Nitric Oxide Synthase Expression in Endothelial Cells Exposed to Mechanical Forces

Abstract

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...

Mechanisms of H2O2-induced oxidative stress in endothelial cells

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“…Because these mechanical forces play key roles in regulating wall tone, atherogenesis, angiogenesis, and other features of vascular homeostasis, understanding their impact is important. Using perfusion systems that expose cultured endothelial cells to controlled levels of phasic flow with or without distension, studies have revealed effects of pulsatility on NO synthase gene expression, [25][26][27] endothelin, and other signaling cascades 28 that tend to favor a vasorelaxant response. Recent studies used more physiological waveforms ( Figure 4A) and assessed post-translational changes in signaling proteins.…”

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