Project Summary The success of lung transplantation is limited by high rates of primary graft dysfunction due to ischemia-reperfusion injury (IRI) characterized by robust inflammation, vascular permeability, and alveolar damage. IRI is also a risk factor for late graft rejection (bronchiolitis obliterans), the major cause of mortality beyond one year of transplant. TRPV4 is a transmembrane calcium channel expressed in numerous cell types including vascular endothelial cells (ECs), alveolar epithelial cells, macrophages, and neutrophils. TRPV4 activation can induce lung endothelial/epithelial barrier dysfunction, a critical feature of IRI, and our data suggests that TRPV4 activity in endothelial cells (ECs) is a significant mediator of lung IRI and that inhibition of TRPV4 activity is significantly protective. We also show that TRPV4 activity may be affected by ATP released by pannexin (Panx1) channels on ECs. Thus, our proposal will test the overall hypothesis that endothelial TRPV4 channel signaling is a critical mediator of lung IRI by inducing endothelial barrier disruption, vascular permeability and leukocyte infiltration. Aim 1 will determine if TRPV4 activity on ECs mediates lung IRI leading to endothelial/epithelial barrier dysfunction, vascular inflammation, and leukocyte infiltration. A potential role for TRPV4 in alveolar epithelial cells, macrophages, and neutrophils will also be evaluated during lung IRI. Aim 2 will define mechanisms for a Panx1/TRPV4 axis in ECs that mediates lung IRI by testing the hypothesis that TRPV4 is activated by ATP released by Panx1 channels after IR. We will also determine if TRPV4 activity is induced by NADPH oxidase-derived reactive oxygen species after IR. Aim 3 will utilize a murine orthotopic lung transplant model to decipher the role of TRPV4 in donor versus recipient cells as well as in ECs after transplantation. In addition, a clinically relevant, large animal porcine lung transplant model will be used to determine if pharmacologic inhibition of Panx1 will prevent lung IRI after transplantation. There currently are no preventative therapies for IRI, and our studies will provide novel insight into mechanisms of lung IRI and will define TRPV4 channels as a novel therapeutic target for the prevention of IRI after lung transplantation.