Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals.

Abstract-Cultured vascular endothelium displays profound morphological adaptations to shear stress that include planar cell polarity (PCP) that is directed downstream. Endothelial cells in blood vessels are also polarized; however, the direction of polarity is vessel specific, and shear-independent mechanisms have been inferred. The regulation of endothelial PCP is therefore controversial. We report that the direction of PCP in blood vessels is age and vessel specific; nonetheless, it is caused by shear-related regulation of glycogen synthase kinase-3␤ (GSK-3␤), a profound regulator of endothelial microtubule stability. When GSK-3␤ is inhibited, PCP reverses direction. Endothelium is the only cell type studied to date that can reverse direction of polarity. Tight regulation of GSK-3␤, microtubule dynamics, and cell polarity was also required for the striking morphological responses of endothelium to shear stress (cell elongation and orientation with shear). Finally, the cytoskeletal polarity displayed in blood vessels is associated with polarized (shear-directed) cell mitoses that have important effects on endothelial repair. Vascular endothelium therefore displays a novel mode of mechanosensitive PCP that represents the first example of a single cell type that can reverse direction of polarity. Key Words: shear stress Ⅲ endothelium Ⅲ microtubules Ⅲ planar cell polarity V ascular endothelial cells display profound morphological adaptations to shear stress that include cell elongation in the shear direction that is driven by a novel reorganization of actin in stress fibers, 1 redistribution of focal adhesion complexes, 1,2 and partial disassembly and reassembly of cell-cell junctional complexes. 3 Imposition of shear stress on cultured endothelium also induces planar cell polarity (PCP) in the downstream direction. 4 PCP occurs when cell organelles, cytoskeleton, and/or adhesion complexes exhibit unidirectional organization along an axis that lies in the plane of a cell monolayer. This process was manifest in sheared endothelium in vitro as redistribution of microtubules and the microtubule-organizing center (MTOC) (from which microtubules emanate) to the downstream side of the cell nuclei, a hallmark of PCP. 5 PCP has been of great interest to cell biologists because it provides directionality to cell functions. PCP in vertebrate cells has been studied primarily during directional migration at wound margins where it is initiated by integrin activation and multiple downstream pathways that involve the Rho GTPase, Cdc42, adenomatous polyposis coli (APC), glycogen synthase kinase-3␤ (GSK-3␤), and mammalian Diaphanous-1 (mDia1). 6,7 Polarization of the microtubule system is important because motor proteins (kinesins and dyneins) can deliver cargoes (eg, cell membrane and protein) along microtubules to and from the cell anterior and posterior and because microtubules regulate the reorganization of cell adhesion complexes. 8 This process is enhanced by microtubule stabilization through posttranslational modificati...