Summary Acute lung injury in the form of acute respiratory distress syndrome (ARDS) or ischemia-reperfusion injury (IRI) after lung transplant remains a major clinical issue associated with high morbidity and mortality. Currently, no methods exist for targeted treatment and rapid rehabilitation of lungs affected by ARDS. The majority of treatment strategies remain supportive in nature and are associated with continued poor outcomes. We have demonstrated breakthrough methods for treatment or prevention of acute lung injury via normothermic perfusion of isolated lungs in the form of ex vivo lung perfusion (EVLP) for marginal donor lungs or in vivo lung perfusion (IVLP) for ARDS. IVLP combines the benefits of extracorporeal membrane oxygenation and EVLP to provide a platform upon which injured lungs can be treated in vivo with targeted therapies in an isolated fashion without the potential risks of systemic treatment. We have demonstrated the rehabilitative capacity of isolated lung perfusion with Steen solution whereby sepsis-induced ARDS as well as IRI of transplanted donation after circulatory death (DCD) lungs are attenuated. We have also demonstrated that Steen solution directly preserves pulmonary endothelial barrier function and that inhibition of Panx1 or TRPV4 channels attenuates lung IRI via endothelial barrier protection. Thus, this project will test the overall hypothesis that rehabilitation of injured lungs by isolated, normothermic perfusion with Steen solution can be augmented by Panx1- or TRPV4-targeted therapy aimed at preserving endothelial barrier function to attenuate vascular inflammation and improve lung function. We will test our hypothesis using porcine and murine models of IVLP and EVLP of lungs injured by ARDS or IRI in three specific aims. Aim 1 will determine if isolated perfusion with Steen solution will rehabilitate injured lungs and if inhibition of Panx1 or TRPV4 during perfusion will augment rehabilitation and endothelial barrier function. Aim 1A will use a porcine, LPS-induced ARDS model to determine if IVLP provides durable lung rehabilitation up to 24 hours after IVLP. Aim 1B will determine if IVLP rehabilitates lungs injured in a porcine model of gastric aspiration-induced ARDS. Aim 1C will determine if EVLP rehabilitates murine lungs injured by ARDS caused by SARS-CoV-2 infection. Aim 2 will use a porcine lung transplant model to determine if rehabilitation of DCD lungs by EVLP can be augmented by treatment with Panx1 or TRPV4 inhibitor for successful transplant. Aim 3 will use in vitro models of LPS-induced injury to determine protective mechanisms of Steen solution involving direct anti-inflammatory effects on alveolar-capillary barrier or leukocytes. Our recent studies demonstrate the potential of IVLP for the treatment of severe ARDS, representing a major paradigm shift in the management of ARDS. If successful, our proposed studies will define IVLP as a novel platform for targeted therapy of severe ARDS and...