Yin Zhao

dNK derived IFN-γ mediates VSMC migration and apoptosis via the induction of LncRNA MEG3: A role in uterovascular transformation

INTRODUCTION Appropriate spiral artery remodeling is critical for successful fetal development and pregnancy outcomes. The vascular smooth muscle cell (VSMC) loss and separation, involving cell apoptosis and migration, plays an important role in this process. Decidual natural killer cells (dNK)-derived interferon gamma (IFN-γ), a key regulator of uterine arterial remodeling, can facilitate separation of VSMC layers, however, the specific mechanisms of it action are unknown. Long non-coding RNA MEG3 functions as tumor suppressor by regulating apoptosis and migration. Moreover, IFN-γ has been shown to influence cell vitality through regulating MEG3 expression. However, the functional role of dNK derived IFN-γ and MEG3 on VSMC viability, as well as the relationship between IFN-γ and MEG3 in VSMCs, has not been completely elaborated. METHODS The up-regulation strategies and reagent treatment were employed to detect the effects of MEG3 and dNK/IFN-γ on VSMC proliferation, apoptosis and migration. At the same time, MEG3, p53 and matrix metalloproteinase 2 (MMP-2) expressions were investigated. RESULTS dNK/IFN-γ treatment led to up-regulation of MEG3 expression in VSMCs. Both MEG3 over-expression and dNK/IFN-γ treatment inhibited VSMC proliferation, stimulated VSMC migration and resulted in a small but significant induction of VSMC apoptosis, as well as promoted p53 and MMP-2 expression in VSMCs. DISCUSSION MEG3 is regulated by dNK-derived IFN-γ and regulates VSMC migration and apoptosis. Therefore, it may be an important positive regulator in VSMC loss from the maternal uterine spiral arteries during vascular transformation.

Edaravone protects against cobalt chloride-induced dysfunctions in apoptosis and invasion in trophoblast cells

This study was performed to investigate the effects of edaravone on hypoxia‐induced trophoblast cell proliferation, apoptosis, and invasion. The trophoblast cell line HTR‐8 (H8) was treated with cobalt chloride (CoCl2) with or without a 1‐hr edaravone pretreatment, followed by assessment of intracellular reactive oxygen species (ROS) levels, cell proliferation, apoptosis, migration, and invasion. Metrics of apoptosis included measurement of cysteine‐aspartic acid protease 3 (CASP3) activity as well as BAX and BCL2 expression. Migration and invasion phenotypes were complemented with expression analysis of matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2) at the transcript and protein levels. CoCl2 inhibited the proliferation of H8 cells, promoted apoptosis, and up‐regulated CASP3 activation and BAX expression while inhibiting BCL2 expression. CoCl2 treatment also reduced the invasiveness of H8 cells by inhibiting MMP2 activity. Edaravone significantly increased H8 cell proliferation; inhibited apoptosis by down‐regulating CASP3 activation and BAX production while promoting BCL2 stability; and ameliorated the migration and invasion phenotypes associated with CoCl2 treatment. These results suggest that edaravone may rescue hypoxia‐induced abnormalities in H8 cell proliferation, apoptosis, and invasion, thereby protecting the trophoblast lineage against hypoxia. Mol. Reprod. Dev. 83: 576–587, 2016.

EPHB4, a down stream target of IFN‐γ/STAT1 signal pathway, regulates endothelial activation possibly contributing to the development of preeclampsia

Preeclampsia is characterized by endothelial activation and excessive inflammation, of which interferon (IFN)‐γ is a potent inducer. Eph receptor B4 (EPHB4) also involved in endothelial activation in inflammation. Their role and relationship in preeclampsia remain unclear.

Idiopathic polyhydramnios at term and pregnancy outcomes: a multicenter observational study

Abstract Aim: To assess pregnancy outcomes in pregnancies with idiopathic polyhydramnios at term. Methods: We conducted a retrospective cohort study of 106 225 term pregnancies from 37 hospitals in China. Maternal and fetal outcomes in pregnancies with idiopathic polyhydramnios were compared with pregnancies with normal amniotic fluid. The primary outcome was intra-uterine fetal death (IUFD). Results: In all, 307 out of 106 225 (0.3%) had idiopathic polyhydramnios at term, 276 of which were mild and 31 of which were moderate-severe. Compared to term pregnancies with normal amniotic fluid, pregnancies idiopathic polyhydramnios was associated with over 24-fold higher risk for IUFD (adjusted odds ratio [aOR] 24.4, 95% confidence interval (CI) 7.3–82.0), macrosomia (aOR 2.8, 95%CI 2.0–3.8), malpresentation (aOR 2.5, 95%CI 1.7–3.7), cesarean delivery (aOR 2.5, 95%CI 1.7–3.7) and low APGAR scores at 5 min (aOR 4.3, 95%CI 2.4–7.8), which increased with severity of idiopathic polyhydramnios. Conclusion: Term pregnancies with idiopathic polyhydramnios, especially moderate–severe ones are at a significantly increased rate for adverse pregnancy outcome. Increased antepartum surveillance of fetal well-being and timed delivery are warranted.

