MiR-181b, endothelial cells, and vascular inflammation

描述（由申请人提供）：内皮细胞（EC）的激活和功能障碍与多种血管炎性疾病状态有关，包括动脉粥样硬化-西方社会发病率和死亡率的主要原因。越来越多的研究强调了在包括血管内皮在内的动脉粥样硬化组织中NF-kB通路激活增强的关键作用。促炎细胞因子和促动脉粥样硬化危险因素如高脂血症可导致NF-kB活化，这种作用赋予内皮细胞促粘附、促血栓形成的特性。因此，抑制血管内皮的炎症反应可能为限制动脉粥样硬化血栓形成提供一种新的治疗方法。MicroRNAs （miRNAs）是一种小的单链非编码rna，能够通过与mRNA靶标的3‘非翻译区（3’- utr）的碱基配对来抑制基因表达，并参与多种病理生理过程，包括免疫和炎症反应的调节。然而，microrna在动脉粥样硬化相关的内皮细胞活化中的作用仍然不明确。我们在内皮细胞（ECs）中采用了微阵列分析方法，发现miR-181b的表达在TNF-a和动脉粥样硬化易感小鼠的血管内皮中迅速降低，这些观察结果在体内的人类炎症范式中得到了概括。根据我们的初步研究，我们发现miR-181b过表达抑制tnf -a诱导的nf - kb应答靶标，包括粘附分子（如VCAM-1、E-selectin）、趋化因子（如CX3CL1、CXCL1）和促炎介质（如COX-2、PAI-1），这些靶标对病变形成、组成或血栓形成的易感至关重要。功能获得和功能丧失研究表明，miR-181b能有效抑制白细胞粘附内皮单层，而抑制miR-181b则具有相反的效果。机制上，我们发现MiR-181b抑制NF-？通过独特地结合importin-a3的3'UTR，一种参与NF-？B，并简化其表达式。最后，全身静脉注射miR-181b模拟体内EC活化和白细胞积累。这些观察结果为核心假设提供了基础，即miR-181b可能是EC激活和血管稳态的关键调节剂。为了更好地了解miR-181b在NF-kB信号传导和EC激活中的确切作用，提出了三个目标。在Aim1中，我们将描述在内皮细胞中调控miR-181b表达的上游机制。在Aim 2中，我们将确定miR-181b调节NF-kB信号传导和EC激活能力的分子基础。在Aim 3中，我们将探讨miR-181b表达改变对急性和慢性血管炎症的影响。这些研究结果将提供关于miR-181b在EC生物学、血管炎症和动脉粥样硬化血栓形成中的功能的见解，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.