MiR-181b, endothelial cells, and vascular inflammation

描述(申请人提供)：血管内皮细胞(EC)的激活和功能障碍与各种血管炎症疾病状态有关，包括动脉粥样硬化--这是西方社会发病率和死亡率的主要原因。越来越多的研究表明，在包括血管内皮细胞在内的动脉粥样硬化组织中，增强的核因子-kB途径激活起着关键作用。促炎细胞因子和致动脉粥样硬化的危险因素如高脂血症导致核因子-kB的激活，这种作用赋予内皮细胞促黏附和促血栓形成的特性。因此，抑制血管内皮细胞的炎症反应可能为限制动脉粥样硬化血栓形成提供新的治疗途径。MicroRNAs(MiRNAs)是一种小的、单链的、非编码的RNAs，能够通过碱基配对的方式抑制基因的表达，参与多种病理生理过程，包括免疫和炎症反应的调节。然而，microRNAs在动脉粥样硬化相关的内皮激活中的作用仍然不清楚。我们在内皮细胞(ECs)中进行了微阵列分析方法，并发现miR-181b的表达随着肿瘤坏死因子-a的反应而迅速减少，在容易发生动脉粥样硬化的小鼠的血管内皮细胞中-观察到的结果在体内的人类炎症模式中得到了概括。基于我们的初步研究，我们发现miR-181b的过表达抑制了肿瘤坏死因子-a诱导的核因子-kB反应靶点，包括黏附分子(例如VCAM-1、E-选择素)、趋化因子(例如CX3CL1、CXCL1)和促炎介质(例如COX-2、PAI-1)，这些分子对病变的形成、组成或血栓形成的易感性至关重要。功能获得和功能丧失研究表明，miR-181b有效地抑制白细胞与内皮单分子层的黏附，而抑制miR-181b具有相反的作用。从机制上讲，我们发现MIR-181b通过与Importin-A3的3‘UTR特异结合而抑制了NF-βB途径的激活，Importin-A3是一种参与核转位的蛋白质，并降低了其表达。最后，全身静脉注射miR-181b模拟减少EC激活和体内白细胞聚集。这些观察结果为中心假设提供了基础，即miR-181b可能作为EC激活和血管内稳态的关键调节因子。为了更好地了解miR-181b在NF-kB信号和EC激活中的确切作用，我们提出了三个目标。在Aim1中，我们将描述调控内皮细胞miR-181b表达的上游机制。在目标2中，我们将确定miR-181b调节NF-kB信号和EC激活的分子基础。在目标3中，我们将探讨miR-181b表达改变在急性和慢性血管炎症中的作用。这些研究的结果将对miR-181b在EC生物学、血管炎症和动脉粥样硬化血栓形成中的作用提供深入的认识，并可能为抗炎治疗提供新的靶点。

DESCRIPTION (provided by applicant): Endothelial cell (EC) activation and dysfunction have been linked to a variety of vascular inflammatory disease states including atherosclerosis-the major cause of morbidity and mortality in Western Societies. Accumulating studies highlight a critical role for enhanced NF-kB pathway activation in atherosclerotic tissues including the vascular endothelium. Proinflammatory cytokines and proatherogenic risk factors such as hyperlipidemia lead to NF-kB activation, an effect that confers pro-adhesive, pro-thrombotic properties to ECs. Therefore, suppressing the inflammatory response in the vascular endothelium may provide a novel therapeutic approach to limit atherothrombosis. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs capable of repressing gene expression by base pairing to the 3' untranslated regions (3'-UTRs) of mRNA targets and are involved in a variety of pathophysiological processes including the regulation of immune and inflammatory responses. However, the role of microRNAs in atherosclerotic-associated endothelial activation remains poorly defined. We undertook a microarray profiling approach in endothelial cells (ECs) and identified that miR-181b expression is rapidly reduced in response to TNF-a and in the vascular endothelium from atherosclerotic-prone mice - observations that are recapitulated in human inflammatory paradigms in vivo. Based on our preliminary studies, we find that miR-181b overexpression inhibited TNF-a-induced NF-kB-responsive targets including adhesion molecules (e.g. VCAM-1, E-selectin), chemokines (e.g. CX3CL1, CXCL1), and proinflammatory mediators (e.g. COX-2, PAI-1) that are critical to lesion formation, composition, or pre-disposition to thrombosis. Gain and loss-of function studies reveal that miR-181b potently inhibited leukocyte adhesion to endothelial monolayers, whereas inhibition of miR-181b had the opposite effect. Mechanistically, we find that MiR-181b suppressed the activation of the NF-?B pathway by binding uniquely to the 3'UTR of importin-a3, a protein involved in the nuclear translocation of NF-?B, and reduced its expression. Finally, systemic intravenous administration of miR-181b mimics reduced EC activation and leukocyte accumulation in vivo. These observations provide the foundation for the central hypothesis that miR-181b may serve as a critical regulator of EC activation and vascular homeostasis. To better understand the precise role of miR-181b in NF-kB signaling and EC activation, three aims are proposed. In Aim1, we will delineate the upstream mechanisms governing miR-181b expression in ECs. In Aim 2, we will determine the molecular basis for miR-181b's ability to regulate NF-kB signaling and EC activation. In Aim 3, we will explore the effect of altered miR-181b expression on acute and chronic vascular inflammation. The results of these studies will provide insights regarding miR-181b function in EC biology, vascular inflammation, and atherothrombosis and may provide new targets for anti-inflammatory therapy.