OBJECTIVETo study the expression of matrix metalloproteinases (MMPs ) in the decidual stromal cells (DSCs) and extravillous cytotrophoblasts (EVCT) in human early pregnancy and explore the change of MMPs in endometrial stromal cell (ESC) decidualization and its impact on implantation and placentation.

METHODSThe decidua and villi from 5 women with early pregnancy and mid-secretory endometrium from 5 normal women were collected and cultured in vitro, and the supernatants of the culture media were collected after 48 hours of incubation. The expression of the MMPs in the ESCs, DSCs and EVCTs was detected using Luminex xMAP system simultaneously and the difference in MMPs expression and their correlations were analyzed with SPSS10.0 software.

RESULTSThe MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, and MMP-9) were expressed in ESCs, DSCs and EVCTs, while MMP-12 was not found in ESCs and MMP-13 not in DSCs. Expressions of MMP-8, MMP-12, and MMP-13 were lowered. Compared with the ESCs, DSCs and EVCTs showed significantly lowered expressions of MMP-1, MMP-3, and MMP-7 (P<0.05), whereas expression of MMP-2 and MMP-9 increased significantly, and the high expressions of MMP-1, MMP-3, and MMP-7 was especially obvious in the DSCs. The expressions of MMP-1, MMP-3, and MMP-7, however, were significantly decreased in the EVCTs in comparison with the DSCs. Significant correlations were noted between MMP-1, MMP-3, and MMP-7, and MMP-2 was closely correlated with MMP-9. MMP-8 was significantly lower and MMP-12 and MMP-13 showed no obvious variation in the cell culture.

CONCLUSIONMMPs are secreted by ESCs, DSCs and EVCTs. Diverse MMPs play an important role in proliferation and differentiation of the ESC to affect embryo implantation and placentation. All MMPs establish a balance to co-regulate the process of pregnancy.

Adult ; Cells, Cultured ; Decidua ; cytology ; enzymology ; Endometrium ; cytology ; enzymology ; Female ; Humans ; Isoenzymes ; metabolism ; Matrix Metalloproteinases ; metabolism ; Pregnancy ; Pregnancy Trimester, First ; Stromal Cells ; cytology ; enzymology ; Trophoblasts ; cytology ; enzymology

This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expression in HCMV-infected EVT in vitro. A human trophoblast cell line (HPT-8) was chosen in this study. HCMV with 100 TCID50 was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by virus titration. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in infected HPT-8 cells was decreased (P<0.05), and the levels of VIP mRNA and protein, and the concentration were raised to the normal (P>0.05). Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.
Antiviral Agents ; pharmacology ; Cell Line ; Cytomegalovirus ; drug effects ; physiology ; Flavanones ; pharmacology ; Humans ; Trophoblasts ; cytology ; drug effects ; metabolism ; virology ; Vasoactive Intestinal Peptide ; metabolism ; Virus Inactivation ; drug effects

The human organic anion transporter 4 (hOAT4) has been identified as the fourth isoform of OAT family. hOAT4 contributes to move several negatively charged organic compounds between cells and their extracellular milieu. The functional characteristics and regulatory mechanisms of hOAT4 remain to be elucidated. It is well known that caveolin plays a role in modulating proteins having some biological functions. To address this issue, we investigated the co-localization and interaction between hOAT4 and caveolin-1. hOAT4 and caveolin-1 (mRNA and protein expression) were observed in cultured human placental trophoblasts isolated from placenta. The confocal microscopy of immuno-cytochemistry using primary cultured human trophoblasts showed hOAT4 and caveolin-1 were co-localized at the plasma membrane of the cell. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions and immune-precipitates from the trophoblasts. When synthesized cRNA of hOAT4 along with scrambled- or antisense-oligodeoxynucleotide (ODN) of Xenopus caveolin-1 were co-injected to Xenopus oocytes, the [3H]estrone sulfate uptake was significantly decreased by the co-injection of antisense ODN but not by scrambled ODN. These findings suggest that hOAT4 and caveolin-1 share a cellular expression in the plasma membrane and caveolin-1 up-regulates the organic anionic compound uptake by hOAT4 under the normal physiological condition.
Animals ; Caveolin 1/genetics/*metabolism ; Cells, Cultured ; Female ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Microscopy, Confocal ; Models, Biological ; Oocytes/metabolism ; Organic Anion Transporters/genetics/*metabolism ; Placenta/cytology ; Protein Binding ; Reverse Transcriptase Polymerase Chain Reaction ; Trophoblasts/cytology/*metabolism ; Xenopus

BACKGROUNDPlacental multidrug resistance-associated protein 2 (MRP2), encoded by ABCC2 gene in human, plays a significant role in regulating drugs' transplacental transfer rates. Studies on placental MRP2 regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the roles of epigenetic mechanisms in regulating placental drug transporters are still unclear. This study aimed to investigate the effect of histone deacetylases (HDACs) inhibition on MRP2 expression in the placental trophoblast cell line and to explore whether HDAC1/2/3 are preliminarily involved in this process.

