Abstract-In vivo, endothelial cells (ECs) are subjected to a complex mechanical environment composed of shear stress, pressure, and circumferential stretch. The aim of this study was to subject bovine aortic ECs to a pulsatile pressure oscillating from 70 to 130 mm Hg (mean of 100 mm Hg) in combination with pulsatile shear stresses from 0.1 to 6 dyne/cm 2 (1 dyne/cm 2 ϭ0.1 N/m 2 ) with or without a cyclic circumferential stretch of 4% for 1, 4, and 24 hours. The effect of highly reversing oscillatory shear stress (range Ϫ3 to ϩ3 dyne/cm 2 , mean of 0.3 dyne/cm 2 ) typical of regions prone to the development of atherosclerotic plaques was also studied at 4 and 24 hours. Endothelin-1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) mRNA expression was time and mechanical force dependent. ET-1 mRNA was maximal at 4 hours and decreased to less than static culture expression at 24 hours, whereas ecNOS mRNA increased over time. Pressure combined with low shear stress upregulated ET-1 and ecNOS mRNA compared with static control. Additional increase in expression for both genes was observed under a combination of higher shear stress and pressure. A cyclic circumferential stretch of 4% did not induce a further increase in ET-1 and ecNOS mRNA at either low or high shear stress. Oscillatory shear stress with pressure induced a higher expression of ET-1 mRNA but lower expression of ecNOS mRNA compared with unidirectional shear stress and pressure. We have shown that the combination of pressure and oscillatory shear stress can downregulate ecNOS levels, as well as upregulate transient expression of ET-1, compared with unidirectional shear stress. These results provide a new insight into the exact role of mechanical forces in endothelial dysfunction in regions prone to the development of atherosclerosis. (Arterioscler Thromb Vasc Biol. 1998;18:686-692.) Key Words: mechanical stress Ⅲ hemodynamics Ⅲ vascular endothelium Ⅲ nitric oxide synthase Ⅲ endothelin T he fluid mechanics of blood are known to transiently regulate vascular tone. In addition to short-term action, fluid mechanics have also been shown to regulate vascular remodeling in the case of long-term changes in pressure (hypertension) and flow rate (pregnancy, arteriovenous shunts, and exercise).1 More importantly, it has been demonstrated that the localization of the atherosclerotic plaque is correlated with regions characterized by oscillatory shear stress environment. 2 This regulation has been demonstrated in part to occur via two molecules secreted by the ECs and which have been identified as ET-1 and NO.ET-1 is a 21-amino acid peptide acting as a powerful vasoconstrictor and an SMC mitogen.3 ET-1 is thought to play an active role in SMC proliferation during remodeling of arteries. ET-1 mRNA expression has been shown to be transiently stimulated at 1 to 4 hours in cultured porcine aortic ECs exposed to a shear stress of 5 dyne/cm 2 (1 dyne/cm 2 ϭ0.1 N/m 2 ) and 30 minutes in BAECs exposed to 15 dyne/cm 2 . 4,5 Longer time exposure to 15 dyne/cm 2...

Abstract-Nitric oxide (NO) has been demonstrated to play a central role in vascular biology and pathobiology. The expression of endothelial NO synthase (eNOS) is regulated in part by blood flow-induced mechanical factors. The purpose of this study was to evaluate how the expression of eNOS mRNA correlates with the activation of its promoter in both arterial and venous endothelial cells (ECs) exposed to mechanical forces, ie, shear stress and cyclic circumferential stretch. Bovine aortic ECs (BAECs) and EA hy.926, a cell line derived from human umbilical vein ECs, were grown on the inside of elastic tubes and subjected to combinations of pressure, pulsatile shear stress, and cyclic circumferential stretch for 24 hours. Two patterns of shear stress were used: unidirectional (mean of 6, ranging from 3 to 9 dyne/cm 2 ) and oscillatory (mean of 0.3, ranging from Ϫ3 to ϩ3 dyne/cm 2 ). The expression of eNOS mRNA was quantified by Northern blot analysis. Activation of the promoter was assessed by luciferase activity after the cells were transiently transfected before the flow experiments with a plasmid construct containing the fully functional eNOS promoter coupled to a luciferase reporter gene. Expression of eNOS mRNA was increased and promoter activity was enhanced by unidirectional shear stress compared with static control. Oscillatory shear slightly upregulated eNOS mRNA in BAECs, whereas it downregulated eNOS mRNA in EA hy.926. In both BAECs and EA hy.926, there was a good correlation between the increase in eNOS mRNA expression and promoter activation by unidirectional shear stress. In contrast, in both BAECs and EA hy.926 cells exposed to shear stress, cyclic stretch did not change eNOS mRNA expression, but the activation of eNOS promoter was significantly lower. Moreover, when ECs were exposed to oscillatory shear stress, there was a dramatic activation of the eNOS promoter. These results demonstrate that unidirectional shear stress increases eNOS mRNA expression via a transcriptional mechanism. However, oscillatory shear stress and cyclic stretch appear to control eNOS expression through posttranscriptional regulatory events. NO has been demonstrated to interfere with key events involved in atherogenesis.1 Not only is NO the most potent vasodilator, it also inhibits platelet aggregation and leukocyte adhesion to ECs and suppresses vascular smooth muscle cell proliferation and migration. The production of NO, as well as the expression of eNOS by ECs, has been shown to be dependent on mechanical factors.2-4 Shear stress and cyclic circumferential stretch have been demonstrated to increase NO release as well as upregulate eNOS mRNA and protein. 2,3More recently, using an in vitro tube model in which pressure, shear stress, and cyclic circumferential stretch can be combined, we have shown that shear stress represents the major mechanical factor inducing an increase in eNOS expression in BAECs. Pressure and cyclic stretch, however, did not significantly alter changes in eNOS expression in cells exposed to shear str...

