A Modified Parallel Plate Flow Chamber to Study Local Endothelial Response to Recirculating Disturbed Flow

Jason Matthew Sedlak

doi:10.17918/00000037

Cardiovascular disease is the leading cause of death around the world, including the stiffening of artery walls known as atherosclerosis. Atherosclerosis develops at arterial sites where endothelial cells (ECs) are exposed to low time averaged hemodynamic shear stress, particularly in regions of recirculating disturbed flow. While the effects of disturbed hemodynamics are greatly studied, the complexity of in vivo geometry and how the resulting spatial transitions between atheroprotective and atherogenic hemodynamics affect EC dysfunction are not as well understood. The core objective of this dissertation is to explore the association between local heterogeneity in blood flow and EC function, through functional phenotype assessment and correlation to computational fluid dynamics modelling of flow within a custom microfluidic device. Computational fluid dynamic (CFD) modelling informed an in vitro parallel plate flow chamber gasket modification for protruding baffles in order to produce segments of large recirculating flow contiguous with segments of steady laminar flow. After experimental validation using bovine aortic endothelial cells (BAECs), four regions of interest were identified: at the apex of the baffles (DFG-High), within the recirculation (DFG-Recirc), the low shear stress transition from DFG Recirc to DFG-Low (DFG-Low), and the center lane of moderate shear stress bulk flow which was bounded by the previous three conditions (DFG-2Pa). Then, BAECs within these regions were assessed by immunofluorescent imaging for adaption to in vitro flow via changes to morphology, cell quiescence, and monolayer permeability and junction integrity. Surprisingly, cells in disturbed flow device regions exposed to atheroprotective shear stress (DFG-2Pa) did not consistently align or decrease permeability as expected and demonstrated low levels of nitric oxide bioavailability (DFG-High & DFG-2Pa). Finally, the relationship between coordinate-specific measurements of F-actin alignment and CFD-derived shear stress features was investigated using supervised partial least square regression (PLSR) principal component analysis. In samples with an overall-low degree of alignment, shear stress magnitude contributed the most of any other variable to the PLSR model (18.9% of the 67.7% total variance in alignment explained). Conversely, in samples with an overall-high degree of alignment, the shear stress gradient components parallel and perpendicular to the net direction of flow were equally as effective compared to shear stress magnitude (12.4%, 11.8%, and 12.7%, respectively, of the 69.24% total variance in alignment explained). These results demonstrate cells in flow unexpectedly adopting a hybrid phenotype between atheroprotective and atheroprone, with post-hoc analysis suggesting local shear stress gradients play a determining role. This research supports advancing understanding of EC mechanotransduction within complex atheroma environment hemodynamics to focus research topics and develop predictive clinical diagnostic tools. Keywords: Atherosclerosis, Disturbed flow, Endothelial, Mechanotransduction, Shear stress gradient, Transverse flow

A Modified Parallel Plate Flow Chamber to Study Local Endothelial Response to Recirculating Disturbed Flow

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A Modified Parallel Plate Flow Chamber to Study Local Endothelial Response to Recirculating Disturbed Flow

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