METHODSThe human choriocarcinoma-derived trophoblast cell line (Bewo cells) was treated with the HDAC inhibitors-trichostatin A (TSA) at different concentration gradients of 0.5, 1.0, 3.0, and 5.0 μmol/L. Cells were harvested after 24 and 48 h treatment. Small interfering RNA (siRNA) specific for HDAC1/HDAC2/HDAC3 or control siRNA was transfected into cells. Total HDAC activity was detected by colorimetric assay kits. HDAC1/2/3/ABCC2 messenger RNA (mRNA) and protein expressions were determined by real-time quantitative polymerase chain reaction and Western-blot analysis, respectively. Immunofluorescence for MRP2 protein expression was visualized and assessed using an immunofluorescence microscopy and ImageJ software, respectively.

RESULTSTSA could inhibit total HDAC activity and HDAC1/2/3 expression in company with increase of MRP2 expression in Bewo cells. Reduction of HDAC1 protein level was noted after 24 h of TSA incubation at 1.0, 3.0, and 5.0 μmol/L (vs. vehicle group, all P < 0.001), accompanied with dose-dependent induction of MRP2 expression (P = 0.045 for 1.0 μmol/L, P = 0.001 for 3.0 μmol/L, and P < 0.001 for 5.0 μmol/L), whereas no significant differences in MRP2 expression were noted after HDAC2/3 silencing. Fluorescent micrograph images of MRP2 protein were expressed on the cell membrane. The fluorescent intensities of MRP2 in the control, HDAC2, and HDAC3 siRNA-transfected cells were week, and no significant differences were noticed among these three groups (all P > 0.05). However, MRP2 expression was remarkably elevated in HDAC1 siRNA-transfected cells, which displayed an almost 3.19-fold changes in comparison with the control siRNA-transfected cells (P < 0.001).

CONCLUSIONSHDACs inhibition could up-regulate placental MRP2 expression in vitro, and HDAC1 was probably to be involved in this process.

Cell Line ; Histone Deacetylase 1 ; metabolism ; Histone Deacetylase 2 ; metabolism ; Histone Deacetylase Inhibitors ; pharmacology ; Histone Deacetylases ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Microscopy, Fluorescence ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; RNA, Messenger ; Trophoblasts ; cytology ; metabolism

OBJECTIVETo investigate the molecular mechanisms of effective ingredients of Astragalus-Salvia compound (ASC) in increasing placental blood supply, to provide thought for establishing an effective prevention and treatment of insufficient placental blood supply caused complications of pregnancy.

METHODSThe effective ingredients of ASC was isolated and abstracted and the rat model of inhibited nitric oxide synthesis was established by using L-Arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. The mRNA expression of vascular endothelial growth factor (VEGF) in placental trophocyte and primordial micrangial endothelial cell was determined by using in situ hybridization at 12th day of pregnancy.

RESULTSThe VEGF mRNA expression was significantly lower in nitric oxide synthesis inhibited model rats than that in rats non-modeled, or in model rats treated by ASC abstracts or nitroglycerin (P < 0.05, P < 0.01).

CONCLUSIONEffective ingredients of ASC may improve VEGF mRNA expression in placental trophocyte and vascular endothelial cell in early pregnancy, and thus be favorable for placental vascular net formation, trophocyte infiltration and mother-placental circulation constitution for increasing placental blood supply to prevent and treat the insufficient placental blood supply caused diseases such as pregnancy-induced hypertension syndrome.

Animals ; Astragalus membranaceus ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; Female ; Male ; Nitric Oxide ; biosynthesis ; Placenta ; cytology ; metabolism ; Pregnancy ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Trophoblasts ; metabolism ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo study the expression of Toll-like receptors (TLRs) mRNA in human trophoblast HTR-8/SVneo cells and the changes in indoleamine 2,3-dioxygenase (IDO) mRNA expression in response to TLR ligand stimulation.

