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, alveolar damage, and vascular permeability. IRI is also a risk factor for late graft rejection (bronchiolitis obliterans), the major cause of mortality beyond one year of transplant. ATP is a nucleotide released in large amounts after acute lung injury such as IRI and serves as a “danger signal” to mediate inflammation. Recent studies reveal that cells can actively release ATP in a controlled manner through pannexin 1 (Panx1) channels to signal through purinergic P2X or P2Y receptors. Our data suggest that Panx1 on endothelial cells (ECs) is an important mediator of lung injury and vascular inflammation after IR and may be a major source of extracellular ATP after IR. Thus this proposal will test the overall hypothesis that EC Panx1 signaling is a major, early inflammatory mediator of lung IRI via release of ATP resulting in endothelial barrier dysfunction, vascular inflammation and leukocyte infiltration. Specific Aim 1 will determine if EC Panx1 signaling mediates lung IRI via ATP release and whether the P2X7 receptor is a major determinant of IRI. Specific Aim 2 will determine if Panx1 signaling mediates leukocyte activation and infiltration during lung IRI. The role of Panx1 and specific purinergic receptors on alveolar macrophages will be deciphered as well as whether Panx1 mediates neutrophil transendothelial migration. Specific Aim 3 will determine if pharmacologic inhibition of Panx1 will prevent IRI after lung transplantation using a murine orthotopic lung transplant model. This transplant model will also be utilized to decipher the role of Panx1 signaling in donor versus recipient cells as well as in ECs and alveolar macrophages after transplantation. There currently are no preventative therapies for IRI, and our studies will provide novel insight into mechanisms or lung IRI and will define Panx1 as a novel therapeutic target for the prevention of IRI after lung transplantation.