Ex vivo maintenance of endothelial cell barrier integrity via gap junction modification to prevent early ischemic injury in solid organ transplantation

项目摘要 器官移植（Tx）是终末期器官衰竭患者的主要治疗方法。然而， 存在妨碍移植物长期存活的因素。众所周知，在移植过程中， 供体器官保存和再灌注阶段，为器官晚期衰竭做准备。根据来自 我们的实验室，由记忆T细胞介导的缺血再灌注损伤（IRI）和早期同种免疫， 在影响内皮细胞（EC）的最普遍和不可避免的早期损伤中， contact.因此，我们的科学前提围绕着同种异体移植物的内皮细胞作为这些损害的中心。 内皮细胞含有一层完整的排列整齐的内皮细胞，这些内皮细胞通过细胞间连接连接， 他们沟通，形成一个保护屏障。在IRI和早期同种异体免疫期间，EC屏障被破坏。 破坏，使EC倾向于不适当的抗原呈递，最终导致晚期移植物衰竭。 因此，EC屏障对于移植物保护至关重要。内皮细胞间的通讯依赖于 间隙连接包括连接蛋白43（Cx43）。Cx43缝隙连接是否在EC保护中发挥作用 屏障和抑制EC免疫原性对早期损伤允许治疗靶点，形成 我们提案的核心问题。根据我们的初步数据，我们假设稳定和/或 在内皮细胞中过度表达Cx43缝隙连接将减轻早期移植物损伤。为了研究这个假设，我们 具体目的1 -阐明缝隙连接蛋白Cx43在EC中的作用 体外IRI期间的活化/免疫原性。具体目标2 -证明临床前能力， 通过维持Cx43下调体内移植心脏中EC活化/免疫原性 通过对供体心脏的预处理来降低蛋白质水平。为了实现这些目标，我们将采用体外模型 模拟冷缺血和热再灌注损伤，研究Cx43缝隙连接在缺血再灌注损伤中的作用。 EC健康和免疫原性。我们还将采用临床相关的体内脑死亡小鼠模型， IRI和同种免疫心脏Tx，并利用一种新颖独特的供体器官预处理策略， Cx43调节剂。这些创新和高风险高回报的研究将是第一个定义 Cx43缝隙连接在EC中的特异性和集中性影响，与器官保存和 再灌注此外，它将提供一个概念验证，以靶向EC中的Cx43间隙连接， 移植使用我们独特的预处理策略，这将为移植的范式转变奠定基础。 在器官Tx期间的当前护理标准。

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.