MicroRNA-181b and Sepsis

 描述（由申请人提供）：累积研究强调了内皮细胞（EC）炎症和功能障碍在脓毒性休克和脓毒症诱导的肺损伤发病机制中的关键作用，这是危重患者死亡的主要原因。促炎介质的过度产生可导致侧支血管功能障碍，这种作用赋予EC促粘附性、促渗透性和促血栓形成特性。因此，调节血管内皮中的这些事件可能提供一种新的治疗方法来限制脓毒症和脓毒症诱导的肺损伤的后遗症。MicroRNA（miRNAs）是一类小的非编码RNA，在转录后水平抑制靶基因的表达，参与多种生物学反应。然而，microRNA在脓毒症相关的内皮功能障碍中的作用仍然不清楚。使用微阵列分析方法在EC中，我们发现miR-181 b表达在内毒素血症小鼠的血管内皮中迅速降低-观察结果在体内脓毒症患者中重现。我们的研究发现，miR-181 b抑制控制2个关键信号通路的靶点，即NF-κ B和AKT/eNOS，这些通路控制与脓毒症和脓毒症诱导的肺损伤有关的EC粘附、血管通透性和促炎介质。在EC中的初步功能研究显示，miR-181 b有效地抑制对白细胞粘附、EC渗透性和凝血酶诱导的EC炎症的作用。MiR-181 b通过与importin-a3的3 'UTR结合而在EC中独特地抑制NF-κ B途径的活化，importin-a3是一种参与EC而不是白细胞中NF-κ B核转位的蛋白质。相比之下，miR-181 b通过直接靶向磷酸酶PHLPP 2诱导eNOS磷酸化，已知磷酸酶PHLPP 2抑制AKT磷酸化。最后，通过全身静脉内施用miR-181 b模拟物作为“替代疗法”来治疗小鼠减少了内毒素血症诱导的EC炎性标志物、白细胞积聚、肺损伤，并显著改善了存活率。因此，我们推测miR-181 b可能是脓毒症和脓毒症诱导的肺损伤中内皮细胞和血管功能障碍的关键稳态调节因子。为了进一步了解miR-181 b在脓毒症中的保护作用，我们提出：在Aim 1中，描述脓毒症过程中调控miR-181 b表达的近端事件。在Aim 2中，使用包括脓毒症和脓毒症诱导的肺损伤患者的人血浆样本在内的炎症刺激物，我们将剖析miR-181 b调节NF-κ B和AKT信号转导以响应EC功能障碍的机制。在Aim 3中，我们将探索改变miR-181 b表达或其靶点（使用importin-a3或PHLPP 2的遗传、siRNA或药理学方法）对脓毒症和EC功能的体内实验模型的影响和时机。成功完成拟议的研究将确定有关miR-181 b在EC炎症，血管渗漏和微血管血栓形成中功能的重要见解，并可能为脓毒症诱导的肺损伤提供离散的新靶点。

 DESCRIPTION (provided by applicant): Accumulating studies highlight a critical role for endothelial cell (EC) inflammation and dysfunction in the pathogenesis of septic shock and sepsis-induced lung injury, leading causes of death among critically ill patients. Excessive generation of pro-inflammatory mediators can lead to collateral vascular dysfunction, an effect that confers pro-adhesive, pro-permeability, and pro-thrombotic properties to ECs. Therefore, modulating these events in the vascular endothelium may provide a novel therapeutic approach to limit the sequelae of sepsis and sepsis-induced lung injury. MicroRNAs (miRNAs) are small, non-coding RNAs that suppress the expression of target genes at the post-transcriptional level and are involved in a range of biological responses. However, the role of microRNAs in sepsis-associated endothelial dysfunction remains poorly defined. Using a microarray profiling approach in ECs, we identified that miR-181b expression is rapidly reduced in the vascular endothelium from endotoxemic mice - observations that are recapitulated in human subjects with sepsis in vivo. Our studies have uncovered that miR-181b inhibits targets that control 2 key signaling pathways, NF-kB and AKT/eNOS, that govern EC adhesion, vascular permeability, and proinflammatory mediators implicated in sepsis and sepsis-induced lung injury. Preliminary functional studies in ECs reveal that miR-181b potently inhibits effects on leukocyte adhesion, EC permeability, and thrombin-induced EC inflammation. MiR-181b suppresses the activation of the NF-kB pathway uniquely in ECs by binding to the 3'UTR of importin-a3, a protein involved in NF-kB nuclear translocation in ECs and not leukocytes. In contrast, miR-181b induces eNOS-phosphorylation by directly targeting the phosphatase PHLPP2, known to inhibit AKT-phosphorylation. Finally, treatment of mice by systemic intravenous administration of miR-181b mimics as "replacement therapy" reduces endotoxemia-induced EC inflammatory markers, leukocyte accumulation, lung injury, and markedly improves survival. Thus, we hypothesize that miR-181b may serve as a critical homeostatic regulator of ECs and vascular dysfunction in sepsis and sepsis-induced lung injury. To further understand the protective role of miR-181b in sepsis, we propose: in Aim1, to delineate the proximal events during sepsis regulating miR-181b expression. In Aim2, using inflammatory stimuli including human plasma samples from patients with sepsis and sepsis-induced lung injury, we will dissect the mechanisms by which miR-181b regulates NF-kB and AKT signaling in response to EC dysfunction. In Aim3, we will explore the effect and timing of altered miR-181b expression, or its targets (using genetic, siRNA, or pharmacological approaches to importin-a3 or PHLPP2), on experimental models of sepsis and EC function in vivo. Successful completion of the proposed studies will identify significant insights regarding miR-181b function in EC inflammation, vascular leak, and microvascular thrombosis, and may provide discrete, novel targets for sepsis-induced lung injury.