| OBJECTIVES(1)In order to explore the effects of PPAR? agonist rosiglitazone on the expression of PPAR? and the expression and secretion of ANGPTL4 in trophoblasts HTR8/SVneo,HUVECs and placental explants,and the role of PPAR? in them.(2)To further investigate the molecular regulatory mechanism of rosiglitazone-induced the expression and secretion of ANGPTL4 in HTR8/SVneo,HUVECs cells and placental explants.(3)To study the expression PPAR? and ANGPTL4 in placental tissues and the circulating activators levels of PPAR? and the secretion of ANGPTL4 in serum among normal pregnant women and patients with preeclampsia,and to analyze the correlation between PPAR? and ANGPTL4.(4)To investigate the roles of ANGPTL4 and PPAR? in the proliferation and hydrogen peroxide-induced apoptosis in HTR8/SVneo cells.(5)To explore the roles of ANGPTL4 and PPAR? in the migration and invasion of HTR8/SVneo cells and the growth of placental explants.(6)To investigate the role of ANGPTL4 and PPAR? in endothelial cell angiogenesis.METHODS(1)HUVECs,HTR8/SVneo and placental explants were treated with different concentrations of rosiglitazone.The mRNA and protein expression of PPAR? and ANGPTL4 and the secretion of ANGPTL4 were detected by Western blotting,qRTPCR,ELISA and immunofluorescence.Silencing the expression of PPAR? with small interfering RNA,the roles of PPAR? in the expression and secretion ANGPTL4 induced by rosiglitazone was further examined through the above experiments.(2)The potential PPAR?/RXR? binding sites on the promoter of ANGPTL4 were predicted by software.Specific PPAR?/RXR? binding site on ANGPTL4 promoter was identified in HTR8/SVneo cells,HUVECs and placental explants through ChIP assay.(3)The placental tissue,blood and clinical data of 30 normal pregnant women and 30 patients with preeclampsia diagnosed by department of Obstetrics and Gynecology in Ren Ji Hospital affiliated to Shanghai Jiao Tong University School of Medicine were collected.The expression PPAR? and ANGPTL4 in placental tissues and the circulating activators levels of PPAR? and the secretion of ANGPTL4 in serum were detected by immunohistochemistry,Western blotting,qRT-PCR and ELISA,and the correlation between PPAR? and ANGPTL4 was statistically analyzed.(4)HTR8/SVneo cells transfected with si-Con or si-ANGPTL4 was treated with rosiglitazone.The effects of rosiglitazone and ANGPTL4 on the apoptosis and proliferation of HTR8/SVneo cells and the expression of genes related to cell proliferation and apoptosis were detected by TUNEL,CCK-8,Western blotting and qRT-PCR.In addition,the expression of caspase-3(apoptosis-related protein)and cyclin D1(proliferation-related protein)in placental tissues among two groups of pregnant women was examined by immunohistochemistry.(5)HTR8/SVneo cells transfected with si-Con or si-ANGPTL4 was treated with rosiglitazone.The roles of rosiglitazone and ANGPTL4 in the migration and invasion of trophoblast cells and placental explant outgrowth and the expression of MMP2 and MMP9 were examined by wound healing assay,transwell invasion assay,placental explant model and Western blotting.Meanwhile,the expression of MMP2 and MMP9 in placental tissues among two groups of pregnant women was detected by immunohistochemistry.(6)HUVECs transfected with si-Con or si-ANGPTL4 cells was treated with rosiglitazone.The effects of rosiglitazone and ANGPTL4 on the angiogenesis in HUVECs and the expression and secretion of VEGF were assessed through tube formation assay,Western blotting,qRT-PCR and ELISA.The expression of PPAR?,ANGPTL4,CD31 and VEGF in placental tissues among two groups of pregnant women was analyzed by immunohistochemistry.RESULTS(1)Rosiglitazone upregulated the mRNA and protein expression of ANGPTL4 and PPAR? and the secretion of ANGPTL4 in HUVECs,HTR8/SVneo cells and placental explants in a concentration-dependent manner.Silencing PPAR? inhibited rosiglitazone-induced the expression and secretion of ANGPTL4.