Abstract 1377: Long Term Cyclic Strain Down-Regulates the Expression of the Catalytic NADPH Oxidase Subunit Nox4 in Human Endothelial Cells
Endothelial cells in vivo are constantly exposed to mechanical stimuli such as shear stress and cyclic strain. In endothelial cells, a Nox4-containg-NAD(P)H oxidase complexes have been identified as major sources of superoxide anion (•O2-) formation. We tested the hypothesis whether long-term cyclic strain regulates endothelial oxidative stress. In this study, we analysed the effect of cyclic strain on •O2- formation by ESR technique and DHE fluorescence, on NO formation by Griess reaction and on NAD(P)H oxidase subunit expression by RT-PCR and Western blot in human endothelial cells. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to 5% and 12% cyclic strain for up to 24 h using the flexcell system. Long-term application of 12% cyclic strain results in down regulation of•O2- formation measured by DHE fluorescence (con: 49.701±2.032 RLU/mg protein, 12%: 35.294±2.008 RLU/mg protein, n=3; *P<0,05) and ESR technique (con: 17,9±1,5 μM CP•/mg protein, 12%: 13,2±1,7 μM CP•/mg protein, n=6, *P<0,05). In addition Nox4 expression was reduced on mRNA (Nox4: 36±5%; n=5, *P<0,05) and protein (15±4%, n=3, *P<0,05) level. Down-regulation of Nox4 was further confirmed by promoter analysis using dual luciferase assay. In parallel, endothelial NO formation (con: 0,31±0,02μM NO; 12%: 1,88±0,23 μM NO, n=6, *P<0,05) and eNOS (296±64%, n=6, *P<0,05) were increased, but reduction of oxidative stress was not changed by eNOS inhibition with L-NAME. In contrast, Cu/Zn SOD, MnSOD and catalase expression was decreased after application of chronic cyclic strain. Short-term application of cyclic strain had no effect on gene expression. The start of the transcription of Nox4 was calculated using race assay. Analysis of the basal Nox4 promoter activity shows a minimum of 355 bp upstream of the transcription start. In conclusion, we demonstrate a reduction of oxidative stress by chronic application of cyclic strain to endothelial cells. This novel mechanism might contribute to a positive balance between NO and superoxide anions, resulting in a vasoprotective potential of physiological cyclic strain.