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 器官移植是终末期器官衰竭患者的主要治疗方法。然而，障碍 使得移植物无法长期存活。在移植过程中，对同种异体移植物的早期损伤是公认的 供体器官保存和再灌流阶段，为器官晚期衰竭做好准备。基于来自 我们的实验室，由记忆T细胞介导的缺血再灌注损伤(IRI)和早期同种异体免疫 在影响内皮细胞(ECs)的最普遍和不可避免的早期损伤中，作为第一点 联系。因此，我们的科学前提是围绕着同种异体移植物的内皮细胞，作为这些侮辱的中心。 内皮细胞包含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在一起，并使 他们相互沟通，形成一道保护性屏障。在IRI和早期同种免疫期间，EC屏障是 破坏，使内皮细胞不适当的抗原提呈，最终导致晚期移植物失败。 因此，EC屏障对于移植物保护至关重要。内皮细胞与细胞间的通讯依赖于 缝隙连接包括连接蛋白43(Cx43)。Cx43缝隙连接是否在保护EC中发挥作用 屏障和抑制EC对早期损伤的免疫原性，从而形成治疗靶点，形成 这是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 在内皮细胞中过表达Cx43缝隙连接将减轻移植物早期损伤。为了研究这一假设，我们 提出两个目标：特异性目标1-阐明Cx43缝隙连接蛋白在EC中的作用 体外IRI时的激活/免疫原性。具体目标2-展示临床前能力 维持Cx43下调移植心脏内皮细胞活化/免疫原性 通过对供者心脏的预处理来提高蛋白质水平。与这些目标相同，我们将使用体外模型 模拟冷缺血和热再灌注损伤，研究Cx43缝隙连接在脑缺血再灌注损伤中的作用 EC健康性和免疫原性。我们还将采用临床相关的活体脑死亡小鼠模型 IRI和同种免疫心脏TX，并利用一种新的和独特的供体器官前处理策略来传递 Cx43调节剂。这些创新和高风险、高回报的研究将首先定义 Cx43缝隙连接在内皮细胞中的特异性和焦点影响，与器官保存和 再灌流。此外，它将提供一个概念验证，以在ECs中的Cx43间隙连接为目标 使用我们独特的预处理策略进行移植，这将为 器官移植期间的现行护理标准。

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.