||Nicotine has been widely demonstrated as one of major risk factors for cardiovascular lesions such as atherosclerosis. However, the definite mechanism of how nicotine causes endothelial dysfunction remains unclear due to lack of hemodynamic factor in most of prior in vitro studies. In this study, we aimed to investigate the effectiveness of hemodynamic stress on nicotine-mediated human endothelial inflammation using human umbilical vein endothelial cells (HUVECs) and parallel-plate flow system to simulated blood flow-induced laminar shear stress (LSS). HUVECs were first sheared by 12 dynes/cm2 using medium with and without 10-4 M nicotine for 0 (static culture), 6, 12, and 24 h, respectively, and followed by conductions of monocyte adhesion assay, detection of reactive oxygen species (ROS) generated, and analysis of inflammation-related molecular expression. Our results showed that the monocyte adhesion rate (MDR), which was represented as the level of cell inflammation, was not affected by LSS within 24 h in the absence of nicotine. As compared to the shearing cultures without nicotine, the MDR of the group treated by both nicotine and LSS remarkably increased about 1.8- and 2.6-fold (P < 0.05 for each) while the exposure time was set at 12 and 24 h, respectively. Moreover, the inflammation level of HUVECs treated with both LSS and nicotine for 24 h significantly increased about 1.4 fold (P < 0.05) as compared to the group with nicotine alone. These results were consistent with the data obtained from the ROS measurement that the level of ROS generated from the cells with both LSS and nicotine exhibited 2.55- and 4.22-fold (P < 0.05 for each) higher than the groups with nicotine or LSS alone, respectively. To investigate the mechanism of enhanced endothelial inflammation caused by synergistic effect of LSS and nicotine, expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were examined. We found that expressions of both ICAM-1 and VCAM-1 increased 1.3 and 1.9-fold while cells were exposed to 10-4 M nicotine for 24 h, whereas LSS enabled to further increased the ICAM-1 but decreased the VCAM-1 expression of nicotine-treated HUVECs after sheared with 12 dynes/cm2 for 24 h, This result suggested that LSS plays different roles in modulating ICAM-1 and VCAM-1 expressions in endothelial cells in the presence of nicotine. In summary, we have demonstrated that the level of endothelial inflammation can be significantly increased by synergistic effect of nicotine and LSS, and exhibited that high level of LSS (≥ 12 dynes/cm2) plays a detrimental role in nicotine-mediated endothelial injury.|
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