MicroRNA-181b and Sepsis

 描述(由申请人提供)：越来越多的研究表明，内皮细胞(EC)炎症和功能障碍在败血症休克和败血症所致肺损伤的发病机制中起着关键作用，这是危重患者死亡的主要原因。过度产生促炎介质可导致侧支血管功能障碍，这种作用使内皮细胞具有亲黏附、促通透性和促血栓形成特性。因此，在血管内皮细胞中调节这些事件可能提供一种新的治疗方法来限制脓毒症及其所致肺损伤的后遗症。MicroRNAs(MiRNAs)是一种小的、非编码的RNAs，它在转录后水平抑制靶基因的表达，并参与一系列生物反应。然而，microRNAs在脓毒症相关的内皮功能障碍中的作用仍不清楚。在内皮细胞中使用微阵列分析方法，我们发现miR-181b在内毒素血症小鼠的血管内皮细胞中的表达迅速减少-这一观察结果在体内感染的人类受试者中得到了概括。我们的研究发现，miR-181b抑制控制两个关键信号通路--NF-kB和AKT/eNOS的靶点，这两个通路控制着EC黏附、血管通透性和促炎介质，参与了脓毒症和脓毒症所致的肺损伤。初步的内皮细胞功能研究显示，miR-181b可有效抑制白细胞黏附、内皮细胞通透性和凝血酶诱导的内皮细胞炎症。MIR-181b通过与Importin-A3的3‘非编码区结合来抑制内皮细胞中NF-kB途径的激活，Importin-A3是一种参与内皮细胞而不是白细胞中NF-kB核转位的蛋白。相反，miR-181b通过直接靶向磷酸酶PHLPP2诱导eNOS磷酸化，已知PHLPP2抑制AKT-磷酸化。最后，全身静脉注射miR-181b对小鼠的治疗类似于“替代疗法”，减少了内毒素血症诱导的EC炎症标志物、白细胞聚集、肺损伤，并显著提高了存活率。因此，我们假设miR-181b可能在脓毒症和脓毒症所致肺损伤中作为内皮细胞和血管功能障碍的关键动态平衡调节因子。为了进一步了解miR-181b在脓毒症中的保护作用，我们建议：在AIM1中，描述脓毒症过程中调节miR-181b表达的近端事件。在AIM2中，使用包括脓毒症患者和脓毒症所致肺损伤患者的血浆样本在内的炎症刺激，我们将剖析miR-181b调节NF-kB和AKT信号以应对EC功能障碍的机制。在AIM3中，我们将探索改变miR-181b表达或其靶点(使用遗传、siRNA或药理学方法导入蛋白-A3或PHLPP2)对体内脓毒症和EC功能的实验模型的影响和时机。这项拟议研究的成功完成将确定miR-181b在EC炎症、血管泄漏和微血管血栓形成中的重要作用，并可能为脓毒症诱导的肺损伤提供离散的、新的靶点。

 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.