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Mechanisms Regulating Smooth Muscle Cell Phenotype by Atheroprone and Atheroprotective Hemodynamics on Endothelial Cells

Boerboom, Lauren
Format
Thesis/Dissertation; Online
Author
Boerboom, Lauren
Advisor
Blackman, Brett
Abstract
Atherosclerosis is an inflammatory disease of the arteries that develops preferentially in regions where shear stress, caused by blood flowing within the lumen of the vessel, is disturbed. Recent evidence demonstrates focal fibronectin (FN) expression in the sub-endothelial layer in pre-atherosclerotic and advanced lesions. We have found that atheroprone shear stress promotes FN deposition and inflammatory signaling pathways in endothelial cells (ECs). Recently, our lab discovered that EC platelet endothelial cell adhesion molecule-1 (PECAM), which is important for sensing biomechanical stresses, is necessary for the production, secretion, and assembly of the FN-rich matrix in atheroprone regions. Similar to ECs, underlying vascular smooth muscle cells (SMCs) also display a pro-inflammatory phenotype in regions of atherogenesis, which is a key feature involved in the development of atherosclerosis. Our lab has designed a novel in vitro system that applies human-derived shear stresses to ECs, which are co-cultured with underlying SMCs. This allows for molecular manipulation of individual cell types, while permitting the study of the inherent signaling cross-talk between ECs and SMCs. Such an approach is useful because, while atheroprone shear stress is known to regulate EC signaling and promote a pro-inflammatory environment, the influence of that signaling on SMC phenotype has yet to be investigated. To assess the role of endothelial PECAM and FN signaling in promoting an inflammatory SMC phenotype, the co-culture model was exposed to atheroprone hemodynamic shear stress. siRNA knockdown of either PECAM or FN in ECs reduced SMC inflammatory genes, proteins, and transcription activity. The reduction in SMC inflammation also has the functional consequence of reducing monocyte adhesion. When EC FN is knocked down, the inflammatory SMC phenotype was rescued through the addition of exogenous FN to the EC media. In addition to an increased inflammatory phenotype in response to atheroprone shear stress, SMCs are able to promote inflammation back to ECs. SMC FN was found to regulate EC inflammatory transcription activity and adhesion protein expression. Next, the role of secreted proteins in signaling inflammation from ECs under atheroprone shear stress to SMCs was determined. Cell media collected from EC PECAM and FN knockdown experiments was able to differentially regulate SMC inflammation without the physical presence of ECs. A co-culture recovery experiment confirmed the ability for conditioned media from a control experiment to restore SMC inflammation despite EC FN knockdown. Media was then screened with a multiplexing approach for secreted cytokines differentially regulated by FN. Of the 19 cytokines measured, 12 were significantly inhibited by the knockdown of EC FN. In particular, GRO-α was found to increase SMC inflammation in a FN-dependent manner. This study supports the importance of cytokine signaling in promoting an atheroprone SMC phenotype downstream of the endothelial PECAM and FN pathway. Finally, the atheroprotective role of Kruppel-like factor 4 (KLF4) in the endothelium was investigated. Anti-inflammatory and anti-thrombotic factors were previously shown to be under the regulation of the transcription factor KLF4. To study the role of KLF4 in establishing an atheroprotective phenotype, mono-cultured ECs were exposed to hemodynamic shear stress. KLF4 had increased expression under atheroprotective shear stress compared to atheroprone. Overexpression of KLF4 conferred an atheroprotective EC phenotype even when exposed to an atheroprone shear stress environment. In contrast, knockdown of EC KLF4 resulted in a pro-inflammatory and pro-thrombotic gene program despite exposure to atheroprotective shear stress. Therefore, the atheroprotective environment is not simply an absence of atheroprone stimulation, but actively promotes anti-atherogenic signaling pathways.
Language
English
Published
University of Virginia, Department of Biomedical Engineering, PHD (Doctor of Philosophy), 2013
Published Date
2013-04-16
Degree
PHD (Doctor of Philosophy)
Collection
Libra ETD Repository
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