The Compensatory Role And Mechanism Study Of H3K27me3-modulated BMP2 Signaling In Preeclampsia

BackgroundPreeclampsia(PE)is a common pregnancy-related disease,with an incidence of 2-8%among pregnant women.It is characterized by onset hypertension(≥140/90 mmHg)and proteinuria(urinary protein≥0.3g/24h)after 20 weeks of gestation,and is a major cause of mortality and morbidity among pregnant and perinatal women worldwide.The etiology of preeclampsia is not clear,but studies suggest that it is associated with various factors,including placental abnormalities(formation and development of the placenta),immune factors,preexisting or underlying medical conditions,and environmental factors.The pathogenesis of preeclampsia is also unclear,but the hypothesis of abnormal placental implantation and insufficient spiral artery remodeling is widely accepted.Abnormal placental implantation and insufficient spiral artery remodeling lead to shallow placentation,resulting in hypoxia and increased oxidative stress,endothelial damage,and release of anti-angiogenic and inflammatory factors into the maternal circulation.This cascade of events subsequently precipitates pervasive endothelial dysfunction,leading to impaired function across a spectrum of organ systems.At present,the clinical diagnosis and treatment landscape is bereft of effective targeted therapeutic drugs for preeclampsia.Progression of the disease leaves clinicians with no viable treatment options apart from termination of pregnancy.Consequently,elucidating the pathogenesis and identifying effective therapeutic targets holds immense significance for advancing the diagnosis and treatment of preeclampsia.Bone morphogenetic proteins(BMPs),highly conserved members of the transforming growth factor-beta superfamily,play crucial roles in development,angiogenesis,and reproduction.Although BMPs,including BMP2,BMP4,BMP6,and BMP 10,are essential for trophoblast differentiation during early embryonic development,only BMP2 has to date been identified as a pro-invasive factor that can regulate the invasive behavior of human trophoblasts in vitro.However,the precise cellular origin of BMP2 in the placenta,as well as the potential influence of BMP2 signaling on the pathogenesis of preeclampsia,remain to be conclusively established.The contribution of epigenetic modifications to the regulation of placental development has been gradually unveiled over time.However,research probing the roles of epigenetic regulation in the pathogenesis of preeclampsia(PE)remains sparse.Nonetheless,it has been postulated that uncovering PE-related epigenetic-based targets could aid in identifying potential biomarkers for the onset of PE.Among the most well-characterized epigenetic influences of histone modifications on placental development,the trimethylation of lysine 27 of histone H3(H3K27me3)is acknowledged to partake in the regulation of MMP2/9 expression,both of which correlate with trophoblast invasiveness.One preceding study posited that BMP2 can be modulated by H3K27me3 modification in mouse embryonic fibroblast;notwithstanding,the extent to which H3K27me3 contributes to the dysregulation of placental BMP2 signaling in the context of PE development remains elusive.ObjectiveTo elucidate the cell source of BMP2 in the placenta and its relationship with preeclampsia(PE),explore the upstream and downstream regulatory processes of BMP2,and further investigate its potential therapeutic effects through the utilization of an animal model of preeclampsia.MethodsPlacental tissues from women with healthy pregnancy(HP)or with clinical diagnosis of PE were collected,and a multi-omics analysis of ChIP-seq and RNA-seq was performed to establish the transcriptome and H3K27me3 modification maps of PE placenta.ELISA,Western blot,and qPCR were used to detect the expression levels of BMP2 in serum and placental tissues and to analyze their association with clinical manifestations.Immunofluorescence was used to determine the expression location of BMP2 in the placenta.Human primary Hofbauer cells were sorted by flow cytometry and cultured in vitro.The cells were treated with DZNep,an inhibitor of EZH2,and qPCR results indicated an upregulation of BMP2 expression.These results suggest that H3K27me3 modification regulates BMP2 expression in placental Hofbauer cells.We performed transcriptome sequencing analysis on HTR-8/SVneo cells(a human immortalized extravillous trophoblast cell line)after BMP2 intervention,and identified BMP6 as a differentially expressed gene.We investigated the effects of BMP6 on trophoblast cells and the mediating role of BMP6 in BMP2-induced trophoblast cell invasion through in vitro treatment with recombinant human BMP6,BMP2,and siRNA knockdown,combined with Transwell invasion assay,EDU,TUNEL,villous explants outgrowth,and endothelial-like tube formation assay experiments.Using small molecule inhibitors DMH-1 and SB431542 as well as siRNA