The Role And Mechanism Of Neuropathy Target Esterase In The Dysangiogenesis Of Preeclamptic Placenta

Background and objectives:Placental dysangiogenesis is considered to be one of the main causes of preeclampsia,but its specific mechanism is not fully understood.In recent years,some researches had shown that neuropathy target esterase(NTE)is crucial in the development of placenta.Phosphatidylcholine,the physiological substrates of NTE,could induce preeclampsia symptoms such as hypertension and proteinuria by injected into pregnant mice,and be closely related to the placental dysangiogenesis.Therefore,the purpose of this study was to investigate the expression of NTE in preeclampsia placentas and its effects on the proliferation,migration and capillary-like tube formation of human umbilical vein endothelial cells,as well as its mechanism.Methods:1.Immunohistochemistry and Western blot were used to observe the expression of NTE protein in placenta of normal pregnancy and preeclampsia.2.By transfecting pEGFP-N3-NTE and pGenesil-1-shNTE plasmid,HUVECs were overexpressed or silenced NTE.3.MTS assay was used to observe the effects of overexpression or silence of NTE on proliferation of HUVECs.4.Scratch-wound assay and transwell experiments were used to observe the effects of overexpression or silence of NTE on migration of HUVECs.5.In vitro angiogenesis assay was used to observe the effects of overexpression or silence of NTE on the capillary-like tube formation of HUVECs.6.After transfection of plasmid,ELISA was used to detect sFlt-1 levels of cell supernatants.7.After transfection of plasmid,Thin-layer chromatography(TLC)and phosphatidylcholine assay kit were used to detect the levels of phosphatidylcholine(PC)and glycerophosphocholine(GPC)in HUVECs.8.In vitro angiogenesis assay was used to observe the effects of PC on the capillary-like tube formation of HUVECs.9.In vitro angiogenesis assay was used to observe the effects of lysophosphatidylcholine(LPC)on the capillary-like tube formation of HUVECs.Results:1.Immunohistochemical results showed that NTE protein was expressed in placental vascular(marked with CD31)of both normal pregnancy and preeclampsia.The expression of NTE protein in preeclamptic placenta significantly decreased compared with normal pregnancy placenta.2.Western blot results showed that the expression of NTE protein significantly decreased in preeclamptic placenta(P<0.01)compared with normal pregnancy placenta.3.Real-time quantitative PCR and Western blot results showed that compared with control group,the expressions of NTE mRNA(P<0.001)and NTE protein(P<0.01)in NTE-EGFP group were significantly higher.The expressions of NTE mRNA(P<0.001)and NTE protein(P<0.05)significantly decreased in NTE shRNA group.4.MTS analysis showed that 24 h,48 h and 72 h after transfection,compared with control group,the proliferation of HUVECs in 20% FBS group and NTE-EGFP group were significantly promoted(P<0.05).In NTE shRNA group,the proliferation of HUVECs was significantly inhibited(P<0.05).5.Scratch-wound assay and transwell experiment showed that compared with the other groups,the migration ability of HUVECs in NTE-EGFP group was significantly enhanced(P<0.01).The migration ability of HUVECs in NTE shRNA group significantly decreased(P<0.01).6.In vitro angiogenesis assay showed: 24 h after transfection,we compared the capillary-like tube formation in each group.In NTE-EGFP group,the number of capillary-like tube formation significantly increased(P<0.001)and the area of capillary-like tube formation was significantly larger(P<0.01).In NTE shRNA group,the number(P<0.001)and the area(P<0.05)of capillary-like tube formation significantly reduced.7.ELISA analysis showed: 24 h and 48 h after transfection,compared with the other groups,the levels of sFlt-1 in NTE-EGFP group significantly decreased(P<0.05).The levels of sFlt-1 in NTE shRNA group significantly increased(P<0.05).8.Thin-layer chromatography(TLC)showed: 24 h after transfection,we compared the levels of PC and GPC in each group.In NTE-EGFP group,the level of PC was significantly lower(P<0.01)and the level of GPC was significantly higher(P<0.01).In NTE shRNA group,the level of PC was significantly higher(P<0.001)and the level of GPC was significantly lower(P<0.01).Phosphatidylcholine assay kit showed: 24 h after transfection,compared with the other groups,the level of PC in NTE-EGFP group was significantly lower(P<0.001).The level of PC in NTE shRNA group was significantly higher(P<0.001),consistent with the results of TLC.9.In vitro angiogenesis assay showed: 24 h after PC treatment,we compared the capillary-like tube formation in each group.Compared with the 0 ?M group,the number(P<0.001)and the area(P<0.001)of capillary-like tube formation significantly reduced with the increase of the dose of 0.2?0.5?1.0 ?M group.10.In vitro angiogenesis assay showed: 24 h after LPC treatment,we compared the capillary-like tube formation in each group.Compared with the 0 ?M group,the number(P<0.001)and the area(P<0.001)of capillary-like tube formation significantly reduced with the increase of the dose of 0.2?0.5?1.0 ?M group.Conclusion:1.Compared with normal pregnancy placenta,the expression of NTE protein in preeclamptic placenta and placental vascular significantly decreased.2.After NTE silencing,the proliferation,migration and capillary-like tube formation of HUVECs significantly decreased.3.NTE regulated the angiogenesis of placenta in preeclampsia by adjusting sFlt-1 levels.4.NTE influenced the angiogenesis of placenta in preeclampsia by regulating the metabolism of PC?GPC and LPC.