The Role And Mechanisms Of TET2 On Low Shear Stress Induced HUVECs Pyroptosis

Vascular endothelial cells(VECs)inflammation induced by low shear stress contributes to the initation and progression of atherosclerosis(As).However,its regulation mechanism is still unclear.Recent studies suggest that pyroptosis(an inflammatory procedural cell death)exists through the initation and progression of atherosclerotic lesions.Our previous studies showed that DNA methylolase TET2 is a shear stress sensitive gene.Low shear stress down-regulates TET2.However,the effect of low shear stress on HUVECs pyroptosis and its underlaying mechanism are still unclear.[Objective]: To investigate the role of TET2 in induced VECs pyroptosis and its underlying mechanisms.[Methods]: 1.The parallel plate flow chamber system was used to observe the expression levels of caspase1,GSDMD,NLRP3,AIM,IL-1β and TET2 in HUVECs under low shear stress.2.HUVECs were TET2 incubated with shRNA lentiviral for 96 h to inhibite TET2 expression.The transfection efficiency was detected by immunofluorescence.3 After treatment with TET2 shRNA,the expression levels of caspase1,GSDMD,NLRP3,AIM and IL-1β were detected by Western blot.Immunofluorescence was used to detect the expression of caspase1;Mitochondrial function was detected by electron microscopy,flow cytometry,ATP assay kit and ROS fluorescent probe.[Results]: The results of western blot showed that low shear stress significantly up-regulated the expression of caspase1,GSDMD,NLRP3 and IL-1β in HUVECs.The expression level of TET2 was significantly reduced under low shear stress.Immunofluorescence showed that the transfection efficiency of TET2 lentivirus was as high as 90%,and western bolt confirmed TET2 shRNA has ability to significaintly decreased TET2 expression.TET2 shRNA group showed the higher expressions of caspase1,GSDMD,NLRP3,and IL-1β compared with control group.However,the expression of AIM was no changed.Transmission electron microscopy showed that the cell membrane of TET2 shRNA group was ruptured and the contents of the cells were leaked.Mitochondria in TET2 shRNA group moved to the surrounding area and became shorter.The number of mitochondria was decreased and mitochondrial volume was increased,indicating that TET2 shRNA led to HUVECs mitochondrial damage.Flow cytometry showed that the mitochondrial membrane potential of HUVECs in TET2 shRNA group was abnormal,mainly as follows: most of the cells were distributed in Q3 area,and the ratio of Q2/Q3 was decreased.TET2 shRNA group showed significantly lower mitochondrial ATP production and higher ROS production level than the control group.[Conclusion]: Low shear stress down-regulates VECs TET2,which contributes mitochondrial dysfunction and HUVECs pyroptosis...