EPHB4 Regulates Human Trophoblast Cell Line HTR-8/SVneo Function: Implications for the Role of EPHB4 in Preeclampsia

ABSTRACT Successful pregnancy depends on well-regulated extravillous trophoblast (EVT) invasion into the uterine decidua and moderate uterine spiral artery remodeling. Ephrin receptor B4 (EPHB4) is a membrane-anchored receptor tyrosine kinase that plays an important role in various cellular functions in human normal tissue and tumors. Reportedly, EPHB4 plays important roles during placentation. Still, there is no investigation of EPHB4 modulating trophoblast function. In our study, term placentas of preeclamptic pregnancies showed a significantly increased EPHB4 expression compared to those of uncomplicated pregnancies (n = 15). Exogenous up-regulation of EPHB4 in HTR-8/SVneo cells was performed to investigate the effects of EPHB4 on cell biological behavior. The results showed that EPHB4 enhancement reduced cell proliferation and promoted trophoblast apoptosis; and inhibited cell migration, invasion, and endothelial replacement. Associated factors, such as matrix metalloproteinases, vascular endothelial growth factor, placental growth factor, and soluble Fms-like tyrosine kinase 1 were examined at transcriptional level. Furthermore, cell functional results were confirmed in a placenta-decidua coculture system, showing poor vascular remodeling. Additionally, we detected possible down-stream PI3K-Akt signal pathway involved in EPHB4-mediated function of HTR-8/SVneo cells. Our study demonstrates that EPHB4 overexpression may contribute to trophoblasts dysfunction and impair maternal artery remodeling, as is associated with the pathogenesis of preeclampsia.

NOTCH4 signaling controls EFNB2-induced endothelial progenitor cell dysfunction in preeclampsia

Preeclampsia is a serious complication of pregnancy and is closely related to endothelial dysfunction, which can be repaired by endothelial progenitor cells (EPCs). The DLL4/NOTCH-EFNB2 (ephrinB2) cascade may be involved in the pathogenesis of preeclampsia by inhibiting the biological activity of EPCs. In addition, both NOTCH1 and NOTCH4, which are specific receptors for DLL4/NOTCH, play critical roles in the various steps of angiogenesis. However, it has not been determined which receptor (NOTCH1, NOTCH4, or both) is specific for the DLL4/NOTCH-EFNB2 cascade. Accordingly, we performed a series of investigations to evaluate it. EFNB2 expression was examined when NOTCH4 or NOTCH1 was downregulated, with or without DLL4 treatment. Then, the effects of NOTCH4 on EPC function were detected. Additionally, we analyzed NOTCH4 and EFNB2 expression in the EPCs from preeclampsia and normal pregnancies. Results showed that NOTCH4 downregulation led to decreased expression of EFNB2, which maintained the same level in the presence of DLL4/NOTCH activation. By contrast, NOTCH1 silencing resulted in a moderate increase in EFNB2 expression, which further increased in the presence of DLL4/NOTCH activation. The downregulation of NOTCH4 resulted in an increase of EPC biological activity, which was similar to EFNB2 silencing. NOTCH4 expression, consistent with the EFNB2 level, increased notably in preeclampsia EPCs compared with the controls. These findings suggest that NOTCH4, not NOTCH1, is the specific receptor for the DLL4/NOTCH-EFNB2 cascade. Blockade of this cascade may enhance the angiogenic property of EPCs, and act as a potential target to promote angiogenesis in patients with preeclampsia.

Inhibiting trophoblast PAR-1 overexpression suppresses sFlt-1-induced anti-angiogenesis and abnormal vascular remodeling: a possible therapeutic approach for preeclampsia