treatment,we elucidated the signaling pathways of BMP2 regulating BMP6 expression.Additionally,we used single-cell transcriptomics and immunofluorescence to determine the cellular and spatial localization of BMP2.Recombinant BMP2 was injected during mid-pregnancy in a rat model of preeclampsia induced by adenoviral overexpression of sFlt-1.The rescue effect of BMP2 on the preeclampsia-like phenotype in a rat model of preeclampsia(PE)was assessed.Blood pressure and proteinuria were monitored as indicators of PE severity.Additionally,fetal and placental weight measurements were obtained to assess the impact of BMP2 on fetal growth and placental development.Histological examination of tissue slices stained with hematoxylin and eosin(HE)was performed to further investigate the histopathological changes associated with the preeclampsia phenotype.ResultsBMP2 is upregulated in preeclamptic placentas and its level is negatively correlated with the severity of clinical manifestationsTranscriptome sequencing of placentas from 7 PE and 7 healthy pregnant women revealed an enrichment of BMP2 targets and EZH2 targets.Further analysis showed increased BMP2 expression in PE placentas through public transcriptome databases and qPCR.Correlation analysis showed a negative association between BMP2 expression in the placenta and blood pressure.Importantly,given multiple previous studies showing that BMP2 promotes trophoblast invasion and vascular mimicry,the negative correlation we detected could be interpreted to indicate that increased BMP2 may represent a self-compensatory mechanism for shallow trophoblast invasion in PE.BMP2 locus is modified by H3K27me3 in Hofbauer cell which is the origin of BMP2 in placentaChIP sequencing of placental samples revealed lower signals of H3K27me3 in the genome of preeclampsia(PE)placentas compared to normal placentas.Western blot analysis confirmed reduced H3K27me3 protein levels and increased BMP2 protein levels in PE placentas.Further analysis showed that the H3K27me3 signals levels in the BMP2 TSS were decreased in PE placentas.Additionally,single-cell transcriptome and immunofluorescence analysis confirmed that BMP2 is localized in Hofbauer cells of the placenta.In vitro experiments utilizing primary human Hofbauer cells have shown that DZNep(EZH2 inhibitors)treatment leads to the upregulation of BMP2 expression.These findings suggest that H3K27me3 modification plays a role in regulating BMP2 expression in placental Hofbauer cells.BMP2 promotes trophoblast cell invasion by upregulating BMP6 expressionIn vitro experiments using BMP2 treatment on HTR8/SVneo cells showed upregulation of invasion-related genes,including ID1,MMP2,MMP11 and BMP6 was significantly upregulated and its expression was further confirmed to be time-and dose-dependent through qPCR.ELISA results indicated elevated levels of BMP6 in serum of preeclampsia patients,suggesting it as a potential target in PE.Functional assays,such as transwell,endothelial tube formation,and villous explant culture experiments,demonstrated that recombinant BMP6 promotes trophoblast cell invasion and endothelial-like tube formation.Using small interfering RNA(siRNA)and intervention with recombinant BMP2 confirmed that BMP6 mediates the pro-invasive and endothelial-like tube formation effects of BMP2.Moreover,specific inhibition of ALK2/3 or ALK4/5 demonstrated that only inhibition of ALK2/3 suppressed the upregulation of BMP6 and phosphorylation of SMADs.Knockdown experiments using siRNA targeting ALK3,SMAD2/3,or SMAD4 showed a suppression of BMP2-induced upregulation of BMP6.These findings provide evidence that BMP2 upregulates BMP6 expression through the BMPR1A-SMAD2/3-SMAD4 signaling pathway.Supplementation with recombinant BMP2 alleviates PE-related phenotypes in an Ad Flt1-induced PE rat modelIn vivo administration of recombinant BMP2 alleviated the preeclampsia-like phenotype in the rat model,including hypertension,restricted fetal growth,decreased placental weight,and a decreased ratio of labyrinth zone to junctional zone in the placenta.However,it did not ameliorate kidney damage and did not lead to ectopic bone formation.These findings suggest that BMP2 can rescue the preeclampsia phenotype and may serve as a potential therapeutic target.ConclusionIn conclusion,patients of preeclampsia exhibit decreased H3K27me3 modification,leading to increased expression of BMP2 in Hofbauer cells.BMP2,through the BMPR1ASMAD2/3-SMAD4 signaling pathway,upregulates BMP6 to promote trophoblast cell invasion and endothelial-like tube formation as a compensatory response for shallow trophoblast invasion in preeclampsia.Importantly,administration of recombinant BMP2 shows promising results in partially rescuing the preeclampsia-like phenotype in a rat model.These findings highlight the potential of targeting this signaling pathway as a therapeutic approach for the clinical management of preeclampsia.