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

描述（由申请人提供）： 项目摘要 本提案的目的是确定 microRNA 表达对响应剪切应力而发生的内皮细胞功能特定变化的影响。血流产生的剪切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发挥着重要作用。在动脉树中，内皮细胞受到不同的剪切应力，这会对基因表达和功能产生不同的影响。发生在树的笔直部分的单向剪切应力会引起内皮基因表达的变化，这种变化通常具有抗炎和动脉粥样硬化的作用。相反，发生在动脉树分支点的振荡剪切应力会诱导整体促炎和促动脉粥样硬化反应。 MicroRNA (miRNA) 是最近公认的一类短 (19-25 nt)、单链非编码 RNA，已成为分子生物学研究的主要焦点，因为它们转录后调节参与一系列细胞功能（包括分化、生长、增殖和凋亡）的基因表达。尽管已经证明 miRNA 表达对各种生物过程（包括心脏发生和血管生成）具有重要作用，但目前有关特定 miRNA 在内皮细胞生物学中的作用的数据有限。在对人类内皮细胞进行长期单向剪切应力（24小时，15达因/厘米2）的初步研究中，鉴定出一组miRNA，其表达在这种刺激下显着上调，表明这些miRNA在内皮细胞中调节基因表达和功能方面发挥着重要作用。为了进一步明确 miRNA 表达在调节剪切应力诱导的内皮细胞生物学变化中的作用，将研究最高度调节的 miRNA miR-21 的功能。具体来说，所提出的实验将确定 miR-21-靶基因相互作用对剪切应力诱导的细胞凋亡和炎症分子表达变化的影响。研究将对培养的人内皮细胞进行单向和振荡剪切应力的研究。将评估包含 miR-21 靶基因（例如 PI3K/Akt 和 MAP2K3）的细胞凋亡或炎症信号传导通路的活性。通路参与将通过实验操纵 miR-21、其靶基因或 miRNA-靶基因相互作用下游通路成员的表达来测试。随后，将量化这些操作对内皮细胞凋亡和粘附分子表达的影响。最后，将在主动脉血流改变的小鼠模型中体内研究 miR-21 表达与细胞凋亡或炎症通路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内皮细胞中 miRNA 功能的了解不足，并将增强我们对剪切应力调节血管疾病机制的理解。 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和死亡的主要原因。尽管动脉粥样硬化的病因与糖尿病、高血压、高胆固醇血症和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，而不受干扰的血流区域，这表明血流模式对于疾病过程至关重要。该项目的目的是研究一类新发现的调节分子（称为 microRNA）调节血管基因表达和响应血流功能的机制。该项目的研究结果将提供对新的潜在治疗靶点的深入了解。

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.