Mechanism Study Of Activin A-regulated Trophoblast Invasion And Its Relationship With Preeclampsia And Pregnancy-induced Hypertension

Chapter Ⅰ Activin A induces trophoblast invasion by up-regulatingintegrin β1 expressionBackground:Preeclampsia(PE)and pregnancy-induced hypertension(PIH)are both gestational hypertensive disorders that refer to the development of hypertension with or without proteinuria after 20 weeks of gestation in previously normotensive women,which are common pregnancy complications that adversely affect the health of both mothers and fetuses.The pathogenesis of PE/PIH is complicated,and it has been wildly believed that insufficient trophoblast invasion and unsuccessful decidual spiral arteries remodeling are closely related to the development of PE/PIH.Activin A,a important member of the transforming growth factor-β(TGF-β)superfamily,has been found to be widely expressed at the materal-fetal interface during early pregnancy and promotes trophoblast growth and invasion.However,the molecular mechanism of activin A-induced trophoblast invasion has not been fully elucidated,and studies have shown that it involves a complex regulatory network composed of various molecules related to cell adhesion and extracellular matrix(ECM)remodeling.Integrin β1 is the critical transmembrane cell adhesion molecule in the ECM pathway.Studies have shown that the expression of integrin β1 is significantly elevated in invasive and differentiating cytotrophoblasts of first-trimester human placentae,indicating that integrin β1 plays a vital role in the process of trophoblasts invasion.Therefore,studying whether integrinβ1 mediates activin A-increased trophoblast invasion and the underlying mechanisms will benefit the understanding of the etiology of PE/PIH and the development of new targets for early diagnosis and treatment.Objective:To investigate the mediation role of integrin β1 in activin A-induced trophoblast invasion.Methods:We first investigated the transcriptome profiles of HTR-8/SVneo cells(immortalized human extravillous trophoblast cell line)treated with or without activin A and the activin A up-regulated integrin β1 expression was further verified by RT-qPCR and Western blot.Immunohistochemistry staining was utilized to determine the expression of integrin β1 at the first-trimester maternal-fetal interface.Human recombinant activin A protein and small interfering RNA(siRNA)transfection combined with functional experiments including matrigel-coated transwell invasion assay,MTT assay,and endothelial-like tube formation assay was performed to explore the mediating role of integrinβ1 in activin A-induced trophoblast invasion.The effect of integrin β1 was further validated in isolated and verified human primary extravillous cytotrophoblasts(EVT).Results:Transcriptome sequencing analysis showed that significantly differentially expressed genes after activin A treatment were primarily enriched in extracellular matrix(ECM)-receptor interaction pathway and the expression of integrin β1(ITGB1)was significantly increased.SYBR RT-qPCR and Western blot analysis revealed that activin A has a specific inducing effect on integrin β1 mRNA and protein expression level in a time-dependent manner in HTR-8/SVneo cells.During the first trimester,integrin β1 is highly expressed in cytotrophoblast cells in the maternal-fetal interface.Importantly,integrinβ1 knockdown significantly decreased both basal and activin A-increased HTR-8/SVneo cell invasion and endothelial-like tube formation.Moreover,we isolated and purified human primary EVT cells and validated the mediating role of integrin β1 in activin A-induced trophoblast invasion in human primary EVT cells.Conclusion:Activin A promotes human trophoblast cell invasion and endothelial-like tube formation by up-regulating integrin β1 expression.Chapter Ⅱ Activin A increases integrin β1 expression through ALK4-activated SMAD2/3-SMAD4 signaling in trophoblast cellsBackground:Like other members in transforming growth factor-β(TGF-β)superfamily,the effects of activin A are mediated through canonical SMAD-dependent or SMAD-independent pathway activated by activin type Ⅰ and type Ⅱ receptors.Type Ⅰ receptor was identified and termed as activin receptor-like kinases(ALKs)which has seven variants.In canonical SMAD-dependent pathway,activated type Ⅰ and type Ⅱ receptors phosphorylates SMAD2/3 which then combines with SMAD4 to enter the nucleus and regulate target gene expression.In addition,activin A can also regulate SMAD-independent signaling pathways such as ERK1/2,MAPK,and JNK pathway.In the previous chapter,we found that activin A can induce trophoblast invasion by up-regulating the expression of integrin β1,but the underlying mechanism of this process remains to be explored.Objective:To investigate the underlying mechanism of activin A up-regulated integrinβ1 expression.Methods:HTR-8/SVneo cells and human primary EVT cells were used as experimental models.Cells were treated by human recombinant activin A protein combined with TGF-βtype Ⅰ receptors inhibitor or siRNA transfection.RT-qPCR and Western blot were exhibited to detect mRNA and protein expression levels.Results:Pretreatment with SB431542(the inhibitor of TGF-β type I receptors ALK4/5/7)for 1 hour abolished activin A-activated integrin β1 overexpression.ALK4 knockdown attenuated activin A-induced ITGB1 up-regulation in HTR-8/SVneo