Study Of Shear Stress On KLF2 、Cav-1 And Its Signal Pathway Of Venous Endothelial Cells In Arteriovenous Fistula

Objective:The well-functioning vascular access is necessary to ensure hemodialysis patients for long-term survival.arteriovenous fistula(AVF)as the preferred vascular access has advantages.However,the dysfunction of AVF is affected by many factors.with the study of the AVF dysfunctional mechanism,it is found that changes of hemodynamic and the biological environment may lead to endothelial cell dysfunction,intimal hyperplasia and venous stenosis.The purpose of this study was to investigate the mechanism of shear stress on endothelial cell dysfunction,and to provide theoretical basis for reduction of AVF dysfunction.Methods:(1)The parallel plate flow chamber was used to simulate the hemodynamic changes of AVF,different forces and forms shear stress was produced,and conclusions was analyzed with computational fluid dynamics(CFD)models,the research foundation of in vitro experiments was established.(2)Endothelial cells were inserted into the parallel plate flow chamber.A constant flow pump was used to produce laminar shear stress,low shear stress strength was 4 dyn/cm~2;physiological shear stress strength was 12dyn/cm~2;high shear stress strength was 20dyn/cm~2;an injection pump produced periodic fluid to simulate oscillating shear stress,strength was 0±4 dyn/cm~2.The action time were 0h,4h,12h and 24 h.The expression and distribution of KLF2、CAV-1、ERK、e NOS were detected by immunofluorescence and real-time fluorescence quantitative PCR technique.The expression of these cytokines and proteins was analysised after disruption of caveolae.(3)The expression and distribution of KLF2、CAV-1、ERK、e NOS were detected by immunofluorescence and real-time q PCR after the uremic serum was added to act on endothelial cells.Results:(1)the pressure in the parallel plate flow chamber was drived by flow.The pressure distribution of flow field was no significant difference along z and y direction,and the pressure decreased gradually along the flow direction.In the mode of steady flow,the distribution of shear stress was no significant difference,and the shear stress was positively correlated to the flow rate.In the mode of unsteady flow,the flow rate and the wall shear stress changed periodically with time,and the flow rate has a linear relationship with the wall shear stress.(2)Under the physiological environment,the expression of KLF2、Cav-1、e NOS increased and the expression of ERK decreased in ECs exposed to high sgear stress and physiological shear stress.With the prolongation of the action time,the expression of KLF2 and e NOS increased gradually,while the expression of Cav-1 and ERK decreased gradually.Exposed to oscillatory shear stress and low shear stress,the expression of KLF2、Cav-1、e NOS decreased and the expression of ERK increased,the expression of KLF2 increased gradually with the prolongation of action time,but but it was still obviously lower than that of high shear stress,and the expression of Cav-1、e NOS decreased,while the expression of ERK increased.(3)In uremic environment,the expression of KLF2、Cav-1、e NOS decreased significantly,the expression of ERK increased.The expression of KLF2、Cav-1、e NOS can still be improved exposed in high shear stress,but it is still less than that of physiological state.The expression of KLF2、Cav-1、e NOS exposed in oscillatory shear stress was further reduced,and the expression of ERK is gradually enhanced.(4)Under the action of oscillatory shear stress,the expression of Cav-1 and e NOS was inhibited by MβCD,but the expression of KLF2and ERK increased.Conclusion:Parallel plate flow chamber and CFD technique can be used to simulate AVF flow pattern.Under physiological conditions,high shear stress increased the expression of KLF2、Cav-1、e NOS,but decreased the expression of ERK.Low shear stress and oscillatory shear stress decreased the expression of KLF2、Cav-1、e NOS,and the expression of ERK increased.Under uremic environment,the expression of KLF2、Cav-1、e NOS was significantly decreased,the expression was improved by high shear stress but further decreased by oscillatory shear stress,and the expression of ERK increased,the expression was decreased by high shear stress but increased by oscillatory shear stress.The expression of Cav-1、e NOS decreased and the expression of KLF2、ERK increased by MβCD.Oscillatory shear stress may lead to endothelial cell dysfunction、intimal hyperplasia and AVF dysfunction by KLF2 and Cav-1/e NOS/ERK signaling pathway.