The development of atherosclerosis is thought to be initiated by a dysfunctional state of the vascular endothelium. The proposal that mechanical forces play a role in the localization of this disease has led researchers to develop in vitro models to assess their effects on cultured endothelial cells. The arterial endothelium is exposed simultaneously to circumferential hoop stretch and wall shear stress, yet previous investigations have focused on the isolated effects of either cyclic stretch or shear stress. The influence of physiological levels of combined shear stress and hoop stretch on the morphology and F-actin organization of bovine aortic endothelial cells was investigated. Cells subjected for 24 hours to shear stresses higher than 2 dyne/cm2 or to hoop stretch greater than 2% elongated significantly compared with unstressed controls and oriented along the direction of flow and perpendicular to the direction of stretch. Exposure to more than 4% stretch significantly enhanced the responses to shear stress. Both shear stress and hoop stretch induced formation of stress fibers that were aligned with the cells' long axes. Simultaneous exposure to both stimuli appeared to enhance stress fiber size and alignment. These results indicate that shear stress and hoop stretch synergistically induce morphological changes in endothelial cells, which suggests that circumferential strain might modulate sensitivity of endothelial cells towards shear stress.

Flow and the associated shear stress have been shown to play an active role in the regulation of the structure and function of endothelial cells (EC) in vitro. Although cultured EC subjected to flow exhibit an elongated morphology and a decreased cell growth rate rather like those observed in vivo, there are differences in morphology and growth rate, as well as other characteristics, between in vitro and in vivo EC. This suggests that flow is only one of the many factors affecting EC differentiation in vivo. In this study, a co-culture model system was designed, which includes smooth muscle cells (SMC), a matrix of collagen type I, and a confluent monolayer of EC, and this simplified model of the arterial wall was subjected to a steady, laminar shear stress of 10 and 30 dyn/cm2. Under non-flow conditions, EC exhibited an elongated shape, but with a random orientation. In response to flow, there was an alignment with the direction of flow. This alignment occurred more rapidly at 30 dyn/cm2 than at 10 dyn/cm2. The collagen matrix was found to be primordial in the maintenance of a quiescent endothelium, even in the absence of SMC and flow, suggesting the importance of an organized extracellular matrix (ECM) in the differentiation of cells in vivo.

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Important to the tissue engineering of a substitute blood vessel is an understanding of those factors which regulate vascular biology. A major factor in this regulation is the mechanical environment imposed by the hemodynamics of the vascular system. In this the vascular endothelium plays a critical role, and over the past two decades much has been learned about the influence of hemodynamics on vascular endothelial biology, to a large degree using cell culture to study the effects of flow and cyclic stretch. In our laboratory, such studies are now being extended through the development of a model of the arterial wall involving the co-culture of endothelial cells and smooth muscle cells. The development of such a model and its use in the study of hemodynamic effects represents necessary steps in the evolution of approaches to tissue engineering a blood vessel.

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In this study we investigated the effect of flow and the associated shear stress on the process of endothelial cell division. A shear stress of 7 N/m 2 (70 dyne/cm 2 ) was applied to a monolayer of bovine aortic endothelial cells (BAECs) by using a parallel-plate flow-chamber system. Dividing cells under flow conditions were qualitatively compared with those under static-culture conditions. In addition, the duration of some phases of the cell cycle (ie, mitosis and cytokinesis) was measured for both static and flow conditions. Dividing BAECs in static cultures bowed upward; however, when cells were preconditioned by at least 6 hours of exposure to flow, they stayed relatively attached to the substrate during the entire V ascular endothelial cells (ECs) line the inside of blood vessels and the chambers of the heart. The endothelium, a selective barrier between blood components and the vessel wall, interacts actively with both of them.1 ECs also secrete vasoactive substances (eg, histamine, prostacyclin, and endotheliumderived relaxing factor) and extracellular matrix proteins (eg, fibronectin and glycosaminoglycans).The role that the endothelium plays in the genesis of atherosclerosis has also been recognized.2 Initiating events include intimal smooth muscle cell (SMC) migration and proliferation; synthesis and deposition by SMCs of connective tissue matrix proteins, including collagen, elastin fibers, and proteoglycans; and accumulation of lipids within proliferating SMCs and macrophages, which become foam cells, as well as newly formed connective tissue matrix. In the early stages of atherosclerotic disease, the endothelium is intact but is believed to be a mediator of effects associated with blood flowing through a vessel. This includes hemodynamic effects. It has been shown that hemodynamic features and lesion location are correlated, with the location of atheromas usually at sites where shear stress is low and oscillates with a high residence time.3 Several studies have shown that endothelial morphology is an indicator of the pattern of blood flow. 46In vivo permeability experiments with Evans blue dye have shown that ECs are more permeable to macromolecules at sites that are prone to the formation of atherosclerotic lesions.710 These so-called "blue sites" appear to be correlated with an increased rate of EC proliferation; ie, the "leaky" cells appear to be ECs that are undergoing cell division.11 ' 12Received August 10, 1993; revision accepted January 25, 1994. From the Biomechanics Laboratory and School of Mechanical Engineering, Georgia Institute of Technology, Atlanta.Correspondence to Robert M. Nerem, Biomechanics Laboratory and School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0405. process of cell division. Cell area significantly decreased during division for both static-culture and flow conditions. Finally, although significant changes in the duration of phases were observed in single experiments during which cells were exposed to shear stress, any differ...