We have developed an in-vitro system for studying the dynamic response of vascular endothelial cells to controlled levels of fluid shear stress. Cultured monolayers of bovine aortic endothelial cells are placed in a cone-plate apparatus that produces a uniform fluid shear stress on replicate samples. Subconfluent endothelial cultures continuously exposed to 1-5 dynes/cm2 shear proliferate at a rate comparable to that of static cultures and reach the same saturation density (congruent to 1.0-1.5 X 10(5) cells/cm2). When exposed to a laminar shear stress of 5-10 dynes/cm2, confluent monolayers undergo a time-dependent change in cell shape from polygonal to ellipsoidal and become uniformly oriented with flow. Regeneration of linear "wounds" in confluent monolayer appears to be influenced by the direction of the applied force. Preliminary studies indicate that certain endothelial cell functions, including fluid endocytosis, cytoskeletal assembly and nonthrombogenic surface properties, also are sensitive to shear stress. These observations suggest that fluid mechanical forces can directly influence endothelial cell structure and function. Modulation of endothelial behavior by fluid shear stresses may be relevant to normal vessel wall physiology, as well as the pathogenesis of vascular diseases, such as atherosclerosis.

The effects of hemodynamic forces upon vascular endothelial cell turnover were studied by exposing contact-inhibited confluent cell monolayers to shear stresses of varying amplitude in either laminar or turbulent flow. Laminar shear stresses (range, 8-15 dynes/cm2; 24 hr) induced cell alignment in the direction of flow without initiating the cell cycle. In contrast, turbulent shear stresses as low as 1.5 dynes/cm2 for as short a period as 3 hr stimulated substantial endothelial DNA synthesis in the absence of cell alignment, discernible cell retraction, or cell loss. The results of these in vitro experiments suggest that in atherosclerotic lesion-prone regions of the vascular system, unsteady blood flow characteristics, rather than the magnitude ofwall shear stressperse, may be the major determinant of hemodynamically induced endothelial cell turnover.Hemodynamic forces have been implicated in the initiation, localization, and development of atherosclerotic vascular disease (1, 2). Little is known, however, about the effects of such forces upon the endothelial cell lining of blood vessels, the integrity of which is essential for normal vascular function. In certain areas ofthe aorta and its main branches, blood flow characteristics are both variable and complex. In locations such as the descending thoracic aorta and distal carotid arteries, pulsatile laminar flow is prevalent (3), whereas in other regions, such as coronary arteries and the carotid bifurcation, secondary flows, vortices, and intermittently changing flow directions are encountered (4). The distribution of atherosclerotic lesions in susceptible species, including humans, is closely correlated with the location of disturbed flow in the major vessels (5). Time-dependent flow separation and unsteady secondary flow typically occur in localized regions that are usually well defined and of limited size. Furthermore, turbulence will occur in the largest arteries under conditions of increased flow velocity and cardiac output (4). Thus, shear stresses, which are the direct tractive forces acting on the endothelial cell surface as a result of blood flow, are highly variable in magnitude, frequency, and direction in such regions.Autoradiographic studies in vivo have demonstrated increased endothelial DNA synthesis in localized areas of the aorta and its major branches, suggesting that locally increased endothelial cell turnover, perhaps as a result of injury, may occur near branches and bifurcations (6, 7). Increased cell turnover need not imply denudation of the endothelium and indeed during the initiation and early development of atherosclerotic lesions the endothelium remains a confluent monolayer of cells (8).The role of fluid shear stress in promoting endothelial cell injury and/or turnover is uncertain: both high and low shear stresses have been implicated. High shear stress has been linked to alignment of endothelial cells (9), cell loss (10), increased arterial permeability (11), and enhanced endothelial biosynthetic capabilities (12). Ath...

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In vitro investigations of the responses of vascular endothelium to fluid shear stress have typically been conducted under conditions where the time-mean shear stress is uniform. In contrast, the in vitro experiments reported here have re-created the large gradients in surface fluid shear stress found near arterial branches in vivo; specifically, we have produced a disturbed-flow region that includes both flow separation and reattachment. Near reattachment regions, shear stress is small but its gradient is large. Cells migrate away from this region, predominantly in the downstream direction. Those that remain divide at a rate that is high compared with that of cells subjected to uniform shear. We speculate that large shear stress gradients can induce morphological and functional changes in the endothelium in regions of disturbed flow in vivo and thus may contribute to the formation of atherosclerotic lesions. ( The focal occurrence of atherosclerosis in such regions of disturbed flow 2 ' 7 -8 provides indirect evidence that fluid shear stress gradients may play a role in arterial wall pathology. Our objective was to explore in vitro the specific cellular responses that may be caused by the large shear stress gradients that occur in regions of flow separation and reattachment that are characteristic of arterial bifurcations in vivo. MethodsBovine aortic endothelial cells were grown to confluent densities on 12-mm glass coverslips and exposed to steady fluid forces in a cone-and-plate flow chamber 21 for times up to 48 hours. A separated flow region was created by placing a rectangular strip that was 23.8 mm Because of the small dimensions of the model, exact numerical solutions of the complete Navier-Stokes equations for Reynolds' numbers (Re) between 3 and 100 were obtained using the computational program NEKTON. 22 The cell response discussed here was obtained at an Re of 4 and 7.5 (see figure captions).Endothelial cells committed to the cell cycle (division) were identified by in situ monoclonal antibody detection of bromodeoxyuridine (BrdU) incorporation into cellular DNA during DNA synthesis. Immediately after exposure to flow, the coverslips were incubated in BrdU-labeled medium for 20 hours. The antigen was localized by using the avidin-biotin immunoperoxidase staining method. 23 The originally reported method was significantly modified to improve contrast.