Placental tissue expresses many lymphatic markers. The current study was undertaken to examine if D2-40/podoplanin, a lymphatic endothelial marker, was expressed in the human placenta, and how it is altered developmentally and pathologically. We examined D2-40/podoplanin and VEGFR-3 expressions in placentas from normotensive pregnancies at different gestational ages and in placentas from women with clinically defined preeclampsia. D2-40 expression in systemic lymphatic vessel endothelium served as a positive control. Protein expression for D2-40, VEGFR-3, and β-actin was determined by Western blot in placentas from normotensive (n = 6) and preeclamptic (n = 5) pregnancies. Our results show that D2-40/podoplanin was strongly expressed in the placenta, mainly as a network plexus pattern in the villous stroma throughout gestation. CD31 was limited to villous core fetal vessel endothelium and VEGFR-3 was found in both villous core fetal vessel endothelium and trophoblasts. D2-40/podoplanin expression was significantly decreased, and VEGFR-3 significantly increased in preeclamptic placental tissues compared to normotensive placental controls. Placental villous stroma is a reticular-like structure, and the localization of D2-40 to the stroma suggests that a lymphatic-like conductive network may exist in the human placenta. D2-40/podoplanin is an O-linked sialoglycoprotein. Although little is known regarding biological functions of sialylated glycoproteins within the placenta, placental D2-40/podoplanin may support fetal vessel angiogenesis during placenta development and reduced D2-40/podoplanin expression in preeclamptic placenta may contribute to altered interstitial fluid homeostasis and impaired angiogenesis in this pregnancy disorder.

Increased placental release of soluble VEGF receptor-1 (sFlt-1) is believed to play an important role in the pathogenesis of preeclampsia (PE). Although the reason for increased placental sFlt-1 release in PE is unknown, proteolytic effect has been proposed as one of the mechanisms that mediate sFlt-1 release in the placenta. In this study, using various protease inhibitors, we tested the possible role of proteases in sFlt-1 release by human placenta. Villous explants from normal term placentas were incubated with various protease inhibitors including serine protease inhibitors (PMSF, aprotini, and specific chymotrypsin inhibitor (CI)), cysteine protease inhibitor E-64, metalloendopeptidase inhibitor PAD, and universal metalloprotease (ADAM) inhibitor PTM. Culture medium was collected and measured for sFlt-1 by ELISA. Our results showed that villous tissue treated with CI and PTM produced significantly less sFlt-1 than those of controls. PMSF, aprotini, E-64, and PAD had no effect on sFlt-1 release. We further examined chymotrypsin-like protease/chymase and ADAM10 expressions in tissue sections from normal and PE placentas by immunohistochemistry. We found that immunostaining for chymase and ADAM10 was significantly increased in the layer of syncytiotrophoblasts in PE placentas compared to normal placentas. These results suggest chymotrypsin-like serine protease and ADAM10, but not cysteine protease and metalloendopeptidase, may play a role in inducing sFlt-1 release in PE placentas.

Interleukin-6 (IL-6) and its receptor complex, IL-6 receptor (IL-6R) and gp130, are critical in induction of suppressor of cytokine signalling-3 (SOCS-3) protein, a negative cytokine regulator and anti-inflammatory mediator, in a biological system. Increased inflammatory response is believed to contribute to the placental dysfunction in pre-eclampsia (PE). However, it is not known if altered IL-6 receptor signalling and decreased SOCS-3 expression occur in placentas from PE. To study this, we examined IL-6, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) production by villous tissue from normal and PE placentas. Hypoxia effects on IL-6, sIL-6R and sgp130 production was determined. IL-6R, gp130 and SOCS-3 expression were determined by immunohistochemical staining and by Western blot. Our results showed that under normoxic conditions (21% O(2)), villous tissue from PE placentas produced relative more sgp130, but significantly less IL-6 and sIL-6R (p<0.01) than normal placental tissue. The ratio of sgp130/sIL-6R release was significantly higher by PE placentas than normal placentas, p<0.01. Under hypoxic conditions (2% O(2)), IL-6 production was significantly reduced by both normal (p<0.01) and PE (p<0.05) placental tissue. Hypoxia promoted sgp130 release by normal, but not by PE, placental tissue. Reduced IL-6R and SOCS-3 immunostaining and expression were found in PE placentas. We concluded that increased ratio of sgp130/sIL-6R production and/or reduced sIL-6R production combined with down-regulation of IL-6R and SOCS-3 expression in trophoblasts may lead to less cytokine inhibitory activity in PE placentas, which may account for the increased placental inflammatory response in PE.

Genistein is a phytoestrogen isolated from soya beans. Although soy products are staple food of Asian, the potential effect of genistein on reproduction has not been fully addressed. Lipopolysaccharide (LPS) is an endotoxin found in the cell membrane of gram-negative bacteria. It may cause inflammation and other immune responses. Previous study has shown that LPS may induce pre-mature birth in rodents. In the present study, effect of genistein on LPS-induced preterm birth was investigated. Pregnant ICR mice were gavaged with genistein at 40, 200 and 400 mg/kg body weight/day during E13 to E16. LPS was injected i.p. on E16.5 and the animals were sacrificed at E17. Compared to the control group, an increased incidence of early delivery was observed in the pooled mice under LPS treatment. A rising trend of incidence was also demonstrated dose-dependently with genistein co-treatment. Real-time RT-PCR indicated that the placental crh expression was highly induced by the co-administration of 400 mg/kg genistein and LPS. By contrast, neither genistein nor LPS alone could alter the expression. Increased plasma CRH concentration was also seen in the co-treatment groups. In addition, the mRNA expression of placental CRH-binding protein and plasma progesterone concentration were reduced in these groups. These results indicated that genistein might exacerbate the undesirable effect of LPS on pregnant mice by altering hormonal regulations.

