| Objective To investigate the effects of fluid shear stress on IL-8 protein production of human umbilical vein endothelial cells (HUVECs) and the roles of time course of low shear stress and intensity of fluid shear stress using quantitative sandwich enzyme-linked immunosorbent assay (ELISA); To identify the effect of fluid shear stress on the adhesion of neutrophil-endothelial cells and the roles of intensity of fluid shear stress and its mechanism.Methods (1) HUVECs were exposed to low shear stress (2.09,4.61,6.19dyne/cm2) for 1h﹑2h﹑3h﹑4h﹑5h﹑6h﹑8h﹑10h﹑12h, respectively. Normal static cultured HUVECs were selected as a control. Quantitative sandwich enzyme-linked immunosorbent assay (ELISA) was employed to assay the production of IL-8 protein. (2) HUVECs were exposed to different shear stress (2.09,4.61,6.19,8.51,10.50,12.59,14.41,17.22,18.32 dyne/cm2) for 5h or 6h. Normal static cultured HUVECs were selected as a control. Quantitative sandwich ELISA was employed to assay the production of IL-8 protein. (3) HUVECs were exposed to different shear stress(2.09,8.51,12.59,18.32dyne/cm2) for 5h or 6h. Human neutrophils were incubated with the flow media. The adhesion rates of neutrophil-endothelial cells were observed and measured. (4) HUVECs were exposed to different shear stress(2.09,8.51,12.59,18.32dyne/cm2) for 5h or 6h. Human neutrophils were incubated with the flow media. The expression of CD11a/CD18(LFA-1) and CD11b/CD18(Mac-1) were assayed. (5) Human neutrophils were blocked with anti-LFA-1 or anti-Mac-1, or both anti-LFA-1 and anti-Mac-1, then its adhesion with endothelial cells treated with shear stress were observed and measured. We also incubated neutrophil with Ca2+ chealator and measured its adhesion with endothelial cells. Results (1) HUVECs untreated with fluid shear stress secreted very little IL-8 in culture media. IL-8 secretion increased when HUVECs subjected to fluid shear stress for 1h, and it reached the summit when HUVECs exposed to fluid shear stress for 5h. Then IL-8 secretion decreased at 8h of stimulation by shear stress and remained at a constant level throughout the time course of the study( 12h ). The increase of IL-8 secretion by shear stress was time-dependent. The biphasic response of IL-8 protein production was found in experiments in which the applied shear stress was 2.09dyne/cm2, 4.61 dyne/cm2, and 6.19dyne/cm2.(2) HUVECs untreated with fluid shear stress secreted very little IL-8 in culture media. After HUVECs subjected to low fluid shear stress (2.09 dyne/cm2) for 5h or 6h, IL-8 protein production increased near 6 or 7 times as that of HUVECs subjected to high fluid shear stress (18.32 dyne/cm2). The linear regression equations between IL-8 protein production (y) and shear stress (dyne/cm2 , x ) are y= 760.12─36.06x, γ=—0.978( for 5 h) ; y= 781.87 ─36.66x, γ=—0.980( for 6 h). (3) The adhesion rate of neutrophil-endothelial cells which was not treated with fluid shear stress is very low. After 5h or 6h of exposure to low shear stress ( 2.09 dyne/cm2) , the adhesion rate is very high. The neutrophil-endothelial cells adhesion rate was force intensity-dependent. After HUVECs subjected to low shear stress ( 2.09 dyne/cm2) for 5h or 6h, the neutrophil-endothelial cells adhesion rate increased near 2 times as that of HUVECs subjected to high shear stress ( 18.32 dyne/cm2). (4) The expression of LFA-1 and Mac-1 increased when neutrophil incubated with flow media of 2.09 dyne/cm2. The expression decreased while the shear stress increased. The expression was force intensity-dependent. There is negative relationship between expression of LFA-1 and Mac-1 and intensity of fluid shear stress. After neutrophil was incubated with anti-LFA-1 or anti-Mac-1 antibody alone, the neutrophil-endothelial cells adhesion rate did not change significantly compared with control group. After incubated with both anti-LFA-1 and anti-Mac-1 antibody, the neutrophil-endothelial cells adhesion rate decreased significantly. After incubated with Ca2+ chealator, t... |
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