The Relationship Between Excessive Autophagy And Placenta Dysfunction In Early Onset Preeclampsia

Objective: Through observe the expressions of LC3and Beclin-1oftrophoblast cell and vascular endothelial cell in human placenta, speculatethat excessive autophagy maybe existed in trophoblast cell and vascularendothelial cell in early-onset preeclampsia (EOPE) placenta tissue. Andthen, simulated an oxidative stress environment by GO, trophoblast celland vascular endothelial cell as the research objects, to probe thepathogenesis and potential molecular mechanism of excessive autophagyinvolved in placenta dysfunction in EOPE.Methods:1.18cases of placenta tissues were collected from womansuffer from EOPE (<34gestational weeks) and20cases were collectedfrom normal pregnant women, all the cases were under the supervision ofGynecology and Obstetrics Department in The First Affiliated Hospital ofChongqing Medical University from January1,2010to May1,2012.Immunohistochemistry was employed to evaluate the expression of LC3and Beclin-1in placenta tissues; transmission electron microscope wasused to observe the autophagosome occurred in placenta tissues; tissue immunofluorescence double staining was used to detect the expressionrelationship between nitrotyrosine and LC3and Beclin-1in placenta tissue.2. GO was used to simulate an oxidative stress environment,immunofluorescence staining, qRT-PCR and WB were applied to detectthe expression of LC3and Beclin-1in human extravillous trophoblast cellline (HTR8/SVneo) and human umbilical vein endothelial cell (HUEVCs),the autophagosome was observed by transmission electron microscope.Hoechst33258was conducted to observe the occurrence of apoptosis, theexpression of cleaved caspase-3was detected by WB. The proteinexpressions of matrix metalloproteinase9(MMP9) and vascular endothelialgrowth factors receptor2(VEGFR2) were measured by WB, transwellinvasion assay was used to detect the invasion ability of HTR8/SVneo, tubeformation assay was applied to detect the tube formation ability.Results:1. The occurrence of autophagy was increased in EOPE placentatissues.(1) Immunohistochemistry result showed that the expressions ofLC3and Beclin-1in trophoblast and vascular endothelial cell of EOPEplacentas were both significantly increased compared with normalplacentas (P <0.05).(2) Typical autophagosome were observed in EOPEplacenta by transmission electron microscope.(3) Immunofluorescencedouble staining showed that the expression of nitrotyrosine observed inEOPE placentas was stronger than normal placentas, and similar result ofLC3and Beclin-1was obtained.2. Excessive autophagy induced by oxidative stress impaired the function of HTR8/SVneo and HUVECs.(1)The expressions of LC3and Beclin-1were increased in HTR8/SVneo inGO treated group detected by qRT-PCR and WB, statistical significancewas existed (P <0.05); immunofluorescence result showed thatautophagosome were significantly increased in GO treated group (P <0.05);while autophagosome were observed in GO treated group by transmissionelectron microscope.(2) The apoptotic cell stained by hoechst33258wasnot increased in GO treated group, and no difference was seen in the cellsamong the four groups; moreover, the expression of cleaved caspase-3wassimilar among the four groups in WB analysis result (P>0.05).(3) WBand immunofluorescence result showed that the expression of MMP9wassignificantly reduced in GO treated group compaired with other groups (P<0.05); moreover, transwell invasion assay showed that the invasionability of HTR8/SVneo was impaired in GO treated group (P <0.05), theinvasion ability in3-MA+GO treated group was increased compaired withGO treated group (P <0.05). In HUVECs, WB and immunofluorescenceresult showed that the expression of VEGFR2was significantly reduced inGO treated group compaired with other groups (P <0.05), the expressionof VEGFR2was increased in3-MA+GO treated group compaired with GOtreated group (P <0.05); tube formation assay showed that the tubeformation ability of HUVECs was impaired in GO treated group (P <0.05), and the tube formation ability in3-MA+GO treated group was increasedcompaired with GO treated group (P <0.05).Conclusion:1. There was excessive autophagy in EOPE placentatissues.2. Excessive autophagy impaired the invasion ability ofHTR8/SVneo and the tube formation ability of HUVECs.3. The excessiveautophagy of trophoblast cell and vascular endothelial cell may be involvedin the pathogenesis of EOPE placental dysfunction.