The etiology and pathophysiology of preeclampsia are not fully understood. However, oxidative stress has been strongly linked to the occurrence of this multi-system disease. This has led to many theories of the pathogenesis of preeclampsia involving placental oxidative stress. In this study, we hypothesized that polymorphisms of anti-oxidant genes in the placental tissue contributed to susceptibility to preeclampsia. Polymorphisms in copper/zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (MnSOD), glutathione-S-transferase M1 (GSTM1), and glutathione-S-transferase T1 (GSTT1) in the umbilical cord tissue were assayed by polymerase chain reaction (PCR) in 23 nulliparous preeclampsia cases and 32 nulliparous normotensive controls. Corresponding enzyme activity levels and an oxidative stress biomarker (8-isoprostane) of the placental tissue were also measured. In addition, maternal plasma 8-isoprostane levels were also determined. Our results showed that no significant differences in polymorphism frequency of the tested genes, enzyme activity levels or 8-isoprostane levels in the placental tissue were detected between the cases and controls. However, maternal plasma 8-isoprostane level was significantly higher in the cases than in the controls (105.8 vs. 27.9 pg/ml, p=0.03). In conclusion, our study showed that polymorphisms of CuZn-SOD, MnSOD, GSTM1 and GSTT1 in the placental tissue were not associated with preeclampsia.

Our previous study has demonstrated that thymic stromal lymphopoietin (TSLP) stimulates trophoblast proliferation and invasion, suggesting TSLP plays an important role in the placentation in early human pregnancy, but the intracellular molecular mechanism is currently unknown. The present study is undertaken to investigate whether the STAT3-c-Myc signaling pathway is involved in TSLP-mediated trophoblast proliferation. Primary human first-trimester trophoblasts were treated with TSLP only, or TSLP combined with different signaling inhibitors (STAT3, STAT5, AKT, and ERK). The levels of STAT3 tyrosine phosphorylation and c-Myc expression were determined by using Western blot analysis, and the proliferation of trophoblasts was analyzed by BrdU cell proliferation assay. JEG-3 cells were transfected with siRNA targeting to c-Myc, and the proliferation was determined in JEG-3 cells treated with TSLP only, or TSLP combined with c-Myc silencing. It was revealed that treatment with TSLP significantly enhanced STAT3 phosphorylation and c-Myc expression in human trophoblasts. The effect of TSLP upregulation on trophoblast proliferation was abrogated completely by either STAT3 inhibitor or c-Myc siRNA silence. We further found that the upregulation of TSLP on c-Myc expression was abrogated completely by the STAT3 inhibitor, which suggests that the intracellular STAT3 phosphorylation is an upstream signal of c-Myc expression in the TSLP-stimulated trophoblast proliferation. These results suggest that TSLP may upregulate c-Myc expression through activation of STAT3 pathway, thereby inducing trophoblast proliferation.

Gestational trophoblastic disease (GTD) is a heterogeneous group of pregnancy-related disorders. Hydatidiform mole (HM) is the most common type of GTD, whereas gestational choriocarcinoma is the most aggressive. Non-receptor tyrosine kinase c-Src contributes to the transformation to a malignant phenotype in various cancers. However, the role of c-Src in the pathogenesis of GTD remains largely unknown.

Placenta-derived chymotrypsin-like protease (CLP/chymase) promotes endothelial P-selectin and E-selectin expression, which may be responsible for the increased neutrophil/endothelial interactions in preeclampsia (PE). However, little is known about this protease expression and production in human placenta. This study was undertaken to determine the distribution and gene expression of CLP in human placenta. Human placental tissues were obtained immediately after delivery from normal and PE pregnancies. We examined (1) CLP/chymase immunoactivity by immunohistochemical staining of villous tissue sections; (2) trophoblast mRNA and protein expression for chymase by RT-PCR and Western blot analysis; (3) chymase cDNA sequencing in isolated trophoblast cells (TCs); and (4) release of CLP by placental villous tissue cultured under 2% and 20% O(2). We found (1) CLP expression is mainly localized in the epithelial layer of syncytiotrophoblasts; (2) both mRNA and protein expression are significantly (p<0.05) upregulated in TCs isolated from PE vs. normal placentas; (3) TC chymase cDNA sequence and the deduced amino acid sequence are 100% identical to that reported for the human heart; and (4) villous tissue releases more chymotrypsin when cultured with 2% O(2). We conclude that (1) the DNA and protein sequence for chymase in placental trophoblast cells are the same as those reported in the human heart; (2) CLP/chymase expression is upregulated in TCs during PE; and (3) lowered oxygen condition promotes CLP release by placental TCs. Since chymase is a potent non-ACE angiotensin II producing enzyme, our data suggest that if placenta-derived CLP/chymase is released into the maternal circulation, it may contribute to the cardiovascular complications associated with PE.