Modulation of Endothelial Cell Function by the Shear Stress-Responsive miR-155

描述（由申请人提供）：本提案的目的是定义microRNA表达对响应剪切应力力而发生的内皮细胞功能特异性变化的影响。由血流产生的剪切应力在调节血管张力、血管重构和动脉粥样硬化病变的局灶性发展中起重要作用。在动脉树中，内皮细胞暴露于不同的剪切应力，其诱导对基因表达和功能的不同影响。单向剪切应力，发生在树的直的部分，elevening内皮基因表达的变化，通常是抗炎和动脉粥样硬化保护。相反，在动脉树中的分支点处发生的振荡剪切应力诱导总体促炎和促动脉粥样硬化反应。microRNA（miRNAs）是近年来发现的一类短（19-25 nt）单链非编码RNA，由于其在转录后水平调控细胞分化、生长、增殖和凋亡等多种功能基因的表达，已成为分子生物学研究的热点。虽然已经证明了miRNA表达在各种生物学过程中的重要作用，包括心脏发生和血管生成，但目前关于特定miRNA在内皮细胞生物学中作用的数据有限。在人内皮细胞经受长时间单向剪切应力（24小时，15达因/cm 2）的初步研究中，鉴定了一组miRNA，其表达响应于该刺激而显著上调，表明这些miRNA在调节内皮细胞中的基因表达和功能中是重要的。为了进一步确定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，将研究一种高度剪切响应性miRNA miR-155的功能。具体而言，拟议的研究将确定miR-155-靶基因相互作用对内皮细胞凋亡，屏障功能和迁移的影响。为了研究miR-155调节细胞凋亡的机制，我们将重点关注SHIP 1/PI 3 K/Akt通路。为了研究miR-155调节内皮细胞单层通透性和迁移的机制，我们将重点关注RhoA/Rho激酶途径。我们将通过实验操作miR-155、其靶基因或miRNA-靶基因相互作用下游途径成员的表达来测试miR- 155对这些关键途径的影响。随后，将量化这些操作对内皮细胞凋亡、单层渗透性和迁移的影响。最后，将在体内研究剪切诱导的miR-155表达和内皮细胞调节途径活性之间的关联，在小鼠模型中改变主动脉血流。我们预计，这些研究将有助于解决我们对内皮细胞中miRNA功能的认识不足，并将增强我们对剪切应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。尽管动脉粥样硬化的病因与危险因素有关，如糖尿病、高血压、高胆固醇血症和吸烟，但炎症过程优先发生在与低血流和受干扰血流相关的动脉区域，而不影响未受干扰的血流区域，表明血流模式对疾病过程至关重要。该项目的目的是研究一类重要的新型调节分子（称为microRNA）调节血管基因表达和功能以响应流量的机制。

DESCRIPTION (provided by applicant): 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 posttranscriptional 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 one highly shear-responsive miRNA, miR-155, will be studied. Specifically, the proposed research will define the impact of miR-155-target gene interaction on endothelial cell apoptosis, barrier function and migration. To study the mechanism by which miR-155 modulates apoptosis, we will focus on the SHIP1/PI3K/Akt pathway. To study the mechanism by which miR-155 modulates endothelial monolayer permeability and migration, we will focus on the RhoA/Rho kinase pathway. We will test the influence of miR- 155 on these critical pathways by experimentally manipulating expression of miR-155, 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, monolayer permeability, and migration will be quantified. Finally, the association between shear-induced miR-155 expression and activity of endothelial cell regulatory pathways 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 greatest cause of mortality in the U.S. 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 an important new class of regulatory molecules, known as microRNAs, modulate vascular gene expression and function in response to flow.