The mortality rate for acute respiratory distress syndrome (ARDS) remains unacceptably high (35-40%), and there are no FDA-approved drug treatments. Overwhelming data demonstrate that ARDS patient outcomes are significantly worse among the elderly population. However, we do not yet understand the mechanisms that account for this age-associated predisposition. The NADPH oxidase (Nox) enzymes are an important cellular source of reactive oxygen species (ROS) generation and signaling. We previously demonstrated that Nox4-dependent ROS plays a critical role in mediating endothelial cell (EC) barrier function during acute responses to lung injury. However, a growing body of evidence supports a link between excessive Nox4-derived ROS and numerous age-related diseases. We have demonstrated that in response to lung injury, aged mice exhibit persistently elevated Nox4/ROS levels that result in susceptibility to severe pre-clinical ARDS and failure to resolve injury. We discovered highly divergent induction of Nox4/ROS in “young” control vs. senescent ECs; control ECs exhibit a rapid and transient induction of Nox4, whereas senescent ECs exhibit persistently elevated levels of Nox4/ROS ROS leading to impaired barrier function. We identified a novel mechanism by which Nox4 expression can be rapidly altered in ECs via post-translational modification. Further, senescent ECs exhibit defective ubiquitin-mediated degradation of Nox4, which promotes sustained Nox4/ROS levels. Finally, we identified a small molecular weight splice variant of Nox4 (Nox4D) to be expressed only in the lungs of aged mice with severe pre-clinical ARDS and in the lungs of ARDS patients from 3 different cohorts. Finally, targeting of Nox4/D-dependent ROS in senescence/aging provided the most significant therapeutic benefit (as compared to young), and may be the key to improving outcomes for elderly ARDS patients. The proposed studies will test the central hypothesis that age-dependent persistent Nox4/D expression promotes redox imbalance leading to senescence- associated barrier disruptive EC phenotypes and greater inflammatory injury, ultimately resulting in the increased susceptibility and impaired resolution of ARDS in aging. Aim 1 studies will determine the role of Nox4 in mediating age-dependent barrier disruptive EC phenotypes and severity/resolution of pre-clinical ARDS. Aim 2 studies will investigate the association of Nox4D in ARDS patients and its functional role in ALI in vivo. Aim 3 studies will evaluate post-translational mechanisms contributing to the persistence of Nox4/D in senescent ECs. The proposed studies will provide insight into the roles of Nox4/D in regulating EC barrier and inflammatory responses, and how age-dependent dysfunction of Nox4/D contributes to ARDS pathogenesis. These studies will offer a novel mechanistic link that may help to explain the increased incidence and mortality of ARDS associated with the elderly and will provide proof-of-concept for therap...