The Impact of miR-21 Expression on Endothelial Cell Apoptosis and Inflammation

描述(由申请人提供)： 项目概述本研究的目的是确定在剪切力作用下，microRNA的表达对内皮细胞功能的特定改变的影响。血流产生的剪应力在调节血管张力、血管重塑和动脉粥样硬化病变的局灶性发展中起着重要作用。在动脉树中，内皮细胞暴露在不同的剪切力下，对基因表达和功能产生不同的影响。单向剪应力发生在树的直线部分，引起内皮基因表达的变化，这通常是抗炎和动脉粥样硬化保护的。相反，发生在动脉树分支点的振荡切应力会诱导整体的促炎和动脉粥样硬化反应。MicroRNAs(MiRNAs)是最近发现的一类短小(19-25nT)、单链、非编码RNA，由于它们在转录后调节一系列细胞功能相关基因的表达，包括分化、生长、增殖和凋亡，因此已成为分子生物学研究的主要焦点。虽然已经证明miRNA的表达在包括心脏生成和血管生成在内的各种生物学过程中都发挥着重要的作用，但目前关于特定miRNA在内皮细胞生物学中的作用的数据仍然有限。在对人内皮细胞承受持续的单向剪切应力(24小时，15dynes/cm2)的初步研究中，发现了一组miRNAs，它们的表达显著上调，表明这些miRNAs在调控内皮细胞的基因表达和功能方面起着重要作用。为了进一步确定miRNA表达在调节切应力引起的内皮细胞生物学变化中的作用，我们将研究调控最高的miRNA miR-21的功能。具体地说，拟议的实验将确定miR-21靶基因相互作用对剪应力诱导的细胞凋亡和炎症分子表达变化的影响。将在单向和振荡剪切应力作用下对培养的人内皮细胞进行研究。将评估含有miR-21靶基因的凋亡或炎症信号通路的活性，如PI3K/Akt和MAP2K3。将通过实验操纵miR-21、其目标基因或miRNA-靶基因相互作用下游的途径成员的表达来测试通路的参与。随后，将量化这些操作对内皮细胞凋亡和黏附分子表达的影响。最后，在小鼠主动脉血流改变的模型中，将在体内研究miR-21表达与细胞凋亡或炎症途径活性之间的相互作用。我们预计，这些研究将有助于解决我们对血管内皮细胞中miRNAs功能的认识不足，并将加强我们对剪切力调节血管疾病的机制的理解。 公共卫生相关性： 冠状动脉粥样硬化性心脏病是一种炎症性疾病，是美国最大的死亡原因，也是美国退伍军人发病率和死亡率的主要原因。尽管动脉粥样硬化的病因与糖尿病、高血压、高胆固醇血症、吸烟等危险因素有关，但炎症过程优先发生在与低血流和血流紊乱相关的动脉区域，而不发生在未受干扰的血流区域，表明血流模式在疾病过程中是必不可少的。该项目的目的是研究一类新发现的调控分子，即microRNAs，通过其调节血管基因表达和功能以响应血流的机制。该项目的发现将为新的潜在治疗靶点提供洞察力。

DESCRIPTION (provided by applicant): Project Summary The purpose of this proposal is to define the influence of microRNA expression on specific changes in endothelial cell function that occur in response to shear stress forces. Shear stress forces, generated by blood flow, play an important role in the regulation of vascular tone, vascular remodeling, and the focal development of atherosclerotic lesions. In the arterial tree, endothelial cells are exposed to different shear stress forces that induce distinct effects on gene expression and function. Unidirectional shear stress, which occurs in the straight part of the tree, elicits a change in endothelial gene expression that is generally anti-inflammatory and atheroprotective. In contrast, oscillatory shear stress, which occurs at branch points in the arterial tree, induces an overall pro-inflammatory and proatherosclerotic response. MicroRNAs (miRNAs) are a recently recognized class of short (19-25 nt), single stranded, noncoding RNAs that have become a major focus in molecular biology research because they posttranscriptionally regulate the expression of genes involved in an array of cell functions, including differentiation, growth, proliferation, and apoptosis. Although an important role for miRNA expression has been demonstrated for various biological processes, including cardiogenesis and angiogenesis, data on the role of specific miRNAs in endothelial cell biology is currently limited. In preliminary studies of human endothelial cells subjected to prolonged unidirectional shear stress (24 hrs, 15 dynes/cm2), a group of miRNAs was identified whose expression was significantly upregulated in response to this stimulus, suggesting that these miRNAs are important in regulating gene expression and function in endothelial cells. To further define the role of miRNA expression in modulating shear stress-induced changes in endothelial cell biology, the function of the most highly regulated miRNA, miR-21, will be studied. Specifically, the proposed experiments will define the impact of miR-21-target gene interaction on shear stress-induced changes in apoptosis and inflammatory molecule expression. Studies will be performed on cultured human endothelial cells subjected to unidirectional and oscillatory shear stress. The activity of apoptotic or inflammation signaling pathways containing miR-21 target genes, such as PI3K/Akt and MAP2K3, will be assessed. Pathway involvement will be tested by experimentally manipulating expression of miR-21, its target gene, or members of the pathway that are downstream of the miRNA-target gene interaction. Subsequently, the effect of these manipulations on endothelial cell apoptosis and adhesion molecule expression will be quantified. Finally, the interaction between miR-21 expression and apoptotic or inflammatory pathway activity will be studied in vivo, in a mouse model of altered aortic flow. We anticipate that these studies will help address a deficit in our knowledge about the function of miRNAs in endothelial cells and will enhance our understanding of the mechanisms by which shear stress forces modulate vascular disease. PUBLIC HEALTH RELEVANCE: Coronary atherosclerotic heart disease is an inflammatory disease that is the largest cause of mortality in the U.S and a major cause of morbidity and mortality among the nation's veteran population. Although the etiology of atherosclerosis is related to risk factors, such as diabetes, hypertension, hypercholesterolemia, and smoking, the inflammatory process occurs preferentially in arterial regions associated with low and disturbed blood flow while sparing the undisturbed flow regions, indicating that blood flow patterns are essential to the disease process. The purpose of this project is to examine the mechanisms by which a newly identified class of regulatory molecules, known as microRNAs, modulate vascular gene expression and function in response to flow. Findings from this project will provide insight into new potential therapeutic targets.