Effects of Exercise Induced Endothelial Cell Shear Stress on eNOS and Nitrotyrosine Expression in Different Sites of a Bifurcated Model

Abstract

Introduction Endothelial dysfunction leads to the progression of coronary artery disease. Moreover, atherosclerosis tends to develop at specific areas in the vascular system such as bifurcated regions due to the disruption of unidirectional flow. Endothelial shear stress (ESS) involves mechanical forces produced by alterations in blood flow patterns triggering biochemical signaling pathways that influence the production of nitric oxide and reactive oxygen species (ROS); therefore, affecting vascular homeostasis. Although exercise-induced ESS might act as a protective mechanism against atherosclerosis, the role of exercise-induced ESS in regions susceptible to the development of atherosclerosis such as bifurcated regions has not been explored. The purpose of this experiment was to determine the role of exercise induced ESS in bifurcated vascular regions on eNOS and nitrotyrosine expression. We hypothesized a lower expression of eNOS and nitrotyrosine at the bifurcation compared to the areas before and after the bifurcation. Methods Commercially available human umbilical vein endothelial cells (HUVEC; Sigma-Aldrich, St Louis, MO) were cultured until 90-100% confluence. Moderate exercise-induced ESS in vivo was obtained from our previous reports. Cultured cells were exposed to resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour at moderate intensity exercise induced ESS (50 dynes/cm2) on parallel fluidic units using bifurcated flow chambers (Ibidi USA, Fitchburg, WI). After exposure to ESS, cells were fixed and immunocytochemistry was used to assess the expression of nitric oxide synthase (eNOS) and nitro-tyrosine, a marker of endothelial oxidative stress, in the areas before, at the bifurcation, in both top and bottom walls of it, and after the bifurcation. A Wilcoxon signed rank test was used to assess differences in protein expression before and after the bifurcation using the SPSS 26 (IBM, Armonk, NY). Significance was considered at p≤0.05. Results The analysis of eNOS and nitrotyrosine fluorescence intensity resulted in a non-statistically significant difference (p>0.05) in the expression of both proteins at and after the bifurcation. There was a decrease in eNOS expression at the top, bottom and after the bifurcation (58%, 54% and 59% respectively). Similarly there was a decrease in the expression of nitrotyrosine at the top, bottom and after the bifurcation (58%, 49% and 54% respectively). Discussion Our results suggest that eNOS and nitrotyrosine expression is reduced in areas following a bifurcation, possibly due to decreases in ESS. Additionally, our results suggest that moderate physical activity reduces the loss of eNOS expression at bifurcated sites indicating exercise might act as a protective mechanism in preventing endothelial dysfunction at atherosclerotic prone regions. Even though eNOS expression was decreased at sites of non-unidirectional flow, levels of eNOS expression during exercise were higher compared to resting conditions in areas of unidirectional flow, which we have previously reported.