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.

PROJECT NARRATIVE Acute respiratory distress syndrome (ARDS) accounts for 75,000 deaths, 3.6 million hospital days, and $5 billion in healthcare costs in the U.S. alone per year. Our objective is to use experimental and computational tools in a teaching environment to assess the role that the cellular enzyme NADPH oxidase 4 (NOX4) plays in driving injurious cellular pathways in the lung that lead to ARDS. We will also develop a mathematical model of lung cell energy production to address changes in individual processes that contribute to overall function, with outcomes expected to improve understanding of ARDS, and to motivate investigation of specific therapeutic targets to mitigate ARDS morbidity, mortality, and financial burdens.