cells.In addition,SMAD2/3 phosphorylation increased significantly after activin A treatment,and SB431542 pretreatment completely abolished activin A-induced SMAD2/3 phosphorylation in HTR-8/SVneo cells,suggesting that activin A activates canonical SMAD-dependent pathway through ALKs.Knockdown of common SMAD4 completely abolished the activin A-induced integrin β1 overexpression in HTR-8/SVneo cells.Furthermore,activin A up-regulated integrin β1 expression was also attenuated after the depletion of ALK4 or SMAD4 in primary EVT cells.Conclusion:Activin A increases integrin β1 expression through ALK4-activated SMAD2/3-SMAD4 signaling in human trophoblast cells.Chapter Ⅲ First-trimester maternal serum activin A and cytokines profile in IVF patients with PE/PIHBackground:PE/PIH are common pregnancy complications characterized by new-onset hypertension with or without proteinuria after 20 weeks of gestation,which has serious adverse effects on perinatal and even long-term health and safety of both mothers and infants.Unfortunately,the clinical intervention of PE/PIH stagnates in the passive treatment after symptoms appear.As a result of the lack of effective therapies,in recent years,guidelines place greater emphasis on early warning,early detection and early intervention.However,there is a lack of first-trimester predictive/diagnostic biomarkers for PE/PIH.Compared to naturally conceived pregnancies,women underwent assisted reproductive technology(ART)including in vitro fertilization and embryo transfer(IVF-ET)tend to have higher morbidities of PE/PIH.Therefore,it is urgent to explore definitive biomarkers for first-trimester prediction of PIH in this population.Insufficient trophoblast invasion and unsuccessful decidual spiral arteries remodeling during first-trimester play a vital role in the development of PE/PIH.Various cytokines,including activin A,participate in regulating trophoblast invasion process during early pregnancy and can be secreted into the maternal blood circulation.Studies have shown that maternal serum activin A concentration in PE/PIH patients is increased,and second-trimester maternal serum activin A combined with placental growth factor(PlGF)and uterine artery pulsatility index are useful in predicting PE.However,it has not been elucidated whether first-trimester maternal serum activin A or combined with other cytokines can be an effective early predictive or diagnosis biomarker for PE/PIH in the IVF population.Moreover,considering activin A influences the secretion of multiple cytokines and the dysregulation of which are closely related to PE/PIH,it would be of great significance to study the relationship between cytokines and PE/PIH to develop a multi-factor model of cytokines which have higher predictive/diagnostic value for PE/PIH.Objective:To investigate first-trimester maternal serum activin A and cytokines profile in PE/PIH patients who conceived after IVF-ET,and study their predictive/diagnostic value for PE/PIH.Methods:Case-control studies were designed based on an ongoing large prospective ART cohort conducted at the Reproductive Hospital Affiliated to Shandong University.60 PE/PIH patients including 14 early-onset PE/PIH patients and 59 matched normal controls were randomly selected from this cohort.First-trimester maternal serum activin A levels were were detected using ELISA assay kit,and the difference between control group(N=59)with PE/PIH group(N=60)and early-onset PE/PIH group(N=14)were compared.Second,33 PE/PIH patients and 33 matched normal controls were also selected from the cohort and human cytokine screening panel interrogating 48 cytokines were carried out to compare the difference of cytokine profiles between PE/PIH patients and controls.Predictive/diagnostic value of activin A and cytokines for PE/PIH was analyzed by logistic regression analysis and receiver operator characteristic curve(ROC).Results:In IVF population,compared with normal pregnancies,first-trimester serum activin A levels significantly increased in patients with subsequent early-onset PE/PIH,but not significantly increased in patients with subsequent PE/PIH.Increased first-trimester serum activin A concentration was significantly associated with elevated risks of subsequent early-onset PE/PIH(unadjusted OR 9.53,95%CI:1.87-48.54;adjusted OR 14.23,95%CI:2.49-81.37)and first-trimester serum activin A has a good predictive/diagnostic value for subsequent early-onset PE/PIH in IVF population(AUC:0.729).Moreover,first-trimester serum cytokine profiling showed that 29 cytokines were significantly reduced in PE/PIH patients compared with the control group.Decreased concentrations of 17 cytokines in first-trimester maternal serum were associated with increased risk of subsequent PE/PIH,including IL-2Rα,M-CSF,IL-6,IL-2,β-NGF,IL-7,IL-12(p70),SCF,IL-10,IL-9,MIG,GM-CSF,LIF,IL-lα,MCP-3,IL-4,HGF.Combined detection of first-trimester maternal serum IL-7,MIG and SCF has an early prediction/diagnostic value for PE/PIH(AUC:0.821).Conclusion:In IVF pregnancies,first-trimester serum activin A could be a potential predictive/diagnostic marker for early-onset PE/PIH.Low serum levels of immune-related cytokines during the first trimester of pregnancy may be risk factors for PE/PIH.Combined detection of first-trimester maternal serum IL-7,MIG and SCF could be valuable for the early prediction/diagnosis of PE/PIH in IVF pregnancies.