METHODSThe expressions of TLRs and IDO mRNA in human HTR-8/SVneo cells were tested by RT-PCR, and the changes in IDO mRNA levels after exposure to TLR3, TLR4, TLR7/8, and TLR9 ligands were quantitatively analyzed with real-time PCR.

RESULTSIDO and TLR1-10 mRNAs were expressed in HTR-8/SVneo cells. As the cell culture time extended, IDO mRNA expression level tended to increase within 48 h. After stimulation with the TLR ligands, the expression of TLR-3 mRNA was down-regulated while the expression of TLR-4, 7, 8, and 9 mRNA up-regulated. Stimulation of the cells with poly(I:C) lowered the expression of IDO mRNA while IFN-γ increased its expression.

CONCLUSIONSThe expression of IDO mRNA is associated with the nutrition of the maternal-fetal interface. Stimulation with the TLR ligands affects the expression of IDO and TLR mRNA expressions in the cells, which verifies the functional activity of TLRs and suggests a role of IDO in TLR pathway-dependent antiviral immunity.

Cell Line ; Female ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; metabolism ; Interferon-gamma ; pharmacology ; Ligands ; Poly I-C ; pharmacology ; RNA, Messenger ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Trophoblasts ; cytology ; metabolism

To investigate the expressions of Fas and Fas ligand (FasL) in human placenta, we studied the expressions of Fas and FasL in placenta with RT-PCR, immunoblotting and immunostaining. We observed amplified products of Fas and FasL transcripts, the band of Fas (52 kDa) and multiple bands of FasL (42-52 kDa) in pla-centa. Fas and FasL localized mainly on fetal vessels and on syncytiotrophoblasts respectively. The differential distribution of Fas and FasL in human placenta may reflect intrinsic expressions of them by trophoblasts during differentiation. The increased expression of Fas in trophoblasts may promote apoptosis of placenta in pathologic condition such as preeclampsia.
Antigens, CD95/biosynthesis/*genetics ; Fas Ligand Protein ; *Gene Expression ; Gene Expression Profiling ; Glycosylation ; Humans ; Immunoblotting/methods ; Membrane Glycoproteins/biosynthesis/*genetics ; Placenta/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Trophoblasts/cytology/metabolism

OBJECTIVE: To investigate the role of focal adhesion kinase (FAK) in extracellular signal-regulated kinase (ERK) signaling pathway mediated invadsion of trophoblasts. METHODS: We established a human extravillous cytotrophoblasts in vitro invasion model. Different concentrations of herbimycin A(FAK inhibitor)and PD98059 (ERK inhibitor) were given to observe the influence on the growth of trophoblast cells, FAK, ERK phosphorylation, and trophoblast invasion abilities. RESULTS: The expression of phosphorylated FAK in the extravillous cytotrophoblasts (EVCT) was inhibited by herbimycin A in a concentration-dependent manner and expression of phosphorylated ERK1/2 was also partially reduced. PD98059 had no effect on the expression of phosphorylated FAK. Herbimycin A and PD98059 suppressed the in vitro invasion of EVCT to various degrees. CONCLUSION ERK signaling pathway may be the common pathway for many invasive signals,and play a key role in the regulation of trophoblast invasion.
Benzoquinones ; pharmacology ; Cell Division ; physiology ; Cell Movement ; physiology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Phosphorylation ; Rifabutin ; analogs & derivatives ; Signal Transduction ; physiology ; Trophoblasts ; cytology ; physiology

The present study aims to investigate the effects of soluble endoglin (sEng) on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy. Cytotrophoblasts of normal 6 to 8-week pregnancy were cultured by trypsin digestion method, and were incubated with cell culture medium without (control group) and with 10 μg/L sEng (sEng group), respectively for 24 h. The invasive ability was determined by transwell invasion assay, and expressions of MMP-2, MMP-9 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that the invasive ability of cytotrophoblasts in sEng group was lower than that in control group (P < 0.05). Compared with control group, the expressions of MMP-2 and MMP-9 mRNA and protein of cytotrophoblasts were significantly lower (P < 0.05). In conclusion, sEng may participate in the genesis of preeclampsia by affecting the invasive ability of cytotrophoblasts through regulation of the expression of MMP-2 and MMP-9.
Antigens, CD ; pharmacology ; Cell Movement ; drug effects ; physiology ; Cells, Cultured ; Endoglin ; Female ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Placentation ; physiology ; Pre-Eclampsia ; physiopathology ; Pregnancy ; Pregnancy Trimester, First ; RNA, Messenger ; genetics ; metabolism ; Receptors, Cell Surface ; Trophoblasts ; cytology