MECHANICAL REMODELING OF THE ENDOTHELIAL SURFACE AND ACTIN CYTOSKELETON INDUCED BY FLUID-FLOW

Objective: Tile mechanism by which cultured endothelial cells respond to shear stress is controversial. The cell surface and cytoskeleton ar e in involved, but their roles are undefined. In this study, previousl y unknown changes in the surface detail and actin cytoskeleton of bovi ne aortic endothelial cells were identified. Methods: Actin filament c ontent and filament number in resting and flow-oriented cells were det ermined by biochemical assays. The three-dimensional organization of t he actin cytoskeleton in cells was defined in the confocal microscope and in tile electron microscope after rapid-freezing, freeze-drying, a nd metal coating of detergent-permeabilized cells. Results: Endothelia l cells have smooth apical membranes in situ. However, cultured cells exhibit surface microvilli which increase tile apical surface area, ex posing the ruffled surface to forces from fluid flow and potentially e nhancing cell interactions with blood-borne white cells. Stereoscopic micrographs show that stress fibers are integrated into a complex dist ributed cytoplasmic structural actin network (DCSA). This lattice is f ormed by actin filaments that frequently cross and connect to each oth er, stress fibers, and microfilaments and microtubules. The cytoskelet ons of cells cultured in static media lack apparent order when compare d to cytoskeletons from cells which have been exposed to 24 hours of l aminar flow. Conclusions: The DCSA physically connects tile apical and basal cell membranes and fills the volume between nucleus and membran e, providing a mechanism for transmitting mechanical forces across cel ls and a signaling pathway ay from membrane to nucleus. Stress fibers increase tile mechanical modulus of die DCSA, although this increase i s probably-unnecessary to withstand the increase in shear stress cause d by blood flow in vivo. This implies that actin rearrangements are no t required for mechanical integrity, but serve an alternate function.