STUDY QUESTION Is it possible to improve vascular remodeling by inhibiting the excessive expression of protease-activated receptor 1 (PAR-1) in trophoblast of abnormal placenta? SUMMARY ANSWER Inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, offering an alternative therapeutic approach for preeclampsia. WHAT IS KNOWN ALREADY PAR-1 is high-affinity receptor of thrombin. Thrombin increases sFlt-1 secretion in trophoblast via the activation of PAR-1. It is reported that the expression of both thrombin and PAR-1 expression are increased in placentas of preeclampsia patients compared with normal placentas. STUDY DESIGN, SIZE, DURATION Trophoblast cells were transfected with PAR-1 short hairpin RNA (shRNA) or PAR-1 overexpression plasmids in vitro. Tube formation assays and a villus-decidua co-culture system were used to study the effect of PAR-1 inhibition on placental angiogenesis and vascular remodeling, respectively. Placentas from rats with preeclampsia were transfected with PAR-1 shRNA to confirm the effect of inhibiting PAR-1 overexpression in placenta. PARTICIPANTS/MATERIALS, SETTING, METHODS The trophoblast cell line HTR-8/SVneo was transfected with PAR-1 shRNA or PAR-1 overexpression plasmids. After 48 h, supernatant was collected and the level of sFlt-1 secretion was measured by ELISA. Human umbilical cord epithelial cells and a villus-decidua co-culture system were treated with conditioned media to study the effect of PAR-1 inhibition on tube formation and villi vascular remodeling. A preeclampsia rat model was established by intraperitoneal injection of L-NAME. Plasmids were injected into the placenta of the preeclampsia rats and systolic blood pressure was measured on Days 15 and 19. The effect of different treatments was evaluated by proteinuria, placental weights, fetal weights and fetal numbers in study and control groups. The level of serum sFlt-1 in rats with preeclampsia was also measured. Changes in the placenta microvessels were studied by histopathological staining. MAIN RESULTS AND THE ROLE OF CHANCE PAR-1 shRNA inhibited PAR-1 expression and significantly suppressed sFlt-1 expression in trophoblasts. Soluble Flt-1 level in the supernatant was suppressed by PAR-1 inhibition plasmid transfection and increased by PAR-1 overexpression plasmids (46.93 ± 5.22 vs. 25.21 ± 4.18 vs. 67.84 ± 3.58 ng/ml, P < 0.01). Tube formation assays showed that conditioned media from shPAR-1 transfected cells resulted in an increase in the total number of branching points compared with that of blank controls (P < 0.05). The villus-decidua co-culture system confirmed down-regulation of PAR-1 was conducive to angiogenesis and vascular remodeling. Transfecting placenta with PAR-1 shRNA plasmids improved placental vascular development and ameliorated the symptoms of preeclampsia in rats. After treatment with shRNA, blood pressure was controlled (140.83 ± 1.08 vs. 123.6 ± 1.47 mmHg, P < 0.001) and proteinuria levels were decreased (4.48 ± 0.36 vs. 2.64 ± 0.25 μg/μl, P < 0.01). sFlt-1 protein levels were significantly higher in preeclampsia group than in the control group (1.44 ± 0.33 vs. 2.92 ± 0.85 ng/ml, P < 0.001), but was reduced (0.92 ± 0.06 ng/ml, vs. PE, P < 0.001) in the treatment group. The histopathological changes of the placental microvessels showed that in the preeclampsia group, the number of blood vessels was reduced, while in treatment group, the placental microvasculature was improved (P < 0.001). LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Despite our promising results, the evaluation of kidney damage was studied only by proteinuria measurement. Histochemistry of kidney damage will be supplemented in a further study. WIDER IMPLICATIONS OF THE FINDINGS The data showed that inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, potentially offering an alternative therapeutic approach for preeclampsia. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81100442 and 81771605 for Y.Z. and 81179584 for L.Z.) and the Hubei Province Health and Family Planning Scientific Research Project (Grant No. WJ2017 M093 for Y.Z.). The authors declare that there is no conflict of interest.

Notch1 Impairs Endothelial Progenitor Cell Bioactivity in Preeclampsia

Aberrant vasculature and endothelial dysfunction on both the maternal and the fetal side are thought to play a role in the pathogenesis of preeclampsia, a hypertensive complication during pregnancy. Endothelial progenitor cells (EPCs) have the capacity for endothelial repair. The Dll4/Notch signaling pathway suppresses the functions of EPCs in the pathogenesis of preeclampsia. Notch1 was found to be one of the specific receptors for ligands of the Delta 4 and play critical roles in angiogenesis. However, the roles of Notch1 with regard to EPCs and preeclampsia have yet to be completely characterized. The aim of this study is to determine whether Notch1 also has a negative influence on the regulation of EPC activity. Accordingly, we analyzed the differences between the preeclampsia group and the control group in terms of the number of EPCs and colony-forming units (CFUs) and their Notch1 expressions. The influence of the Notch1 signaling pathway on functions of EPCs was determined by repeating the assays in the presence of Notch1 downregulation. The number of EPCs and CFUs was significantly lower in patients with preeclampsia compared to healthy controls. Additionally, there was a notable increase in Notch1 expression in EPCs of patients with preeclampsia compared to controls. The downregulation of Notch1 promoted the proliferation, differentiation, migration, and adhesion of EPCs and the ability to form human umbilical vein endothelial cell tubes. These findings suggested that decrease and dysfunction of EPCs may be involved in the pathogenesis of preeclampsia. Inhibition of Notch1, which promoted EPC-mediated angiogenesis in vitro, may be an alternative therapeutic approach to promoting vasculogenesis in patients with preeclampsia.