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.
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.
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