(2)There are three potential PPAR?/RXR? binding sites in the ANGPTL4 promoter region,PPRE1(-1822 /-1808),PPRE2(-809/-795)and PPRE3(-233/-209),respectively.It was confirmed via ChIP assay that the binding site of PPAR?/RXR? in the promoter of ANGPTL4 was PPRE3(-233 /-209).(3)Compared with normal pregnant women,the expression of ANGPTL4 and PPAR? in placental tissues among patients with preeclampsia were significantly reduced.Similarly,the circulating activators levels of PPAR? and the secretion of ANGPTL4 in serum in preeclampsia patients were significantly less than that of normal pregnant women.Statistical analysis also showed that the mRNA expression of PPAR? in placental tissues of the two groups was positively correlated with the expression and secretion levels of ANGPTL4 in corresponding placental tissue and serum.(4)Compared with the control group,the results of TUNEL,CCK-8,Western blotting and qRT-PCR showed that rosiglitazone treatment significantly promoted the proliferation of HTR8/SVneo and the expression of cyclin D1,c-Myc,Bcl-2 and pHH3 and inhibited hydrogen peroxide-induced HTR8/SVneo apoptosis and the expression of cleaved PARP,cleaved caspase-3,cleaved caspase-9,caspase-3 and caspase-9.Silencing ANGPTL4 expression blocked these effects of rosiglitazone on HTR8/SVneo.The increased expression of caspase-3 and the decreased expression of cyclin D1 in preeclampsia placenta with the low expression of PPAR? and ANGPTL4 were further confirmed through immunohistochemistry compared with that of normal pregnant women.(5)The results of wound healing assay,transwell invasion assay and placental explant assay demonstrated that rosiglitazone significantly promoted the migration and invasion of HTR8/SVneo cells and the growth of placental explants compared with the control group.Western blotting and gelatin zymography assay showed that rosiglitazone significantly promoted the expression of MMP2 and MMP9 and inhibited the expression of TIMP-1 and TIMP-2 in HTR8/SVneo cells.Silencing ANGPTL4 expression blocked these effects of rosiglitazone.In addition,the decreased expression of MMP2 and MMP9 in preeclampsia placenta with the low expression of PPAR? and ANGPTL4 was confirmed via immunohistochemistry compared with that of normal pregnant women.(6)Tube formation assay indicated that rosiglitazone obviously promoted the angiogenesis in HUVECs compared with the control group.The results of Western blotting,qRT-PCR and ELISA showed that rosiglitazone significantly enhanced the expression and secretion of VEGF in HUVECs compared with the control group.The silence of ANGPTL4 blocked these effects of rosiglitazone.More importantly,the decreased expression of VEGF and CD31 in preeclampsia placenta with the low expression of PPAR? and ANGPTL4 was further verified by immunohistochemistry compared with that of normal pregnant women.CONCLUSIONS(1)Rosiglitazone induces the expression and secretion of ANGPTL4 through PPAR?.(2)ANGPTL4 is a target gene of PPAR?,and PPAR?/RXR? heterodimer can bind to the promoter of ANGPTL4 and promote its transcriptional activation.(3)The expression of PPAR? and ANGPTL4 in placental tissues and the circulating activators levels of PPAR? and the secretion of ANGPTL4 in serum were decreased in preeclampsia patients compared with normal pregnant women.(4)ANGPTL4 mediates rosiglitazone-induced the survival and proliferation of trophoblast HTR8/SVneo.(5)ANGPTL4 is an important mediator for rosiglitazone-induced migration and invasion of HTR8/SVneo and the growth of placental explants.(6)ANGPTL4 is involved in angiogenesis induced by rosiglitazone in HUVECs. |
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