Abstract Resistin was identified as a link between obesity and insulin resistance and is associated with many diseases in mice. Deciphering the related development and molecular mechanism is necessary for the treatment of these diseases. Previous studies have revealed that increased resistin levels are correlated with lipid accumulation and play a role in non-alcoholic fatty liver disease (NAFLD) development. However, the exact mechanisms underlying these processes remain unclear. To further clarify whether acute elevated resistin level exacerbated liver steatosis, a high-fat diet-induced NAFLD animal model was used and treated with or without resistin for 6 days. We discovered that resistin altered mitochondrial morphology, decreased mitochondrial content, and increased lipid accumulation in HFD mice. qRT-PCR and western blot analysis showed that acute elevated resistin significantly altered the gene expression of mitochondrial biogenesis and liver lipid metabolism molecules in HFD mice. Consequently, in vitro experiments verified that resistin reduced the mitochondrial content, impaired the mitochondrial function and increased the lipid accumulation of palmitate-treated HepG2 cells. Additionally, we demonstrated that resistin upregulated proinflammatory factors, which confirmed that resistin promoted the development of inflammation in NAFLD mice and palmitate-treated HepG2 cells. Signaling-transduction analysis demonstrated that acute elevated resistin aggravated liver steatosis through AMPK/PGC-1α pathway in male mice. This reveals a novel pathway through which lipogenesis is induced by resistin and suggests that maintaining mitochondrial homeostasis may be key to treatments for preventing resistin-induced NAFLD aggravation.

Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT‐induced VSMC loss. In this study, we first examined the location of MEG3 in human first‐trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR‐8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT‐induced VSMC loss, such as tumor necrosis factor‐α (TNF‐α), Fas ligand (FasL), and tumor necrosis factor‐α‐related apoptosis‐inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3‐upregulated HTR‐8/SVneo to explore the role of MEG3 on EVT‐mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR‐8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR‐8/SVneo and the expression of TNF‐α, FasL and TRAIL in HTR‐8/SVneo were reduced following MEG3 overexpression in HTR‐8/SVneo. Furthermore, HTR‐8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3‐upregulated HTR‐8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT‐mediated VSMC loss.

STUDY QUESTIONIs it possible to improve vascular remodeling by inhibiting the excessive expression of protease-activated receptor 1 (PAR-1) in trophoblast of abnormal placenta?SUMMARY ANSWERInhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, offering an alternative therapeutic approach for preeclampsia.WHAT IS KNOWN ALREADYPAR-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, DURATIONTrophoblast 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, METHODSThe 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 CHANCEPAR-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 DATAN/A.LIMITATIONS, REASONS FOR CAUTIONDespite 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 FINDINGSThe 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.

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.

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-gamma), 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-gamma has been shown to influence cell vitality through regulating MEG3 expression. However, the functional role of dNK derived IFN-gamma and MEG3 on VSMC viability, as well as the relationship between IFN-gamma 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-gamma on VSMC proliferation, apoptosis and migration. At the same time, MEG3, p53 and matrix metalloproteinase 2 (MMP-2) expressions were investigated.Results: dNK/IFN-gamma treatment led to up-regulation of MEG3 expression in VSMCs. Both MEG3 over expression and dNK/IFN-gamma 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-gamma 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. (C) 2016 Elsevier Ltd. All rights reserved.

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.

PROBLEM: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.METHOD OF STUDY:Intercellular adhesion molecular (ICAM)-1 was employed as the hallmark of endothelial activation. The serum levels of IFN-γ and the expression of EPHB4 and ICAM-1 were assessed by ELISA, qRT-PCR and WB, respectively. Primary human umbilical vein endothelial cells (HUVECs) were treated with IFN-γ of different concentration or for different times to determine the effect of IFN-γ on EPHB4 and ICAM-1 expression. Overexpression and shRNA constructs, chromatin immunoprecipitation (ChIP) and luciferase assays were conducted to clarify the regulation mechanism of IFN-γ/STAT1 on EPHB4 resulting in HUVECs activation. Endothelial-trophoblast co-culture model was used to illustrate the role of EPHB4 in the process of activated endothelial cells resisting trophoblast invasion.RESULTS:IFN-γ, EPHB4 and ICAM-1 expression were elevated in preeclampsia. IFN-γ induced HUVECs activation through EPHB4 expression. ChIP and luciferase assays revealed that IFN-γ promoted EPHB4 transcription by STAT-1 binding to EPHB4 promoter. EPHB4 probably involved in resisting trophoblasts displacement by IFN-γ-activated HUVECs.CONCLUSION:This study uncovered the character of EPHB4-regulating endothelial activation in the pathogenesis of 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.

SUMMARY 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 (CoCl 2 ) 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. CoCl 2 inhibited the proliferation of H8 cells, promoted apoptosis, and up‐regulated CASP3 activation and BAX expression while inhibiting BCL2 expression. CoCl 2 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 CoCl 2 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. © 2016 Wiley Periodicals, Inc .

Axl encodes the tyrosine-protein kinase receptor, participating in the proliferation and migration of many cells. This study examined the role of Axl in functions of endothelial progenitor cells (EPCs). Axl was detected by RT-PCR and Western blotting in both placentas and EPCs from normal pregnancy and preeclampsia patients. The Axl inhibitor, BMS777-607, was used to inhibit the Axl signalling pathway in EPCs. Cell proliferation, differentiation, migration and adhesion were measured by CCK-8 assay, cell differentiation assay, Transwell assay, and cell adhesion assay, respectively. Results showed the expression levels of Axl mRNA and protein were significantly higher in both placentas and EPCs from preeclampsia patients than from normal pregnancy (P<0.05). After treatment with BMS777-607, proliferation, differentiation, migration and adhesion capability of EPCs were all significantly decreased. Our study suggests Axl may play a role in the function of EPCs, thereby involving in the pathogenesis of preeclampsia.

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.

Introduction: Preeclampsia is a hypertensive complication in pregnancy, closely related to endothelial dysfunction. Endothelial progenitor cells (EPCs) have the capacity for endothelial repair. Both Ephrin-B2 and DII4/Notch pathway play critical roles in various steps of angiogenesis. In addition, there is an up-regulation of ephrin-B2 expression consequent to DII4/Notch activation in endothelial cells (ECs). However, the roles of ephrin-B2 and DII4/Notch signaling on EPCs, as well as the relationship between them, have not been completely characterized.Methods: We analyzed expression of ephrin-B2 in the EPCs and placenta from preeclampsia and normal pregnancy. Then up-regulation and down-regulation strategies were employed to detect the effects of ephrin-B2 on EPC proliferation, differentiation, migration and HUVEC-tube formation. The effects of DII4/Notch signaling on EPCs' functions were determined by repeating the assays in the presence of DII4 or DAPT as agonists or antagonists of Notch signaling, respectively.Results: Ephrin-B2 expression increased notably in preeclampsia EPCs and placenta, compared with controls. Up-regulation of ephrin-B2 impaired EPCs' proliferation, differentiation, migration and HUVEC-tube formation capabilities. In contrast, down-regulation of ephrin-B2 in EPCs, resulted in the opposite effects. In addition, activation of DII4/Notch signaling led to increased expression of ephrin-B2 and subsequent inhibition of EPCs activity.Discussion: Ephrin-B2, a downstream of DII4/Notch signaling pathway, might be act as an inhibitor of EPC-mediated vasculogenesis in vitro, as well as a potential target in the effort to promote angiogenesis of patients with preeclampsia. (C) 2015 Elsevier Ltd. All rights reserved.