Project Summary Organ transplantation (Tx) is the mainstay therapy for patients with end-stage organ failure. Yet, barriers exist that preclude long-term graft survival. It is well-established that early injury to the allograft, during the donor organ preservation and reperfusion phases, set the organ up for late failure. Based on studies from our laboratory, ischemia-reperfusion injury (IRI) and early alloimmunity, mediated by memory T cells, are among the most prevalent and inevitable early injuries affecting endothelial cells (ECs) as the first point of contact. Thus, our scientific premise revolves around the allograft's endothelium as central to these insults. The endothelium contains an intact layer of aligned ECs that are joined by cell-to-cell junctions and enables them to communicate and form a protective barrier. During IRI and early alloimmunity, the EC barrier is disrupted, predisposing the ECs to inappropriate antigen presentation ultimately resulting in late graft failure. Thus, the EC barrier is vital for graft protection. Endothelial cell-to-cell communication is dependent upon gap junctions including connexin 43 (Cx43). Whether Cx43 gap junctions play a role in protecting the EC barrier and dampening EC immunogenicity against early injuries allowing for a therapeutic target, forms the central question of our proposal. Based on our preliminary data, we hypothesize that stabilizing and/or overexpressing Cx43 gap junctions in ECs will mitigate early graft injury. To study this hypothesis, we propose two Aims: Specific Aim 1 - Elucidate the role of Cx43 gap junction protein on EC activation/immunogenicity during IRI in vitro. Specific Aim 2 - Demonstrate the preclinical ability to downregulate the EC activation/immunogenicity in transplanted hearts in vivo by maintaining Cx43 protein levels via pre-treatment of donor hearts. Common to these aims, we will employ an in vitro model of injury that simulates cold ischemia and warm reperfusion injury to study the role of Cx43 gap junctions on EC health and immunogenicity. We will also employ clinically relevant in vivo brain-death mouse models of IRI and alloimmune cardiac Tx and utilize a novel and unique donor organ pre-treatment strategy to deliver Cx43 modulating agents. These innovative and high-risk high-reward studies will be the first to define the specific and focused impact of Cx43 gap junction in ECs, associated with organ preservation and reperfusion. Furthermore, it will provide a proof-of-concept to target the Cx43 gap junction in ECs prior to transplantation using our unique pretreatment strategy which will set a stage for a paradigm shift in the current standard of care during organ Tx.