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定义性病理标志。 事实上，脓毒症每年在美国造成的医院死亡人数比任何其他疾病都多， 疾病目前缺乏任何有针对性的药物干预。关键是要了解 炎症影响血管屏障功能的一个重要原因是整个循环系统的内皮细胞不 同质的炎症通过对静脉内皮的作用特异性地影响血管通透性， 而动脉内皮对炎症诱导的渗透性不敏感 调节血压。因此，关于静脉内皮屏障功能如何调节的机制观点是： 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并没有考虑到 对于内皮异质性和独特的细胞粘附和信号传导途径， 细胞类型。嘌呤能信号传导已被鉴定为内皮通透性的关键调节剂;然而， 允许同时调节嘌呤反应和紧密连接的细胞途径尚未被发现。 探讨了我们推测，静脉内皮细胞的嘌呤释放通道泛连接蛋白（Panx）1是一个重要的调节因子。 在炎症刺激如脓毒症时激活的动态信号联系的组分。我们将使用三个 旨在验证这一概念。在目标1中，我们将鉴定靶向Panx 1的激酶信号通路， 静脉通透性该目标将使用Panx 1点突变的新型小鼠模型进行翻译后研究。 修改和测量这些关键修改如何改变盲肠结扎穿刺的病理反应 (CLP)脓毒症模型，静脉通透性和ATP释放。目标2，我们将衡量claudin 11的影响 （cldn）11对Panx 1的细胞内分布和组织所需的其他组件的影响 响应TNF α刺激的静脉通透性的嘌呤能信号传导。基于我们以前的工作，我们 发现cldn 11在静脉内皮中唯一表达， 对TNF α/脓毒症的反应;这与密蛋白5形成对比。这是一个独特的信令中心， 其受钙变化调节能力和紧密连接的性质调节Panx 1功能 在静脉内皮上。最后，在目标3中，我们将确定内部化和周转的作用， Panx 1/Cldn 11枢纽的组成部分调节静脉通透性。翻转和重置 信号复合物是细胞对炎症反应的重要组成部分。为此，我们提供 有证据表明，小窝蛋白1促进炎症刺激后Panx 1和cldn 11的再循环， 这是如何发生的机制。所有建议都强调了实现这些目标的可行性。 敲除小鼠在手，加上强有力的初步数据和高生产力的前一个融资周期 两个调查员之间。

|| 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.