The Regulation Of Endothelium-dependent Vasodilation By Placental Extracellular Vesicles Delivered Neprilysin

Background:Preeclampsia(PE)is a disease of placenta-origin.Abnormal placentation and peripheral vascular endothelial dysfunction induced by placenta-derived substances is important basis of PE pathogenesis,and extracellular vesicles secreted by the placenta(PL-EVs)may be involved in the regulation of placentation and peripheral vascular endothelial function by placenta.This study aims to investigate the molecular mechanism underlying the regulation of trophoblastic and vascular endothelial cellular function by placenta via PL-EVs,thus to provide new insights and experimental evidences for the prevention and treatment of PE.Methods:(1)Extraction of NPL-EVs and PEPL-EVs by ultracentrifugation.Heparin and/or PL-EVs were injected into CD1pregnant mice via tail vein.The blood pressure of mice was monitored by the noninvasive blood pressure system.Urine albumin of mice was detected by ELIZA.The kidney tissues were stained with PAS staining.PL-EVs was stained with PKH67 or R-18 for EVs-uptake assay and membrane fusion assay.(2)The endothelium-dependent vasodilation of mouse uterine artery was detected by Wire Myograph.Frozen sections of mice placenta were detected by Immunofluorescence staining.The proliferation and apoptosis of HTR8/SVneo cells were detected by EdU staining,cck-8 and flow cytometry.The invasive ability of HTR8/SVneo cells were detected by Matrigel invasion assay.The proliferation and tube formation of HUVEC cells were detected by tube formation assay,cck-8and Western blot.The nitric oxide synthesis of HUVEC cells were detected by Griess Reagent and Western blot.(3)The Neprilysin expression of PL-EVs was detected by LC-MS/MS,Western blot and silver staining.The Neprilysin expression in placentas and HUVEC cells were detected by Western blot.Frozen sections of human placentas and mice uterine arteries were detected by Immunofluorescence staining.Recombinant Neprilysin protein was injected into CD1 pregnant mice via tail vein.The blood pressure of mice was monitored by the noninvasive blood pressure system.The kidney tissues were stained with PAS staining.The endothelium-dependent vasodilation of mouse uterine artery was detected by Wire Myograph.The affinity among Neprilysin and ANP,CNP,AngⅡ,ET-1 and bradykinin were detected by ForteBio molecular interaction analysis system.Results:(1)PEPL-EVs treatment significantly increased the arterial systolic pressure and urinary albumin levels in pregnant mice.While Fetal birth weight and placental weight were significantly reduced in PEPL-EVs treatment group.Morphological changes such as glycogen accumulation and glomerular swelling were were observed in kidney of PEPL-EVs treatment group.The treatment of heparin partially alleviates these abnormalities.PL-EVs were mainly uptake by trophoblast cells and vascular endothelial cell.The treatment of heparin effectively inhibits the uptake of PL-EVs by target cells,but fail to inhibits the membrane fusion between PL-EVs and target cells.(2)PEPL-EVs treatment significantly reduced the junction zone area of mice placenta,as well as the endothelium-dependent vasodilation of pregnant mice uterine artery.The treatment of heparin partially alleviates these abnormalities.PEPL-EVs treatment significantly suppresses the invasion of HTR8/SVneo cells,while treatment of heparin partially alleviates these abnormalities.PL-EVs treatment significantly increased phosphorylation of eNOS~ser1177er1177 in HUVEC cells,which Induced NO synthesis.The expression level of Neprilysin was significantly higher in PE placentas and PEPL-EVs than those from uncomplicated pregnancies.Neprilysin was primarily found in syncytiotrophoblasts of human placenta.PL-EVs carry Neprilysin into vascular endothelial cells and cause intracellular NEP levels to rise.Recombinant Neprilysin protein treatment significantly reduced endothelium-dependent vasodilation of pregnant mice uterine artery,which increased the arterial systolic pressure.The affinity of Neprilysin and CNP is the highest among Neprilysin,ANP,CNP,AngⅡ,ET-1 and bradykinin.Conclusion:(1)PEPL-EVs could be uptake by trophoblasts and vascular endothelial cells trough HSPG pathway,and cause PE-like hallmark in pregnant mice.(2)PEPL-EVs treatment cause abnormal placentation trough inhibition of invasion in trophoblasts.(3)PEPL-EVs treatment inhibits the endothelium-dependent vasodilation of pregnant mice uterine artery,but independent of NO synthesis by vascular endothelial cells.(4)Abnormally up-regulated NEP in syncytiotrophoblasts is transported to peripheral vascular endothelial cells by PL-EVs to degrade CNP,and thus inhibit the endothelium-dependent vasodilation,which could be a potential mechanism for PE development.