The Role Mechanism Of TET2 In Shear Stress Induced EndMT

[Background]Atherosclerosis(As)is a chronic inflammatory prolifera-tive response caused by arterial intimal injury,which occurs in areas of low shear stress such as branching and bifurcation of arteries.Previous studies have shown that TET2 is DNA hydroxymethylase.Low shear stress down-regulates the expression of TET2,and over-expression of TET2 inhibits low shear stress-induced atherosclerosis lesions.[Purpose]To investigate the effect of TET2 on endothelial mesenchy-mal transformation induced by low shear stress and its mechanism.[Method]The parallel plate flow chamber system was perfused for 6hours under low shear stress(4dyn/cm~2)and physiological laminar shear stress(15dyn/cm~2)to observe the effect of low shear stress on EndoMT and TET2 expression.TET2 shRNA lentivirus was transfected into human umbilical vein endothelial cells(HUVECs)to construct low expression of TET2 vascular endothelial cells,to observe the effect of TET2 shRNA on EndoMT and the expression of target genes MYC and AXIN2 downstream of Wnt/?-Catenin signaling pathway.LV-TET2lentivirus was transfected into HUVECs to construct high expression of TET2 vascular endothelial cells.The parallel plate flow chamber system was used for 6 hours under low shear stress conditions to observe the effect of TET2 overexpression on EndoMT under low shear stress.Western Blot immunoblotting was used to detect the expression of vascular endothelial cell surface antigen CD31,VE-Cadherin and Pecam1,and the expression of mesenchymal cell surface antigen Vimentin,?-SMA and FSP-1,expression of target genes MYC and AXIN2downstream of Wnt/?-Catenin signaling pathway as well as expression of TET2.Detection of morphological and structural changes of vascular endothelial cells by phalloidin staining,scratch test to detect the effect of TET2 shRNA on vascular endothelial cell migration,MTT assay to detect the proliferation of vascular endothelial cells,flow cytometry was used to detect changes in the vascular endothelial cell cycle.[Results]Compared with the physiological laminar shear stress group,the expression of VE-Cadherin in the vascular endothelial cell surface of the low shear stress group was significantly decreased.However,the surface antigen Vimentin expression was significantly up-regulated.It indicates that low shear stress promotes endothelial mesenchymal transition.At the same time,the expression of TET2 in the low shear stress group was lower than that in the physiological laminar flow group,indicating that low shear stress can down-regulate the expression of TET2.Western blot analysis showed that TET2 shRNA down-regulated the expression of endothelial cell markers CD31,VE-Cadherin,and Pecam1,and up-regulated the expression of stromal cell markers Vimentin,?-SMA and FSP-1,indicating that TET2 shRNA promotes EndoMT.TET2 shRNA up-regulated the expression of MYC and AXIN2 in the downstream of Wnt/?-Catenin signaling pathway,indicating that down-regulation of TET2 can activate Wnt/?-Catenin signaling pathway.Under low shear stress,the expression of VE-Cadherin and CD31 was increased in LV-TET2 group,while the expression of stromal cell markers Vimentin and?-SMA was down-regulated,It is shown that LV-TET2 inhibits low shear stress induced EndoMT.The results of rhodamine ghost pen ring peptide staining showed that TET2shRNA group HUVECs microfilament thickening,stress fiber increased,cytoskeletal rearrangement.Cell scratch test results showed that the migration ability of HUVECs in the TET2 shRNA group was enhanced.The results of MTT assay showed that there was no significant difference in HUVECs proliferation between the TET2 shRNA group and the control group.Flow cytometry results showed that the cell cycle of the TET2 shRNA group was not significantly different from that of the control group.[Conclusion]Low shear stress down-regulates the expression of TET2,Which up-regulates the expression of MYC and AXIN2,thereby inducing EndoMT.