Moderate Exercise‐Induced Endothelial Shear Stress Increases eNOS Expression in vitro

Abstract

Background Sedentary lifestyle is a risk factor in the development of coronary artery disease. Moreover, endothelial cell dysfunction has been shown to be the first step in the pathogenesis of coronary artery disease. Endothelial shear stress (ESS) involves mechanoreceptors that sense different forces produced through the alterations in blood flow, triggering a biochemical response leading to cellular adaptations. Exercise induced ESS might act as a protective mechanism in preventing endothelial cell dysfunction. Although exercise has been shown to improve endothelial cell function, the role of varying exercise intensities on endothelial cell homeostasis has not been explored. The purpose of this study was to determine the effects of pulsatile exercise induced-ESS varying in exercise intensity on endothelial cell function determined through oxidative stress and pro-vasodilatory protein expression. Hypothesis It was hypothesized that exercise induced ESS would increase nitric oxide synthase (eNOS) expression and reduce oxidative stress. Methods Commercially available human umbilical vein endothelial cells (HUVEC; Sigma-Aldrich, St Louis, MO) were cultured until 90-100% confluence. Exercise-induced ESS in vivo was obtained from our previous reports. Cultured cells were exposed to pulsatile resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour at resting ESS, low-intensity exercise-induced ESS (35 dynes/cm2), or moderate-intensity exercise-induced ESS (50 dynes/cm2) on parallel fluidic units (Ibidi USA, Fitchburg, WI.). After ESS, cells were fixed and immunocytochemistry was used to assess endothelial cell function by measuring the expression of eNOS and nitrotyrosine, a marker of endothelial oxidative stress. A Friedman test was used to assess differences in protein expression using the SPSS 25.0 (IBM, Armonk, NY). Significance was considered at p<0.05. Results There was a higher expression of eNOS in cells exposed to moderate ESS (4.39 ± 2.78 AU) compared to cells exposed to low ESS (3.66 ± 0.72 AU) and resting ESS (1.52 ± 0.41 AU). The analysis of nitrotyrosine content resulted in a non-statistically significant difference between ESS conditions (p=1.00). Conclusion Our results suggest that exposure to moderate intensity exercise induced ESS increases the expression of eNOS content in vitro. Higher intensities of exercise have been shown to result in a greater amount reactive oxygenated species (ROS) produced; however, exercise has also been shown to combat these effects through the increase of antioxidant production with the capability of scavenging free radicals. It is possible that the fixing of the cells immediately after ESS exposure did not allow the time needed for antioxidants to scavenge the ROS produced leading to no changes in oxidative stress. Further studies need to be performed investigating the effects of exercise induced ESS on endothelial function.