeability that characterize ARDS, and 2) develop a mechanistic computational model of lung tissue bioenergetics for predicting functional implications of alterations in mitochon- drial processes. The key outcomes are i) a mechanistic and quantitative understanding of the role of NOX4 in modifying mitochondrial function in ARDS, and ii) a comprehensive computational model for integrating bioen- ergetics data and for identifying potential therapeutic targets to protect against ARDS or mitigate its progression.