Role of endothelial JAK/STAT signaling in the regulation of vascular leakage

因长期或加重炎症导致的持续血管渗漏会导致器官损害，持久 后遗症和死亡率增加。血管内皮细胞屏障功能长期丧失的机制 人们对此了解甚少。我们认为内皮细胞的转录变化，特别是STAT3- 依赖的基因调控，可能解释了这些长期的变化。STAT3可以促进对 内皮细胞。而抑制其磷酸化(由JAK激酶介导)则是抗炎和 降低内皮细胞通透性，内皮细胞STAT3基因敲除实际上会增加血管渗漏。这个 这项授权的中心假设是，长期的STAT3磷酸化促进了促炎作用 STAT3转录反应涉及糖基化反应的改变，包括降解 透明质酸和肌动蛋白细胞骨架的剧烈变化。这一假设挑战了当前的 通过细胞因子信号转导了解STAT3在内皮炎症反应中的作用 通过一种新的机制持续的血管渗漏。更好地了解这条途径不仅可以 解释多个临床观察，也有望对未来的抗病毒治疗产生重大的积极影响 通过识别新的药理靶点来预防和/或逆转全身炎症治疗 血管渗漏不影响病原体可能需要的其他免疫反应 清除或组织愈合。我们将结合体外和体内实验的三个不同的目标： 确定STAT3依赖的转录调节因子在内皮屏障丧失中的特定作用。我们确认了 IL-6下游依赖STAT3的新基因，包括内皮糖催化分解代谢酶和 肌动蛋白细胞骨架的调节器。透明质酸和肌动蛋白应激纤维的丢失与IL-6- 诱导HUVEC屏障功能丧失。我们将确定这些候选基因的任何因果作用。 确定内皮细胞STAT3磷酸化在促进体内屏障功能丧失中的需求。 磷酸化和非磷酸化的STAT3可能具有不同的甚至相反的作用。我们将使用 建立脓毒症模型以评估小鼠的存活、血管渗漏和内皮细胞糖基化 STAT3(STAT3iEKO)和gp130iEKO基因敲除小鼠以及携带AFP基因的小鼠细胞骨架的调节 可诱导的内皮特异性敲击蛋白Y705F突变(STAT3iE-Y705F)。 确定导致STAT3持续激活的反馈环在屏障功能中的作用。我们会 确定IL-6诱导的STAT3持续激活如何导致血管长时间渗漏 干扰涉及SOCS3和TC45的负反馈环路。我们将确定潜在的后 负调控因子SOCS3的翻译修饰可能导致持续的STAT3激活。至 评估这些环在体内的作用，我们将通过培育产生内皮特异的SOCS3敲除 SOCS3f1小鼠到cdh5-CreERT2小鼠。

Sustained vascular leakage due to prolonged or exacerbated inflammation leads to organ damage, lasting sequelae and increased mortality. The mechanisms that mediate long-term loss of endothelial barrier function are poorly understood. We propose that transcriptional changes in the endothelium, and in particular STAT3- dependent gene regulation, may explain these long-term changes. STAT3 can promote opposing effects on the endothelium. While inhibition of its phosphorylation (mediated by JAK kinases) is anti-inflammatory and reduces endothelial permeability, endothelial STAT3 knockout actually increases vascular leakage. The central hypothesis of this grant is that prolonged STAT3 phosphorylation promotes a pro-inflammatory STAT3 transcriptional response that involves changes in the glycocalyx, including degradation of hyaluronic acid, and drastic alterations in the actin cytoskeleton. This hypothesis challenges the current understanding of the role of STAT3 in the endothelial inflammatory response by linking cytokine signaling to sustained vascular leakage via a novel mechanism. A better understanding of this pathway not only can explain multiple clinical observations, but also is expected to have a significant positive impact on future anti- inflammatory treatments by identifying novel pharmacological targets to prevent and/or revert the systemic vascular leakage without affecting other arms of the immune response that may be required for pathogen clearing or tissue healing. We will combine in vitro and in vivo experimentation in the three separate aims to: Identify the specific role of STAT3-dependent transcriptional mediators in endothelial barrier loss. We identified novel STAT3-dependent genes downstream of IL-6, including endothelial glycocalyx catabolic enzymes and modulators of the actin cytoskeleton. Loss of hyaluronan and actin stress fibers temporally correlate with IL-6- induced loss of barrier function in HUVEC. We will determine any causal roles for these candidate genes. Determine the requirement of endothelial STAT3 phosphorylation in promoting barrier function loss in vivo. Phosphorylated and non-phosphorylated STAT3 may have different and even opposing roles. We will use established models of sepsis to assess mice survival, vascular leakage and endothelial glycocalyx and cytoskeletal regulation of STAT3 (STAT3iEKO) and gp130iEKO knockout mice, as well as mice harboring an inducible, endothelial-specific knockin Y705F mutation (STAT3iE-Y705F). Determine the role of feedback loops leading to STAT3 sustained activation in barrier function. We will determine how an IL-6-induced sustained activation of STAT3 can lead to prolonged vascular leakage by interfering with negative feedback loops involving SOCS3 and TC45. We will identify potential post- translational modifications of the negative regulator SOCS3 that could lead to sustained STAT3 activation. To assess the role of these loops in vivo, we will create endothelial-specific SOCS3 knockouts by breeding SOCS3fl mice to cdh5-CreERT2 mice.