Purinergic Regulation of Veinous Endothelial Permeability

||摘要 内皮细胞屏障的破坏被认为是多种疾病的一个决定性的病理标志。 事实上，在美国，脓毒症每年导致的医院死亡人数比任何其他疾病都多，而且 目前，这种疾病缺乏任何有针对性的药物干预。对于了解如何 炎症影响血管屏障功能是整个循环系统的内皮细胞不受影响 同质的。炎症通过对静脉内皮细胞的影响来特别影响血管的通透性， 而动脉内皮细胞对炎症诱导的通透性不敏感，相反，主要是 调节血压。因此，一种关于静脉内皮屏障功能如何调节的机械论观点是 对人类健康和疾病至关重要。我们目前对血管屏障功能的理解不能解释 对于内皮的异质性和每个内皮特有的独特的细胞黏附和信号通路 单元类型。嘌呤能信号已被认为是内皮通透性的关键调节因子；然而， 允许同时调节嘌呤反应和紧密连接的细胞通路尚未被 探索过了。我们假设静脉内皮细胞中的嘌呤释放通道pAnnexin(Panx)1是一种 在炎症刺激(如败血症)时激活的动态信号网络的组成部分。我们将使用三个 旨在测试这一概念。在目标1中，我们将确定针对Panx1的激酶信号通路 静脉通透性。这个目标将使用带有Panx1点突变的新的小鼠模型来进行翻译后 修改和测量这些关键修改如何改变盲肠结扎穿刺术的病理反应 (CLP)脓毒症模型、静脉通透性和ATP释放。目标2，我们将测量Claudin 11的影响 (CLDN)11关于Panx1的细胞内分布和所需其他成分的组织 嘌呤能信号转导反应中的TNFa刺激静脉通透性。基于我们以前的工作，我们 发现cldn11在静脉内皮细胞中唯一表达，这种血管内皮选择性地被分解。 对TNFa/脓毒症的反应；这与claudin5形成对比。这里假设cldn11是唯一的信令中枢，因为 它受钙离子变化调节能力，紧密连接的性质调节Panx1功能 静脉内皮细胞。最后，在目标3中，我们将确定内部化和更替的角色 Panx1/Cldn11中枢在调节静脉通透性中的成分。营业额和重置 信号复合体是细胞对炎症反应的重要组成部分。在这一目标中，我们提供 小窝蛋白1促进炎性刺激后Panx1和cldn11循环的证据，并探讨 这可能发生的机制。所有提出的建议都强调了实现这些目标的可行性 基因敲除小鼠在手，再加上强劲的初步数据和之前高生产率的融资周期 两名调查员之间的关系。

|| ABSTRACT Breakdown of the endothelial cell barrier is considered a defining pathological hallmark of multiple diseases. Indeed, sepsis accounts for more hospital deaths per year than any other condition in the United States, and the disease is currently devoid of any targeted pharmacological intervention. Critical to understanding how inflammation affects vascular barrier function is that endothelial cells throughout the circulatory system are not homogenous. Inflammation specifically affects vascular permeability through effects on the venous endothelium, whereas the arterial endothelium is not susceptible to inflammation-induced permeability and instead primarily regulates blood pressure. Thus, a mechanistic view of how venous endothelial barrier function is regulated is essential to human health and disease. Our current understanding of vascular barrier function does not account for endothelial heterogeneity and the unique cell adhesion and signaling pathways specific to each endothelial cell type. Purinergic signaling has been identified as a key regulator of endothelial permeability; however the cellular pathway allowing for simultaneous regulation of the purine response and tight junctions has not been explored. We hypothesize that the purine release channel pannexin (Panx)1 in venous endothelium is a component of a dynamic signaling nexus activated upon inflammatory stimuli such as sepsis. We will use three aims to test this concept. In Aim 1, we will identify kinase signaling pathways targeting Panx1 that regulate venous permeability. This aim will use novel mouse models with point mutations in Panx1 for post-translational modification and measure how these key modifications alter pathological responses to cecal ligation puncture (CLP) sepsis model, venous permeability, and ATP release. Aim 2, we will measure the impact of claudin11 (cldn)11 on the intracellular distribution of Panx1 and organization of other components required for purinergic signaling in response TNFa stimulated venous permeability. Based on our previous work, we found cldn11 to be uniquely expressed across the venous endothelium that was selectively broken down in response to TNFa/sepsis; this contrasted with claudin5. Here was posit cldn11 is a unique signaling hub due to its ability to be regulated by changes in calcium, and that property of the tight junction regulates Panx1 function on venous endothelium. Last, in Aim 3 we will determine roles for internalization and turnover of components of the Panx1/Cldn11 hub in regulation of venous permeability. Turnover and reseting the signaling complex is an important component to the cellular response to inflammation. In this aim we provide evidence that caveolin1 facilitates recycling of both Panx1 and cldn11 after inflammatory stimuli, and probe the mechanisms of how this may occur. The feasibility of accomplishing these aims is underscored by all proposed knockout mice being in hand, coupled with strong preliminary data and a highly productive previous funding cycle between the two investigators.