Exercise‐Induced Shear Stress Upregulates eNOS Protein in vitro

Abstract

BackgroundEndothelial cell dysfunction has been shown to be the first step in the pathogenesis of cardiovascular disease. A risk factor in the development of cardiovascular disease is physical inactivity. Endothelial shear stress (ESS) involves mechanoreceptors on the surface of endothelial cells that are capable of sensing different forces, triggering a biochemical response leading to cellular adaptations. Exercise‐induced ESS might act as a protective mechanism in preventing endothelial cell dysfunction. Endothelial nitric oxide synthase (eNOS), a key enzyme involved maintaining vascular homeostasis has been shown to be upregulated in response to higher shear stress levels. 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 in vitro exercise‐induced ESS during 3 different exercise intensities on eNOS expression.MethodsCommercially 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 2 hours, followed by 1 hour at resting ESS, low‐intensity exercise‐induced ESS (35 dynes/cm2), moderate‐intensity exercise‐induced ESS (50 dynes/cm2), or high‐intensity exercise‐induced ESS (75 dynes/cm2) on a closed circuit pump and channeled slide (Ibidi pump system (PumpControl Software 1.5.4) and slides, Ibidi Inc., Fitchburg, WI). A set of cells were exposed to physiologically low shear stress levels (10 dynes/cm2) for 3 hours as a control. Following ESS conditions, total protein was extracted, and western immunoblotting was performed to measure eNOS protein expression with GAPDH used as the normalizing protein. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.ResultsLow and moderate intensity ESS increased eNOS protein expression compared to physiologically low conditions (P<0.05). There was a trend showing high intensity ESS increasing eNOS expression compared to physiologically low conditions (p<0.10). Additionally, moderate intensity ESS showed a significant increase in eNOS compared to resting conditions (P<0.05).ConclusionsOur data shows that exercise‐induced ESS upregulates eNOS expression. Moderate intensity exercise was shown to produce the highest levels of eNOS expression. Our results from previous studies also show that moderate intensity exercise is capable of activating eNOS, which may act as a protective mechanism to prevent atherosclerosis development.