An In Vitro Study On Endothelial Cell Function Modulated By Exercise-Induced Vascular Wall Shear Stress

Exercise training is a noninvasive important approach to prevent cardiovascular diseases,which can alter the magnitude and frequency of pulsatile blood flow wall shear stress(WSS)in an intensity-dependent manner.Endothelial cells lining the innermost layer of vascular wall can recognize different WSS signals,and transduce these mechanical signals into the interior of cells to modulate the release of vasoactive substances,such as nitric oxide(NO)and reactive oxygen species(ROS).These vasoactive substances are closely related to the improvement of endothelial function and the maintenance of vascular homeostasis.Although the pivotal role of WSS induced by exercise in endothelial function has been recognized,the regulation law and the mechanobiological mechanisms of WSS induced by different intensity exercise in endothelial function are poorly understood.Since systems in vitro can simulate WSS signals in vivo and facilitate the monitoring of cellular responses,in this study,we constructed multi-component parallel-plate flow chamber(PPFC)systems to reproduce the WSS waveforms in the common carotid artery before and immediately after different intensity exercise,and then investigated the role and the mechanisms of WSS in the responses of intracellular NO and ROS in endothelial cells.This study will provide possibility for selecting appropriate exercise intensity to prevent cardiovascular diseases.The main research contents and results are as follows:(I)Based on the measured data and the hemodynamic modeling method,the waveforms and key characteristics of WSS in the common carotid artery before and immediately after different intensity exercise were acquired.The waveforms of center-line blood velocity and inner diameter in the common carotid arteries of 11 subjects were measured with a color Doppler ultrasound,and the heart rates and brachial pressures were synchronously measured using an electronic sphygmomanometer in resting state and immediately after acute moderate(55%maximal heart rate)and high(75%maximal heart rate)intensity aerobic cycling exercise.Based on the above measured hemodynamic variables,the WSS waveforms in resting state,immediately after moderate and high intensity exercise were calculated using the elastic and the rigid tube models,respectively.The results demonstrated that the elastic deformation of arterial wall was an important factor in the calculation of WSS,and acute aerobic exercise significantly increased the magnitude and frequency of WSS in an intensity-dependent manner.(2)Rectangular and nonrectangular PPFC systems in vitro were constructed to mimic the WSS waveforms with bidirectional oscillation characteristics in common carotid artery before and immediately after exercise.Firstly,according to the in vivo WSS waveforms before and immediately after different intensity exercise,a five-element lumped parameter model,which could generate bidirectional oscillating flow phenomenon was developed.Then,the parameter values were determined through numerical simulation.Finally,with the appropriate components simulating compliance,fluid inertia and fluid resistance,the PPFC system was fabricated to reproduce the key characteristics of WSS waveforms in vivo before and immediately after different intensity exercise.The PPFC in the system consisted of rectangular and nonrectangular structures,respectively.The constant WSS profile created at the bottom of the rectangular flow chamber was convenient for collecting cellular protein after stimulated with the specific magnitude and frequency of WSS,while the gradual increasing WSS profile formed at the bottom of the nonrectangular flow chamber was suitable and efficient for studying the effect of different magnitude and same frequency of WSS on cellular responses.(3)Intracellular NO and ROS responses and interplay between NO and ROS were clarified under WSS induced by different intensity exercise.Using the above assembled multi-component nonrectangular PPFC system,the dynamics of intracellular NO and ROS in human umbilical vein endothelial cells were detected under the combination of three types of WSS magnitudes(low,moderate,high)or/and three types of WSS frequencies(1.3 Hz,1.8 Hz,2.5 Hz),and the relationships between intracellular NO and ROS under abovementioned WSS waveforms were also investigated.The experimental results indicated that the increase of WSS magnitude alone or WSS frequency alone elevated the levels of intracellular NO and ROS.However,with the simultaneous increases of WSS magnitude and frequency,intracellular ROS levels were continuously raised,while intracellular NO was significantly elevated at 1.8 Hz and moderate magnitude WSS,and then decreased with the increase of WSS magnitude and frequency exceeding a certain extent.Meanwhile,the results also suggested that modest elevated ROS promoted NO production under moderate intensity exercise-induced WSS,whereas excessive ROS attenuated NO levels in endothelial cells exposed to high intensity exercise-induced WSS.(4)The effect of autophagy on intracellular NO and ROS expression was revealed under different intensity exercise-induced WSS.In order to clarify the mechanobiological mechanisms of intracellular NO and ROS production,using the fabricated multi-component rectangular PPFC system,we investigated the regulatory role of autophagy on the phosphorylation of endothelial NO synthase(eNOS)and the production of NO and ROS.By detecting the autophagy associated proteins with Western Blot and tracing the formation and degradation of autophagosome using mRFP-EGFP-LC3 plasmid,we found that the variation trends of autophagy as well as eNOS Ser1177 phosphorylation and NO production were consistent under moderate and high intensity exercise-induced WSS,and they all exhibited highest levels under WSS induced by moderate intensity exercise.The phosphorylation of eNOS at Ser1177 and the production of NO in cells were significantly decreased after autophagy inhibitor chloroquine application,and they were markedly increased after treatment with autophagy activator rapamycin,revealing that autophagy upregulated the production of NO through eNOS Ser1177 phosphorylation under different intensity exercise-induced WSS.Additionally,the intracellular ROS level was elevated with the pretreatment of chloroquine and was decreased after incubation with rapamycin,indicating autophagy inhibited intracellular ROS production under different intensity exercise-induced WSS.In conclusion,the fabricated PPFC systems provide an effective platform to investigate the responses of intracellular NO and ROS and their mechanobiological mechanisms under different intensity exercise-induced WSS.This paper not only reveals the effect of moderate and high intensity exercise on vascular endothelial cell function from the perspective of cell mechanobiology,but aslo offers the possibility for selecting appropriate exercise intensity to prevent cardiovascular diseases.