Preliminary Study On The Impact Of Impinging Flow On Subcellular Localization Of P120 Catenin And Transcription Factor Kaiso In Human Umbilical Vein Endothelial Cells

Objective:The specific mechanism in the formation and progression of intracranial aneurysm is still being explored.The injury of vascular endothelial cells and inflammation induced by hemodynamic factors plays a key role in the pathogenesis of intracranial aneurysm.Our previous studies showed that the expression of P120catenin(P120-catenin,p120ctn)and transcription factor Kaiso decreased in cultured vascular endothelial cells under the impinging flow.The aim of this study was to determine the subcellular localization of p120 ctn and Kaiso in cultured human umbilical vein endothelial cells(HUVEC)under the impinging flow,and explore the mechanism of p120 ctn and Kaiso involved in the regulation of endothelial cell damage induced by hemodynamics.Methods:Human umbilical vein endothelial cells(HUVEC)and HUVEC transfected with shRNA CTNND1 recombinant lentiviral vector were cultured in vitro.Then,these two kinds of cells were challenged under impinging flow of velocity of 500ml/min on HUVEC 3 hour and 6 hours in vitro.The expression of p120 ctn and Kaiso in the cytoplasm and nucleus of the cell was determined by Western blot and immunocytochemistry.Result:(1)After 6 hours of impinging flow,the morphology of cells and the adhesion between cells maintained at the impact area,but the cell density decreased significantly,cells became round,and most of them were dispersed at adjacent region,while the density of cells in the downstream area increased,and the alignment of some cells was approximately the same as the direction of the impinging flow.(2)Western blot analysis showed that p120 ctn and Kaiso were both expressed in cytoplasm and nucleus of HUVEC in the control group.Compared with the control group,expression of p120 ctn decreased in cytoplasm and increased in the nucleus for HUVEC in the experimental group as the increase of impinging flow time,whichshowed statistical significance(P<0.05);and there was no significant change in Kaiso expression in cytoplasm,but increased in nucleus(P<0.05).Compared with normal HUVEC,the expression of p120 ctn protein was significantly reduced after transfection with shRNA CTNND1 recombinant lentiviral vector(HUVEC),which was statistically significant(P<0.05).(3)Immunocytochemistry showed that p120 ctn was mainly expressed in the cytoplasm,Kaiso was expressed mainly around the nucleus,and a small amount was found in the nucleus of HUVEC in the control group.Compared with the control group,with the increasing of impinging flow time,the expression of p120 ctn in cytoplasm was decreased,and the expression was significantly increased in the nucleus;the expression of Kaiso was decreased around the nucleus,but there was an increasing tendency in the nucleus.Using shRNA CTNND1 recombinant lentivirus transfected HUVEC,the expression of p120 ctn was significantly decreased,and the expression of Kaiso in the nucleus was decreased.Conclusion:Under the action of impinging flow,the p120 ctn in the HUVEC may undergo a cytoplasmic shuttle as the increase of the loading time,which was from the cytoplasm into the nucleus.After knockdown of p120 ctn gene,the expression of Kaiso decreased in the nucleus under the impinging flow.