Atherosclerosis is a chronic inflammatory disease that results from dysfunction in the endothelial layer. Plaques preferentially form in regions of low and oscillatory wall shear stresses that form in complex geometries of the vasculature including areas of high curvature, branching, and bulbous regions. These regions are associated with elevated adhesion markers, permeability to lipids, and macrophage infiltration. Hemodynamic regulation of endothelial cell (EC) migration and permeability play a significant role in the focal development of atherosclerosis. Several key molecular mechanisms regulate both EC migration and permeability in response to shear stress. We developed novel tools to investigate the molecular mechanisms of both real-time EC migration as well as barrier function in the presence of hemodynamic shear stress.

Hemodynamic regulation of directional EC migration implies an essential role of shear stress in governing EC polarity. We have characterized the global patterns of EC migration in confluent monolayers as a function of shear stress direction and exogenous pleiotropic factors. Results demonstrate that the presence of mitogenic factors significantly affects the flow-induced dynamics of movement by prolonging the onset of monolayer quiescence up to 4 days. Shear stress induced morphology, however, is independent of exogenous growth factors. In conjunction with increased motility, exogenous growth factors contributed to the directed migration of ECs in the flow direction. ECs exposed to arterial flow in serum/growth factor-free media and then supplemented with growth factors rapidly increased directional migration to 85% of cells migrating in the direction of flow and increased the distance traveled with the flow direction. This response was modulated by the directionality of flow and inhibited by the expression of dominant-negative Par6, a major downstream effector of Cdc42-induced polarity.

The investigation of endothelial permeability in the context of hemodynamic shear stress is necessary to elucidate the molecular events that are early precursors of atherosclerosis. We developed a transwell model of permeability that incorporates the microscope mounted flow device to apply physiological shear stress. Using this tool, we demonstrated that shear stress induced permeability is dependent on PAK activation and localization to the cell junctions. Further, PAK activation and flow induced permeability are enhanced by atherogenic extracellular matrices, specifically fibronectin. A second model of endothelial barrier function was integrated with the microscope flow device to provide real-time barrier integrity measurements with applied hemodynamic flow. Results demonstrate that atheroprone flow induces barrier disruption in comparison to atheroprotective flow profiles and previously published laminar flow models of similar time-average shear stress. When challenged by either histamine or thrombin, ECs in the presence of both atheroprone and atheroprotective flow exhibited barrier enhancement when compared to static controls demonstrating the protective effects of shear stress, regardless of level. Additionally, we established that PAK activation and association with PPIX are necessary for shear stress induced barrier disruption.

The adherens junction regulates shear stress induced EC permeability by maintaining the junctional integrity of the monolayer as well as signaling downstream effectors of cell contractility. We demonstrated that shear stress induced permeability is dependent on expression of a major adherens junction protein, β-catenin. ECs deficient in β-catenin are unable to activate Erkl/2 in response to flow, a major mediator of cell contractility that is downstream of PAK. Shear stress induced PAK activation, however, was P-catenin independent. Therefore, we hypothesized that P-catenin was regulating a signaling complex that is an intermediary between PAK activation and Erkl/2 signaling. We demonstrated that knock down of P-catenin resulted in decreased activity in the PAK-βPIX-GIT1-MEK signaling complex that activates Erkl/2 and cell contractility. Further investigation found that P-catenin modulates EC barrier function in response to the vasoactive agents, histamine and thrombin as well. In static conditions, a knock down of P-catenin reduced the barrier disruption stimulated by both thrombin and histamine. Shear stress induced barrier response was further reduced by a deficiency in P-catenin. These data further suggest that β-catenin regulates the signaling complex PAK-βPIX-GIT1-MEK and downstream cell contractility.

As a whole, the endothelial phenotype, as characterized by EC migration, polarity, and permeability, was a critical modulator of atherogenesis and required the incorporation of physiological shear stress to discover relevant molecular mechanisms.