Shear stress-induced morphological adaptations of endothelial cells, reorganization of cell adhesion complexes and the actin cytoskeleton

grantor: University of TorontoEndothelial cells exhibit profound morphological responses to shear stress derived from blood flow. Adaptations in cell shape and orientation necessitate reorganization of cell-substrate adhesions, cell-cell adhesions and the cytoskeleton; however, reorganization of these structures, in response to chronic exposure to shear stress, has not been explicitly examined. Therefore, the objective of this thesis was to understand the mechanisms underlying endothelial cell shape change in response to chronic exposure to shear stress. In particular, studies focused on the adaptive responses of cell adhesion complexes and associated cytoskeleton. Cultured porcine aortic endothelial cells were exposed to laminar shear stress (15 dynes/cm2) for 8, 16, 24, 48 or 96 hours, using a parallel-plate flow chamber. Shear-induced effects on lateral adherens junctions, basal focal adhesions and the actin cytoskeleton were assessed via indirect immunofluorescence, confocal microscopy and biochemical techniques. Results demonstrated that shear-induced endothelial cell shape was associated with changes in the morphology, distribution and protein composition of endothelial adherens junctions and focal adhesions. Initial stages of cell shape change were characterized by partial disassembly of linear adherens junctions and formation of punctate 'arrowhead' focal adhesions. As shape change proceeded and cells and stress fibers aligned with flow, adherens junctions reorganized into punctate adherens plaques that localized to the ends of stress fibers inserting into the lateral plasma membrane. Further, focal adhesion complexes reorganized into fine, linear structures associated with the ends of basally inserting stress fibers. Finally, actin assembly occurred preferentially at both ends of stress fibers in a polarized manner. Prolonged exposure to shear stress (96 hours) caused punctate adherens plaques to reassemble back into linear adherens junctions. Additionally, focal adhesions were heterogeneous with respect to morphology, distribution and protein composition. This heterogeneity revealed focal adhesion organization, and its association with motility, that were specific to shear stressed endothelium. Finally, actin assembly remained polarized, but occurred preferentially at one end of stress fibers. In summary, shear-induced cell shape change is associated with dramatic reorganization of endothelial adherens junctions, focal adhesions and the cytoskeleton. Adaptation to shear stress is associated with redistribution of these complexes that characterize achievement of a new steady-state.Ph.D